Delta-5 Desaturase And Its Use In Making Polyunsaturated Fatty Acids

Abstract

Isolated nucleic acid fragments and recombinant constructs comprising such fragments encoding delta-5 desaturase along with a method of making long chain polyunsaturated fatty acids (PUFAs) using this delta-5 desaturase in plants and oleaginous yeast are disclosed.

Description

[0001] This application claims the benefit of U.S. Provisional Patent Application 60/801,119, filed May 17, 2006, the entire contents of which are herein incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention is in the field of biotechnology. More specifically, this invention pertains to the identification of nucleic acid fragments encoding a delta-5 fatty acid desaturase enzyme and the use of this desaturase in making long chain polyunsaturated fatty acids (PUFAs).

BACKGROUND OF THE INVENTION

[0003] The importance of PUFAs is undisputed. For example, certain PUFAs are important biological components of healthy cells and are recognized as: "essential" fatty acids that cannot be synthesized de novo in mammals and instead must be obtained either in the diet or derived by further desaturation and elongation of linoleic acid (LA;18:2 omega-6) or .alpha.-linolenic acid (ALA; 18:3 omega-3); constituents of plasma membranes of cells, where they may be found in such forms as phospholipids or triacylglycerols; necessary for proper development (particularly in the developing infant brain) and for tissue formation and repair; and, precursors to several biologically active eicosanoids of importance in mammals (e.g., prostacyclins, eicosanoids, leukotrienes, prostaglandins). Additionally, a high intake of long-chain omega-3 PUFAs produces cardiovascular protective effects (Dyerberg, J. et al., Amer. J. Clin. Nutr., 28:958-966 (1975); Dyerberg, J. et al., Lancet, 2(8081)117-119 (July 15, 1978); Shimokawa, H., World Rev. Nutr. Diet, 88:100-108 (2001); von Schacky, C. and Dyerberg, J., World Rev. Nutr. Diet, 88:90-99 (2001)). And, numerous other studies document wide-ranging health benefits conferred by administration of omega-3 and/or omega-6 PUFAs against a variety of symptoms and diseases (e.g., asthma, psoriasis, eczema, diabetes, cancer).

[0004] A variety of different hosts including plants, algae, fungi and yeast are being investigated as means for commercial PUFA production. Genetic engineering has demonstrated that the natural abilities of some hosts (even those natively limited to LA and ALA fatty acid production) can be substantially altered to result in high-level production of various long-chain omega-3/omega-6 PUFAs. Whether this is the result of natural abilities or recombinant technology, production of arachidonic acid (ARA; 20:4 omega-6), eicosapentaenoic acid (EPA; 20:5 omega-3) and docosahexaenoic acid (DHA; 22:6 omega-3) may all require expression of a delta-5 desaturase.

[0005] Most delta-5 desaturase enzymes identified so far have the primary ability to convert dihomo-gamma-linolenic acid (DGLA; 20:3 omega-6) to ARA, with secondary activity in converting eicosatetraenoic acid (ETA; 20:4 omega-3) to EPA (where DNA is subsequently synthesized from EPA following reaction with an additional C.sub.20/22 elongase and a delta-4 desaturase), The delta-5 desaturase has a role in both the delta-6 desaturase/delta-6 elongase pathway (which is predominantly found in algae, mosses, fungi, nematodes and humans and which is characterized by the production of gamma-linolenic acid (GLA; 18:3 omega-6) omegaand/or stearidonic acid (STA; 18:4 omega-3))omega and the delta-9 elongase/delta-8 desaturase pathway (which operates in some organisms, such as euglenoid species and which is characterized by the production of eicosadienoic acid (EDA; 20:2 omega-6)omega and/or eicosatrienoic acid (ETrA; 20:3 omega-3)) omega (FIG. 1).

[0006] Based on the role delta-5 desaturase enzymes play in the synthesis of e.g., ARA, EPA and DHA, there has been considerable effort to identify and characterize these enzymes from various sources. As such, numerous delta-5 desaturases have been disclosed in both the open literature (e.g., GenBank Accession No. AF199596, No. AF226273, No. AF320509, No. AB072976, No. AF489588, No. AJ510244, No. AF419297, No. AF07879, No. AF067654 and No. AB022097) and the patent literature (e.g., U.S. Pat. Nos. 5,972,664 and 6,075,183). Also, commonly owned, co-pending application having Provisional Application No. 60/801,172 (filed May 17, 2006) discloses amino acid and nucleic acid sequences for a delta-5 desaturase enzyme from Euglena gracilis, while commonly owned, co-pending application having Provisional Application No. 60/915733 (BB1614) (filed May 3, 2007) discloses amino acid and nucleic acid sequences for a delta-5 desaturase enzyme from Euglena anabaena.

[0007] The instant invention concerns the identification and isolation of additional genes encoding delta-5 desaturases from Peridinium sp. CCMP626 that would be suitable for heterologous expression in a variety of host organisms for use in the production of omega-3/omega-6 fatty acids.

SUMMARY OF THE INVENTION

[0008] The present invention concerns an isolated polynucleotide comprising: [0009] (a) a nucleotide sequence encoding a polypeptide having delta-5 desaturase activity, wherein the polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 01100% amino acid identity, based on the Clustal W method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:2; [0010] (b) a nucleotide sequence encoding a polypeptide having delta-5 desaturase activity, wherein the nucleotide sequence has at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity, based on the BLASTN method of alignment, when compared to a nucleotide sequence as set forth in SEQ ID NO:1 or SEQ ID NO:3;

[0011] (c) a nucleotide sequence encoding a polypeptide having delta-5 desaturase activity, wherein the nucleotide sequence hybridizes under stringent conditions to a nucleotide sequence as set forth in SEQ ID NO:1 or SEQ ID NO:3; or [0012] (d) a complement of the nucleotide sequence of (a), (b) or (c), wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.

[0013] In a second embodiment, the invention concerns a recombinant DNA construct comprising any of the isolated polynucleotides of the invention operably linked to at least one regulatory sequence.

[0014] In a third embodiment, the invention concerns a cell comprising in its genome the recombinant DNA construct of the invention. Such cells can be plant cells or yeast cells.

[0015] In a fourth embodiment, the invention concerns a method for transforming a cell, comprising transforming a cell with a recombinant construct of the invention or an isolated polynucleotide of the invention and selecting those cells transformed with the recombinant construct or the isolated polynucleotide.

[0016] In a fifth embodiment, the invention concerns transgenic seed comprising in its genome the recombinant construct of the invention or a transgenic seed obtained from a plant made by a method of the invention. Also of interest is oil or by-products obtained from such transgenic seeds. In a sixth embodiment, the invention concerns a method for making long-chain polyunsaturated fatty acids in a plant cell comprising: [0017] (a) transforming a cell with the recombinant construct of the invention; and [0018] (b) selecting those transformed cells that make long-chain polyunsaturated fatty acids.

[0019] In a seventh embodiment, the invention concerns a method for producing at least one polyunsaturated fatty acid in an oilseed plant cell comprising: [0020] (a) transforming an oilseed plant cell with a first recombinant DNA construct comprising an isolated polynucleotide encoding at least one delta-5 desaturase polypeptide, operably linked to at least one regulatory sequence and at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8 desaturase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a delta-9 desaturase, a delta-9 elongase, a C.sub.14/16 elongase, a C.sub.16/18 elongase, a C.sub.18/20 elongase and a C.sub.20/22 elongase; [0021] (b) regenerating an oilseed plant from the transformed cell of step (a); and [0022] (c) selecting those seeds obtained from the plants of step (b) having an altered level of polyunsaturated fatty acids when compared to the level in seeds obtained from a nontransformed oilseed plant.

[0023] In an eighth embodiment, the invention concerns an oilseed plant comprising in its genome the recombinant construct of the invention. Suitable oilseed plants include, but are not limited to, soybean, Brassica species, sunflower, maize, cotton, flax and safflower.

[0024] In a ninth embodiment, the invention concerns an oilseed plant comprising: [0025] (a) a first recombinant DNA construct comprising an isolated polynucleotide encoding at least one delta-5 desaturase polypeptide, operably linked to at least one regulatory sequence; and [0026] (b) at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8 desaturase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a delta-9 desaturase, a delta-9 elongase, a C.sub.14/16 elongase, a C.sub.16/18 elongase, a C.sub.16/20 elongase and a C.sub.20/22 elongase.

[0027] Also of interest are transgenic seeds obtained from such oilseed plants as well as oil or by-products obtained from these transgenic seeds. A preferred by-product is lecithin.

[0028] In a tenth embodiment, the invention concerns food or feed incorporating an oil or seed of the invention or food or feed comprising an ingredient derived from the processing of the seeds.

[0029] In an eleventh embodiment, the invention concerns progeny plants obtained from obtained from a plant made by the method of the invention or an oilseed plant of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS

[0030] FIG. 1 illustrates the omega-3/omega-6 fatty acid biosynthetic pathway.

[0031] FIG. 2 shows a chromatogram of the lipid profile of a Peridinium sp. CCMP626 cell extract as described in Example 1.

[0032] FIG. 3 shows a portion of an alignment between and among delta-5 desaturase proteins and delta-8 desaturase proteins using a Clustal W analysis (MegAlign.TM. program of DNASTAR software).

[0033] FIG. 4 graphically represents the relationship between SEQ ID NOs:1, 2, 4, 5, 6, 8 and 10, each of which relates to the Peridinium sp. CCMP626 delta-5 desaturase.

[0034] FIG. 5A illustrates the cloning strategy utilized for amplification of the Peridinium sp. CCMP626 delta-5 desaturase gene (RD5). FIG. 5B is a plasmid map of pZUF17, while FIG. 5C is a plasmid map of pDMW368.

[0035] FIG. 6 provides plasmid maps for the following: (A) pKUNF12T6E; (B) pRD5S; and, (C) pZURD5S.

[0036] FIGS. 7A and 7B show a comparison of the DNA sequence of the Peridinium sp. CCMP626 delta-5 desaturase gene (designated as "RD5"; SEQ ID NO:1) and the synthetic gene (designated as "RD5S"; SEQ ID NO:3) codon-optimized for expression in Yarrowia lipolytica.

[0037] FIGS. 8A and 8B show a Clustal V alignment (with default parameters) of a Pavlova lutheri delta-8 desaturase (SEQ ID NO:18), a Pavlova salina delta-8 desaturase (SEQ ID NO:66), a Euglena gracilis delta-8 desaturase (SEQ ID NO:16) and two different Rhizopus stolonifer delta-6 fatty acid desaturases (SEQ ID NOs:53 and 65).

[0038] FIG. 9 provides a plasmid map for pY98.

[0039] FIG. 10A provides the fatty acid profiles for Yarrowia lipolytica expressing pY98 (SEQ ID NO:76; comprising a Mortierella alpina delta-5 desaturase gene designated as "MaD5") or pDMW368 (SEQ ID NO:23; comprising the Peridinium sp. CCMP626 delta-5 desaturase gene designated as "RD5") and fed various substrates. FIG. 10B provides a comparison of the omega-3 and omega-6 substrate specificity of MaD5 versus RD5.

[0040] FIG. 11 provides plasmid maps for the following: (A) pKR916; (B) pKR1038; and, (C) pKR328.

[0041] FIG. 12A provides the average fatty acid profiles for ten events having the highest delta-5 desaturase activity when the Mortierella alpina enzyme (MaD5) is transformed into soybean embryos. FIG. 126 provides the average fatty acid profiles for ten events having the highest delta-5 desaturase activity when the Peridinium sp. CCMP626 enzyme (RD5) is transformed into soybean embryos. Fatty acids are identified as 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic acid), LA, ALA, EDA, SCI, DGLA, ARA, ERA, JUP, ETA and EPA. Fatty acids listed as "others" include: 18:2 (5,9), GLA, STA, 20:0, 20:1(11), 20:2 (7,11) or 20:2 (8,11) and DPA. Each of these "other" fatty acids is present at a relative abundance of less than 3.0% of the total fatty acids. Fatty acid compositions for an individual embryo were expressed as the weight percent (wt. %) of total fatty acids and the average fatty acid composition is an average of six individual embryos for each event.

[0042] FIG. 13 provides the activity of the delta-5 desaturase for the "correct' substrates (i.e., DGLA and ETA) versus the "wrong" substrates (i.e., EDA and ERA). The activity of the delta-5 desaturase for the "correct" substrates ("Correct % delta-5 desat") is plotted on the x-axis and the activity of the delta-5 desaturase for the "wrong" substrates ("Wrong % delta-5 desat") is plotted on the y-axis for MaD5(see FIG. 12A) or RD5 (see FIG. 126).

[0043] The invention can be more fully understood from the following detailed description and the accompanying sequence descriptions, which form a part of this application.

[0044] The following sequences comply with 37 C.F.R. .sctn.1.821-1.825 ("Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures--the Sequence Rules") and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. .sctn.1.822.

[0045] SEQ ID NOs:1-26, 50, 51, 53-56, 63-72 and 75-76 are ORFs encoding genes or proteins (or portions thereof), or plasmids, as identified in Table 1.

TABLE-US-00001 TABLE 1 Summary Of Nucleic Acid And Protein SEQ ID Numbers Nucleic acid Protein Description and Abbreviation SEQ ID NO. SEQ ID NO. Peridinium sp. CCMP626 delta-5 1 2 desaturase ("RD5") (1392 bp) (463 AA) Synthetic delta-5 desaturase, derived from 3 2 Peridinium sp. CCMP626, codon-- (1392 bp) (463 AA) optimized for expression in Yarrowia lipolytica ("RD5S") Peridinium sp. CCMP626--fragment of 4 5 pT-12-D5 (563 bp) (187 AA) Peridinium sp. CCMP626--fragment of 6 -- pT-RD5-5'C2 (693 bp) Peridinium sp. CCMP626--5' sequence 7 -- relative to SEQ ID NO: 4 (511 bp) Peridinium sp. CCMP626--fragment of 8 -- pT-RD5-5'2.sup.nd (358 bp) Peridinium sp. CCMP626--5' sequence 9 -- relative to SEQ ID NO: 6 (161 bp) Peridinium sp. CCMP626--fragment of 10 -- pT-RD5-3' (299 bp) Peridinium sp. CCMP626--3' sequence 11 -- relative to SEQ ID NO: 4 (247 bp) Pythium irregulare delta-5 desaturase -- 12 (GenBank Accession No. AAL13311) (456 AA) Phytophthora megasperma delta-5 -- 13 desaturase (GenBank Accession No. (477 AA) CAD53323) Phaeodactylum tricornutum delta-5 -- 14 desaturase (GenBank Accession No. (469 AA) AAL92562) Dictyostelium discoideum delta-5 -- 15 desaturase (GenBank Accession No. (467 AA) XP_640331) Euglena gracilis delta-8 desaturase (PCT -- 16 Publications No. WO 2006/012325 and (421 AA) No. WO 2006/012326) Pavlova lutheri (CCMP459) delta-8 17 18 desaturase (1269 bp) (423 AA) Conserved Region 1 -- 19 (7 AA) Conserved Region 2 -- 20 (7 AA) Thalassiosira pseudonana delta-8 -- 21 sphingolipid desaturase (GenBank (476 AA) Accession No. AAX14502) Plasmid pZUF17 22 -- (8165 bp) Plasmid pDMW368 23 -- (8480 bp) Plasmid pKUNF12T6E 24 -- (12,649 bp).sup. Synthetic C.sub.18/20 elongase gene derived 25 26 from Thraustochytrium aureum (U.S. (819 bp) (272 AA) Pat. No. 6,677,145), codon-optimized for expression in Yarrowia lipolytica ("EL2S") Plasmid pRD5S 50 -- (4112 bp) Plasmid pZURD5S 51 -- (8480 bp) Rhizopus stolonifer delta-6 fatty acid -- 53 desaturase (NCBI Accession No. (459 AA) AAX22052) Pavlova lutheri delta-8 desaturase-- 54 -- portion of cDNA insert from clone (695 bp) eps1c.pk002.f22 (5' end of cDNA insert) Pavlova lutheri delta-8 desaturase--fully 55 -- sequenced EST eps1c.pk002.f22: fis (full (1106 bp) insert sequence) Pavlova lutheri delta-8 desaturase-- -- 56 translation of nucleotides 1-864 of fully (287 AA) sequenced EST eps1c.pk002.f22: fis (full insert sequence; SEQ ID NO: 55) Pavlova lutheri delta-8 desaturase--full 5' 63 -- end sequence from genome walking (1294 bp) Pavlova lutheri delta-8 desaturase-- 64 -- assembled sequence (1927 bp) Rhizopus stolonifer delta-6 fatty acid -- 65 desaturase (NCBI Accession No. (459 AA) ABB96724) Pavlova salina delta-8 desaturase -- 66 (427 AA) Mortierella alpina delta-5 desaturase 67 68 (1338 bp) (446 AA) Plasmid pY5-22 69 -- (6473 bp) Plasmid pY5-22GPD 70 -- (6970 bp) Yarrowia lipolytica glyceraldehyde-3- 71 -- phosphate dehydrogenase promoter (968 bp) (GPD) Plasmid pYZDE2-S 72 -- (8630 bp) Plasmid pKR136 75 -- (6339 bp) Plasmid pY98 76 -- (8319 bp) Euglena gracilis delta-9 elongase 77 -- ("EgD9e") (774 bp) Euglena gracilis delta-8 desaturase 78 -- ("EgD8") (1263 bp) Plasmid pKR906 81 -- (4311 bp) Plasmid pKR72 82 -- (7085 bp) Plasmid pKS102 83 -- (2540 bp) Plasmid pKR197 84 -- (4359 bp) Plasmid pKR911 85 -- (5147 bp) Plasmid pKR680 86 -- (6559 bp) Plasmid pKR913 87 -- (9014 bp) Plasmid pKR767 88 -- (5561 bp) Plasmid pKR916 89 -- (11,889 bp).sup. Plasmid pKR974 90 -- (5661 bp) Saprolegnia diclina delta-5 desaturase 91 -- ("SdD5") (1413 bp) Plasmid pKR1033 92 -- (5621 bp) Plasmid pKR1038 93 -- (11,949 bp).sup. Plasmid pKR328 94 -- (8671 bp)

[0046] SEQ ID NOs:27-29 correspond to AP primer, Smart IV oligonucleotide primer and CDSIII 5' primer, respectively, used for Peridinium sp. CCMP626 cDNA synthesis.

[0047] SEQ ID NOs:30-33 correspond to degenerate oligonucleotide primers 5-1A, 5-1 B, 5-1C and 5-1D, respectively, that encode Conserved Region 1.

[0048] SEQ ID NOs:34-37 correspond to degenerate oligonucleotide primers 5-4AR, 5-4BR, 5-4CR and 5-4DR, respectively, that encode Conserved Region 2.

[0049] SEQ ID NOs:38-42 correspond to primers ODMW520, ODMW521, DNR CDS 5', ODMW541 and ODMW542, respectively, used for 5' RACE.

[0050] SEQ ID NOs:43-45 correspond to primers ODMW523, AUAP and ODMW524, respectively, used for 3' RACE.

[0051] SEQ ID NOs:46-49 correspond to primers YL807, YL810, YL808 and YL809, respectively, used for amplification of the full length cDNA of RD5.

[0052] SEQ ID NO:52 corresponds to primer 17, used for sequencing the Pavlova lutheri (CCMP459) cDNA library.

[0053] SEQ ID NOs:57 and 58 correspond to primers SeqE and SeqW, respectively, used for sequencing Pavlova lutheri (CCMP459) clones.

[0054] SEQ ID NOs:59 and 60 correspond to the universal primer AP1 and primer GSP PvDES, respectively, used for amplification of genomic Pavlova lutheri (CCMP459) DNA.

[0055] SEQ ID NOs:61 and 62 correspond to primers M13-28Rev and PavDES seq, respectively, used for sequencing Pavlova lutheri (CCMP459) genomic inserts.

[0056] SEQ ID NOs:73 and 74 are primers GPDsense and GPDantisense, respectively, used for amplifying the GPD promoter.

[0057] SEQ ID NOs:79 and 80 correspond to primers oEugEL1-1 and oEugEL1-2, respectively, used to amplify a Euglena gracilis delta-9 elongase (EgD9e).

DETAILED DESCRIPTION OF THE INVENTION

[0058] All patents, patent applications, and publications cited herein are incorporated by reference in their entirety. This specifically includes the following Applicants' Assignee's co-pending applications: U.S. Pat. No. 7,125.672, U.S. Pat. No. 7,189,559, U.S. Pat. No. 7,192,762, U.S. Pat. No. 7,198,937, U.S. Pat. No. 7,202,356, U.S. patent applications Ser. No. 10/840,579 and Ser. No. 10/840,325 (filed May 6, 2004), U.S. patent application Ser. No. 10/869,630 (filed Jun. 16, 2004), U.S. patent application Ser. No. 10/882,760 (filed Jul. 1, 2004), U.S. patent applications Ser. No. 10/985,254 and Ser. No. 10/985,691 (filed Nov. 10, 2004), U.S. patent application Ser. No. 11/024,544 (filed Dec. 29, 2004), U.S. patent application Ser. No. 11/166,993 (filed Jun. 24, 2005), U.S. patent application Ser. No. 11/183,664 (filed Jul. 18, 2005), U.S. patent application Ser. No. 11/185,301 (filed Jul. 20, 2005), U.S. patent application Ser. No. 11/190,750 (filed Jul. 27, 2005), U.S. patent application Ser. No. 11/198,975 (filed Aug. 8, 2005), U.S. patent application Ser. No. 11/225,354 (filed Sep. 13, 2005), U.S. patent application Ser. No. 11/253,882 (filed Oct. 19, 2005), U.S. patent applications Ser. No. 11/264784 and Ser. No. 11/264,737 (filed Nov. 1, 2005), U.S. patent application Ser. No. 11/265761 (filed Nov. 2, 2005), U.S. patent application Ser. No. 11/737,772 (filed Apr. 20, 2007), U.S. patent application Ser. No. 11/787,772 (filed Apr. 17, 2007), U.S. patent application Ser. No. 11/740,298 (filed Apr. 26, 2007), U.S. Patent Applications No. 60/801,172 and No. 60/801,119 (filed May 17, 2006), U.S. Patent Application No. 60/853,563 (filed Oct. 23, 2006), U.S. Patent Application No. 60/855,177 (filed Oct. 30, 2006), U.S. patent applications Ser. No. 11/601,563 and Ser. No. 11/601,564 (filed Nov. 16, 2006), U.S. patent application Ser. No. 11/635,258 (filed Dec. 7, 2006), U.S. patent application Ser. No. 11/613,420 (filed Dec. 20, 2006), U.S. Patent Application No. 60/909,790 (filed Apr. 3, 2007), U.S. Patent Application No. 60/911,925 (filed Apr. 16, 2007), U.S. Patent Application No. 60/910,831 (filed Apr. 10, 2007) and U.S. Patent Application No. 60/915,733 (BB1614) (filed May 3, 2007). This additionally includes the following Applicants' Assignee's co-pending applications: PCT Publication No. US 2004/0172682, concerning the production of PUFAs in plants; and, U.S. Pat. No. 7,129,089, concerning annexin promoters and their use in expression of transgenes in plants.

[0059] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a plant" includes a plurality of such plants, reference to "a cell" includes one or more cells and equivalents thereof known to those skilled in the art, and so forth.

[0060] In accordance with the subject invention, Applicants identify a novel Peridinium sp. CCMP626 delta-5 desaturase enzyme and gene encoding the same that may be used for the manipulation of biochemical pathways for the production of healthful PUFAs. Thus, the subject invention finds many applications.

[0061] PUFAs, or derivatives thereof, made by the methodology disclosed herein can be used as dietary substitutes, or supplements, particularly infant formulas, for patients undergoing intravenous feeding or for preventing or treating malnutrition. Alternatively, the purified PUFAs (or derivatives thereof) may be incorporated into cooking oils, fats or margarines formulated so that in normal use the recipient would receive the desired amount for dietary supplementation. The PUFAs may also be incorporated into infant formulas, nutritional supplements or other food products and may find use as anti-inflammatory or cholesterol lowering agents. Optionally, the compositions may be used for pharmaceutical use (human or veterinary).

[0062] Supplementation of humans or animals with PUFAs produced by recombinant means can result in increased levels of the added PUFAs, as well as their metabolic progeny. For example, treatment with EPA can result not only in increased levels of EPA, but also downstream products of EPA such as eicosanoids (i.e., prostaglandins, leukotrienes, thromboxanes). Complex regulatory mechanisms can make it desirable to combine various PUFAs, or add different conjugates of PUFAs, in order to prevent, control or overcome such mechanisms to achieve the desired levels of specific PUFAs in an individual.

Definitions

[0063] In this disclosure, a number of terms and abbreviations are used. The following definitions are provided.

[0064] "Open reading frame" is abbreviated ORF.

[0065] "Polymerase chain reaction" is abbreviated PCR.

[0066] "American Type Culture Collection" is abbreviated ATCC.

[0067] "Polyunsaturated fatty acid(s)" is abbreviated PUFA(s).

[0068] "Triacylglycerols" are abbreviated TAGS.

[0069] The term "invention" or "present invention" as used herein is not meant to be limiting to any one specific embodiment of the invention but applies generally to any and all embodiments of the invention as described in the claims and specification.

[0070] The term "fatty acids" refers to long chain aliphatic acids (alkanoic acids) of varying chain lengths, from about C.sub.12 to C.sub.22 (although both longer and shorter chain-length acids are known). The predominant chain lengths are between C.sub.16 and C.sub.22. The structure of a fatty acid is represented by a simple notation system of "X:Y", where X is the total number of carbon (C) atoms in the particular fatty acid and Y is the number of double bonds. Additional details concerning the differentiation between "saturated fatty acids" versus "unsaturated fatty acids", "monounsaturated fatty acids" versus "polyunsaturated fatty acids" (or "PUFAs"), and "omega-6 fatty acids" (omega-6 or n-6) versus "omega-3 fatty acids" (omega-3 or n-3) are provided in U.S. Patent Publication No, 2005/0136519.

[0071] Fatty acids are described herein by a simple notation system of "X:Y", wherein X is number of carbon (C) atoms in the particular fatty acid and Y is the number of double bonds. The number following the fatty acid designation indicates the position of the double bond from the carboxyl end of the fatty acid with the "c" affix for the cis-configuration of the double bond (e.g., palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1, 9c), petroselinic acid (18:1, 6c), LA (18:2, 9c,12c), GLA (18:3, 6c,9c,12c) and ALA (18:3, 9c,12c,15c)). Unless otherwise specified, 18:1, 18:2 and 18:3 refer to oleic, LA and ALA fatty acids, respectively. If not specifically written as otherwise, double bonds are assumed to be of the cis configuration. For instance, the double bonds in 18:2 (9,12) would be assumed to be in the cis configuration.

[0072] Nomenclature used to describe PUFAs in the present disclosure is shown below in Table 2. In the column titled "Shorthand Notation", the omega-reference system is used to indicate the number of carbons, the number of double bonds and the position of the double bond closest to the omega carbon, counting from the omega carbon (which is numbered 1 for this purpose). The remainder of the Table summarizes the common names of omega-3 and omega-6 fatty acids and their precursors, the abbreviations that will be used throughout the specification and each compounds' chemical name.

TABLE-US-00002 TABLE 2 Nomenclature of Polyunsaturated Fatty Acids And Precursors Shorthand Common Name Abbreviation Chemical Name Notation Myristic -- tetradecanoic 14:0 Palmitic Palmitate hexadecanoic 16:0 Palmitoleic -- 9-hexadecenoic 16:1 Stearic -- octadecanoic 18:0 Oleic -- cis-9-octadecenoic 18:1 Linoleic LA cis-9,12-octadecadienoic 18:2 omega-6 .gamma.-Linoleic GLA cis-6,9,12- 18:3 omega-6 octadecatrienoic Eicosadienoic EDA cis-11,14-eicosadienoic 20:2 omega-6 Dihomo-.gamma.- DGLA cis-8,11,14- 20:3 omega-6 Linoleic eicosatrienoic Arachidonic ARA cis-5,8,11,14- 20:4 omega-6 eicosatetraenoic .alpha.-Linolenic ALA cis-9,12,15- 18:3 omega-3 octadecatrienoic Stearidonic STA cis-6,9,12,15- 18:4 omega-3 octadecatetraenoic Eicosatrienoic ETrA or cis-11,14,17- 20:3 omega-3 ERA eicosatrienoic Sciadonic SCI cis-5,11,14-eicosatrienoic 20:3b omega-6 Juniperonic JUP cis-5,11,14,17- 20:4b omega-3 eicosatetraenoic Eicosa- ETA cis-8,11,14,17- 20:4 omega-3 tetraenoic eicosatetraenoic Eicosa- EPA cis-5,8,11,14,17- 20:5 omega-3 pentaenoic eicosapentaenoic Docosa- DPA cis-7,10,13,16,19- 22:5 omega-3 pentaenoic docosapentaenoic Docosa- DHA cis-4,7,10,13,16,19- 22:6 omega-3 hexaenoic docosahexaenoic

[0073] The terms "triacylglycerol", "oil" and "TAGs" refer to neutral lipids composed of three fatty acyl residues esterified to a glycerol molecule (and such terms will be used interchangeably throughout the present disclosure herein). Such oils can contain long chain PUFAs, as well as shorter saturated and unsaturated fatty acids and longer chain saturated fatty acids. Thus, "oil biosynthesis" generically refers to the synthesis of TAGs in the cell.

[0074] "Percent (%) PUFAs in the total lipid and oil fractions" refers to the percent of PUFAs relative to the total fatty acids in those fractions. The term "total lipid fraction" or "lipid fraction" both refer to the sum of all lipids (i.e., neutral and polar) within an oleaginous organism, thus including those lipids that are located in the phosphatidylcholine (PC) fraction, phosphatidyletanolamine (PE) fraction and triacylglycerol (TAG or oil) fraction. However, the terms "lipid" and "oil" will be used interchangeably throughout the specification.

[0075] A metabolic pathway, or biosynthetic pathway, in a biochemical sense, can be regarded as a series of chemical reactions occurring within a cell, catalyzed by enzymes, to achieve either the formation of a metabolic product to be used or stored by the cell, or the initiation of another metabolic pathway (then called a flux generating step). Many of these pathways are elaborate.sub.; and involve a step by step modification of the initial substance to shape it into a product having the exact chemical structure desired.

[0076] The term "PUFA biosynthetic pathway" refers to a metabolic process that converts oleic acid to LA, EDA, GLA, DGLA, ARA, ALA, STA, ETrA, ETA, EPA, DPA and DHA. This process is well described in the literature (e.g., see PCT Publication No. WO 2006/052870). Briefly, this process involves elongation of the carbon chain through the addition of carbon atoms and desaturation of the molecule through the addition of double bonds, via a series of special desaturation and elongation enzymes (i.e., "PUFA biosynthetic pathway enzymes") present in the endoplasmic reticulin membrane. More specifically, "PUFA biosynthetic pathway enzymes" refer to any of the following enzymes (and genes which encode said enzymes) associated with the biosynthesis of a PUFA, including: a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a delta-9 desaturase, a delta-8 desaturase, a delta-9 elongase, a C.sub.14/16 elongase, a C.sub.16/18 elongase, a C.sub.18/20 elongase and/or a C.sub.20/22 elongase.

[0077] The term "omega-3/omega-6 fatty acid biosynthetic pathway" refers to a set of genes which, when expressed under the appropriate conditions encode enzymes that catalyze the production of either or both omega-3 and omega-6 fatty acids. Typically the genes involved in the omega-3/omega-6 fatty acid biosynthetic pathway encode PUFA biosynthetic pathway enzymes. A representative pathway is illustrated in FIG. 1, providing for the conversion of myristic acid through various intermediates to DHA, which demonstrates how both omega-3 and omega-6 fatty acids may be produced from a common source. The pathway is naturally divided into two portions where one portion will generate omega-3 fatty acids and the other portion, only omega-6 fatty acids. That portion that only generates omega-3 fatty acids will be referred to herein as the omega-3 fatty acid biosynthetic pathway, whereas that portion that generates only omega-6 fatty acids will be referred to herein as the omega-6 fatty acid biosynthetic pathway.

[0078] The term "functional" as used herein in context with the omega-3/omega-6 fatty acid biosynthetic pathway means that some (or all) of the genes in the pathway express active enzymes, resulting in in vivo catalysis or substrate conversion. It should be understood that "omega-3/omega-6 fatty acid biosynthetic pathway" or "functional omega-3/omega-6 fatty acid biosynthetic pathway" does not imply that all the genes listed in the above paragraph are required, as a number of fatty acid products will only require the expression of a subset of the genes of this pathway.

[0079] The term "delta-6 desaturase/delta-6 elongase pathway" will refer to a PUFA biosynthetic pathway that minimally includes at least one delta-6 desaturase and at least one C.sub.18/20 elongase, thereby enabling biosynthesis of DGLA and/or ETA from LA and ALA, respectively, with GLA and/or STA as intermediate fatty acids. With expression of other desaturases and elongases, ARA, EPA, DPA and DHA may also be synthesized.

[0080] The term "delta-9 elongase/delta-8 desaturase pathway" will refer to a PUFA biosynthetic pathway that minimally includes at least one delta-9 elongase and at least one delta-8 desaturase, thereby enabling biosynthesis of DGLA and/or ETA from LA and ALA, respectively, with EDA and/or ETrA as intermediate fatty acids. With expression of other desaturases and elongases, ARA, EPA, DPA and DHA may also be synthesized.

[0081] The term "intermediate fatty acid" refers to any fatty acid produced in a fatty acid metabolic pathway that can be further converted to an intended product fatty acid in this pathway by the action of other metabolic pathway enzymes. For instance, when EPA is produced using the delta-9 elongase/delta-8 desaturase pathway, EDA, ETrA, DGLA, ETA and ARA can be produced and are considered "intermediate fatty acids" since these fatty acids can be further converted to EPA via action of other metabolic pathway enzymes.

[0082] The term "by-product fatty acid" refers to any fatty acid produced in a fatty acid metabolic pathway that is not the intended fatty acid product of the pathway nor an "intermediate fatty acid" of the pathway. For instance, when EPA is produced using the delta-9 elongase/delta-8 desaturase pathway, sciadonic acid (SGI) and juniperonic acid (JUP) also can be produced by the action of a delta-5 desaturase on either EDA or ETrA, respectively. They are considered to be "by-product fatty acids" since neither can be further converted to EPA by the action of other metabolic pathway enzymes.

[0083] The term "desaturase" refers to a polypeptide that can desaturate, i.e., introduce a double bond, in one or more fatty acids to produce a fatty acid or precursor of interest. Despite use of the omega-reference system throughout the specification to refer to specific fatty acids, it is more convenient to indicate the activity of a desaturase by counting from the carboxyl end of the substrate using the delta-system. Of particular interest herein are delta-5 desaturases that catalyze the conversion of DGLA to ARA and/or ETA to EPA. Other desaturases include: 1.) delta-17 desaturases that desaturate a fatty acid between the 17th and 18th carbon atom numbered from the carboxyl-terminal end of the molecule and which, for example, catalyze the conversion of ARA to EPA and/or DGLA to ETA; 2.) delta-6 desaturases that catalyze the conversion of LA to GLA and/or ALA to STA; 3.) delta-12 desaturases that catalyze the conversion of oleic acid to LA; 4.) delta-15 desaturases that catalyze the conversion of LA to ALA and/or GLA to STA; 5.) delta-4 desaturases that catalyze the conversion of DPA to DHA; 6.) delta-8 desaturases that catalyze the conversion of EDA to DGLA and/or ETrA to ETA; and, 7.) delta-9 desaturases that catalyze the conversion of palmitate to palmitoleic acid (16:1) and/or stearate to oleic acid. In the art, delta-15 and delta-17 desaturases are also occasionally referred to as "omega-3 desaturases", "w-3 desaturases", and/or "omega-3 desaturases", based on their ability to convert omega-6 fatty acids into their omega-3 counterparts (e.g., conversion of LA into ALA and ARA into EPA, respectively). In some embodiments, it is most desirable to empirically determine the specificity of a particular fatty acid desaturase by transforming a suitable host with the gene for the fatty acid desaturase and determining its effect on the fatty acid profile of the host.

[0084] The term "delta-5 desaturase" refers to an enzyme that desaturates a fatty acid between the fifth and sixth carbon atom numbered from the carboxyl-terminal end of the molecule. Preferably, a delta-5 desaturase converts dihomo-gamma-linolenic acid [20:3, DGLA] to arachidonic acid [20:4, ARA] or converts eicosatetraenoic acid [20:4, ETA] to eicosapentaenoic acid [20:5, EPA].

[0085] For the purposes herein, the term "RD5" refers to a delta-5 desaturase enzyme (SEQ ID NO:2) isolated from Peridinium sp. CCMP626, encoded by SEQ ID NO:1 herein. Similarly, the term "RD5S" refers to a synthetic delta-5 desaturase derived from Peridinium sp. CCMP626 that is codon-optimized for expression in Yarrowia lipolytica (i.e., SEQ ID NOs:3 and 2).

[0086] The terms "conversion efficiency" and "percent substrate conversion" refer to the efficiency by which a particular enzyme (e.g., a desaturase) can convert substrate to product. The conversion efficiency is measured according to the following formula: ([product]/[substrate+product])*100, where `product` includes the immediate product and all products in the pathway derived from it.

[0087] The term "elongase" refers to a polypeptide that can elongate a fatty acid carbon chain to produce an acid that is 2 carbons longer than the fatty acid substrate that the elongase acts upon. This process of elongation occurs in a multi-step mechanism in association with fatty acid synthase, as described in U.S. Patent Publication No. 2005/0132442 and PCT Publication No. WO 2005/047480. Examples of reactions catalyzed by elongase systems are the conversion of GLA to DGLA, STA to ETA and EPA to DPA. In general, the substrate selectivity of elongases is somewhat broad but segregated by both chain length and the degree and type of unsaturation. For example, a C.sub.14/16 elongase will utilize a C.sub.14 substrate (e.g., myristic acid), a C.sub.16/18 elongase will utilize a C.sub.16 substrate (e.g., palmitate), a C.sub.18/20 elongase (also known as a delta-6 elongase as the terms can be used interchangeably) will utilize a C.sub.16 substrate (e.g., GLA, STA) and a C.sub.20/22 elongase will utilize a C.sub.20 substrate (e.g., EPA). in like manner, a delta-9 elongase is able to catalyze the conversion of LA and ALA to EDA and ETrA, respectively. It is important to note that some elongases have broad specificity and thus a single enzyme may be capable of catalyzing several elongase reactions (e.g., thereby acting as both a C.sub.16/18 elongase and a C.sub.18/20 elongase).

[0088] The term "oleaginous" refers to those organisms that tend to store their energy source in the form of lipid (Weete, In: Fungal Lipid Biochemistry, 2.sup.nd Ed., Plenum, 1980). The term "oleaginous yeast" refers to those microorganisms classified as yeasts that can make oil. Generally, the cellular oil or TAG content of oleaginous microorganisms follows a sigmoid curve, wherein the concentration of lipid increases until it reaches a maximum at the late logarithmic or early stationary growth phase and then gradually decreases during the late stationary and death phases (Yongmanitchai and Ward, Appl. Environ. Microbial., 57:419-25 (1991)). It is not uncommon for oleaginous microorganisms to accumulate in excess of about 25% of their dry cell weight as oil. Examples of oleaginous yeast include, but are no means limited to, the following genera: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.

[0089] The term "conservative amino acid substitution" refers to a substitution of an amino acid residue in a given protein with another amino acid, without altering the chemical or functional nature of that protein. For example, it is well known in the art that alterations in a gene that result in the production of a chemically equivalent amino acid at a given site (but that do not affect the structural and functional properties of the encoded, folded protein) are common. For the purposes of the present invention, "conservative amino acid substitutions" are defined as exchanges within one of the following five groups: [0090] 1. small aliphatic, nonpolar or slightly polar residues: Ala [A], Ser [S], Thr [T] (Pro [P], Gly [G]); [0091] 2. polar, negatively charged residues and their amides: Asp [D], Asn [N], Glu [E], Gln [Q]; [0092] 3. polar, positively charged residues: His [H], Arg [R], Lys [K]; [0093] 4. large aliphatic, nonpolar residues: Met [M], Leu [I], lie [I], Val M (Cys [C]) ; and, [0094] 5. large aromatic residues: Phe [F], Tyr [Y], Trp [W]. Conservative amino acid substitutions generally maintain: 1) the structure of the polypeptide backbone in the area of the substitution; 2) the charge or hydrophobicity of the molecule at the target site; or 3) the bulk of the side chain, Additionally, in many cases, alterations of the N-terminal and C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein.

[0095] As used herein, "nucleic acid" means a polynucleotide and includes single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases. Nucleic acids may also include fragments and modified nucleotides. Thus, the terms "polynucleotide", "nucleic acid sequence", "nucleotide sequence" or "nucleic acid fragment" are used interchangeably and is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. Nucleotides (usually found in their 5'-monophosphate form) are referred to by their single letter designation as follows: "A" for adenylate or deoxyadenylate (for RNA or DNA, respectively), "C" for cytidylate or deosycytidylate, "G" for guanylate or deoxyguanylate, "U" for uridlate, "T" for deosythymidylate, "R" for purines (A or G), "Y" for pyrimidiens (C or T), "K" for G or T, "H" for A or C or T, "I" for inosine, and "N" for any nucleotide.

[0096] The terms "subfragment that is functionally equivalent" and "functionally equivalent subfragment" are used interchangeably herein. These terms refer to a portion or subsequence of an isolated nucleic acid fragment in which the ability to alter gene expression or produce a certain phenotype is retained whether or not the fragment or subfragment encodes an active enzyme. For example, the fragment or subfragment can be used in the design of chimeric genes to produce the desired phenotype in a transformed plant. Chimeric genes can be designed for use in suppression by linking a nucleic acid fragment or subfragment thereof, whether or not it encodes an active enzyme, in the sense or antisense orientation relative to a plant promoter sequence.

[0097] The term "conserved domain" or "motif" means a set of amino acids conserved at specific positions along an aligned sequence of evolutionarily related proteins. While amino acids at other positions can vary between homologous proteins, amino acids that are highly conserved at specific positions indicate amino acids that are essential in the structure, the stability, or the activity of a protein. Because they are identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers, or "signatures", to determine if a protein with a newly determined sequence belongs to a previously identified protein family.

[0098] The terms "homology", "homologous", "substantially similar" and "corresponding substantially" are used interchangeably herein. They refer to nucleic acid fragments wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype. These terms also refer to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotides that do not substantially alter the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment. It is therefore understood, as those skilled in the art will appreciate, that the invention encompasses more than the specific exemplary sequences.

[0099] Moreover, the skilled artisan recognizes that substantially similar nucleic acid sequences encompassed by this invention are also defined by their ability to hybridize (under moderately stringent conditions, e.g., 0.5.times.SSC, 0.1% SDS, 60.degree. C.) with the sequences exemplified herein, or to any portion of the nucleotide sequences disclosed herein and which are functionally equivalent to any of the nucleic acid sequences disclosed herein. Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions.

[0100] The term "selectively hybridizes" includes reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids. Selectively hybridizing sequences typically have about at least 80% sequence identity, or 90% sequence identity, up to and including 100% sequence identity (i.e., fully complementary) with each other.

[0101] The term "stringent conditions" or "stringent hybridization conditions" includes reference to conditions under which a probe will selectively hybridize to its target sequence. Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, optionally less than 500 nucleotides in length.

[0102] Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30.degree. C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60.degree. C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SOS (sodium dodecyl sulphate) at 37.degree. C., and a wash in 1X to 2.times.SSC (20.times.SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55.degree. C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in 0.5.times. to 1.times.SSC at 55 to 60.degree. C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SOS at 37.degree. C., and a wash in 0.1.times.SSC at 60 to 65.degree. C.,

[0103] Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the T.sub.m can be approximated from the equation of Meinkoth et al., Anal. Biochem. 138:267-284 (1984): T.sub.m=81.5.degree. C.+16.6 (log M)+0.41 (% GC) 0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The T.sub.m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. T.sub.m is reduced by about 1.degree. C. for each 1% of mismatching; thus, T.sub.m, hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with .gtoreq.90% identity are sought, the T.sub.m can be decreased 10.degree. C. Generally, stringent conditions are selected to be about 5.degree. C. lower than the thermal melting point (T.sub.m) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4.degree. C. lower than the thermal melting point (T.sub.m); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10.degree. C. lower than the thermal melting point (T.sub.m); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20.degree. C. lower than the thermal melting point (T.sub.m). Using the equation, hybridization and wash compositions, and desired T.sub.m, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T.sub.m of less than 45.degree. C. (aqueous solution) or 32.degree. C. (formamide solution) it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, Part I,

[0104] Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995). Hybridization and/or wash conditions can be applied for at least 10, 30, 60, 90, 120, or 240 minutes.

[0105] A "substantial portion" of an amino acid or nucleotide sequence is that portion comprising enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Basic

[0106] Local Alignment Search Tool; Altschul, S. F., et al., J. Mol. Biol., 215:403-410 (1993)). In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers, Accordingly, a "substantial portion" of a nucleotide sequence comprises enough of the sequence to specifically identify and/or isolate a nucleic acid fragment comprising the sequence. The instant specification teaches the complete amino acid and nucleotide sequence encoding encoding particular algal proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.

[0107] The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the invention herein also includes isolated nucleic acid fragments that are complementary to the complete sequences as reported in the accompanying Sequence Listing, as well as those substantially similar nucleic acid sequences.

[0108] The terms "homology" and "homologous" are used interchangeably herein. They refer to nucleic acid fragments wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype. These terms also refer to modifications of the nucleic acid fragments of the present invention such as deletion or insertion of one or more nucleotides that do not substantially alter the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment. It is therefore understood, as those skilled in the art will appreciate, that the invention encompasses more than the specific exemplary sequences.

[0109] Moreover, the skilled artisan recognizes that homologous nucleic acid sequences encompassed by this invention are also defined by their ability to hybridize, under moderately stringent conditions (e.g., 0.5 .times.SSC, 0.1% SDS, 60.degree. C.) with the sequences exemplified herein, or to any portion of the nucleotide sequences disclosed herein and which are functionally equivalent to any of the nucleic acid sequences disclosed herein.

[0110] "Codon degeneracy" refers to the nature in the genetic code permitting 3Q variation of the nucleotide sequence without effecting the amino acid sequence of an encoded polypeptide. Accordingly, the instant invention relates to any nucleic acid fragment that encodes all or a substantial portion of the amino acid sequence encoding the instant algal polypeptide as set forth in SEQ ID NO:2. The skilled artisan is well aware of the "codon-bias" exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.

[0111] "Chemically synthesized", as related to a sequence of DNA, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of DNA may be accomplished using well-established procedures or, automated chemical synthesis can be performed using one of a number of commercially available machines. "Synthetic genes" can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form gene segments that are then enzymatically assembled to construct the entire gene. Accordingly, the genes can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell, where sequence information is available.

[0112] "Gene" refers to a nucleic acid fragment that expresses a specific protein, and that may refer to the coding region alone or may include regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. "Native gene" refers to a gene as found in nature with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. "Endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign" gene refers to a gene that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, native genes introduced into a new location within the native host, or chimeric genes. A "transgene" is a gene that has been introduced into the genome by a transformation procedure. A "codon-optimized gene" is a gene having its frequency of codon usage designed to mimic the frequency of preferred codon usage of the host cell.

[0113] "Coding sequence refers to a DNA sequence that codes for a specific amino acid sequence. "Suitable regulatory sequences" refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures.

[0114] The term "allele" refers to one of several alternative forms of a gene occupying a given locus on a chromosome. When all the alleles present at a given locus on a chromosome are the same, then that plant is homozygous at that locus. If the alleles present at a given locus on a chromosome differ, then that plant is heterozygous at that locus.

[0115] "Promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.

[0116] A promoter sequence may consist of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancer" is a DNA sequence that can stimulate promoter activity, and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro, J. K., and Goldberg, R. B., Biochemistry of Plants, 15:1-82 (1989).

[0117] "Translation leader sequence" refers to a polynucleotide sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner, R. and Foster, G. D., Mol. Biotechnol., 3:225-236 (1995)).

[0118] The terms "3' non-coding sequences" and "transcription terminator" refer to DNA sequences located downstream of a coding sequence. This includes . polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor. The 3' region can influence the transcription, RNA processing or stability, or translation of the associated coding sequence.

[0119] "RNA transcript" refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from post-transcriptional processing of the primary transcript and is referred to as the mature RNA. "Messenger RNA" or "mRNA" refers to the RNA that is without introns and that can be translated into protein by the cell. "cDNA" refers to a double-stranded DNA that is complementary to, and derived from, mRNA. "Sense" RNA refers to RNA transcript that includes the mRNA and so can be translated into protein by the cell. "Antisense RNA" refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (U.S. Pat. No. 5,107,065; PCT Publication No. WO 99/28508). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5' non-coding sequence, 3' non-coding sequence, or the coding sequence. "Functional RNA" refers to antisense RNA, ribozyme RNA, or other RNA that is not translated and yet has an effect on cellular processes.

[0120] The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

[0121] The term "expression", as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragments of the invention. Expression may also refer to translation of mRNA into a polypeptide.

[0122] "Mature" protein refers to a post-translationally processed polypeptide, i.e., one from which any pre- or propeptides present in the primary translation product have been removed. `Precursor" protein refers to the primary product of translation of mRNA, i.e., with pre- and propeptides still present. Pre- and propeptides may be (but are not limited to) intracellular localization signals.

[0123] "Transformation" refers to the transfer of a nucleic acid molecule into a host organism, resulting in genetically stable inheritance. The nucleic acid molecule may be a plasmid that replicates autonomously, for example, or, it may integrate into the genome of the host organism. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" or "recombinant" or "transformed" organisms.

[0124] The terms "plasmid", "vector" and "cassette" refer to an extra chromosomal element often carrying genes that are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA fragments. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell. "Expression cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.

[0125] The term "percent identity", as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in: 1.) Computational Molecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.) Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.) Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and, 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991).

[0126] Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the MegAlign.TM. program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences is performed using the "Clustal method of alignment" which encompasses several varieties of the algorithm including the "Clustal V method of alignment" corresponding to the alignment method labeled Clustal V (described by Higgins and Sharp, CABIOS, 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in the MegAlign.TM. program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). For multiple alignments, the default values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal V method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences using the Clustal V program, it is possible to obtain a "percent identity" by viewing the "sequence distances" table in the same program. Additionally the "Clustal W method of alignment" is available and corresponds to the alignment method labeled Clustal W (described by Higgins and Sharp, CABIOS, 5:151-153 (1989); Higgins, D. G. et al., Comput Appl. Biosci., 8:189-191(1992)) and found in the MegAlign.TM. v6.1 program of the LASERGENE bioinforrnatics computing suite (DNASTAR Inc.). Default parameters for multiple alignment correspond to GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergen Seqs (%)=30, DNA Transition VVeight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB. After alignment of the sequences using the Clustal W program, it is possible to obtain a "percent identity" by viewing the "sequence distances" table in the same program.

[0127] "BLASTN method of alignment" is an algorithm provided by the National Center for Biotechnology Information (NGBI) to compare nucleotide sequences using default parameters.

[0128] It is well understood by one skilled in the art that many levels of sequence identity are useful in identifying polypeptides, from other species, wherein such polypeptides have the same or similar function or activity. Suitable nucleic acid fragments (isolated polynucleotides of the present invention) encode polypeptides that are at least about 70% identical, preferably at least about 75% identical, and more preferably at least about 80% identical to the amino acid sequences reported herein. Preferred nucleic acid fragments encode amino acid sequences that are at least about 85% identical to the amino acid sequences reported herein. More preferred nucleic acid fragments encode amino acid sequences that are at least about 90% identical to the amino acid sequences reported herein. Most preferred are nucleic acid fragments that encode amino acid sequences that are at least about 95% identical to the amino acid sequences reported herein, Although preferred ranges are described above, any integer amino acid identity from 67% to 100% may be useful in describing the present invention, such as 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0129] Suitable nucleic acid fragments not only have the above homologies but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250 amino acids.

[0130] The term "conserved domain" or "motif" means a set of amino acids conserved at specific positions along an aligned sequence of evolutionarily related proteins. While amino acids at other positions can vary between homologous proteins, amino acids that are highly conserved at specific positions indicate amino acids that are essential in the structure, the stability, or the activity of a protein. Because they are identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers, or "signatures", to determine if a protein with a newly determined sequence belongs to a previously identified protein family.

[0131] The term "sequence analysis software" refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. "Sequence analysis software" may be commercially available or independently developed. Typical sequence analysis software will include, but is not limited to: 1.) the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol., 215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.) Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTA program incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the "default values" of the program referenced, unless otherwise specified. As used herein "default values" will mean any set of values or parameters that originally load with the software when first initialized. With regard to the BLASTP algorithm used herein default parameters will include the Robinson and Robinson amino acid frequencies (Robinson A. B., Robinson L. R., Proc. Natl Acad. Sci. U.S.A., 88:8880-8884 (1991)), the BLOSUM62 scoring matrix and the gap cost .DELTA.(g)=11+g.

[0132] The term "plant parts" includes differentiated and undifferentiated tissues including, but not limited to the following: roots, stems, shoots, leaves, pollen, seeds, tumor tissue and various forms of cells and culture (e.g., single cells, protoplasts, embryos and callus tissue). The plant tissue may be in plant or in a plant organ, tissue or cell culture.

[0133] The term "plant organ" refers to plant tissue or group of tissues that constitute a morphologically and functionally distinct part of a plant The term "genome" refers to the following: (1) the entire complement of genetic material (genes and non-coding sequences) present in each cell of an organism, or virus or organelle; (2) a complete set of chromosomes inherited as a (haploid) unit from one parent.

[0134] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989) (hereinafter "Maniatis"); by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., Current Protocols in Molecular Biolociy, published by Greene Publishing Assoc. and Wiley-Interscience, Hoboken, N.J. (1987).

[0135] The terms "recombinant construct", "expression construct", "chimeric construct", "construct", and "recombinant DNA construct" are used interchangeably herein. A recombinant construct comprises an artificial combination of nucleic acid fragments, e.g., regulatory and coding sequences that are not found together in nature. For example, a chimeric construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. Such a construct may be used by itself or may be used in conjunction with a vector. If a vector is used, then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art. For example, a plasmid vector can be used. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells comprising any of the isolated nucleic acid fragments of the invention. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al., EMBO J. 4:2411-2418 (1985); De Almeida et al., Mol. Gen. Genetics 218:78-86 (1989)), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, immunoblotting analysis of protein expression, or phenotypic analysis, among others.

[0136] The term "expression", as used herein, refers to the production of a functional end-product (e.g., a mRNA or a protein [either precursor or mature]).

[0137] The term "introduced" means providing a nucleic acid (e.g., expression construct) or protein into a cell. Introduced includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell, and includes reference to the transient provision of a nucleic acid or protein to the cell. introduced includes reference to stable or transient transformation methods, as well as sexually crossing. Thus, "introduced" in the context of inserting a nucleic acid fragment (e.g., a recombinant DNA construct/expression construct) into a cell, means "transfection" or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid fragment into a eukaryotic or prokaryotic cell where the nucleic acid fragment may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0138] "Mature" protein refers to a post-translationally processed polypeptide (i.e., one from which any pre- or propeptides present in the primary translation product have been removed). "Precursor" protein refers to the primary product of translation of mRNA (i.e., with pre- and propeptides still present). Pre- and propeptides may be but are not limited to intracellular localization signals.

[0139] "Stable transformation" refers to the transfer of a nucleic acid fragment into a genome of a host organism, including both nuclear and organellar genomes, resulting in genetically stable inheritance. In contrast, "transient transformation" refers to the transfer of a nucleic acid fragment into the nucleus, or DNA-containing organelle, of a host organism resulting in gene expression without integration or stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" organisms.

[0140] As used herein, "transgenic" refers to a plant or a cell which comprises within its genome a heterologous polynucleotide. Preferably, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of an expression construct. Transgenic is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. The term "transgenic" as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.

[0141] "Antisense inhibition" refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein. "Co-suppression" refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Pat. No. 5,231,020). Co-suppression constructs in plants previously have been designed by focusing on overexpression of a nucleic acid sequence having homology to an endogenous mRNA, in the sense orientation, which results in the reduction of all RNA having homology to the overexpressed sequence (Vaucheret et al., Plant J. 16:651-659 (1998); Gura, Nature 404:804-808 (2000)). The overall efficiency of this phenomenon is low, and the extent of the RNA reduction is widely variable. More recent work has described the use of "hairpin" structures that incorporate all, or part, of an mRNA encoding sequence in a complementary orientation that results in a potential "stem-loop" structure for the expressed RNA (PCT Publication No. WO 99/53050, published Oct. 21, 1999; PCT Publication No. WO 02100904, published Jan. 3, 2002). This increases the frequency of co-suppression in the recovered transgenic plants. Another variation describes the use of plant viral sequences to direct the suppression, or "silencing", of proximal mRNA encoding sequences (PCT Publication No. WO 98/36083, published Aug. 20, 1998). Both of these co-suppressing phenomena have not been elucidated mechanistically, although genetic evidence has begun to unravel this complex situation (Elmayan et al., Plant Cell 10:1747-1757 (1998)).

An Overview: Microbial Biosynthesis of Fatty Acids and Triacylglycerols

[0142] In general, lipid accumulation in oleaginous microorganisms is triggered in response to the overall carbon to nitrogen ratio present in the growth medium. This process, leading to the de novo synthesis of free palmitate (16:0) in oleaginous microorganisms, is described in detail in PCT Publication No. WO 2004/101757. Palmitate is the precursor of longer-chain saturated and unsaturated fatty acid derivates, which are formed through the action of elongases and desaturases (FIG. 1).

[0143] TAGs (the primary storage unit for fatty acids) are formed by a series of reactions that involve: 1.) the esterification of one molecule of acyl-CoA to glycerol-3-phosphate via an acyltransferase to produce lysophosphatidic acid; 2.) the esterification of a second molecule of acyl-CoA via an acyltransferase to yield 1,2-diacylglycerol phosphate (commonly identified as phosphatidic acid); 3.) removal of a phosphate by phosphatidic acid phosphatase to yield 1,2-diacylglycerol (DAG); and, 4.) the addition of a third fatty acid by the action of an acyltransferase to form TAG. A wide spectrum of fatty acids can be incorporated into TAGs, including saturated and unsaturated fatty acids and short-chain and long-chain fatty acids.

Biosynthesis of Omega Fatty Acids

[0144] The metabolic process wherein oleic acid is converted to omega-3/omega-6 fatty acids involves elongation of the carbon chain through the addition of carbon atoms and desaturation of the molecule through the addition of double bonds. This requires a series of special desaturation and elongation enzymes present in the endoplasmic reticulim membrane. However, as seen in FIG. 1 and as described below, there are often multiple alternate pathways for production of a specific omega-3/omega-6 fatty acid.

[0145] Specifically, all pathways require the initial conversion of oleic acid to LA, the first of the omega-6 fatty acids, by a delta-12 desaturase. Then, using the "delta-6 desaturase/delta-6 elongase pathway", omega-6 fatty acids are formed as follows: (1) LA is converted to GLA by a delta-6 desaturase; (2) GLA is converted to DGLA by a C.sub.18/20 elongase; and, (3) DGLA is converted to ARA by a delta-5 desaturase.

[0146] Alternatively, the "delta-6 desaturase/delta-6 elongase pathway" can be utilized for formation of omega-3 fatty acids as follows: (1) LA is converted to ALA, the first of the omega-3 fatty acids, by a delta-15 desaturase; (2) ALA is converted to STA by a delta-6 desaturase; (3) STA is converted to ETA by a C.sub.18/20 elongase; (4) ETA is converted to EPA by a delta-5 desaturase; (5) EPA is converted to DPA by a C.sub.20/22 elongase; and, (6) DPA is converted to DHA by a delta-4 desaturase. Optionally, omega-6 fatty acids may be converted to omega-3 fatty acids; for example, ETA and EPA are produced from DGLA and ARA, respectively, by delta-17 desaturase activity.

[0147] Alternate pathways for the biosynthesis of omega-3/omega-6 fatty acids utilize a delta-9 elongase and delta-8 desaturase. More specifically, LA and ALA may be converted to EDA and ETrA, respectively, by a delta-9 elongase; then, a delta-8 desaturase converts EDA to DGLA and/or ETrA to ETA.

[0148] It is contemplated that the particular functionalities required to be expressed in a specific host organism for production of omega-3/omega-6 fatty acids will depend on the host cell (and its native PUFA profile and/or desaturase/elongase profile), the availability of substrate, and the desired end product(s). One skilled in the art will be able to identify various candidate genes encoding each of the enzymes desired for omega-3/omega-6 fatty acid biosynthesis. Useful desaturase and elongase sequences may be derived from any source, e.g., isolated from a natural source (from bacteria, algae, fungi, plants, animals, etc.), produced via a semi-synthetic route or synthesized de novo. Although the particular source of the desaturase and elongase genes introduced into the host is not critical, considerations for choosing a specific polypeptide having desaturase or elongase activity include: 1.) the substrate specificity of the polypeptide; 2.) whether the polypeptide or a component thereof is a rate-limiting enzyme; 3.) whether the desaturase or elongase is essential for synthesis of a desired PUFA; and/or, 4.) co-factors required by the polypeptide. The expressed polypeptide preferably has parameters compatible with the biochemical environment of its location in the host cell (see PCT Publication No. WO 2004/101757 for additional details).

[0149] In additional embodiments, it will also be useful to consider the conversion efficiency of each particular desaturase and/or elongase. More specifically, since each enzyme rarely functions with 100% efficiency to convert substrate to product, the final lipid profile of un-purified oils produced in a host cell will typically be a mixture of various PUFAs consisting of the desired omega-3/omega-6 fatty acid, as well as various upstream intermediary PUFAs. Thus, each enzyme's conversion efficiency is also a variable to consider, when optimizing biosynthesis of a desired fatty acid.

[0150] With each of the considerations above in mind, candidate genes having the appropriate desaturase and elongase activities (e.g., delta-6 desaturases, C.sub.18/20 elongases, delta-5 desaturases, delta-17 desaturases, delta-15 desaturases, delta-9 desaturases, delta-12 desaturases, C.sub.14/16 elongases, C.sub.16/18 elongases, delta-9 elongases, delta-8 desaturases, delta-4 desaturases and C.sub.20/22 elongases) can be identified according to publicly available literature (e.g., GenBank), the patent literature, and experimental analysis of organisms having the ability to produce PUFAs. These genes will be suitable for introduction into a specific host organism, to enable or enhance the organism's synthesis of PUFAs.

Sequence Identification of a Novel Peridinium sp. CCMP626 Delta-5 Desaturase

[0151] In the present invention, a nucleotide sequence (SEQ ID NO:1) has been isolated from Peridinium sp. CCMP626 encoding a delta-5 desaturase (SEQ ID NO:2), designated herein as "RD5".

[0152] Comparison of the RD5 nucleotide base and deduced amino acid sequences to public databases reveals that the most similar known sequences are about 67% identical to the amino acid sequence of RD5 reported herein over a length of 463 amino acids using a Clustal W alignment method. More preferred amino acid fragments are at least about 70%-80% identical to the sequences herein, where those sequences that are at least about 80%-90% identical are particularly suitable and those sequences that are at feast about 90%-95% identical are most preferred. Similarly, preferred RD5 encoding nucleic acid sequences corresponding to the instant ORF are those encoding active proteins and which are at least about 70%-80% identical to the nucleic acid sequences of RD5 reported herein, where those sequences that are at least about 80%-90% identical are particularly suitable and those sequences that are at least about 90%-95% identical are most preferred.

[0153] In alternate embodiments, the instant RD5 desaturase sequence can be codon-optimized for expression in a particular host organism. As is well known in the art, this can be a useful means to further optimize the expression of the enzyme in the alternate host, since use of host-preferred codons can substantially enhance the expression of the foreign gene encoding the polypeptide. In general, host-preferred codons can be determined within a particular host species of interest by examining codon usage in proteins (preferably those expressed in the largest amount) and determining which codons are used with highest frequency. Then, the coding sequence for a polypeptide of interest having e.g., desaturase activity can be synthesized in whole or in part using the codons preferred in the host species.

[0154] In one preferred embodiment of the invention herein, RD5 was codon-optimized for expression in Yarrowia lipolytica. This was possible by first determining the Y. lipolytica codon usage profile (see PCT Publication No. WO 04/101757 and U.S. Pat. No. 7,125,672) and identifying those codons that were preferred. Then, for further optimization of gene expression in Y. lipolytica, the consensus sequence around the `ATG` initiation codon was determined. This optimization resulted in modification of 247 by of the 1392 by coding region (17.7%) and optimization of 229 codons of the total 463 codons (49.4%). None of the modifications in the codon-optimized gene ("RD5S"; SEQ ID NO:3) changed the amino acid sequence of the encoded protein (SEQ ID NO:2), As described in Example 11, the codon-optimized gene was 8.9% more efficient desaturating DGLA to ARA than the wildtype gene, when expressed in Y. lipolytica.

[0155] One skilled in the art would be able to use the teachings herein to create various other codon-optimized delta-5 desaturase proteins suitable for optimal expression in alternate hosts (i.e., other than Yarrowia lipolytica), based on the wildtype RD5 sequence. Accordingly, the instant invention relates to any codon-optimized delta-5 desaturase protein that is derived from the wildtype RD5 (i.e., encoded by SEQ ID NO:2). This includes, but is not limited to, the nucleotide sequence set forth in SEQ ID NO:3, which encodes a synthetic delta-5 desaturase protein (i.e., RD5S) that was codon-optimized for expression in Yarrowia lipolytica.

Identification and Isolation of Homologs

[0156] Any of the instant desaturase sequences (i.e., RD5, RD5S) or portions thereof may be used to search for delta-5 desaturase homologs in the same or other bacterial, algal, fungal, or plant species using sequence analysis software. In general, such computer software matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.

[0157] Alternatively, any of the instant desaturase sequences or portions thereof may also be employed as hybridization reagents for the identification of delta-5 homologs. The basic components of a nucleic acid hybridization test include a probe, a sample suspected of containing the gene or gene fragment of interest and a specific hybridization method. Probes of the present invention are typically single-stranded nucleic acid sequences that are complementary to the nucleic acid sequences to be detected. Probes are "hybridizable" to the nucleic acid sequence to be detected. Although the probe length can vary from 5 bases to tens of thousands of bases, typically a probe length of about 15 bases to about 30 bases is suitable. Only part of the probe molecule need be complementary to the nucleic acid sequence to be detected. In addition, the complementarity between the probe and the target sequence need not be perfect. Hybridization does occur between imperfectly complementary molecules with the result that a certain fraction of the bases in the hybridized region are not paired with the proper complementary base.

[0158] Hybridization methods are well defined. Typically the probe and sample must be mixed under conditions that will permit nucleic acid hybridization. This involves contacting the probe and sample in the presence of an inorganic or organic salt under the proper concentration and temperature conditions. The probe and sample nucleic acids must be in contact for a long enough time that any possible hybridization between the probe and sample nucleic acid may occur. The concentration of probe or target in the mixture will determine the time necessary for hybridization to occur. The higher the probe or target concentration, the shorter the hybridization incubation time needed. Optionally, a chaotropic agent may be added (e.g., guanidinium chloride, guanidinium thiocyanate, sodium thiocyanate, lithium tetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate, potassium iodide, cesium trifluoroacetate). If desired, one can add formamide to the hybridization mixture, typically 30-50% (v/v).

[0159] Various hybridization solutions can be employed. Typically, these comprise from about 20 to 60% volume, preferably 30%, of a polar organic solvent. A common hybridization solution employs about 30-50% v/v formamide, about 0.15 to 1 M sodium chloride, about 0.05 to 0.1 M buffers (e.g., sodium citrate, Tris-HCl, PIPES or HEPES (pH range about 6-9)), about 0.05 to 0.2% detergent (e.g., sodium dodecylsulfate), or between 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kdal), polyvinylpyrrolidone (about 250-500 kdal), and serum albumin. Also included in the typical hybridization solution will be unlabeled carrier nucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g., calf thymus or salmon sperm DNA, or yeast RNA), and optionally Thorn about 0.5 to 2% wt/vol glycine. Other additives may also be included, such as volume exclusion agents that include a variety of polar water-soluble or swellable agents (e.g., polyethylene glycol), anionic polymers (e.g., polyacrylate or polymethylacrylate) and anionic saccharidic polymers (e.g., dextran sulfate).

[0160] Nucleic acid hybridization is adaptable to a variety of assay formats. One of the most suitable is the sandwich assay format. The sandwich assay is particularly adaptable to hybridization under non-denaturing conditions. A primary component of a sandwich-type assay is a solid support. The solid support has adsorbed to it or covalently coupled to it immobilized nucleic acid probe that is unlabeled and complementary to one portion of the sequence.

[0161] In additional embodiments, any of the delta-5 desaturase nucleic acid fragments described herein (or any homologs identified thereof) may be used to isolate genes encoding homologous proteins from the same or other bacterial, algal, fungal, or plant species. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to: 1.) methods of nucleic acid hybridization; 2.) methods of DNA and RNA amplification, as exemplified by various uses of nucleic acid amplification technologies [e.g., polymerase chain reaction (PCR), Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR), Tabor, S. et al., Proc. Natl. Acad. Sci. U.S.A., 82:1074 (1985); or strand displacement amplification (SDA), Walker, et al., Proc. Natl. Acad. Sci. U.S.A., 89:392 (1992)]; and 3.) methods of library construction and screening by complementation.

[0162] For example, genes encoding similar proteins or polypeptides to the delta-5 desaturases described herein could be isolated directly by using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from e.g., any desired yeast or fungus using methodology well known to those skilled in the art (wherein those organisms producing ARA [or derivatives thereof] would be preferred). Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis, supra). Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan (e.g., random primers DNA labeling, nick translation or end-labeling techniques), or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part of (or full-length of) the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full-length DNA fragments under conditions of appropriate stringency.

[0163] Typically, in PCR-type amplification techniques, the primers have different sequences and are not complementary to each other. Depending on the desired test conditions, the sequences of the primers should be designed to provide for both efficient and faithful replication of the target nucleic acid. Methods of PCR primer design are common and well known in the art (Thein and Wallace, "The use of oligonucleotides as specific hybridization probes in the Diagnosis of Genetic Disorders", in Human Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986) pp 33-50, IRL: Herndon, Va.; and Rychlik, W., in Methods in Molecular Biology, White, B. A. Ed., (1993) Vol. 15, pp 31-39, PCR Protocols: Current Methods and Applications. Humania: Totowa, N.J.).

[0164] Generally two short segments of the instant sequences may be used in PCR protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. PCR may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the instant nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3' end of the mRNA precursor encoding eukaryotic genes.

[0165] Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al., Proc. Natl. Acad. Sci. U.S.A., 85:8998 (1988)) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3' or 5' end. Primers oriented in the 3' and 5' directions can be designed from the instant sequences. Using commercially available 3' RACE or 5' RACE systems (GibcoIBRL, Gaithersburg, Md.), specific 3' or 5' cDNA fragments can be isolated (Ohara et al., Proc. Natl. Acad. Sci. U.S.A., 86:5673 (1989); Loh et al., Science, 243:217 (1989)).

[0166] In other embodiments, any of the delta-5 desaturase nucleic acid fragments described herein (or any homologs identified thereof) may be used for creation of new and improved fatty acid desaturases. As is well known in the art, in vitro mutagenesis and selection, chemical mutagenesis, "gene shuffling" methods or other means can be employed to obtain mutations of naturally occurring desaturase genes. Alternatively, improved fatty acids may be synthesized by domain swapping, wherein a functional domain from any of the delta-5 desaturase nucleic acid fragments described herein are exchanged with a functional domain in an alternate desaturase gene to thereby result in a novel protein.

Methods for Production of Various Omega-3 and/or Omega-6 Fatty Acids

[0167] It is expected that introduction of chimeric genes encoding the delta-5 desaturases described herein. (i.e., RD5, RD5S or other mutant enzymes, codon-optimized enzymes or homologs thereof), under the control of the appropriate promoters will result in increased production of ARA and/or EPA in the transformed host organism, respectively. As such, the present invention encompasses a method for the direct production of PUFAs comprising exposing a fatty acid substrate (i.e., DGLA or ETA) to the desaturase enzymes described herein (e.g., RD5, RD5S), such that the substrate is converted to the desired fatty acid product (i.e., ARA or EPA, respectively).

[0168] More specifically, it is an object of the present invention to provide a method for the production of ARA in a host cell (e.g., oleaginous yeast, soybean), wherein the host cell comprises: [0169] (i) an isolated nucleotide molecule encoding a delta-5 desaturase polypeptide having at least 67% identity when compared to a polypeptide having an amino acid sequence as set forth in SEQ ID NO:2, based on BLASTP algorithms or Clustal W alignment methods; and, [0170] (ii) a source of dihomo-.gamma.-linoleic acid; wherein the host cell is grown under conditions such that the delta-5 desaturase is expressed and the DGLA is converted to ARA, and wherein the ARA is optionally recovered.

[0171] The person of skill in the art will recognize that the broad substrate range of the delta-5 desaturase may additionally allow for the use of the enzyme for the conversion of ETA to EPA. Accordingly the invention provides a method for the production of EPA, wherein the host cell comprises: [0172] (i) an isolated nucleotide molecule encoding a delta-5 desaturase polypeptide having at least 67% identity when compared to a polypeptide having an amino acid sequence as set forth in SEQ ID NO:2, based on BLASTP algorithms or Clustal W alignment methods; and, [0173] (ii) a source of ETA; wherein the host cell is grown under conditions such that the delta-5 desaturase is expressed and the ETA is converted to EPA, and wherein the EPA is optionally recovered.

[0174] Alternatively, each delta-5 desaturase gene and its corresponding enzyme product described herein can be used indirectly for the production of omega-3 fatty acids (see U.S. Patent Publication No 2005/0136519). Indirect production of omega-3/omega-6 PUFAs occurs wherein the fatty acid substrate is converted indirectly into the desired fatty acid product, via means of an intermediate step(s) or pathway intermediate(s). Thus, it is contemplated that the delta-5 desaturases described herein (e.g., RDS, RD5S or other mutant enzymes, codon-optimized enzymes or homologs thereof) may be expressed in conjunction with additional genes encoding enzymes of the PUFA biosynthetic pathway (e,g., delta-6 desaturases, C.sub.18/20 elongases, delta-17 desaturases, delta-15 desaturases, delta-9 desaturases, delta-12 desaturases, C.sub.14/16 elongases, G.sub.16/18 elongases, delta-9 elongases, delta-8 desaturases, delta-4 desaturases, C.sub.20/22 elongases) to result in higher levels of production of longer-chain omega-3 fatty acids (e.g., EPA, DPA and DHA). The particular genes included within a particular expression cassette will depend on the host cell (and its PUFA profile and/or desaturase/elongase profile), the availability of substrate and the desired end product(s).

[0175] In alternative embodiments, it may be useful to disrupt a host organism's native delta-5 desaturase, based on the complete sequences described herein, the complement of those complete sequences, substantial portions of those sequences, codon-optimized desaturases derived therefrom and those sequences that are substantially homologous thereto.

Plant Expression Systems, Cassettes and Vectors, and Transformation

[0176] In one embodiment, this invention concerns a recombinant construct comprising any one of the delta-5 desaturase polynucleotides of the invention operably linked to at least one regulatory sequence suitable for expression in a plant. A promoter is a DNA sequence that directs cellular machinery of a plant to produce RNA from the contiguous coding sequence downstream (3') of the promoter. The promoter region influences the rate, developmental stage, and cell type in which the RNA transcript of the gene is made. The RNA transcript is processed to produce mRNA which serves as a template for translation of the RNA sequence into the amino acid sequence of the encoded polypeptide. The 5' non-translated leader sequence is a region of the mRNA upstream of the protein coding region that may play a role in initiation and translation of the mRNA. The 3' transcription term ination/polyadenylation signal is a non-translated region downstream of the protein coding region that functions in the plant cell to cause termination of the RNA transcript and the addition of polyadenylate nucleotides to the 3' end of the RNA.

[0177] The origin of the promoter chosen to drive expression of the delta-5 desaturase coding sequence is not important as long as it has sufficient transcriptional activity to accomplish the invention by expressing translatable mRNA for the desired nucleic acid fragments in the desired host tissue at the right time. Either heterologous or non-heterologous (i.e., endogenous) promoters can be used to practice the invention. For example, suitable promoters include, but are not limited to: the alpha prime subunit of beta conglycinin promoter, the Kunitz trypsin inhibitor 3 promoter, the annexin promoter, the glycinin Gyl promoter, the beta subunit of beta conglycinin promoter, the P34/Gly Bd m 30K promoter, the albumin promoter, the Leg Al promoter and the Leg A2 promoter.

[0178] The annexin, or P34, promoter is described in PCT Publication No. WO 2004/071178 (published Aug. 26, 2004). The level of activity of the annexin promoter is comparable to that of many known strong promoters, such as: (1) the CaMV 355 promoter (Atanassova et al., Plant Mol. Biol. 37:275-285 (1998); Battraw and Hall, Plant Mol. Biol. 15:527-538 (1990); Holtorl et al., Plant Mol. Biol. 29:637-646 (1995); Jefferson et al., EMBO J. 6:3901-3907 (1987); Wilmink et al., Plant Mol. Biol. 28:949-955 (1995)); (2) the Arabidopsis oleosin promoters (Plant et al., Plant Mol. Biol. 25:193-205 (1994); Li, Texas A&M University Ph.D. dissertation, pp. 107-128 (1997)); (3) the Arabidopsis ubiquitin extension protein promoters (Callis et al., J Biol. Chem. 265(21):12485-93 (1990)); (4) a tomato ubiquitin gene promoter (Rollfinke et al., Gene. 211(2):267-76 (1998)); (5) a soybean heat shock protein promoter (Schoffl et al., Mol Gen Genet. 217(2-3):246-53 (1989)); and, (6) a maize H3 histone gene promoter (Atanassova at al., Plant Mol Biol. 37(2):275-85 (1989)).

[0179] Another useful feature of the annexin promoter is its expression profile in developing seeds. The annexin promoter is most active in developing seeds at early stages (before 10 days after pollination) and is largely quiescent in later stages. The expression profile of the annexin promoter is different from that of many seed-specific promoters, e.g., seed storage protein promoters, which often provide highest activity in later stages of development (Chen et al., Day. Genet. 10:112-122 (1989); Ellerstrom et al., Plant Mol. Biol. 32:1019-1027 (1996); Keddie at al., Plant Mol. Biol. 24:327-340 (1994); Plant et al., (supra); Li, (supra)). The annexin promoter has a more conventional expression profile but remains distinct from other known seed specific promoters. Thus, the annexin promoter will be a very attractive candidate when overexpression, or suppression, of a gene in embryos is desired at an early developing stage. For example, it may be desirable to overexpress a gene regulating early embryo development or a gene involved in the metabolism prior to seed maturation.

[0180] Following identification of an appropriate promoter suitable for expression of a specific delta-5 desaturase coding sequence, the promoter is then operably linked in a sense orientation using conventional means well known to those skilled in the art.

[0181] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J. at al., In Molecular Cloning: A Laboratory Manual; 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y., 1989 (hereinafter "Sambrook et al., 1989") or Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl, K., Eds.; In Current Protocols in Molecular Biology; John Wiley and Sons: New York, 1990 (hereinafter "Ausubel et al., 1990").

[0182] Once the recombinant construct has been made, it may then be introduced into a plant cell of choice by methods well known to those of ordinary skill in the art (e.g., transfection, transformation and electroporation). Oilseed plant cells are the preferred plant cells. The transformed plant cell is then cultured and regenerated under suitable conditions permitting expression of the long-chain PUFA which is then optionally recovered and purified.

[0183] The recombinant constructs of the invention may be introduced into one plant cell; or, alternatively, each construct may be introduced into separate plant cells. Expression in a plant cell may be accomplished in a transient or stable fashion as is described above.

[0184] The desired long-chain PUFAs can be expressed in seed. Also within the scope of this invention are seeds or plant parts obtained from such transformed plants.

[0185] Plant parts include differentiated and undifferentiated tissues including, but not limited to the following: roots, sterns, shoots, leaves, pollen, seeds, tumor tissue and various forms of cells and culture (e.g., single cells, protoplasts, embryos and callus tissue). The plant tissue may be in plant or in a plant organ, tissue or cell culture.

[0186] The term "plant organ" refers to plant tissue or a group of tissues that constitute a morphologically and functionally distinct part of a plant. The term "genome" refers to the following: (1) the entire complement of genetic material (genes and non-coding sequences) that is present in each cell of an organism, or virus or organelle; and/or (2) a complete set of chromosomes inherited as a (haploid) unit from one parent.

[0187] Thus, this invention also concerns a method for transforming a cell, comprising transforming a cell with the recombinant construct of the invention and selecting those cells transformed with the recombinant construct of the invention.

[0188] Also of interest is a method for producing a transformed plant comprising transforming a plant cell with the delta-5 desaturase polynucleotides of the instant invention and regenerating a plant from the transformed plant cell.

[0189] Methods for transforming dicots (primarily by use of Agrobacterium tumefaciens) and obtaining transgenic plants have been published, among others, for; cotton (U.S. Pat. No. 5,004,863; U.S. Pat. No. 5,159,135); soybean (U.S. Pat. No. 5,569,834; U.S. Pat. No. 5,416,011); Brassica (U.S. Pat. No. 5,463,174); peanut (Cheng et al. Plant Cell Rep. 15:653-657 (1996); McKently et al. Plant. Cell Rep. 14:699-703 (1995)); papaya (Ling, K. et al. Bio/technology 9:752-758 (1991)); and pea (Grant at al. Plant Cell Rep. 15:254-258 (1995)). For a review of other commonly used methods of plant transformation see Newell, C. A. (Mol. Biotechnol. 16:53-65 (2000)). One of these methods of transformation uses Agrobacterium rhizogenes (Tepfler, M. and Casse-Delbart, F. Microbiol. Sci. 4:24-28 (1987)). Transformation of soybeans using direct delivery of DNA has been published using PEG fusion (PCT Publication No. WO 92/17598), electroporation (Chowrira, G. M. et al., Mol. Biotechnol. 3:17-23 (1995); Christou, P. et al., Proc. Natl. Acad. Sci. U.S.A. 84:3962-3966 (1987)), microinjection and particle bombardement (McCabe, D. E. et. al., Bio/Technology 6:923 (1988); Christou et al., Plant Physiol. 87:671-674 (1988)).

[0190] There are a variety of methods for the regeneration of plants from plant tissue. The particular method of regeneration will depend on the starting plant tissue and the particular plant species to be regenerated. The regeneration, development and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (Weissbach and Weissbach, In: Methods for Plant Molecular Biology, (Eds.), Academic: San Diego, Calif. (1988)). This regeneration and growth process typically includes the steps of selection of transformed cells and culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants, Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants. A transgenic plant of the present invention containing a desired polypeptide is cultivated using methods well known to one skilled in the art.

[0191] In addition to the above discussed procedures, practitioners are familiar with the standard resource materials which describe specific conditions and procedures for: the construction, manipulation and isolation of macromolecules (e.g., DNA molecules, plasmids, etc.); the generation of recombinant DNA fragments and recombinant expression constructs; and, the screening and isolating of clones. See, for example: Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor: NY (1989); Maliga et al., Methods in Plant Molecular Biology, Cold Spring Harbor: NY (1995); Birren et al., Genome Analysis: Detecting Genes, Vol.1, Cold Spring Harbor: NY (1998); Birren et al., Genome Analysis: Analyzing DNA, Vol. 2, Cold Spring Harbor: NY (1998); Plant Molecular Biology: A Laboratory Manual, eds. Clark, Springer: NY (1997).

[0192] Examples of oilseed plants include, but are not limited to: soybean, Brassica species, sunflower, maize, cotton, flax and safflower.

[0193] Examples of PUFAs having at least twenty carbon atoms and four or more carbon-carbon double bonds include, but are not limited to, omega-3 fatty acids such as EPA, DPA and DHA and the omega-6 fatty acid ARA. Seeds obtained from such plants are also within the scope of this invention as well as oil obtained from such seeds.

[0194] Thus, in one embodiment this invention concerns an oilseed plant comprising:

[0195] (a) a first recombinant DNA construct comprising an isolated polynucleotide encoding a delta-5 desaturase polypeptide, operably linked to at least one regulatory sequence; and,

[0196] (b) at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8 desaturase, a delta-9 desaturase, a delta-9 elongase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a C.sub.14/16 elongase, a C.sub.16/18 elongase, a C.sub.18/20 elongase and a C.sub.20/22 elongase.

[0197] Additional desaturases are discussed, for example, in U.S. Pat. Nos. 6,075,183, 5,968,809, 6,136,574, 5,972,664, 6,051,754, 6,410,288 and PCT Publication Nos. WO 98/46763, WO 98/46764, WO 00/12720 and WO 00/40705.

[0198] The choice of combination of cassettes used depends in part on the PUFA profile and/or desaturase/elongase profile of the oilseed plant cells to be transformed and the long-chain PUFA which is to be expressed.

[0199] In another aspect, this invention concerns a method for making long-chain PUFAs in a plant cell comprising: [0200] (a) transforming a cell with the recombinant construct of the invention; and, [0201] (b) selecting those transformed cells that make long-chain PUFAs.

[0202] In still another aspect, this invention concerns a method for producing at least one PUFA in a soybean cell comprising: [0203] (a) transforming a soybean cell with a first recombinant DNA construct comprising: [0204] (i) an isolated polynucleotide encoding a delta-5 desaturase polypeptide, operably linked to at least one regulatory sequence; and, [0205] (ii) at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8 desaturase, a delta-9 desaturase, a delta-9 elongase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a C.sub.14/16 elongase, a C.sub.16/18 elongase, a C.sub.18/20 elongase and a C.sub.20/22 elongase; [0206] (b) regenerating a soybean plant from the transformed cell of step (a); and, [0207] (c) selecting those seeds obtained from the plants of step (b) having an altered level of PUFAs when compared to the level in seeds obtained from a nontransforrned soybean plant. In other preferred embodiments, the at least one additional recombinant DNA construct encodes a polypeptide having delta-9 elongase activity, e.g., the delta-9 elongase isolated or derived from Isochrysis galbana (GenBank Accession No. AF390174; IgD9e) or the delta-9 elongase isolated or derived from Euglena gracilis.

[0208] In other preferred embodiments, the at least one additional recombinant DNA construct encodes a polypeptide having delta-8 desaturase activity. For example, PCT Publication No. WO 2005/103263 (published Apr. 22, 2005) discloses amino acid and nucleic acid sequences for a delta-8 desaturase enzyme from Pavlova salina (see also U.S. Publication No. 2005/0273885). Sayanova et al. (FEBS Lett. 580:1946-1952 (2006)) describes the isolation and characterization of a cDNA from the free living soil amoeba Acanthamoeba castellanii that, when expressed in Arabidopsis, encodes a C.sub.20 delta-8 desaturase. Also, Applicants' Assignee's co-pending application having Provisional Application No. 60/795,810 filed Apr. 28, 2006 (Attorney Docket No. BB-1566) discloses amino acid and nucleic acid sequences for a delta-8 desaturase enzyme from Pavlova lutheri (CCMP459). U.S Provisional Application No. 60/853,563 (filed Oct. 23, 2006; Attorney Docket No. BB-1574) discloses amino acid and nucleic add sequences for a delta-8 desaturase enzyme from Tetruetreptia pomquetensis CCMP1491, Eutreptiella sp. CCMP389 and Eutreptiella cf.sub.--gymnastica CCMP1594.

Microbial Expression Systems, Cassettes and Vectors, and Transformation

[0209] The delta-5 desaturase genes and gene products described herein (i.e., the Peridinium sp. CCMP626 delta-5 desaturase, or other mutant enzymes, codon-optimized enzymes or homologs thereof) may also be produced in heterologous microbial host cells, particularly in the cells of oleaginous yeasts (e.g., Yarrowia lipolytica).

[0210] Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct chimeric genes for production of any of the gene products of the instant sequences. These chimeric genes could then be introduced into appropriate microorganisms via transformation to provide high-level expression of the encoded enzymes. Vectors or DNA cassettes useful for the transformation of suitable microbial host cells are well known in the art. The specific choice of sequences present in the construct is dependent upon the desired expression products (supra), the nature of the host cell and the proposed means of separating transformed cells versus non-transformed cells. Typically, however, the vector or cassette contains sequences directing transcription and translation of the relevant gene(s), a selectable marker and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5' of the gene that controls transcriptional initiation (e.g., a promoter) and a region 3' of the DNA fragment that controls transcriptional termination (i.e., a terminator). It is most preferred when both control regions are derived from genes from the transformed microbial host cell, although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a production host.

[0211] Initiation control regions or promoters which are useful to drive expression of the instant delta-5 desaturase ORFs in the desired microbial host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of directing expression of these genes in the selected host cell is suitable for the present invention. Expression in a microbial host cell can be accomplished in a transient or stable fashion. Transient expression can be accomplished by inducing the activity of a regulatable promoter operably linked to the gene of interest. Stable expression can be achieved by the use of a constitutive promoter operably linked to the gene of interest. As an example, when the host cell is yeast, transcriptional and translational regions functional in yeast cells are provided, particularly from the host species (e.g., see PCT Publication Nos. WO 2004/101757 and WO 2006/052870 for preferred transcriptional initiation regulatory regions for use in Yarrowia lipolytica). Any one of a number of regulatory sequences can be used, depending upon whether constitutive or induced transcription is desired, the efficiency of the promoter in expressing the ORF of interest, the ease of construction and the like.

[0212] Nucleotide sequences surrounding the translational initiation codon `ATG` have been found to affect expression in yeast cells. If the desired polypeptide is poorly expressed in yeast, the nucleotide sequences of exogenous genes can be modified to include an efficient yeast translation initiation sequence to obtain optimal gene expression. For expression in yeast, this can be done by site-directed mutagenesis of an inefficiently expressed gene by fusing it in-frame to an endogenous yeast gene, preferably a highly expressed gene. Alternatively, one can determine the consensus translation initiation sequence in the host and engineer this sequence into heterologous genes for their optimal expression in the host of interest.

[0213] The termination region can be derived from the 3' region of the gene from which the initiation region was obtained or from a different gene. A large number of termination regions are known and function satisfactorily in a variety of hosts (when utilized both in the same and different genera and species from where they were derived). The termination region usually is selected more as a matter of convenience rather than because of any particular property, Preferably, when the microbial host is a yeast cell, the termination region is derived from a yeast gene (particularly Saccharomyces, Schizosaccharomyces, Candida, Yarrowia or Kluyveromyces). The 3'-regions of mammalian genes encoding .gamma.-interferon and .alpha.-2 interferon are also known to function in yeast. Termination control regions may also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary; however, it is most preferred if included. Although not intended to be limiting, termination regions useful in the disclosure herein include: .about.100 by of the 3' region of the Yarrowia lipolytica extracellular protease (XPR; GenBank Accession Na M17741); the acyl-coA oxidase (Aco3: GenBank Accession No. AJ001301 and No. CAA04661; Pox3: GenBank Accession No, XP.sub.--503244) terminators; the Pex20 (GenBank Accession No. AF054613) terminator; the Pex16 (GenBank Accession No. U75433) terminator; the Lip1 (GenBank Accession No. 250020) terminator; the Lip2 (GenBank Accession No. AJ012632) terminator; and the 3-oxoacyl-coA thiolase (OCT; GenBank Accession No. X69988) terminator.

[0214] As one of skill in the art is aware, merely inserting a gene into a cloning vector does not ensure that it will be successfully expressed at the level needed. In response to the need for a high expression rate, many specialized expression vectors have been created by manipulating a number of different genetic elements that control aspects of transcription, translation, protein stability, oxygen limitation and secretion from the microbial host cell. More specifically, some of the molecular features that have been manipulated to control gene expression include: (1) the nature of the relevant transcriptional promoter and terminator sequences; (2) the number of copies of the cloned gene and whether the gene is plasmid-borne or integrated into the genome of the host cell; (3) the final cellular location of the synthesized foreign protein; (4) the efficiency of translation and correct folding of the protein in the host organism; (5) the intrinsic stability of the mRNA and protein of the cloned gene within the host cell; and (6) the codon usage within the cloned gene, such that its frequency approaches the frequency of preferred codon usage of the host cell. Each of these types of modifications are encompassed in the present invention, as means to further optimize expression of the delta-5 desaturase described herein.

[0215] Once the DNA encoding a polypeptide suitable for expression in an appropriate microbial host cell (e.g., oleaginous yeast) has been obtained (e.g., a chimeric gene comprising a promoter, ORF and terminator), it is placed in a plasmid vector capable of autonomous replication in a host cell, or it is directly integrated into the genome of the host cell. Integration of expression cassettes can occur randomly within the host genome or can be targeted through the use of constructs containing regions of homology with the host genome sufficient to target recombination within the host locus. Where constructs are targeted to an endogenous locus, all or some of the transcriptional and translational regulatory regions can be provided by the endogenous locus.

[0216] In the present invention, the preferred method of expressing genes in Yarrowia lipolytica is by integration of linear DNA into the genome of the host; and, integration into multiple locations within the genome can be particularly useful when high level expression of genes are desired [e.g., in the Ura3 locus (GenBank Accession No. AJ306421), the Leu2 gene locus (GenBank Accession No. AF260230), the Lys5 gene (GenBank Accession No. M34929), the Aco2 gene locus (GenBank Accession No. AJ001300), the Pox3 gene locus (Pox3: GenBank Accession No. XP.sub.--503244; or, Aco3: GenBank Accession No. AJ001301), the delta-12 desaturase gene locus (PCT Publication No. WO2004/104167), the bpi gene locus (GenBank Accession No. Z50020) and/or the Lip2 gene locus (GenBank Accession No. AJ012632)].

[0217] Advantageously, the Ura3 gene can be used repeatedly in combination with 5-fluoroorotic acid (5-fluorouracil-6-carboxylic acid monohydrate; "5-FOA") selection (infra), to readily permit genetic modifications to be integrated into the Yarrowia genome in a facile manner.

[0218] Where two or more genes are expressed from separate replicating vectors, it is desirable that each vector has a different means of selection and should lack homology to the other construct(s) to maintain stable expression and prevent reassortment of elements among constructs. Judicious choice of regulatory regions, selection means and method of propagation of the introduced construct(s) can be experimentally determined so that all introduced genes are expressed at the necessary levels to provide for synthesis of the desired products.

[0219] Constructs comprising the gene of interest may be introduced into a microbial host cell by any standard technique. These techniques include transformation (e.g., lithium acetate transformation [Methods in Enzymology, 194:186-187 (1991)]), protoplast fusion, bolistic impact, electroporation, microinjection, or any other method that introduces the gene of interest into the host cell. More specific teachings applicable for oleaginous yeasts (i.e., Yarrowia lipolytica) include U.S. Pat. No. 4,880,741 and U.S. Pat. No. 5,071,764 and Chen, D. C. et al. (Appl. Microbiol. Biotechnol., 48(2)232-235 (1997)).

[0220] For convenience, a host cell that has been manipulated by any method to take up a DNA sequence (e.g., an expression cassette) will be referred to as "transformed" or "recombinant" herein. Thus, the term "transformed" and "recombinant" are used interchangeably herein. The transformed host will have at least one copy of the expression construct and may have two or more, depending upon whether the gene is integrated into the genome, amplified or is present on an extrachromosomal element having multiple copy numbers.

[0221] The transformed host cell can be identified by various selection techniques, as described in PCT Publication Nos. WO 2004/101757 and WO 2006/052870. Preferred selection methods for use herein are resistance to kanamycin, hygromycin and the amino glycoside G418, as well as ability to grow on media lacking uracil, leucine, lysine, tryptophan or histidine. In alternate embodiments, 5-FOA is used for selection of yeast Ura- mutants. The compound is toxic to yeast cells that possess a functioning URA3 gene encoding orotidine 5'-monophosphate decarboxylase (OMP decarboxylase); thus, based on this toxicity, 5-FOA is especially useful for the selection and identification of Ura.sup.- mutant yeast strains (Bartel, P. L. and Fields, S., Yeast 2-Hybrid System, Oxford University: New York, v. 7, pp 109-147, 1997). More specifically, one can first knockout the native Ura3 gene to produce a strain having a Ura+ phenotype, wherein selection occurs based on 5-FOA resistance. Then, a cluster of multiple chimeric genes and a new Ura3 gene can be integrated into a different locus of the Yarrowia genome to thereby produce a new strain having a Ura+ phenotype. Subsequent integration produces a new Ura3- strain (again identified using 5-FOA selection), when the introduced Ura3 gene is knocked out. Thus, the Ura3 gene (in combination with 5-FOA selection) can be used as a selection marker in multiple rounds of transformation.

[0222] Following transformation, substrates suitable for the instant delta-5 desaturase (and, optionally other PUFA enzymes that are co-expressed within the host cell) may be produced by the host either naturally or transgenically, or they may be provided exogenously.

[0223] Microbial host cells for expression of the instant genes and nucleic acid fragments may include hosts that grow on a variety of feedstocks, including simple or complex carbohydrates, fatty acids, organic acids, oils and alcohols, and/or hydrocarbons over a wide range of temperature and pH values. Based on the needs of the Applicants' Assignee, the genes described in the instant invention will be expressed in an oleaginous yeast (and in particular Yarrowia lipolytica); however, it is contemplated that because transcription, translation and the protein biosynthetic apparatus is highly conserved, any bacteria, yeast, algae and/or fungus will be a suitable microbial host for expression of the present nucleic acid fragments. Preferred microbial hosts, however, are oleaginous yeasts. These organisms are naturally capable of oil synthesis and accumulation, wherein the oil can comprise greater than about 25% of the cellular dry weight, more preferably greater than about 30% of the cellular dry weight, and most preferably greater than about 40% of the cellular dry weight. Genera typically identified as oleaginous yeast include, but are not limited to: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces. More specifically, illustrative oil-synthesizing yeasts include: Rhodosporidium toruloides, Lipomyces starkeyii, L. lipoferus, Candida revkaufi, C. pulcherrima, C. tropicalis, C. utilis, Trichosporon pullans, T. cutaneum, Rhodotorula glutinus, R. graminis, and Yarrowia lipolytica (formerly classified as Candida lipolytica).

[0224] Most preferred is the oleaginous yeast Yarrowia lipolytica; and, in a further embodiment, most preferred are the Y. lipolytica strains designated as ATCC #20362, ATCC #8862, ATCC #18944, ATCC #76982 and/or LGAM S(7)1 (Papanikolaou S., and Aggelis G., Bioresour. Technol. 82(1):43-9 (2002)).

[0225] Historically, various strains of Y. lipolytica have been used for the manufacture and production of: isocitrate lyase; lipases; polyhydroxyalkanoates; citric acid; erythritol; 2-oxoglutaric acid; .gamma.-decalactone; .gamma.-dodecalatone; and pyruvic acid. Specific teachings applicable for engineering ARA, EPA and DHA production in Y. lipolytica are provided in U.S. patent application Ser. No. 11/264,784 (WO 2006/055322), U.S. patent application Ser. No. 11/265,761 (WO 2006/052870) and U.S. patent application Ser. No. 11/264,737 (WO 2006/052871), respectively.

[0226] Other preferred microbial hosts include oleaginous bacteria, algae and other fungi; and, within this broad group of microbial hosts, of particular interest are microorganisms that synthesize omega-3/omega-6 fatty acids (or those that can be genetically engineered for this purpose [e.g., other yeast such as Saccharomyces cerevisiae]). Thus, for example, transformation of Mortierella alpina (which is commercially used for production of ARA) with any of the present delta-5 desaturase genes under the control of inducible or regulated promoters could yield a transformant organism capable of synthesizing increased quantities of DGLA. The method of transformation of M. alpina is described by Mackenzie et al. (Appl. Environ. Microbial., 66:4655 (2000)). Similarly, methods for transformation of Thraustochytriales microorganisms are disclosed in U.S. Pat. No. 7,001,772.

[0227] Based on the teachings described above, in one embodiment this invention is drawn to a method of producing either ARA or EPA, respectively, comprising: [0228] (a) providing an oleaginous yeast comprising: [0229] (i) a first recombinant DNA construct comprising an isolated polynucleotide encoding a delta-5 desaturase polypeptide, operably linked to at least one regulatory sequence; and, [0230] (ii) a source of desaturase substrate consisting of either DGLA or ETA, respectively; and, [0231] (b) growing the yeast of step (a) in the presence of a suitable fermentable carbon source wherein the gene encoding the delta-5 desaturase polypeptide is expressed and DGLA is converted to ARA or ETA is converted to EPA, respectively; and, [0232] (c) optionally recovering the ARA or EPA, respectively, of step (b). Substrate feeding may be required.

[0233] Of course, since naturally produced PUFAs in oleaginous yeast are limited to 18:2 fatty acids (i.e., LA), and less commonly, 18:3 fatty acids (i.e., ALA), in more preferred embodiments of the present invention the oleaginous yeast will be genetically engineered to express multiple enzymes necessary for long-chain PUFA biosynthesis (thereby enabling production of e.g., ARA, EPA, DPA and DHA), in addition to the delta-5 desaturases described herein.

[0234] Specifically, in one embodiment this invention concerns an oleaginous yeast comprising:

[0235] (a) a first recombinant DNA construct comprising an isolated polynucleotide encoding a delta-5 desaturase polypeptide, operably linked to at least one regulatory sequence; and,

[0236] (b) at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of: a delta-4 desaturase, a delta-5 desaturase, delta-6 desaturase, a delta-9 desaturase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a delta-9 elongase, a C.sub.14/16 elongase, a C.sub.16/18 elongase, a C.sub.18/20 elongase and a C.sub.20/22 elongase.

[0237] In particularly preferred embodiments, the at least one additional recombinant

[0238] DNA construct encodes a polypeptide having delta-9 elongase activity, e.g., the delta-9 elongase isolated or derived from Isochrysis galbana (GenBank Accession No. AF390174; IgD9e or IgD9eS) or the delta-9 elongase isolated or derived from Euglena gracilis.

Metabolic Engineering of Omega-3 and/or Omega-6 Fatty Acid Biosynthesis in Microbes

[0239] Methods for manipulating biochemical pathways are well known to those skilled in the art; and, it is expected that numerous manipulations will be possible to maximize omega-3 and/or omega-6 fatty acid biosynthesis in oleaginous yeasts, and particularly, in Yarrowia lipolytica. This manipulation may require metabolic engineering directly within the PUFA biosynthetic pathway or additional coordinated manipulation of various other metabolic pathways.

[0240] In the case of manipulations within the PUFA biosynthetic pathway, it may be desirable to increase the production of LA to enable increased production of omega-6 and/or omega-3 fatty acids. Introducing and/or amplifying genes encoding delta-9 and/or delta-12 desaturases may accomplish this. To maximize production of omega-6 unsaturated fatty acids, it is well known to one skilled in the art that production is favored in a host microorganism that is substantially free of ALA; thus, preferably, the host is selected or obtained by removing or inhibiting delta-15 or omega-3 type desaturase activity that permits conversion of LA to ALA. Alternatively, it may be desirable to maximize production of omega-3 fatty acids (and minimize synthesis of omega-6 fatty acids). In this example, one could utilize a host microorganism wherein the delta-12 desaturase activity that permits conversion of oleic acid to LA is removed or inhibited; subsequently, appropriate expression cassettes would be introduced into the host, along with appropriate substrates (e.g., ALA) for conversion to omega-3 fatty acid derivatives of ALA (e.g., STA, ETrA, ETA, EPA, DPA, DHA).

[0241] In alternate embodiments, biochemical pathways competing with the omega-3 and/or omega-6 fatty acid biosynthetic pathways for energy or carbon, or native PUFA biosynthetic pathway enzymes that interfere with production of a particular PUFA end-product, may be eliminated by gene disruption or down-regulated by other means (e.g., antisense mRNA).

[0242] Detailed discussion of manipulations within the PUFA biosynthetic pathway as a means to increase ARA, EPA or DHA (and associated techniques thereof) are presented in PCT Publication Nos. WO 2006/055322, WO 2006/052870 and WO 2006/052871, respectively, as are desirable manipulations in the TAG biosynthetic pathway and the TAG degradation pathway (and associated techniques thereof).

[0243] Within the context of the present invention, it may be useful to modulate the expression of the fatty acid biosynthetic pathway by any one of the strategies described above. For example, the present invention provides methods whereby genes encoding key enzymes in the delta-9 elongase/delta-8 desaturase biosynthetic pathway are introduced into oleaginous yeasts for the production of omega-3 and/or omega-6 fatty acids. It will be particularly useful to express the present the delta-5 desaturase genes in oleaginous yeasts that do not naturally possess omega-3 and/or omega-6 fatty acid biosynthetic pathways and coordinate the expression of these genes, to maximize production of preferred PUFA products using various means for metabolic engineering of the host organism.

Microbial Fermentation Processes for PUFA Production

[0244] The transformed host cell is grown under conditions that optimize expression of chimeric desaturase genes and produce the greatest and the most economical yield of desired PUFAs. In general, media conditions that may be optimized include the type and amount of carbon source, the type and amount of nitrogen source, the carbon-to-nitrogen ratio, the amount of different mineral ions, the oxygen level, growth temperature, pH, length of the biomass production phase, length of the oil accumulation phase and the time and method of cell harvest. Yarrowia lipolytica are generally grown in complex media (e.g., yeast extract-peptone-dextrose broth (YPD)) or a defined minimal media that lacks a component necessary for growth and thereby forces selection of the desired expression cassettes (e.g., Yeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.)).

[0245] Fermentation media in the present invention must contain a suitable carbon source. Suitable carbon sources are taught in PCT Publication No. WO 2004/101757. Although it is contemplated that the source of carbon utilized in the present invention may encompass a wide variety of carbon-containing sources, preferred carbon sources are sugars, glycerol, and/or fatty acids. Most preferred is glucose and/or fatty acids containing between 10-22 carbons. Nitrogen may be supplied from an inorganic (e.g., (NH.sub.4).sub.2SO.sub.4) or organic (e.g., urea or glutamate) source. In addition to appropriate carbon and nitrogen sources, the fermentation media must also contain suitable minerals, salts, cofactors, buffers, vitamins and other components known to those skilled in the art suitable for the growth of the oleaginous host and promotion of the enzymatic pathways necessary for PUFA production. Particular attention is given to several metal ions (e.g., Mn.sup.+2, Co.sup.+2, Zn.sup.+2, Mg.sup.+2) that promote synthesis of lipids and PUFAs (Nakahara, T, et al., Ind. Appl. Single Cell Oils, D. J. Kyle and R. Colin, eds. pp 61-97 (1992)).

[0246] Preferred growth media in the present invention are common commercially prepared media, such as Yeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.). Other defined or synthetic growth media may also be used and the appropriate medium for growth of the transformant host cells will be known by one skilled in the art of microbiology or fermentation science. A suitable pH range for the fermentation is typically between about pH 4.0 to pH 8.0, wherein pH 5.5 to pH 7.5 is preferred as the range for the initial growth conditions. The fermentation may be conducted under aerobic or anaerobic conditions, wherein microaerobic conditions are preferred.

[0247] Typically, accumulation of high levels of PUFAs in oleaginous yeast cells requires a two-stage process, since the metabolic state must be "balanced" between growth and synthesis/storage of fats. Thus, most preferably, a two-stage fermentation process is necessary for the production of PUFAs in oleaginous yeast (e.g., Yarrowia lipolytica). This approach is described in PCT Publication No. WO 2004/101757, as are various suitable fermentation process designs (i.e., batch, fed-batch

and continuous) and considerations during growth. Purification and Processing of PUFA Oils

[0248] PUFAs may be found in the host microorganisms and plants as free fatty acids or in esterified forms such as acylglycerols, phospholipids, sulfolipids or glycolipids, and may be extracted from the host cells through a variety of means well-known in the art. One review of extraction techniques, quality analysis and acceptability standards for yeast lipids is that of Z. Jacobs (Critical Reviews in Biotechnology, 12(5/6):463-491 (1992)). A brief review of downstream processing is also available by A. Singh and O. Ward (Adv. Appl. Microbiol., 45:271-312 (1997)).

[0249] In general, means for the purification of PUFAs may include extraction with organic solvents, sonication, supercritical fluid extraction (e.g., using carbon dioxide), saponification and physical means such as presses, or combinations thereof. One is referred to the teachings of PCT Publication No. WO 2004/101757 for additional details. Methods of isolating seed oils are well known in the art: (Young et al., Processing of Fats and Oils, In The Lipid Handbook, Gunstone et al., eds., Chapter 5 pp 253-257; Chapman & Hall: London (1994)). For example, soybean oil is produced using a series of steps involving the extraction and purification of an edible oil product from the oil-bearing seed. Soybean oils and soybean byproducts are produced using the generalized steps shown in Table 3.

TABLE-US-00003 TABLE 3 Generalized Steps for Soybean Oil and Byproduct Production Process Impurities Removed and/or Step Process By-Products Obtained # 1 soybean seed # 2 oil extraction meal # 3 degumming lecithin # 4 alkali or physical refining gums, free fatty acids, pigments # 5 water washing soap # 6 bleaching color, soap, metal # 7 (hydrogenation) # 8 (winterization) stearine # 9 deodorization free fatty acids, tocopherols, sterols, volatiles # 10 oil products

[0250] More specifically, soybean seeds are cleaned, tempered, dehulled and flaked, thereby increasing the efficiency of oil extraction. Oil extraction is usually accomplished by solvent (e.g., hexane) extraction but can also be achieved by a combination of physical pressure and/or solvent extraction. The resulting oil is called crude oil. The crude oil may be degummed by hydrating. phospholipids and other polar and neutral lipid complexes that facilitate their separation from the nonhydrating, triglyceride fraction (soybean oil). The resulting lecithin gums may be further processed to make commercially important lecithin products used in a variety of food and industrial products as emulsification and release (i.e., antisticking) agents. Degummed oil may be further refined for the removal of impurities (primarily free fatty acids, pigments and residual gums). Refining is accomplished by the addition of a caustic agent that reacts with free fatty acid to form soap and hydrates phosphatides and proteins in the crude oil. Water is used to wash out traces of soap formed during refining. The soapstock byproduct may be used directly in animal feeds or acidulated to recover the free fatty acids. Color is removed through adsorption with a bleaching earth that removes most of the chlorophyll and carotenoid compounds. The refined oil can be hydrogenated, thereby resulting in fats with various melting properties and textures. Winterization (fractionation) may be used to remove stearine from the hydrogenated oil through crystallization under carefully controlled cooling conditions. Deodorization (principally via steam distillation under vacuum) is the last step and is designed to remove compounds which impart odor or flavor to the oil. Other valuable byproducts such as tocopherols and sterols may be removed during the deodorization process. Deodorized distillate containing these byproducts may be sold for production of natural vitamin E and other high-value pharmaceutical products. Refined, bleached, (hydrogenated, fractionated) and deodorized oils and fats may be packaged and sold directly or further processed into more specialized products. A more detailed reference to soybean seed processing, soybean oil production and byproduct utilization can be found in Erickson, Practical Handbook of Soybean Processing and Utilization, The American Oil Chemists' Society and United Soybean Board (1995). Soybean oil is liquid at room temperature because it is relatively low in saturated fatty acids when compared with oils such as coconut, palm, palm kernel and cocoa butter.

[0251] Plant and microbial oils containing PUFAs that have been refined andfor purified can be hydrogenated, to thereby result in fats with various melting properties and textures. Many processed fats (including spreads, confectionary fats, hard butters, margarines, baking shortenings, etc.) require varying degrees of solidity at room temperature and can only be produced through alteration of the source oil's physical properties. This is most commonly achieved through catalytic hydrogenation.

[0252] Hydrogenation is a chemical reaction in which hydrogen is added to the unsaturated fatty acid double bonds with the aid of a catalyst such as nickel. For example, high oleic soybean oil contains unsaturated oleic, LA and linolenic fatty acids and each of these can be hydrogenated. Hydrogenation has two primary effects. First, the oxidative stability of the oil is increased as a result of the reduction of the unsaturated fatty acid content. Second, the physical properties of the oil are changed because the fatty acid modifications increase the melting point resulting in a semi-liquid or solid fat at room temperature.

[0253] There are many variables which affect the hydrogenation reaction, which in turn alter the composition of the final product. Operating conditions including pressure, temperature, catalyst type and concentration, agitation and reactor design are among the more important parameters that can be controlled. Selective hydrogenation conditions can be used to hydrogenate the more unsaturated fatty acids in preference to the less unsaturated ones, Very light or brush hydrogenation is often employed to increase stability of liquid oils. Further hydrogenation converts a liquid oil to a physically solid fat. The degree of hydrogenation depends on the desired performance and melting characteristics designed for the particular end product. Liquid shortenings (used in the manufacture of baking products, solid fats and shortenings used for commercial frying and roasting operations) and base stocks for margarine manufacture are among the myriad of possible oil and fat products achieved through hydrogenation. A more detailed description of hydrogenation and hydrogenated products can be found in Patterson, H. B. W., Hydrogenation of Fats and Oils: Theory and Practice. The American Oil Chemists' Society (1994).

[0254] Hydrogenated oils have become somewhat controversial due to the presence of trans-fatty acid isomers that result from the hydrogenation process. Ingestion of large amounts of trans-isomers has been linked with detrimental health effects including increased ratios of low density to high density lipoproteins in the blood plasma and increased risk of coronary heart disease.

PUFA-Containing Oils for Use in Foodstuffs

[0255] The market place currently supports a large variety of food and feed products, incorporating omega-3 and/or omega-6 fatty acids (particularly ARA, EPA and DHA). It is contemplated that the plant/seed oils, altered seeds and microbial oils of the invention comprising PUFAs will function in food and feed products to impart the health benefits of current formulations. Compared to other vegetable oils, the oils of the invention are believed to function similarly to other oils in food applications from a physical standpoint (for example, partially hydrogenated oils such as soybean oil are widely used as ingredients for soft spreads, margarine and shortenings for baking and frying).

[0256] Plant/seed oils, altered seeds and microbial oils containing omega-3 and/or omega-6 fatty acids as described herein will be suitable for use in a variety of food and feed products including, but not limited to: food analogs, meat products, cereal products, baked foods, snack foods and dairy products. Additionally, the present plant/seed oils, altered seeds and microbial oils may be used in formulations to impart health benefit in medical foods including medical nutritionals, dietary supplements, infant formula as well as pharmaceutical products. One of skill in the art of food processing and food formulation will understand how the amount and composition of the plant and microbial oils may be added to the food or feed product. Such an amount will be referred to herein as an "effective" amount and will depend on the food or feed product, the diet that the product is intended to supplement or the medical condition that the medical food or medical nutritional is intended to correct or treat.

[0257] Food analogs can be made using processes well known to those skilled in the art. There can be mentioned meat analogs, cheese analogs, milk analogs and the like. Meat analogs made from soybeans contain soy protein or tofu and other ingredients mixed together to simulate various kinds of meats. These meat alternatives are sold as frozen, canned or dried foods. Usually, they can be used the same way as the foods they replace. Meat alternatives made from soybeans are excellent sources of protein, iron and B vitamins. Examples of meat analogs include, but are not limited to: ham analogs, sausage analogs, bacon analogs, and the like.

[0258] Food analogs can be classified as imitation or substitutes depending on their functional and compositional characteristics. For example, an imitation cheese need only resemble the cheese it is designed to replace. However, a product can generally be called a substitute cheese only if it is nutritionally equivalent to the cheese it is replacing and meets the minimum compositional requirements for that cheese. Thus, substitute cheese will often have higher protein levels than imitation cheeses and be fortified with vitamins and minerals.

[0259] Milk analogs or nondairy food products include, but are not limited to, imitation milks and nondairy frozen desserts (e.g., those made from soybeans and/or soy protein products).

[0260] Meat products encompass a broad variety of products. In the United States "meat" includes "red meats" produced from cattle, hogs and sheep. In addition to the red meats there are poultry items which include chickens, turkeys, geese, guineas, ducks and the fish and shellfish. There is a wide assortment of seasoned and processed meat products: fresh, cured and fried, and cured and cooked. Sausages and hot dogs are examples of processed meat products. Thus, the term "meat products" as used herein includes, but is not limited to, processed meat products.

[0261] A cereal food product is a food product derived from the processing of a cereal grain. A cereal grain includes any plant from the grass family that yields an edible grain (seed). The most popular grains are barley, corn, millet, oats, quinoa, rice, rye, sorghum, triticale, wheat and wild rice. Examples of a cereal food product include, but are not limited to: whole grain, crushed grain, grits, flour, bran, germ, breakfast cereals, extruded foods, pastas, and the like.

[0262] A baked goods product comprises any of the cereal food products mentioned above and has been baked or processed in a manner comparable to baking (i.e., to dry or harden by subjecting to heat). Examples of a baked good product include, but are not limited to: bread, cakes, doughnuts, bars, pastas, bread crumbs, baked snacks, mini-biscuits, mini-crackers, mini-cookies, and mini-pretzels. As was mentioned above, oils of the invention can be used as an ingredient.

[0263] A snack food product comprises any of the above or below described food products.

[0264] A fried food product comprises any of the above or below described food products that has been fried.

[0265] A health food product is any food product that imparts a health benefit. Many oilseed-derived food products may be considered as health foods.

[0266] A beverage can be in a liquid or in a dry powdered form.

[0267] For example, there can be mentioned non-carbonated drinks such as fruit juices, fresh, frozen, canned or concentrate; flavored or plain milk drinks, etc. Adult and infant nutritional formulas are well known in the art and commercially available (e,g, Similace, Ensure.RTM., Jevity.RTM., and Alimentum.RTM. from Ross Products Division, Abbott Laboratories).

[0268] Infant formulas are liquids or reconstituted powders fed to infants and young children. "Infant formula" is defined herein as an enteral nutritional product which can be substituted for human breast milk in feeding infants and typically is composed of a desired percentage of fat mixed with desired percentages of carbohydrates and proteins in an aquous solution (e.g., see U.S. Pat. No. 4,670,285). Based on the worldwide composition studies, as well as levels specified by expert groups, average human breast milk typically contains about 0.20% to 0.40% of total fatty acids (assuming about 50% of calories from fat); and, generally 25. the ratio of DHA to ARA would range from about 1:1 to 1:2 (see, e.g., formulations of Enfamil LIPIL.TM. (Mead Johnson & Company) and Similac Advance.TM. (Ross Products Division, Abbott Laboratories)). Infant formulas have a special role to play in the diets of infants because they are often the only source of nutrients for infants; and, although breast-feeding is still the best nourishment for infants, infant formula is a close enough second that babies not only survive but thrive.

[0269] A dairy product is a product derived from milk. A milk analog or nondairy product is derived from a source other than milk, for example, soymilk as was discussed above. These products include, but are not limited to: whole milk, skim milk, fermented milk products such as yogurt or sour milk, cream, butter, condensed milk, dehydrated milk, coffee whitener, coffee creamer, ice cream, cheese, etc.

[0270] Additional food products into which the PUFA-containing oils of the invention could be included are, for example, chewing gums, confections and frostings, gelatins and puddings, hard and soft candies, jams and jellies, white granulated sugar, sugar substitutes, sweet sauces, toppings and syrups, and dry-blended powder mixes.

PUFA-Containing Oils for Use in Health Food Products and Pharmaceuticals

[0271] A health food product is any food product that imparts a health benefit and include functional foods, medical foods, medical nutritionals and dietary supplements. Additionally, the plant/seed oils, altered seeds and microbial oils of the invention may be used in standard pharmaceutical compositions (e.g., the long-chain PUFA containing oils could readily be incorporated into the any of the above mentioned food products, to thereby produce a functional or medical food). More concentrated formulations comprising PUFAs include capsules, powders, tablets, softgels, gelcaps, liquid concentrates and emulsions which can be used as a dietary supplement in humans or animals other than humans.

PUFA-Containing Oils for Use in Animal Feeds

[0272] Animal feeds are generically defined herein as products intended for use as feed or for mixing in feed for animals other than humans. The plant/seed oils, altered seeds and microbial oils of the invention can be used as an ingredient in various animal feeds.

[0273] More specifically, although not limited therein, it is expected that the oils of the invention can be used within pet food products, ruminant and poultry food products and aquacultural food products. Pet food products are those products intended to be fed to a pet (e.g., dog, cat, bird, reptile, rodent). These products can include the cereal and health food products above, as well as meat and meat byproducts, soy protein products, grass and hay products (e.g., alfalfa, timothy, oat or brome grass, vegetables). Ruminant and poultry food products are those wherein the product is intended to be fed to an animal (e.g., turkeys, chickens, cattle, swine). As with the pet foods above, these products can include cereal and health food products, soy protein products, meat and meat byproducts, and grass and hay products as listed above. Aquacultural food products (or "aquafeeds") are those products intended to be used in aquafarming, i.e., which concerns the propagation, cultivation or farming of aquatic organisms and/or animals in fresh or marine waters.

EXAMPLES

[0274] The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

General Methods

[0275] Standard recombinant DNA and molecular cloning techniques used in the Examples are well known in the art and are described by: 1.) Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989) (Maniatis); 2.) T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1984); and 3.) Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience Hoboken, N.J. (1987).

[0276] Materials and methods suitable for the maintenance and growth of microbial cultures are well known in the art. Techniques suitable for use in the following examples may be found as set out in Manual of Methods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, Eds), American Society for Microbiology: Washington, D.C. (1994)); or by Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, 2nd ed., Sinauer Associates: Sunderland, Mass. (1989). All reagents, restriction enzymes and materials used for the growth and maintenance of microbial cells were obtained from Aldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.), unless otherwise specified. E. coli (XL1-Blue) competent cells were purchased from the Stratagene Company (San Diego, Calif.). E. coli strains were typically grown at 37.degree. C. on Luria Bertani (LB) plates.

[0277] General molecular cloning was performed according to standard methods (Sambrook et al., supra). DNA sequence was generated on an ABI Automatic sequencer using dye terminator technology (U.S. Pat. No. 5,366,860; EP 272,007) using a combination of vector and insert-specific primers. Sequence editing was performed in Sequencher (Gene Codes Corporation, Ann Arbor, Mich.). All sequences represent coverage at least two times in both directions. Unless otherwise indicated herein comparisons of genetic sequences were accomplished using DNASTAR software (DNASTAR Inc., Madison, Wis.).

[0278] The meaning of abbreviations is as follows: "sec" means second(s), "min" means minute(s), "h" means hour(s), "d" means day(s), ".mu.L`" means microliter(s), "mL" means milliliter(s), "L" means liter(s), ".mu.M" means micromolar, "mM" means millimolar, "M" means molar, "mmol" means millimole(s), ".mu.mole" mean micromole(s), "g" means gram(s), ".mu.g" means microgram(s), "ng" means nanogram(s), "U" means unit(s), "bp" means base pair(s) and "kB" means kilobase(s)

Transformation and Cultivation of Yarrowia lipolytica

[0279] Yarrowia lipolytica strain ATCC #20362 was purchased from the American Type Culture Collection (Rockville, Md.). Y. lipolytica strains were usually grown at 28.degree. C. on YPD agar (1% yeast extract, 2% bactopeptone, 2% glucose, 2% agar).

[0280] Transformation of Y. lipolytica was performed according to the method of Chen, D. C. et al. (Appl. Microbial Biotechnol., 48(2):232-235 (1997)), unless otherwise noted. Briefly, Yarrowia was streaked onto a YPD plate and grown at 30.degree. C. for approximately 18 hr. Several large loopfuls of cells were scraped from the plate and resuspended in 1 mL of transformation buffer containing: 2.25 mL of 50% PEG, average MW 3350; 0.125 mL of 2 M Li acetate, pH 6.0; 0.125 mL of 2 M DTT; and 50 .mu.g sheared salmon sperm DNA. Then, approximately 500 ng of linearized plasmid DNA was incubated in 100 .mu.l of resuspended cells, and maintained at 39.degree. C. for 1 hr with vortex mixing at 15 min intervals. The cells were plated onto selection media plates and maintained at 30.degree. C. for 2 to 3 days. For selection of transformants, minimal medium ("MM") was generally used; the composition of MM is as follows: 0.17% yeast nitrogen base (DIFCO Laboratories, Detroit, Mich.) without ammonium sulfate or amino acids, 2% glucose, 0.1% praline, pH 6.1). Supplements of uracil were added as appropriate to a final concentration of 0.01% (thereby producing "MMU" selection media, prepared with 20 g/L agar).

[0281] Alternatively, transformants were selected on 5-fluoroorotic acid ("FOA"; also 5-fluorouracil-6-carboxylic acid monohydrate) selection media, comprising: 0.17% yeast nitrogen base (DIFCO Laboratories) without ammonium sulfate or amino acids, 2% glucose, 0.1% proline, 75 mg/L uracil, 75 mg/L uridine, 900 mg/L FOA (Zymo Research Corp., Orange, Cali.f) and 20 g/L agar.

Fatty Acid Analysis of Yarrowia lipolytica

[0282] For fatty acid analysis, cells were collected by centrifugation and lipids were extracted as described in Bligh, E. G. & Dyer, W. J. (Can. J. Biochem. Physiol., 37:911-917 (1959)). Fatty acid methyl esters were prepared by transesterification of the lipid extract with sodium methoxide (Roughen, G., and Nishida I., Arch Biochem Biophys., 276(1):38-46 (1990)) and subsequently analyzed with a Hewlett-Packard 6890 GC fitted with a 30-m.times.0.25 mm (i.d.) HP-INNOWAX (Hewlett-Packard) column. The oven temperature was from 170.degree. C. (25 min hold) to 185.degree. C. at 3.5.degree. C./min.

[0283] For direct base transesterification, Yarrowia culture (3 mL) was harvested, washed once in distilled water, and dried under vacuum in a Speed-Vac for 5-10 min. Sodium methoxide (100 .mu.l of 1%) was added to the sample, and then the sample was vortexed and rocked for 20 min. After adding 3 drops of 1 M NaCl and 400 .mu.l hexane, the sample was vortexed and spun. The upper layer was removed and analyzed by GC as described above.

Example 1

Peridinium sp. CCMP626 Lipid Profile and RNA Isolation

[0284] Peridinium sp. CCMP626 (red algae) was purchased from The Provasoli-Guillard National Center for Culture of Marine Phytoplankton (Boothbay Harbor, Me.). About 200 mg cells were dried for 5 min under vacuum, resuspended in 100 .mu.L of trimethylsulfonium hydroxide (TMSH) and incubated at room temperature for 15 min with shaking. After this, 0.5 mL of hexane was added and the vials were incubated for 15 min at room temperature with shaking. Fatty acid methyl esters (5 .mu.L injected from hexane layer) were separated and quantified using a Hewlett-Packard 6890 Gas Chromatograph fitted with an Omegawax 320 fused silica capillary column (Catalog #24152, Supelco Inc., Bellefonte, Pa.). The oven temperature was programmed to hold at 220.degree. C. for 2.7 min, increase to 240.degree. C. at 20.degree. C./min and then hold for an additional 2.3 min. Carrier gas was supplied by a Whatman hydrogen generator. Retention times were compared to those for methyl esters of standards commercially available (Catalog #U-99-A, Nu-Chek Prep, Inc., Elysian, Minn.) and the resulting chromatogram is shown in FIG. 2.

[0285] The GC profile demonstrated that Peridinium sp. CCMP626 cells produced large amounts of both ARA and EPA, thus suggesting the presence of a delta-5 desaturase with high efficiency. Since only a trace amount of GLA was detected, it was hypothesized that Peridinium sp. CCMP626 utilizes a delta-9 elongase/delta-8 desaturase pathway for PUFA production.

[0286] Total RNA was extracted from CCMP626 cells. Specifically, cells from 1 L of culture were collected by centrifugation, quick frozen in liquid N.sub.2 and stored at -80.degree. C. The cell pellet was resuspended in 1 mL of Trizol reagent (Invitrogen, Carlsbad, Calif.), mixed with 0.6 mL of glass beads (0.5 mm), and the mixture was homogenized at the highest setting on a Biospec Mini leadbeater (Bartlesville, Okla.) for 3 min. Total RNA was then isolated, according to Invitrogen's protocol for Trizol. In this way, total RNA (34 .mu.g) was obtained from 1 L culture. The total RNA sample was used for preparation of cDNA.

Example 2

Peridinium sp. CCMP626 cDNA Synthesis

[0287] cDNA was synthesized directly from the Peridinium sp. CCMP626 total RNA as follows. Specifically, the total RNA was primered with adapter primer AP (SEQ ID NO:27) from Invitrogen's 3'-RACE kit (Carlsbad, Calif.), in the presence of the Smart IV oligonucleotide (SEQ ID NO:28) from the BD-Clontech Creator.TM. Smart.TM. cDNA library kit (Mississauga, ON, Canada). The reverse transcription was done with Superscript II reverse transcriptase from the 3'-RACE kit according to the protocol of the Creator.TM. Smart.TM. cDNA library kit.

[0288] The 1.sup.st strand cDNA synthesis mixture was used as template for PCR amplification, using primer AP (SEQ ID NO:27) as the 3' primer and CDSIII 5' primer (SEQ ID NO:29) as the 5' primer (supplied with the BD-Clontech Creator Smart.TM. cDNA library kit). Amplification was carried out with Clontech Advantage cDNA polymerase mix at 94.degree. C. for 30 sec, followed by 20 cycles of 94.degree. C. for 10 sec and 68.degree. C. for 6 min. A final extension at 68.degree. C. for 7 min was performed.

Example 3

Isolation of a Portion of the Coding Region of the Peridinium sp. CCMP626 Delta-5 Desaturase Gene

[0289] The present Example describes the identification of a portion of the Peridinium sp. CCMP626 gene encoding delta-5 desaturase (designated herein as "RD5" [i.e., red algae D5] and corresponding to SECS ID NOs:1 and 2), by use of primers derived from conserved regions of other known delta-5 and delta-8 desaturase sequences.

[0290] Various considerations were made when evaluating which desaturases might enable design of degenerate primers suitable to isolate the Peridinium sp. CCMP626 delta-5 desaturase. Specifically, the Applicants knew that only delta-5, delta-6 and delta-8 desaturase sequences comprise a conserved `HPGG` motif at their N-terminus (wherein the `HPGG` domain is part of the well-known cytochrome B5 domain); in contrast, delta-9 desaturases possess a `HPGG` motif of the cytochrome B5 domain at their C-terminus, while both delta-17 and delta-12 desaturases lack the cytochrome B5 domain. Based on the GC results described in FIG. 2, it was assumed that a delta-9 elongase/delta-8 desaturase pathway operated in Peridinium sp. CCMP626; thus, among the desaturases sharing the N-terminal conserved `HPGG` motif, only delta-5 and delta-8 desaturases were expected within the organism. Finally, although only a few delta-8 desaturase sequences are known, numerous delta-5 desaturase are publicly available. The Applicants selected those delta-5 desaturase sequences that possessed lower homology to "traditional" delta-5 desaturase genes and that also shared high homology to one another.

[0291] Based on the above, the four delta-5 desaturases and two delta-8 desaturases shown below in Table 3 were aligned, using the method of Clustal W (slow, accurate, Gonnet option; Thompson et al., Nucleic Acids Res., 22:4673-4680 (1994)) of the MegAlign.TM. program of DNASTAR software.

Table 3

Delta-5 and Delta-8 Desaturases Aligned to Identify Regions of Conserved

TABLE-US-00004 [0292] Amino Acids SEQ ID Desaturase Organism Reference NO: delta-5 Pythium irregulare GenBank Accession No. 12 AAL13311 delta-5 Phytophthora GenBank Accession No. 13 megasperma CAD53323 delta-5 Phaeodactylum GenBank Accession No. 14 tricornutum AAL92562 delta-5 Dictyostelium GenBank Accession No. 15 discoideum XP_640331 delta-8 Euglena gracilis PCT Publications No. WO 16 2006/012325 and No. WO 2006/012326 delta-8 Pavlova lutheri Example 12 (infra) 18

[0293] FIG. 3 shows a portion of the resulting alignment, containing several stretches of conserved amino acid sequence among the 6 different organisms. Based on this alignment, two sets of degenerate oligonucleotides were designed to amplify a portion of the coding region of the delta-5 desaturase gene from Peridinium sp. CCMP626, corresponding to the regions of FIG. 3 that are labeled as "Conserved Region 1" and "Conserved Region 2". Specifically, the conserved amino acid sequence GHH(IN)YTN (SEQ ID NO:19) was designed to correspond to Conserved Region 1, while the conserved amino acid sequence NFQ(V/A)(S/N)HV (SEQ ID NO:20) was designed to correspond to Conserved Region 2. In order to reduce the degeneracy of the oligonucleotides, 4 sets of oligonucleotides (i.e., 5-1A, 5-1B, 5-1C and 5-1D) were designed to encode Conserved Region 1; and, 4 sets of oligonucleotides (i.e., 5-4AR, 5-4BR, 5-4CR and 5-4DR) were designed to encode the anti-sense strand of Conserved Region 2.

TABLE-US-00005 TABLE 4 Degenerate Oligonucleotides Used To Amplify The Delta-5 Desaturase Gene From Peridinium sp. CCMP626 Oligo- nucleotide Name Sequence SEQ ID NO 5-1A GGHCAYCAYRTBTAYACAAA SEQ ID NO: 30 5-1B GGHCAYCAYRTBTAYACCAA SEQ ID NO: 31 5-1C GGHCAYCAYRTBTAYACGAA SEQ ID NO: 32 5-1D GGHCAYCAYRTBTAYACTAA SEQ ID NO: 33 5-4AR ACRTGRYTNACYTGRAAGTT SEQ ID NO: 34 5-4BR ACRTGRYTNACYTGRAAATT SEQ ID NO: 35 5-4CR ACRTGNGANACYTGRAAGTT SEQ ID NO: 36 5-4DR ACRTGNGANACYTGRAAATT SEQ ID NO: 37 [Note: The nucleic acid degeneracy code used for SEQ ID NOs: 30 to 37 was as follows: R = A/G; Y = C/T; B = G/T/C; and H = A/C/T.]

[0294] Based on the full-length sequences of the delta-5 sequences of FIG. 3, it was hypothesized that the Peridinium sp. CCMP626 delta-5 gene fragment amplified as described above would be about 630 by in length (lacking about 210 amino acids at its N-terminal and 70 amino acids at its C-terminal).

[0295] A total of sixteen different PCR amplifications were conducted, as all combinations of the primers were tested (i.e., primer 5-1A was used with each of 5-4AR, 5-4BR, 5-4CR and 5-4DR, individually; similarly, primer 5-1 B was used with each 5-4AR, 5-4BR, 5-4CR and 5-4DR; etc.). The PCR amplifications were carried out in a 50 .mu.l total volume comprising: PCR buffer (containing 10 mM KCl, 10 mM (NH.sub.4).sub.2SO.sub.4, 20 mM Tris-HCl (pH 8.75), 2 mM MgSO.sub.4, 0.1% Triton X-100), 100 .mu.g/mL BSA (final concentration), 200 .mu.M each deoxyribonucleotide triphosphate, 10 pmole of each primer, 10 ng cDNA of Peridinium sp. CCMP626 and 1 .mu.l of Taq DNA polymerase (Epicentre Technologies, Madison, Wis.). The thermocycler conditions were set for 35 cycles at 95.degree. C. for 1 min, 56.degree. C. for 30 sec and 72.degree. C. for 1 min, followed by a final extension at 72.degree. C. for 10 min.

[0296] The PCR products were purified using a Qiagen PCR purification kit (Valencia, Calif.). One fragment of the approximate expected size was then further purified following gel electrophoresis in 1% (w/v) agarose and then cloned into the pGEM-T-easy vector (Promega, Madison, Wis.). The ligated DNA was used to transform cells of E. coli DH10B and transformants were selected on LB (1% bacto-tryptone, 0.5% bacto-yeast extract and 1% NaCl) agar containing ampicillin (100 .mu.g/mL). Analysis of the plasmid DNA from a group of 12 transformants confirmed the presence of the insert with the expected size (plasmids were designated as "pT-12-D1", "pT-12-D2", "pT-12-D3", etc. to "pT-12-D12").

[0297] Sequence analyses showed that pT-12-D5 contained a 563 by fragment (SEQ ID NO:4), which encoded 187 amino acids (SEQ ID NO:5) (including amino acids that corresponded to Conserved Region 1 and 2). Identity of the Peridinium sp. CCMP626 sequence was determined by conducting BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., J. Mol. Biol., 215:403-410 (1993)) searches for similarity to sequences contained in the BLAST "nr" database (comprising all non-redundant GenBank GDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the SWISS-PROT protein sequence database, EMBL and DDBJ databases). The sequence was analyzed for similarity to all publicly available DNA sequences contained in the "nr" database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). SEQ 1D NO:4 was compared for similarity to all publicly available protein sequences contained in the "nr" database, using the BLASTX algorithm (Gish, W. and States, D. J., Nature Genetics, 3:266-272 (1993)) provided by the NCBI.

[0298] The results of the BLASTX comparison summarizing the sequence to which SEQ ID NO:4 has the most similarity are reported according to the % identity, % similarity and Expectation value. "% Identity" is defined as the percentage of amino acids that are identical between the two proteins. "% Similarity" is defined as the percentage of amino acids that are identical or conserved between the two proteins. "Expectation value" estimates the statistical significance of the match, specifying the number of matches, with a given score, that are expected in a search of a database of this size absolutely by chance. Thus, the translated amino acid sequence of SEQ ID NO:4 (i.e., SEQ ID NO:5) had 64% identity and 78% similarity with the amino acid sequence of the delta-8-sphingolipid desaturase of Thalassiosira pseudonana (GenBank Accession No, AAX14502; SEQ ID NO:21), with an Expectation value of 2E-64; additionally, the partial fragment of SEQ ID NO:4 had 65% identity and 78% similarity with the delta-5 fatty acid desaturase of Phaeodactylum tricornutum (GenBank Accession No. AAL92562; SEQ ID NO:14), with an Expectation value of 7E-62.

Example 4

Isolation of the 5` Coding Region of the Peridinium sp. CCMP626 Delta-5 Desaturase Gene

[0299] To isolate the N-terminal portion of the putative delta-5 desaturase identified in Example 3, a modified 5' RACE technique based on RACE protocols from two different companies (i.e., Invitrogen and BD-Clontech) was utilized.

[0300] Briefly, the double-stranded cDNA of Peridinium sp. CCMP626 (Example 2) was used as the template in a 5' RACE experiment, comprising two separate.rounds of PCR amplification. In the first round of PCR amplification, the oligonucleotide primers consisted of a gene specific oligonucleotide (i.e., ODMW520; SEQ ID NO:38) and the generic oligonucleotide CDSIII 5' primer (SEQ ID NO:29) from the BD-Clontech Creator.TM. Smart.TM. cDNA library kit. The PCR amplifications were carried out in a 50 .mu.l total volume, comprising: 25 .mu.l of LA Tag.TM. pre-mix (TakeRa Bio Inc., Otsu, Shiga, 520-2193, Japan), 10 pmole of each primer and 1 .mu.l of Taq DNA polymerase (Epicentre Technologies, Madison, Wis.). The thermocycler conditions were set for 35 cycles at 95.degree. C. for 1 min, 56.degree. C. for 30 sec and 72.degree. C. for 1 min, followed by a final extension at 72.degree. C. for 10 min.

[0301] The second round of PCR amplification used 1 .mu.l of the product from the first round PCR reaction as template. Primers consisted of a gene specific oligonucleotide (i.e., ODMW521; SEQ ID NO:39) and the generic oligonucleotide DNR CDS 5' (SEQ ID NO:40), supplied with BD-Clontech's Creator.TM. Smart.TM. cDNA library kit. Amplification was conducted as described above.

[0302] The products of the second round PCR reaction were electrophoresed in 1% (w/v) agarose. Products between 400 by and 800 by were then purified from the gel and cloned into the pGEM-T-easy vector (Promega, Madison, Wis.). The ligated DNA was used to transform E. coli DH10B and transformants were selected on LB agar containing ampicillin (100 .mu.g/mL).

[0303] Analysis of the plasmid DNA from one transformant comprising the 5' region of the putative delta-5 desaturase gene confirmed the presence of the expected plasmid, designated pT-RD5-5'C2. Sequence analyses showed that pT-RD5-5'C2 contained a fragment of 693 by (SEQ 1D NO:6), which over with 182 by from the 5' end of the 563 by fragment of pT-12-D5 (Example 3; SEQ ID NO:4) and additionally provided 511 by of 5' upstream sequence (SEQ ID NO:7) (FIG. 4). The sequence of pT-RD5-5'C2 also corrected the sequence corresponding to Conserved Region 1, resulting from use of a degenerate oligonucleotide for initial PCR amplification of the 563 by fragment in pT-12-D5 (Example 3). However, there was no translation initiation codon in the extended 693 by fragment of SEQ ID NO:6.

[0304] A second round of the modified 5' RACE was carried out as described above, except that oligonucleotides ODMW541 (SEQ ID NO:41) and ODMW542 (SEQ ID NO:42) were used as gene-specific primers. Products between 200 by and 400 bp were then purified from a gel and cloned into the pGEM-T-easy vector (Promega, Madison, Wis.). The ligated DNA was transformed into E. coli DH10B and transformants were selected on LB agar containing ampicillin (100 .mu.g/mL).

[0305] Analysis of the plasmid DNA from one transformant comprising the 5' region of the putative delta-5 desaturase gene confirmed the presence of the expected plasmid, designated pT-RD5-5'2.sup.nd . Sequence analyses showed that pT-RD5-5'2.sup.nd contained a fragment of 358 by (SEQ ID NO:8), which over-lapped with 197 by of the 5' end of the DNA fragment in pT-RD5-5'C2 described above and additionally provided 161 by of 5' upstream sequence (SEQ ID NO:9). One-hundred sixteen (116) by of the 5' extended fragment encoded the N-terminal portion of the putative delta-5 desaturase gene, including the translation initiation codon, thus providing the complete 5' sequence of the gene.

Example 5

Isolation of the 3' Coding Region of the Peridinium sp. CCMP626 Delta-5 Desaturase Gene

[0306] To isolate the C-terminal portion of the putative delta-5 desaturase identified in Example 3, a 3' RACE technique was utilized. The methodology was described above in Example 4; however, the primers used on both the first and second round of PCR amplification were as shown below in Table 5.

TABLE-US-00006 TABLE 5 Oligonucleotide Primers Used For 3' RACE PCR Gene Specific Generic Amplification Oligonucleotide Oligonucleotide 1.sup.st Round ODMW523 AUAP (SEQ ID NO: 43) (SEQ ID NO: 44) 2.sup.nd Round ODMW524 AUAP (SEQ ID NO: 45) (SEQ ID NO: 44) *Primer AUAP was supplied in Invitrogen's 3'-RACE kit (Carlsbad, CA).

[0307] Following isolation and purification of products (Le., 200-500 bp), the fragments were cloned into the pGEM-T-easy vector (Promega) and transformed into E. coli DH10B, as in Example 4.

[0308] Analysis of the plasmid DNA from one transformant comprising the 3' region of the delta-5 desaturase gene confirmed the presence of the expected plasmid, designated pT-RD5-3'. Sequence analyses showed that pT-RD5-3' contained a fragment of 299 by (SEQ ID NO:10), which over-lapped with 52 by from the 3' end of the 563 by fragment of pT-12-D5 (Example 3, SEQ ID NO:4) and provided 247 by of additional 3' downstream sequence (SEQ ID NO:11). The first 202 by of the extended 247 by fragment included within pT-RD5-3' encoded the C-terminal coding region (including the translation stop codon) of the putative delta-5 desaturase gene. The sequence of pT-RD5-3' also corrected the sequence corresponding to Conserved Region 2, resulting from use of a degenerate oligonucleotide for initial PCR amplification of the 563 by fragment in pT-12-D5 (Example 3):

[0309] After 2 rounds of 5' RACE and one round of 3' RACE, the DNA sequence of the entire putative Peridinium sp. CCMP626 delta-5 desaturase (RD5) coding region was determined. As shown in FIG. 4, the RD5 CDS was 1392 by in length (SEQ ID NO:1) and encoded a polypeptide with 463 amino acids (SEQ ID NO:2), based on alignment of SEQ ID NOs:4, 6, 8 and 10. The results of BLASTP sequence analysis algorithms using the full length RD5 gene as the query sequence showed that it shared 67% identity and 81% similarity with the delta-5 fatty acid desaturase of Phaeodactylum tricornutum (GenBank Accession No. AAL92562; SEQ ID NO:14), with an Expectation value of 0.0. Additionally, the full length RD5 gene shared 64% identity and 76% similarity with the delta-8-sphingolipid desaturase of Thalassiosira pseudonana (GenBank Accession No. AAX14502; SEQ ID NO:21), with an Expectation value of 2E-178.

Example 6

Generation of Construct pDMW368, Comprising RD5

[0310] The present Example describes the generation of pDMW368, comprising a chimeric FBAIN::RD5::Pex20 gene (FIG. 5C). This was designed to integrate the chimeric gene into the genome of Yarrowia lipolytica and then study the function of the Peridinium sp. CCMP626 delta-5 desaturase in Yarrowia lipolytica.

[0311] Based on the full length cDNA of RD5 (SEQ ID NO:1), oligonucleotides YL807 and YL810 (SEQ ID NOs:46 and 47, respectively) were used as primers to amplify the first portion of RD5 (FIG. 5A). Primer YL807 contained a NcoI site and primer YL810 contained a BamH1 site. Then, primers YL808 and YL809 (SEQ ID NOs:48 and 49, respectively) were used as primers to amplify the second portion of RD5. Primer YL809 contained a BamH1 site, while primer YL808 contained a NotI site. The PCR reactions, using primer pairs YL807/YL810 or YL808/YL809, with Peridinium sp. CCMP626 cDNA (Example 2) as template, were individually carried out in a 50 .mu.l total volume comprising: PCR buffer (containing 10 mM KCl, 10 mM (NH.sub.4).sub.2SO.sub.4, 20 mM Tris-HCl (pH 8.75), 2 mM MgSO.sub.4, 0.1% Triton X-100), 100 .mu.g/mL BSA (final concentration), 200 .mu.M each deoxyribonucleotide triphosphate, 10 pmole of each primer and 1 .mu.l of Pfu DNA polymerase (Stratagene, San Diego, Calif.). The thermocycler conditions were set for 35 cycles at 95.degree. C. for 1 min, 56.degree. C. for 30 sec and 72.degree. C. for 1 min, followed by a final extension at 72.degree. C. for 10 min.

[0312] The individual PCR products were purified using a Qiagen PCR purification kit. The PCR products from the reaction amplified with primers YL807/YL810 were digested with NcoI and BamH1, while the PCR products from the reaction amplified with primers YL808/YL809 were digested with BamHI and NotI. The NcoI/BamH1- and the BamH1/NotI-digested DNA fragments were purified following gel electrophoresis in 1% (w/v) agarose, and then directionally ligated with NcoI/NotI-digested pZUF17 (FIG. 5B; SEQ ID NO:22; comprising a synthetic delta-17 desaturase gene ["D17st"] derived from Saprolegnia diclina (U.S. Patent Publication No. 200310196217 A1), codon-optimized for expression in Yarrowia lipolytica (PCT Publication No. WO 2004/101757)). The product of this ligation was pDMW368 (FIG. 5C; SEQ ID NO:23, which thereby contained the following components:

TABLE-US-00007 TABLE 6 Components Of Plasmid pDMW368 RE Sites And Nucleotides Within SEQ ID Description Of Fragment NO: 23 And Chimeric Gene Components EcoR I/BsiW I FBAIN::RD5::Pex20, comprising: (6063-318) FBAIN: Yarrowia lipolytica FBAIN promoter (PCT Publication No. WO 2005/049805; U.S. Pat. No. 7,202,356) RD5: Peridinium sp. CCMP626 delta-5 desaturase (SEQ ID NO: 1 described herein) Pex20: Pex20 terminator sequence of Yarrowia Pex20 gene (GenBank Accession No. AF054613) 1354-474 ColE1 plasmid origin of replication 2284-1424 ampicillin-resistance gene (Amp.sup.R) for selection in E. coli 3183-4487 Yarrowia autonomous replication sequence (ARS18; GenBank Accession No. A17608) 6020-4533 Yarrowia Ura 3 gene (GenBank Accession No. AJ306421)

The term "FBAIN promoter" or "FBAIN promoter region" refers to the 5' upstream untranslated region in front of the `ATG` translation initiation codon of the Yarrowia lipolytica fructose-bisphosphate aldolase enzyme (E.C. 4.1.2.13) encoded by the fba1 gene and that is necessary for expression, plus a portion of 5' coding region that has an intron of the fba1 gene.

Example 7

Generation of Yarrowia lipolytica Strain M4 to Produce About 8% DGLA of Total Lipids

[0313] The present Example describes the construction of strain M4, derived from Yarrowia lipolytica ATCC #20362, capable of producing 8% DGLA relative to the total lipids. This strain was engineered to express the delta-6 desaturase/delta-6 elongase pathway, via introduction of construct pKUNF12T6E (FIG. 6A; SEQ ID NO:24). This construct was generated to integrate four chimeric genes (comprising a delta-12 desaturase, a delta-6 desaturase and two C.sub.18/20 elongases) into the Ura3 loci of wild type Yarrowia strain ATCC #20362, to thereby enable production of DGLA. Thus, pKUNF12T6E contained the following components:

TABLE-US-00008 TABLE 7 Description of Plasmid pKUNF12T6E (SEQ ID NO: 24) RE Sites And Nucleotides Within SEQ ID Description Of Fragment NO: 24 And Chimeric Gene Components AscI/BsiWI 784 bp 5' portion of Yarrowia Ura3 gene (GenBank (9420-8629) Accession No. AJ306421) SphI/PacI 516 bp 3' portion of Yarrowia Ura3 gene (GenBank (12128-1) Accession No. AJ306421) SwaI/BsiWI FBAIN::EL1S::Pex20, comprising: (6380-8629) FBAIN: Yarrowia lipolytica FBAIN promoter (PCT Publication No. WO 2005/049805; U.S. Pat. No. 7,202,356; labeled as "Fba1 + intron" in Figure) EL1S: codon-optimized elongase 1 gene (PCT Publication No. WO 2004/101753), derived from Mortierella alpina (GenBank Accession No. AX464731) Pex20: Pex20 terminator sequence from Yarrowia Pex20 gene (GenBank Accession No. AF054613) BglII/SwaI TEF::delta-6S::Lip1, comprising: (4221-6380) TEF: Yarrowia lipolytica TEF promoter (GenBank Accession No. AF054508) delta-6S: codon-optimized delta-6 desaturase gene (PCT Publication No. WO 2004/101753), derived from Mortierella alpina (GenBank Accession No. AF465281) Lip1: Lip1 terminator sequence from Yarrowia Lip1 gene (GenBank Accession No. Z50020) PmeI/ClaI FBA::F.delta-12::Lip2, comprising: (4207-1459) FBA: Yarrowia lipolytica FBA promoter (PCT Publication No. WO 2005/049805; U.S. Pat. No. 7,202,356; labeled as "FBA1" in Figure) F.delta-12: Fusarium moniliforme delta-12 desaturase gene (PCT Publication No. WO 2005/047485) Lip2: Lip2 terminator sequence from Yarrowia Lip2 gene (GenBank Accession No. AJ012632) ClaI/PacI TEF::EL2Syn::XPR2, comprising: (1459-1) TEF: Yarrowia lipolytica TEF promoter (GenBank Accession No. AF054508) EL2Syn: codon-optimized elongase gene (SEQ ID NO: 25), derived from Thraustochytrium aureum (U.S. Pat. No. 6,677,145) XPR2: ~100 bp of the 3' region of the Yarrowia Xpr gene (GenBank Accession No. M17741)

[0314] Plasmid pKUNF12T6E was digested with AscI/SphI, and then used for transformation of wild type V. lipolytica ATCC #20362 according to the General Methods. The transformant cells were plated onto FOA selection media plates and maintained at 30.degree. C. for 2 to 3 days. The FOA resistant colonies were picked and streaked onto MM and MMU selection plates. The colonies that could grow on MMU plates but not on MM plates were selected as Ura- strains. Single colonies of Ura- strains were then inoculated into liquid MMU at 30.degree. C. and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.

[0315] GC analyses showed the presence of DGLA in the transformants containing the 4 chimeric genes of pKUNF12T6E, but not in the wild type Yarrowia control strain. Most of the selected 32 Ura.sup.- strains produced about 6% DGLA of total lipids. There were 2 strains strains M4 and 13-8) that produced about 8% DGLA of total lipids.

Example 8

Functional Analysis of RD5 Gene in Yarrowia lipolytica Strain M4

[0316] Plasmid pDMW368 (Example 6; comprising a chimeric FBAIN::RD5::Pex20 gene was transformed into strain M4 (Example 7), as described in the General Methods. The transformants were selected on MM plates. After 2 days grown at 30.degree. C., 3 transformants grown on the MM plates were picked and re-streaked onto fresh MM plates. Once grown, these strains were individually inoculated into 3 mL liquid MM at 30.degree. C. and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.

[0317] GC analyses showed that there were about 4.2% DGLA and 2.2% ARA of total lipids produced in all three transformants, wherein the conversion efficiency of DGLA to ARA in these three strains was determined to be about 35% (average). The conversion efficiency was measured according to the following formula: ([product]/[substrate+product])*100, where `product` includes the immediate product and all products in the pathway derived from it. Thus, this experimental data demonstrated that the cloned Peridinium sp. CCMP626 delta-5 desaturase, described herein as SEQ ID NOs:1 and 2, efficiently desaturated DGLA to ARA.

Example 9

Synthesis of a Codon-Optimized Delta-5 Desaturase Gene ("RD5S'') for Expression in Yarrowia lipolytica

[0318] The codon usage of the delta-5 desaturase gene of Peridinium sp. CCMP626 (SEQ ID NOs:1 and 2; RD5) was optimized for expression in Yarrowia lipolytica, in a manner similar to that described in PCT Publication No. WO 2004/101753 and U.S. Pat. No. 7,125,672. Specifically, a codon-optimized delta-5 desaturase gene (designated "RD5S", SEQ ID NO:3) was designed based on the coding sequence of the delta-5 desaturase gene of RD5 (SEQ ID NO:2), according to the Yarrowia codon usage pattern (PCT Publication No. WO 2004/101753), the consensus sequence around the `ATG` translation initiation codon, and the general rules of RNA stability (Guhaniyogi, G. and J. Brewer, Gene, 265(1-2):11-23 (2001)). In addition to modification of the translation initiation site, 247 by of the 1392 by coding region was modified (171%, FIGS. 7A and 7B) and 229 codons were optimized (49.4%). The GC content was increased from 49.3% within the wild type gene (i.e., RD5) to 54.2% within the synthetic gene (Le., RD5S). A NcoI site and NotI sites were incorporated around the translation initiation codon and after the stop codon of RD5S (SEQ ID NO:3), respectively. None of the modifications in the codon-optimized gene changed the amino acid sequence of the encoded protein (SEQ ID NO:2). The designed RD5S gene was synthesized by GenScript Corporation (Piscataway, N.J.) and cloned into pUC57 (GenBank Accession No. Y14837) to generate pRD5S (SEQ ID NO:50; FIG. 6B) (RD5S labeled as "RD5S(626)" in Figure).

Example 10

Generation of Construct pZURD5S, Comprising RD5S

[0319] The present Example describes the construction of plasmid pZURD5S comprising a chimeric FBAIN:RD5S::Pex20 gene. Plasmid pZURD5S (SEQ ID NO:51; FIG. 6C) was constructed by replacing the Nco I/Not I fragment of pZUF17 (FIG. 5B; SEQ ID NO:22) with the Nco I/Not I RD5S fragment from pRD5S (SEQ ID NO:50; FIG. 6B). The product of this ligation was pZURD5S (FIG. 6C; SEQ ID NO:51), which thereby contained the following components:

TABLE-US-00009 TABLE 8 Components Of Plasmid pZURD5S RE Sites And Nucleotides Within SEQ ID Description Of Fragment NO: 51 And Chimeric Gene Components EcoR I/BsiW I FBAIN::RD5S::Pex20, comprising: (7458-1713) FBAIN: Yarrowia lipolytica FBAIN promoter (PCT Publication No. WO 2005/049805; U.S. Pat. No. 7,202,356) RD5S: codon-optimized delta-5 desaturase (SEQ ID NO: 3, described herein as RD5S), derived from Peridinium sp. CCMP626 (labeled as "RD5S(626)" in Figure) Pex20: Pex20 terminator sequence of Yarrowia Pex20 gene (GenBank Accession No. AF054613) 2749-1869 ColE1 plasmid origin of replication 3679-2819 ampicillin-resistance gene (Amp.sup.R) for selection in E. coli 4578-5882 Yarrowia autonomous replication sequence (ARS18; GenBank Accession No. A17608) 7415-5928 Yarrowia Ura 3 gene (GenBank Accession No. AJ306421)

Example 11

Expression of the Codon-Optimized Delta-5 Desaturase ("RD5S") in Yarrowia lipolytica Strain M4

[0320] Plasmid pZURD5S (Example 10; comprising a chimeric FBAIN::RD5S::Pex20 gene) was used for transformation into strain M4 (Example 7), as described in the General Methods. Eight (8) transformants grown on the MM plates were picked and re-streaked onto fresh MM plates. Once grown, these strains were individually inoculated into 3 mL liquid MM at 30.degree. C. and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.

[0321] The GC results showed that there were about 5.4% DGLA and 3.3% ARA of total lipids produced in all 8 transformants. The conversion efficiency whereby RD5S converted DGLA to ARA in these 8 strains was at an average rate of about 38%. The conversion efficiency was measured according to the following formula: ([product]/[substrate+product]*100, where `product` includes the immediate product and all products in the pathway derived from it. Thus, this experimental data demonstrated that the codon-optimized delta-5 desaturase gene (ROSS, as set forth in SEQ ID NO:3) derived from Peridinium sp. CCMP626 was about 8.9% more efficient converting DGLA to ARA than the wild type RD5 (Example 8).

Example 12

Isolation of a Pavlova lutheri (CCMP459) Delta-8 Desaturase

[0322] The present example describes the isolation of the Pavlova lutheri (CCMP459) delta-8 desaturase utilized in Example 3 and in FIG. 3 (also described in U.S. patent application Ser. No. 11/737,772, filed Apr. 20, 2007). This required: synthesis of Pavlova lutheri (CCMP459) cDNA; library construction and sequencing; identification of delta-8 desaturase homologs; and, cloning of a full-length delta-8 desaturase from genomic DNA.

Pavlova lutheri (CCMP459) cDNA Synthesis, Library Construction and Sequencing

[0323] A cDNA library of Pavlova lutheri (CCMP459) was synthesized as described in PCT Publication No. WO 20041071467 (published Aug. 26, 2004). Briefly, frozen pellets of Pav459 were obtained from the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (COMP, West Boothbay Harbor, Me.). These pellets were crushed in liquid nitrogen and total RNA was extracted from Pav459 by using the Qiagen RNeasy.RTM. Maxi Kit (Qiagen, Valencia, Calif.), per the manufacturer's instructions. From this total RNA, mRNA was isolated using oligo dT cellulose resin, which was then used for the construction of a cDNA library using the pSport1 vector (Invitrogen, Carlsbad, Calif.). The cDNA thus produced was directionally cloned (5' Sal/I/3' NotI into pSport1 vector. The Pav459 library contained approximately 6.1.times.10.sup.5 clones per mL, each with an average insert size of approximately 1200 bp. The Pavlova lutheri library was named eps1c.

[0324] For sequencing, clones first were recovered from archived glycerol cultures grown/frozen in 384-well freezing media plates, and inoculated with an automatic QPix.RTM. colony picker (Genetix) in 96-well deep-well plates containing LB+100 mg/mL ampicillin. After growing 20 hrs at 37.degree. C., cells were pelleted by centrifugation and stored at -20.degree. C. Plasmids then were isolated on an Eppendorf 5Prime robot, using a modified 96-well format alkaline lysis miniprep method (Eppendorf PerfectPrep.RTM.). Briefly, a filter and vacuum manifold was used to facilitate removal of cellular debris after acetate precipitation. Plasmid DNA was then bound on a second filter plate directly from the filtrate, washed, dried and eluted.

[0325] Plasmids were end-sequenced in 384-well plates, using vector-primed T7 primer (SEQ ID NO:52) and the ABI BigDye version 3 Prism sequencing kit. For the sequencing reaction, 100-200 ng of template and 6.4 pmoL of primer were used, and the following reaction conditions were repeated 25 times: 96.degree. C. for 10 sec, 50.degree. C. for 5 sec and 60.degree. C. for 4 min. After ethanol-based cleanup, cycle sequencing reaction products were resolved and detected on Perkin-Elmer MI 3700 automated sequencers.

Identification of Delta-8 Desaturase Enzyme Homologs from Pavlova lutheri cDNA Library eps1c

[0326] cDNA clones encoding Pavlova lutheri delta-8 desaturase homologs (hereby called delta-8 desaturases) were identified by conducting BLAST searches for similarity to sequences contained in the BLAST "nr" database (as described in Example 3). The P-value (probability) of observing a match of a cDNA sequence to a sequence contained in the searched databases merely by chance as calculated by BLAST are reported herein as "pLog" values, which represent the negative of the logarithm of the reported P-value. Accordingly, the greater the pLog value, the greater the likelihood that the cDNA sequence and the BLAST "hit" represent homologous proteins.

[0327] The BLASTX search using the nucleotide sequence from clone epslc.pk002.f22 revealed similarity of the protein encoded by the cDNA to the delta-6 desaturase from Rhizopus stolonifer (SEQ ID NO:53) (NCBI Accession No. AAX22052 (GI 60499699), locus AAX22052, CDS AY795076; Lu et al., unpublished). The sequence of a portion of the cDNA insert from clone eps1c.pk002.f22 is shown in SEQ ID NO:54 (5' end of cDNA insert). Subsequently, the full insert sequence (eps1c.pk002.f22:fis) was obtained and is shown in SEQ ID NO:55. Sequence for the deduced amino acid sequence (from nucleotide 1 of SEQ ID NO:55 to the first stop codon at nucleotide 864 of SEQ ID NO:55) is shown in SEQ ID NO:56. Full insert sequencing was carried out using a modified transposition protocol. Clones identified for full insert sequencing were recovered from archived glycerol stocks as single colonies, and plasmid DNA was isolated via alkaline lysis. Plasmid templates were transposed via the Template Generation System (TGS II) transposition kit (Finnzymes Oy, Espoo, Finland), following the manufacturer's protocol. The transposed DNA was transformed into EH10B electro-competent cells (Edge BioSystems, Gaithersburg, Md.) via electroporation. Multiple transformants were randomly selected from each transposition reaction, plasmid DNA was prepared, and templates were sequenced as above (ABI BigDye v3.1) outward from the transposition event site, utilizing unique primers SeqE (SEQ ID NO:57) and SeqW (SEQ ID NO:58).

[0328] Sequence data was collected (ABI Prism Collections software) and assembled using the Phrap sequence assembly program (P. Green, University of Washington, Seattle). Assemblies were viewed by the Consed sequence editor (D. Gordon, University of Washington, Seattle) for final editing.

[0329] The amino acid sequence set forth in SEQ ID NO:56 was evaluated by BLASTP, yielding a pLog value of 19.52 (E value of 3e-20) versus the delta-6 desaturase from Mortierella alpine (NCBI Accession No. BAC82361 (GI 34221934), locus BAC82361, CDS AB070557; Sakuradani and Shimizu, Biosci. Biotechnol. Biochem., 67:704-711 (2003)). Based on the results from the BLASTP comparison to the Mortierella alpine and other fatty acid desaturases, the Pavlova lutheri delta-8 desaturase was not full length and was lacking sequence at the 5' end.

Cloning a Full-Length Delta-8 Desaturase from Pavlova lutheri Genomic DNA

[0330] Genomic DNA was isolated from Pavlova lutheri (CCMP459) using the Qiagen DNeasy.RTM. Plant Maxi Prep Kit according to the manufacturer's protocol. Using 1 maxi column per 1 gm of frozen cell pellet, a total of 122 pg of genomic DNA was isolated from 4 gm of Pavlova lutheri culture. The final concentration of genomic DNA was 22.8 ng/.mu.L. GenomeWalker libraries were synthesized using the Universal GenomeWalker.TM. kit (BD Biosciences Clonetech, Palo Alto, Calif.) following the manufacturer's protocol (Prot #PT3042-1, version PRO3300). Briefly, four restriction digests were set up as per the protocol using 300 ng of genomic DNA per reaction. After phenol clean up, pellets were dissolved in 4 .mu.L of water and adapters were ligated as per the protocol.

[0331] For the primary PCR, the Advantage.RTM.-GC Genornic PCR kit (BD Biosciences Clonetech) was used following the manufacturer's protocol (Prot #PT3090-1, version PR1X433). For each restriction digest, 1 .mu.L of library was combined with 22.8 .mu.L of PCR grade water, 10 .mu.L of 5.times. GC Genornic PCR Reaction Buffer, 2.2 .mu.L of 25 mM Mg(CH.sub.3CO.sub.2).sub.2, 10 .mu.L of GC-Melt (5 M), 1 .mu.L of 50.times. dNTP mix (10 mM each), 1 .mu.L of Advantage-GC Genomic Pol. Mix (50.times.), 1 .mu.L of Universal GenomeWalker.TM. primer API (10 .mu.M, SEQ ID NO:59) and 1 .mu.L of GSP PvDES (10 .mu.M, SEQ ID NO:60). After denaturation at 95.degree. C., the following reaction conditions were repeated 35 times: 94.degree. C. for 30 sec, 68.degree. C. for 6 min. After these reaction conditions, an additional extension at 68.degree. C. was carried out for 6 min followed by cooling to 15.degree. C. until removed.

[0332] The primary PCR reaction for each library was analyzed by agarose gel electrophoresis and DNA bands with molecular weights around 6 kB, 3.5 kb, 2.5 kB and 1.2 kB were observed. DNA bands for each library were purified using the Zymocleae.TM. Gel DNA Recovery Kit (Zymo Research, Orange, Calif.) following the manufacturer's protocol. The resulting DNA was cloned into the pGEM.RTM.-T Easy Vector (Promega) following the manufacturer's protocol and inserts were sequenced using the T7 (SEQ ID NO:52) and M13-28Rev (SEQ ID NO:61) primers as described above. Additional sequence was then obtained using a gene-specific sequencing primer PavDES seq (SEQ ID NO:62) that was derived from the newly acquired sequence data. The full 5' end sequence obtained by genome walking is shown in SEQ ID NO:63. The sequence of the overlapping regions of the genomic sequence (SEQ ID NO:63) and the fully sequenced EST eps1c.pk002.f22:fis (SEQ ID NO:55) were aligned using Sequencher.TM. (Version 4.2, Gene Codes Corporation, Ann Arbor, Mich.) using the Large Gap assembly algorithm. Interestingly, the comparison showed that the EST that was originally sequenced (SEQ ID NO:55) was lacking 459 by when compared to the genomic sequence (SEQ ID NO:63). This missing sequence in the EST appeared to be a deletion rather than an intron as no clear intron splice sites were identified in the genomic DNA at the 5' end of the gene. The genomic sequence for the 5' end (SEQ ID NO:63) was combined with the 3' end of the EST sequence (SEQ ID NO:55) to yield SEQ ID NO:64. Using EditSeq.TM. 6.1 sequence analysis software (DNASTAR Inc., Madison, Wis.), an ORE was identified (SEQ ID NO:17). The amino acid sequence coded for by SEQ ID NO:17 is shown in SEQ ID NO:18.

[0333] The amino acid sequence set forth in SEQ ID NO:18 was evaluated by BLASTP, yielding a pLog value of 35.10 (E value of 8e-36) versus the delta-6 desaturase from Rhizopus stolonifer (SEQ ID NO:65) (NCBI Accession No. ABB96724 (GI 83027409), locus ABB96724, CDS DQ291156; Zhang et al., unpublished). Furthermore, the Pavlova lutheri delta-8 desaturase is 78.0% identical to the Pavlova salina delta-8 desaturase sequence (SEQ ID NO:66) disclosed in PCT Publication No. WO 2005/103253 (published Apr. 22, 2005) using the Jotun Hein method. Sequence percent identity calculations performed by the Jotun Hein method (Hein, J. J., Meth. Enz.,183:626-645 (1990)) were done using the MegAlign.TM. v6.1 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.) with the default parameters for pairwise alignment (KTUPLE=2). The Pavlova lutheri delta-8 desaturase is 76.4% identical to the Pavlova salina delta-8 desaturase sequence using the Clustal V method. Sequence percent identity calculations performed by the Clustal V method (Higgins, D. G. and Sharp, P. M., Comput. Appl. Biosci., 5:151-153 (1989); Higgins et al., Comput. Appl. Biosci., 8:189-191 (1992)) were done using the MegAlign.TM. v6.1 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.) with the default parameters for pairwise alignment (KTUPLE=1, GAP PENALTY=3, WINDOW=5, DIAGONALS SAVED=5 and GAP LENGTH PENALTY=10). BLAST scores and probabilities indicate that the fragment of SEQ ID NO:17 encodes an entire Pavlova lutheri delta-8 desaturase.

[0334] FIGS. 8A and 8B show a Clustal V alignment (with default parameters) of SEQ ID NO:18 (the amino acid sequence of the Pavlova lutheri delta-8 desaturase), SEQ ID NO:66 (the amino acid sequence of Pavlova salina delta-8 desaturase sequence, supra), SEQ ID NO:16 (the amino acid sequence of Euglena gracilis delta-8 desaturase sequence disclosed as SEQ ID NO:2 in PCT Publication No. WO 2006/012325; published Feb. 2, 2006), SEQ ID NO:65 (the amino acid sequence for the Rhizopus stolonifer delta-6 fatty acid desaturase (NCBI Accession No. ABB96724, supra)) and SEQ ID NO:53 (the amino acid sequence for the Rhizopus stolonifer delta-6 fatty acid desaturase (NCBI Accession No. AAX22052, supra)). The results of the Clustal V alignment show that SEQ ID NO:18 is 76.4%, 22.6%, 22.2% and 22.2% identical to SEQ ID NO:66, SEQ 1D NO:16, SEQ ID NO:65 and SEQ ID NO:53, respectively.

Example 13

Transformation of Somatic Soybean Embryo Cultures

Culture Conditions

[0335] Soybean embryogenic suspension cultures (cv. Jack) are maintained in 35 mL liquid medium SB196 (infra) on a rotary shaker, 150 rpm, 26.degree. C. with cool white fluorescent lights on 16:8 hr day/night photoperiod at light intensity of 60-85 .mu.E/m.sup.2/s. Cultures are subcultured every 7 days to 2 weeks by inoculating approximately 35 mg of tissue into 35 mL of fresh liquid SB196 (the preferred subculture interval is every 7 days).

[0336] Soybean embryogenic suspension cultures are transformed with soybean expression plasmids by the method of particle gun bombardment (Klein et al., Nature, 327:70 (1987)) using a DuPont Biolistic PDS1000/HE instrument (helium retrofit) for all transformations.

Soybean Embryogenic Suspension Culture Initiation:

[0337] Soybean cultures are initiated twice each month with 5-7 days between each initiation. Pods with immature seeds from available soybean plants 45-55 days after planting are picked, removed from their shells and placed into a sterilized magenta box. The soybean seeds are sterilized by shaking them for 15 min in a 5% Clorox solution with 1 drop of ivory soap (i.e., 95 mL of autoclaved distilled water plus 5 mL Clorox and 1 drop of soap, mixed well). Seeds are rinsed using 2 1-liter bottles of sterile distilled water and those less than 4 mm are placed on individual microscope slides. The small end of the seed is cut and the cotyledons pressed out of the seed coat. Cotyledons are transferred to plates containing SB1 medium (25-30 cotyledons per plate). Plates are wrapped with fiber tape and stored for 8 weeks. After this time secondary embryos are cut and placed into SB196 liquid media for 7 days.

Preparation of DNA for Bombardment:

[0338] Either an intact plasmid or a DNA plasmid fragment containing the delta-5 desaturase genes of interest and the selectable marker gene are used for bombardment. Fragments from soybean expression plasmids comprising the delta-5 desaturase of the present invention are obtained by gel isolation of digested plasmids. The resulting DNA fragments are separated by gel electrophoresis on 1% SeaPlaque GTG agarose (BioWhitaker Molecular Applications) and the DNA fragments containing gene cassettes are cut from the agarose gel. DNA is purified from the agarose using the GELase digesting enzyme following the manufacturers protocol.

[0339] A 50 .mu.L aliquot of sterile distilled water containing 3 mg of gold particles is added to 5 .mu.L of a 1 .mu.g/.mu.L DNA solution (either intact plasmid or DNA fragment prepared as described above), 50 .mu.L 2.5 M CaCl.sub.2 and 20 .mu.L of 0.1 M spermidine. The mixture is shaken 3 min on level 3 of a vortex shaker and spun for 10 sec in a bench microfuge. After a wash with 400 .mu.L of 100% ethanol, the pellet is suspended by sonication in 40 .mu.L of 100% ethanol. DNA suspension (5 .mu.L) is dispensed to each flying disk of the Biolistic PDS1000/HE instrument disk. Each 5 .mu.L aliquot contains approximately 0.375 mg gold particles per bombardment (i.e., per disk).

Tissue Preparation and Bombardment with DNA:

[0340] Approximately 150-200 mg of 7 day old embryonic suspension cultures is placed in an empty, sterile 60.times.15 mm petri dish and the dish is covered with plastic mesh. Tissue is bombarded 1 or 2 shots per plate with membrane rupture pressure set at 1100 PSI and the chamber is evacuated to a vacuum of 27-28 inches of mercury. Tissue is placed approximately 3.5 inches from the retaining/stopping screen.

Selection of Transformed Embryos:

[0341] Transformed embryos ate selected using hygromycin as the selectable marker. Specifically, following bombardment, the tissue is placed into fresh SB196 media and cultured as described above. Six days post-bombardment, the SB196 is exchanged with fresh SB196 containing 30 mg/L hygromycin. The selection media is refreshed weekly. Four to six weeks post-selection, green, transformed tissue is observed growing from untransformed, necrotic embryogenic clusters. Isolated, green tissue is removed and inoculated into multiwell plates to generate new, clonally propagated, transformed embryogenic suspension cultures.

Embryo Maturation:

[0342] Embryos are cultured for 4-6 weeks at 26.degree. C. in SB196 under cool white fluorescent (Phillips cool white Econowatt F40/CW/RS/EW) and Agro (Phillips F40 Agro) bulbs (40 watt) on a 16:8 hr photoperiod with light intensity of 90-120 .mu.E/m.sup.2s. After this time embryo clusters are removed to a solid agar media, SB166, for 1-2 weeks. Clusters are then subcultured to medium SB103 for 3 weeks. During this period, individual embryos are removed from the clusters and screened for alterations in their fatty acid compositions as described supra.

Media Recipes:

SB 196--FN Lite Liquid Proliferation Medium (Per Liter)

TABLE-US-00010 [0343] SB 196 - FN Lite Liquid Proliferation Medium (per liter) MS FeEDTA - 100x Stock 1 10 mL MS Sulfate - 100x Stock 2 10 mL FN Lite Halides - 100x Stock 3 10 mL FN Lite P, B, Mo - 100x Stock 4 10 mL B5 vitamins (1 mL/L) 1.0 mL 2,4-D (10 mg/L final concentration) 1.0 mL KNO.sub.3 2.83 gm (NH.sub.4).sub.2SO.sub.4 0.463 gm asparagine 1.0 gm sucrose (1%) 10 gm pH 5.8

FN Lite Stock Solutions

TABLE-US-00011 [0344] Stock Number 1000 mL 500 mL 1 MS Fe EDTA 100x Stock Na.sub.2 EDTA* 3.724 g 1.862 g FeSO.sub.4--7H.sub.2O 2.784 g 1.392 g 2 MS Sulfate 100x stock MgSO.sub.4--7H.sub.2O 37.0 g 18.5 g MnSO.sub.4--H.sub.2O 1.69 g 0.845 g ZnSO.sub.4--7H.sub.2O 0.86 g 0.43 g CuSO.sub.4--5H.sub.2O 0.0025 g 0.00125 g 3 FN Lite Halides 100x Stock CaCl.sub.2--2H.sub.2O 30.0 g 15.0 g KI 0.083 g 0.0715 g CoCl.sub.2--6H.sub.2O 0.0025 g 0.00125 g 4 FN Lite P, B, Mo 100x Stock KH.sub.2PO.sub.4 18.5 g 9.25 g H.sub.3BO.sub.3 0.62 g 0.31 g Na.sub.2MoO.sub.4--2H.sub.2O 0.025 g 0.0125 g *Add first, dissolve in dark bottle while stirring

SB1 Solid Medium (Per Liter)

[0345] 1 package MS salts (Gibco/ BRL--Cat. No. 11117-066) [0346] 1 mL B5 vitamins 1000.times. stock [0347] 31.5 g sucrose [0348] 2 mL 2,4-D (20 mg/L final concentration) [0349] pH 5.7 [0350] 8 g TC agar

SB 166 Solid Medium (Per Liter)

[0350] [0351] 1 package MS salts (Gibco/ BRL--Cat. No. 11117-066) [0352] 1 mL B5 vitamins 1000.times. stock [0353] 60 g maltose [0354] 750 mg MgCl.sub.2 hexahydrate [0355] 5 g activated charcoal [0356] pH 5.7 [0357] 2 g gelrite

SB 103 Solid Medium (Per Liter)

[0357] [0358] 1 package MS salts (Gibco/BRL--Cat. No. 11117-066) [0359] 1 mL B5 vitamins 1000.times. stock [0360] 60 g maltose [0361] 750 mg MgCl.sub.2 hexahydrate [0362] pH 5.7 [0363] 2 g gelrite

SB 71-4 Solid Medium (Per Liter)

[0363] [0364] 1 bottle Gamborg's B5 salts with sucrose (Gibco/BRL--Cat. No. 21153-036) [0365] pH 5.7 [0366] 5 g TC agar

2,4-D Stock

[0366] [0367] Obtain prernade from Phytotech Cat. No D 295 concentration 1 mg/mL

B5 Vitamins Stock (Per 100 mL)

[0367] [0368] Store aliquots at -20.degree. C. [0369] 10 g myo-inositol [0370] 100 mg nicotinic acid [0371] 100 mg pyridoxine HCl [0372] 1 g thiamine If the solution does not dissolve quickly enough, apply a low level of heat via the hot stir plate.

Example 14

Functional Analysis of Delta-5 Desaturase (SEQ ID NOs:1 and 2) in Somatic Soybean Embryos

[0373] Mature somatic soybean embryos are a good model for zygotic embryos. While in the globular embryo state in liquid culture, somatic soybean embryos contain very low amounts of triacylglycerol (TAG) or storage proteins typical of maturing, zygotic soybean embryos. At this developmental stage, the ratio of total triacylglyceride to total polar lipid (phospholipids and glycolipid) is about 1:4, as is typical of zygotic soybean embryos at the developmental stage from which the somatic embryo culture was initiated. At the globular stage as well, the mRNAs for the prominent seed proteins, .alpha.'-subunit of .beta.-conglycinin, kunitz trypsin inhibitor 3, and seed lectin are essentially absent. Upon transfer to hormone-free media to allow differentiation to the maturing somatic embryo state, TAG becomes the most abundant lipid class. As well, mRNAs for .alpha.'-subunit of .beta.-conglycinin, kunitz trypsin inhibitor 3 and seed lectin become very abundant messages in the total mRNA population, On this basis, the somatic soybean embryo system behaves very similarly to maturing zygotic soybean embryos in vivo, and is thus a good and rapid model system for analyzing the phenotypic effects of modifying the expression of genes in the fatty acid biosynthesis pathway (see PCT Publication No. WO 2002/00904, Example 3). Most importantly, the model system is also predictive of the fatty acid composition of seeds from plants derived from transgenic embryos.

[0374] Transgenic somatic soybean embryos containing the delta-5 desaturase of the present invention are analyzed in the following way. Fatty acid methyl esters are prepared from single, matured, somatic soy embryos by transesterification. Individual embryos are placed in a vial containing 50 .mu.L of trimethylsulfonium hydroxide (TMSH) and 0.5 mL of hexane and incubated for 30 min at room temperature while shaking. Fatty acid methyl esters (5 .mu.L injected from hexane layer) are separated and quantified using a Hewlett-Packard 6890 Gas Chromatograph fitted with an Omegawax 320 fused silica capillary column (Catalog #24152, Supelco Inc.). The oven temperature are programmed to hold at 220.degree. C. for 2.6 min, increase to 240.degree. C. at 20.degree. C./min and then hold for an additional 2.4 min. Carrier gas is supplied by a Whatman hydrogen generator. Retention times are compared to those for methyl esters of standards commercially available (Nu-Chek Prep, Inc.). Routinely, 5-10 embryos per event are analyzed by GC, using the methodology described above.

Example 15

Co-Expressing Other Promoter/Gene/Terminator Cassette Combinations in Somatic Soybean Embryos

[0375] In addition to the genes, promoters, terminators and gene cassettes described herein, one skilled in the art can appreciate that other promoter/gene/terminator cassette combinations can be synthesized in a way similar to, but not limited to, that described herein. For'instance, PCT Publications No. WO 2004/071467 and No. WO 2004/071178 describe the isolation of a number of promoter and transcription terminator sequences for use in embryo-specific expression in soybean. Furthermore, PCT Publications No. WO 2004/071467, No. WO 2005/047479 and No. WO 2006/012325 describe the synthesis of multiple promoter/gene/terminator cassette combinations by ligating individual promoters, genes and transcription terminators together in unique combinations. Generally, a NotI site flanked by the suitable promoter (e.g., those listed in, but not limited to, Table 9) and a transcription terminator (e.g., those listed in, but not limited to, Table 10) is used to clone the desired gene. NotI sites can be added to a gene of interest such as those listed in, but not limited to, Table 11 using PCR amplification with oligonucleotides designed to introduce NotI sites at the 5' and 3' ends of the gene. The resulting PCR product is then digested with NotI and cloned into a suitable promoter/NotI/terminator cassette.

[0376] In addition, PCT Publications No. WO 2004/071467, No. WO 2005/047479 and No. WO 2006/012325 describe the further linking together of individual gene cassettes in unique combinations, along with suitable selectable marker cassettes, in order to obtain the desired phenotypic expression. Although this is done mainly using different restriction enzymes sites, one skilled in the art can appreciate that a number of techniques can be utilized to achieve the desired promoter/gene/transcription terminator combination. In so doing, any combination of embryo-specific promoter/gene/ transcription terminator cassettes can be achieved. One skilled in the art can also appreciate that these cassettes can be located on individual DNA fragments or on multiple fragments where co-expression of genes is the outcome of co-transformation of multiple DNA fragments.

TABLE-US-00012 TABLE 9 Seed-specific Promoters Promoter Organism Promoter Reference .beta.-conglycinin .alpha.'-subunit soybean Beachy et al., EMBO J., 4: 3047-3053 (1985) kunitz trypsin inhibitor soybean Jofuku et al., Plant Cell, 1: 1079-1093 (1989) Annexin soybean PCT Publication No. WO 2004/071467 glycinin Gy1 soybean PCT Publication No. WO 2004/071467 albumin 2S soybean U.S. Pat. No. 6,177,613 legumin A1 pea Rerie et al., Mol. Gen. Genet., 225: 148-157 (1991) .beta.-conglycinin .beta.-subunit soybean PCT Publication No. WO 2004/071467 BD30 (also called P34) soybean PCT Publication No. WO 2004/071467 legumin A2 pea Rerie et al., Mol. Gen. Genet., 225: 148-157 (1991)

TABLE-US-00013 TABLE 10 Transcription Terminators Transcription Terminator Organism Reference phaseolin 3' bean PCT Publication No. WO 2004/071467 kunitz trypsin inhibitor 3' soybean PCT Publication No. WO 2004/071467 BD30 (also called P34) 3' soybean PCT Publication No. WO 2004/071467 legumin A2 3' pea PCT Publication No. WO 2004/071467 albumin 2S 3' soybean PCT Publication No. WO 2004/071467

TABLE-US-00014 TABLE 11 PUFA Biosynthetic Pathway Genes Gene Organism Reference delta-6 desaturase Saprolegnia diclina PCT Publication No. WO 2002/081668 delta-6 desaturase Mortierella alpina U.S. Pat. No. 5,968,809 elongase Mortierella alpina PCT Publication No. WO 2000/12720; U.S. Pat. No. 6,403,349 delta-5 desaturase Mortierella alpina U.S. Pat. No. 6,075,183 delta-5 desaturase Saprolegnia diclina PCT Publication No. WO 2002/081668 delta-15 desaturase Fusarium PCT Publication No. WO moniliforme 2005/047479 delta-17 desaturase Saprolegnia diclina PCT Publication No. WO 2002/081668 elongase Thraustochytrium PCT Publication No. WO aureum 2002/08401; U.S. Pat. No. 6,677,145 elongase Pavlova sp. Pereira et al., Biochem. J., 384: 357-366 (2004) delta-4 desaturase Schizochytrium PCT Publication No. WO aggregatum 2002/090493 delta-9 elongase Isochrysis galbana PCT Publication No. WO 2002/077213 delta-9 elongase Euglena gracilis U.S. patent application No. 11/601563 delta-8 desaturase Euglena gracilis PCT Publication No. WO 2000/34439; U.S. Pat. No. 6,825,017; PCT Publication No. WO 2004/057001; PCT Publication No. WO 2006/012325 delta-8 desaturase Acanthamoeba Sayanova et al., FEBS Lett., castellanii 580: 1946-1952 (2006) delta-8 desaturase Pavlova salina PCT Publication No. WO 2005/103253 delta-8 desaturase Pavlova lutheri U.S. patent application No. 11/737,772

Example 16

Chlorsulfuron Selection (ALS) and Plant Regeneration

Chlorsulfuron (ALS) Selection:

[0377] Following bombardment, the plant tissue is divided between 2 flasks with fresh SB196 media and cultured as described in Example 13. Six to seven days post-bombardment, the SB196 is exchanged with fresh SB196 containing selection agent of 100 ng/mL chlorsulfuron. The selection media is refreshed weekly. Four to six weeks post selection, green, transformed tissue is observed growing from untransformed, necrotic embryogenic clusters. Isolated, green tissue is removed and inoculated into multiwell plates containing SB196 to generate new, clonally propagated, transformed embryogenic suspension cultures.

Regeneration of Soybean Somatic Embryos into Plants:

[0378] In order to obtain whole plants from embryogenic suspension cultures, the tissue must be regenerated. Embyros are matured as described in Example 13. After subculturing on medium SB103 for 3 weeks, individual embryos are removed from the clusters and screened for alterations in their fatty acid compositions as described in Example 14. It should be noted that any detectable phenotype, resulting from the expression of the genes of interest, can be screened at this stage. This would include, but not be limited to: alterations in fatty acid profile, protein profile and content, carbohydrate content, growth rate, viability, or the ability to develop normally into a soybean plant.

[0379] Matured individual embryos are desiccated by placing them into an empty, small petri dish (35.times.10 mm) for approximately 4 to 7 days. The plates are sealed with fiber tape (creating a small humidity chamber). Desiccated embryos are planted into SB71-4 medium where they are left to germinate under the same culture conditions described above. Germinated plantlets are removed from germination medium and rinsed thoroughly with water and then are planted in Redi-Earth in a 24-cell pack tray, covered with a clear plastic dome. After 2 weeks the dome is removed and plants hardened off for a further week. if plantlets look hardy, they are transplanted to 10'' pots of Redi-Earth with up to 3 plantlets per pot. After 10 to 16 weeks, mature seeds are harvested, chipped and analyzed for fatty acids as described in Example 14.

[0380] Media recipes can be found in Example 13 and chlorsulfuron stock is 1 mg/mL in 0.01 N ammonium hydroxide.

Example 17

Comparing the Substrate Specificity of the Mortierella alpina Delta-5 Desaturase (MaD5) with the Peridinium sp. CCMP626 Delta-5 Desaturase (RD5) in Yarrowia Lipolytica

[0381] The present Example describes comparison of the substrate specificity of a Mortierella alpina delta-5 desaturase (MaD5; SEQ ID NOs:67 and 68), which is described in U.S. Pat. No. 6,075,183 and PCT Publication Nos. WO 2004/071467 and No. WO 2005/047479) to that of RD5 (SEQ ID NO:2) in Yarrowia lipolytica.

[0382] This work included the following steps: (1) construction of Yarrowia expression vector pY98 comprising MaD5; (2) transformation of pY98 and pDMW368 into Yarrowia strain Y2224; and, 3.) comparison of lipid profiles within transformant organisms comprising pY98 or pDMW368 after feeding fatty acid substrates.

Construction of Yarrowia Expression Vector pY98, Comprising MaD5

[0383] Plasmid pY5-22 (SEQ ID NO:69) is a shuttle plasmid that can replicate both in E. coli and Yarrowia lipolytica, containing the following: a Yarrowia autonomous replication sequence (ARS18; GenBank Accession No. M91600); a ColE1 plasmid origin of replication; an ampicillin-resistance gene (AmpR) for selection in E. coli; a Yarrowia URA3 gene (GenBank Accession No. AJ306421) for selection in Yarrowia; and, a chimeric TEF::NcoI/NotI::XPR cassette, wherein "XPR" was .about.100 by of the 3' region of the Yarrowia Xpr gene (GenBank Accession No. M17741). Although the construction of plasmid pY5-22 is not described herein in detail, it was derived from pY5 (previously described in PCT Publication No. WO 2004/101757).

[0384] Plasmid pY5-22GPD (SEQ ID NO:70) was created from pY5-22 (SEQ ID NO:69), by replacing the TEF promoter with the Yarrowia lipolytica GPD promoter (SEQ ID NO:71) using techniques well known to one skilled in the art. The Yarrowia "GPD promoter" refers to the 5' upstream untranslated region in front of the `ATG` translation initiation codon of a protein encoded by the Yarrowia lipolytica glyceraldehyde-3-phosphate dehydrogenase (GPD) gene and that is necessary for expression (PCT Publication No. WO 2005/003310). More specifically, the Yarrowia lipolytica GPD promoter was amplified from plasmid pYZDE2-S (SEQ ID NO:72: which was previously described in U.S. patent application Ser. No. 11/737,772 (the contents of which are hereby incorporated by reference)) using oligonucleotides GPDsense (SEQ ID NO:73) and GPDantisense (SEQ ID NO:74). The resulting DNA fragment was digested with SalI/NotI and cloned into the SalI/NotI fragment of pY5-22 (SEQ ID NO:69), thus replacing the TEF promoter and NcoI/NotI site with the GPD promoter and a unique NotI site, and thereby producing pY5-22GPD (SEQ ID NO:70).

[0385] The Mortierella alpina delta-5 desaturase gene (SEQ ID NO:67) was released from pKR136 (SEQ ID NO:75; which was previously described in PCT Publication No WO 2004/071467 (the contents of which are hereby incorporated by reference)) by digestion with NotI and cloned into the NotI site of pY5-22GPD to produce pY98 (SEQ ID NO:76; FIG. 9).

Transformation of pY98 (Comprising MaD5) and pDMW368 (Comprising RD5) into Yarrowia Strain Y2224 and Comparison of Lipid Profiles

[0386] Strain Y2224 was isolated in the following manner: Yarrowia lipolytica ATCC #20362 cells from a YPD agar plate (1% yeast extract, 2% bactopeptone, 2% glucose, 2% agar) were streaked onto a MM plate (75 mg/L each of uracil and uridine, 6.7 g/L YNB with ammonia sulfate, without amino acid, and 20 g/L glucose) containing 250 mg/L 5-FOA (Zymo Research). Plates were incubated at 28.degree. C. and four of the resulting colonies were patched separately onto MM plates containing 200 mg/mL 5-FOA and MM plates lacking uracil and uridine to confirm uracil Ura3 auxotrophy.

[0387] Strain Y2224 was transformed with pY98 (SEQ ID NO:76, FIG. 9) and pDMW368 (SEQ ID NO:23; FIG. 5C; Example 6) as described in the General Methods.

[0388] Single colonies of transformant Yarrowia lipolytica containing pY98 (SEQ ID NO:76) or pDMW368 (SEQ ID NO:23) were grown in 3 mL MM lacking uracil supplemented with 0.2% tergitol at 30.degree. C. for 1 day. After this, 0.1 mL was transferred to 3 rnL of the same medium supplemented with either EDA, ETrA, DGLA, ETA or no fatty acid. These were incubated for 16 h at 30.degree. C., 250 rpm and then pellets were obtained by centrifugation. Cells were washed once with water, pelleted by centrifugation and air dried. Pellets were transesterified (Roughan, G. and Nishida, I., Arch. Biochem. Biophys., 276(1):38-46 (1990)) with 500 .mu.L of 1% sodium methoxide for 3D min at 50.degree. C. after which 500 .mu.L of 1M NaCl and 100 .mu.L of heptane were added. After thorough mixing and centrifugation, fatty acid methyl esters (FAMEs) were analyzed by GC.

[0389] FAMEs (5 .mu.L injected from hexane layer) were separated and quantified using a Hewlett-Packard 6890 Gas Chromatograph fitted with an Omegawax 320 fused silica capillary column (Catalog No. 24152, Supelco Inc.). The oven temperature was programmed to hold at 220.degree. C. for 2.6 min, increase to 240.degree. C. at 20.degree. C./min and then hold for an additional 2.4 min. Carrier gas was supplied by a Whatman hydrogen generator. Retention times were compared to those for methyl esters of standards commercially available (Nu-Chek Prep, Inc.).

[0390] The fatty acid profiles for Yarrowia lipolytica expressing pY98 (SEQ ID NO:76) or pDMW368 (SEQ ID NO:23) and fed various substrates are shown in FIG. 10A. In FIG. 10A shading indicates the substrates fed and products produced; fatty acids are identified as 16:0 (palmitate), 16:1, 18:0 (stearic acid), 18:1 (oleic acid), LA, GLA, ALA, STA, EDA, SCI (sciadonic acid or cis-5,11,14-eicosatrienoic acid; 20:3 omega-6), DGLA, ARA, ETrA, JUP (juniperonic acid or cis-5,11,14,17-eicosatrienoic acid; 20:4 omega-3), ETA and EPA. Fatty acid compositions were expressed as the weight percent (wt. %) of total fatty acids.

[0391] Percent delta-5 desaturation ("% delta-5 desat") of RD5 and MaD5 for each substrate is shown in FIG. 10B and was calculated by dividing the wt. % for product (either SCI, JUP, ARA or EPA) by the sum of the wt. % for the substrate and product (either EDA and SCI, ETrA and JUP, DGLA and ARA, or ETA and EPA, respectively) and multiplying by 100 to express as a %, depending on which substrate was fed.

[0392] The activities of MaD5 and RD5 are compared using the ratio of the percent delta-5 desaturation ("Ratio Desat R/Ma") in FIG. 10B and are calculated by dividing the percent delta-5 desaturation for RD5 on a particular substrate by the percent delta-5 desaturation for MaD5 on the same substrate.

[0393] The substrate specificity of RD5 and MaD5 for the correct omega-6 fatty acid substrate (i.e., DGLA) versus the by-product fatty acid (i.e., SCI) or the correct omega-3 fatty acid substrate (i.e., ETA) versus the by-product fatty acid (i.e., JUP) is also shown in FIG. 10B. Specifically, the substrate specificity ("Ratio Prod/By-Prod") for omega-6 substrates was calculated by dividing the percent delta-5 desaturation (% delta-5 desat) for DGLA by the percent delta-5 desaturation (% delta-5 desat) for EDA and is shown on the same lines as the results for DGLA.

[0394] The substrate specificity ("Ratio Prod/By-Prod") for omega-3 substrates was calculated by dividing the percent delta-5 desaturation (% delta-5 desat) for ETA by the percent delta-5 desaturation (% delta-5 desat) for ETrA and is shown on the same lines as the results for ETA. Furthermore, the ratio of substrate specificity ("Ratio Prod/By-Prod R/Ma") for omega-6 substrates was determined by dividing the substrate specificity for RD5 on the omega-6 substrates (i.e., DGLA/EDA) by that for MaD5. The ratio of substrate specificity ("Ratio Prod/By-Prod R/Ma") for omega-3 substrates was calculated by dividing the substrate specificity for RD5 on the omega-3 substrates (i.e., ETA/ETrA) by that for MaD5.

[0395] The preference of RD5 and MaD5 for omega-6 or omega-3 substrates is compared using the ratio of the percent delta-5 desaturation ("Ratio n-6/n-3") in FIG. 10B and is calculated by dividing the percent delta-5 desaturation for RD5 and MaD5 on a particular omega-6 substrate (either DGLA or EDA) by the percent delta-5 desaturation on the corresponding omega-3 substrate (either ETA or ETrA, respectively).

[0396] From the results in FIG. 10B, it is clear that RD5 is approximately 3.0- to 9.7-fold more active in Yarrowia than MaD5 when DGLA, EDA, ETrA and ETA are used as substrates. The substrate specificity of RD5 compared to MaD5 (RD5/MaD5) for the correct omega-6 substrate (i.e., DGLA versus EDA) is approximately 0.4 and for the omega-3 substrate (i.e., ETA versus ETrA) is approximately 0.6. RD5 also has an approximate 1.4-fold preference for omega-6 substrates EDA and DGLA) over the omega-3 substrates (i.e., ETrA and ETA), respectively.

Example 18

Construction of Soybean Expression Vector pKR916 for Co-Expression of the Mortierella alpina Delta-5 Desaturase (MaD5) with a Delta-9 Elongase Derived From Euglena gracilis (EgD9e) and a Delta-8 Desaturase Derived from Euglena gracilis (EgD8)

[0397] The present Example describes construction of a soybean vector for co-expression of MaD5 (SEQ ID NO:67, Example 17) with EgD9e (SEQ ID NO:77; which is described in U.S. application Ser. No. 11/601,563 (filed Nov. 16, 2006; Attorney Docket No. BB-1562) and EgD8 (SEQ ID NO:78; described as Eg5 in PCT Publication No. WO 2006/012325).

Euglena gracilis Delta-9 Elongase (EgD9e):

[0398] A clone from the Euglena cDNA library (eeg1c), called eeg1c.pk001.n5f, containing the Euglena gracilis delta-9 elongase (EgD9e; SEQ ID NO:77) was used as template to amplifiy EgD9e with oligonucleotide primers oEugEL1-1 (SEQ ID NO:79) and oEugEL1-2 (SEQ ID NO:80) using the VentR.RTM. DNA Polymerase (Catalog No. MO254S, New England Biolabs Inc., Beverly, Mass.) following the manufacturer's protocol. The resulting DNA fragment was cloned into the pCR-Blunt.RTM. cloning vector using the Zero Blunt.RTM. PCR Cloning Kit (Invitrogen Corporation), following the manufacturer's protocol, to produce pKR906 (SEQ ID NO:81).

[0399] A starting plasmid pKR72 (ATCC Accession No. PTA-6019; SEQ ID NO:82, 7085 by sequence), a derivative of pKS123 which was previously described in PCT Publication No. WO 02/008269 (the contents of which are hereby incorporated by reference), contains the hygromycin B phosphotransferase gene (HPT) (Gritz, L. and Davies, J., Gene, 25:179-188 (1983)), flanked by the T7 promoter and transcription terminator (i.e., a T7prom/HPT/T7term cassette), and a bacterial origin of replication (ori) for selection and replication in bacteria (e.g., E. coli). In addition, pKR72 also contains HPT, flanked by the 35S promoter (Odell et al., Nature, 313:810-812 (1985)) and NOS 3' transcription terminator (Depicker et al., J. Mol. Appl. Genet, 1:561-570 (1982)) (i.e., a 35S/HPT/NOS3' cassette) for selection in plants such as soybean. pKR72 also contains a NotI restriction site, flanked by the promoter for the .alpha.' subunit of .beta.-conglycinin (Beachy et al., EMBO J., 4:3047-3053 (1985)) and the 3' transcription termination region of the phaseolin gene (Doyle et al., J. Biol. Chem., 261:9228-9238 (1986)), thus allowing for strong tissue-specific expression in the seeds of soybean of genes cloned into the NotI site.

[0400] The AscI fragment from plasmid pKS102 (SEQ ID NO:83), previously described in PCT Publication No. WO 02/00905 (the contents of which are hereby incorporated by reference), containing a T7prom/hpt/T7term cassette and bacterial ori, was combined with the AscI fragment of plasmid pKR72 (SEQ ID NO:82), containing a .beta.con/NotI/Phas cassette to produce pKR197 (SEQ ID NO:84), previously described in PCT Publication No. WO 04/071467 (the contents of which are hereby incorporated by reference).

[0401] The gene for the Euglena gracilis delta-9 elongase was released from pKR906 (SEQ ID NO:81) by digestion with NotI and cloned into the NotI site of pKR197 (SEQ ID NO:84) to produce intermediate cloning vector pKR911 (SEQ ID NO:85).

Euglena gracilis Delta-8 Desaturase (EgD8):

[0402] Plasmid pKR680 (SEQ ID NO:86), which was previously described in PCT Publication No. WO 2006/012325 (the contents of which are hereby incorporated by reference), contains the Euglena gracilis delta-8 desaturase (EgD8; SEQ ID NO:78; described as Eg5 in WO 2006/012325) flanked by the Kunitz soybean Trypsin Inhibitor (KTi) promoter (Jofuku et al., Plant Cell, 1:1079-1093 (1989)) and the KTi 3' termination region, the isolation of which is described in U.S. Pat. No. 6,372,965, followed by the soy albumin transcription terminator, which was previously described in PCT Publication No. WO 2004/071467 (i.e., a Kti/NotI/Kti3'Salb3' cassette).

[0403] Plasmid pKR680 (SEQ ID NO:86) was digested with BsiWI and the fragment containing EgD8 was cloned into the BsiWI site of pKR911 (SEQ ID NO:85) to produce pKR913 (SEQ ID NO:87).

Mortierella alpina Delta-5 Desaturase (MaD5):

[0404] Plasmid pKR767 (SEQ ID NO:88), which was previously described in PCT Publication No WO 2006/012325 (the contents of which are hereby incorporated by reference), contains the Mortierella alpina delta-5 desaturase (MaD5; SEQ ID NO:67) flanked by the promoter for the soybean glycinin Gyl gene and the pea legumin A2 3' transcription termination region (i.e. a Gy1/MaD5/legA2 cassette; the construction of which is described in WO 2006/012325).

[0405] The Gy1/Mad5/legA2 cassette was released from pKR767 (SEQ 1D NO:88) by digestion with SbfI and the resulting fragment was cloned into the SbfI site of pKR913 (SEQ ID NO:87) to produce pKR916 (SEQ ID NO:89). A schematic depiction of pKR916 is shown in FIG. 11A. In this way, the Euglena gracilis delta-9 elongase (labeled "eug el1" in FIG. 11A) was co-expressed with the Euglena gracilis delta-8 desaturase (labeled "eug d8-sq5" in FIG, 11A) and the Mortierella alpina delta-5 desaturase (labeled "DELTA 5 DESATURASE M ALPINA" in FIG. 11A) behind strong, seed specific promoters.

Example 19

Construction of Soybean Expression Vector pKR1038 for Co-Expression of the Peridinium sp. CCMP626 Delta-5 Desaturase (RD5) with a Delta-9 Elongase Derived from Euglena gracilis (EqD9e) and a Delta-8 Desaturase Derived from Euglena gracilis (EgD8)

[0406] The present Example describes construction of a soybean vector for co-expression of RD5 (SEQ ID NO:1, Example 5) with EgD9e (SEQ ID NO:77, Example 18) and EgD8 (SEQ ID NO:78, Example 18).

[0407] Starting plasmid pKR974 (SEQ ID NO:90) is identical to pKR767 (SEQ ID NO:88, Example 18) except the NotI fragment containing MaD5 was replaced with a NotI fragment containing the Saprolegnia diclina delta-5 desaturase (SdD5; SEQ ID NO:91, which is described in PCT Publication No. WO 2004/071467). In addition, a MfeI site in the legA2 terminator of pKR767 (SEQ ID NO:88) was removed by digestion with MfeI, filling the MfeI site and religating (i.e., CAATTG converted to CAATTAATTG) and therefore, the legA2 terminator of pKR974 (SEQ ID NO:90) is 770 by versus 766 by for pKR767 (SEQ ID NO:88).

[0408] In order to clone RD5 into a soybean expression vector, a NotI restriction site needed to be introduced at the 5' end of the gene. One skilled in the art will realized that there are many ways to introduce restriction sites into genes such as, but not limited to PCR or by subcloning into vectors containing the appropriate sites. In this case, in order to introduce a NotI site at the 5' end of RD5 (SEQ ID NO:1), pDMW368 (SEQ ID NO:23) was digested with MfeI and then partially digested with NcoI. The NcoI/MfeI fragment containing a full length RD5 (SEQ ID NO:1) was cloned into the NcoI/MfeI site of an intermediate cloning vector having a NotI site directly upstream of the NcoI site (i.e., GCGGCCGCAAACCATGG). The resulting plasmid was then digested with NotI and the fragment containing RD5 (SEQ ID NO:1) was cloned into the NotI site of pKR974 (SEQ ID NO:90) to produce pKR1033 (SEQ ID NO:92).

[0409] The Gy1/EgD5/legA2 cassette was released from pKR1033 (SEQ ID NO:92) by digestion with SbfI and the resulting fragment was cloned into the SbfI site of pKR913 (SEQ ID NO:87) to produce pKR1038 (SEQ ID NO:93). A schematic depiction of pKR1038 (SEQ ID NO:93) is shown in FIG. 11B. In this way, the Euglena gracilis delta-9 elongase (labeled "eug ell" in FIG. 1113) could be co-expressed with the Euglena gracilis delta-8 desaturase (labeled "eug d8-sq5" in FIG. 11B) and the Pendinium sp. CCMP626 delta-5 desaturase (labeled "CCMP626 d5 DS" in FIG. 11B) behind strong, seed specific promoters.

Example 20

Co-Expression of the Euglena gracilis Delta-9 Elongase, the Euglena gracilis Delta-8 Desaturase and the Saprolegnia diclina Delta-17 Desaturase With Either the Mortierella alpina Delta-5 Desaturase (pKR916 & pKR328) or The Peridinium CCMP626 Delta-5 Desaturase (pKR1038 & pKR328) In Soybean Somatic Embryos

[0410] The present Example describes the transformation and expression in soybean somatic embryos of pKR916 (SEQ ID NO:89, Example 18; containing EgD9e, EgD8 and MaD5) with pKR328 (SEQ ID NO:94, FIG. 11C, previously described in PCT Publication No. WO 04/071467), containing the Saprolegnia diclina delta-17 desaturase (SdD17) and the hygromycin phosphotransferase gene for selection on hygromycin. The present Example further describes the transformation and expression in soybean somatic embryos of pKR1038 (SEQ ID NO:93, Example 19; containing EgD9e, EgD8 and RD5) with pKR328 (SEQ ID NO:94, FIG. 11C).

[0411] Soybean embryogenic suspension culture (cv. Jack) was transformed with the AscI fragment containing the expression cassette of pKR916 (SEQ ID NO:89) and intact plasmid pKR328 (SEQ ID NO:94), or with the AscI fragment containing the expression cassette of pK1038 (SEQ ID NO:93) and intact plasmid pKR328 (SEQ ID NO:94), as described in Example 13.

[0412] Embryos were matured in soybean histodifferentiation and maturation liquid medium (SHaM liquid media; Schmidt et al., Cell Biology and Morphogenesis, 24:393 (2005)) using a modified procedure. Briefly, after 4 weeks of selection in SB196 as described in Example 13, embryo clusters were removed to 35 mL of SB228 (SHaM liquid media) in a 250 mL Erlenmeyer flask. Tissue was maintained in SHaM liquid media on a rotary shaker at 130 rpm and 26.degree. C. with cool white fluorescent lights on a 16:8 hr day/night photoperiod at a light intensity of 60-85 .mu.E/m.sup.2/s for 2 weeks as embryos matured, Embryos grown for 2 weeks in SHaM liquid media were equivalent in size and fatty acid content to embryos cultured on SB166/SB103 for 5-8 weeks as described in Example 13.

Media Recipes:

SB 228--Soybean Histodifferentiation & Maturation (SHaM) (Per Liter)

TABLE-US-00015 [0413] DDI H.sub.2O 600 mL FN-Lite Macro Salts for SHaM 10X 100 mL MS Micro Salts 1000x 1 mL MS FeEDTA 100x 10 mL CaCl 100x 6.82 mL B5 Vitamins 1000x 1 mL L-Methionine 0.149 g Sucrose 30 g Sorbitol 30 g Adjust volume to 900 mL pH 5.8 Autoclave Add to cooled media (.ltoreq.30.degree. C.): *Glutamine (final concentration 30 mM) 4% 110 mL *Note: Final volume will be 1010 mL after glutamine addition.

Since glutamine degrades relatively rapidly, it may be preferable to add immediately prior to using media. Expiration 2 weeks after glutamine is added; base media can be kept longer without glutamine.

FN-Lite Macro for SHAM 10.times.--Stock #1 (Per Liter)

TABLE-US-00016 [0414] (NH.sub.4).sub.2SO.sub.4 (ammonium sulfate) 4.63 g KNO.sub.3 (potassium nitrate) 28.3 g MgSO.sub.4*7H.sub.20 (magnesium sulfate heptahydrate) 3.7 g KH.sub.2PO.sub.4 (potassium phosphate, monobasic) 1.85 g Bring to volume Autoclave

MS Micro 1000.times.--Stock #2 (Per 1 Liter)

TABLE-US-00017 [0415] H.sub.3BO.sub.3 (boric acid) 6.2 g MnSO.sub.4*H.sub.2O (manganese sulfate monohydrate) 16.9 g ZnSO.sub.4*7H.sub.20 (zinc sulfate heptahydrate) 8.6 g Na.sub.2MoO.sub.4*2H.sub.20 (sodium molybdate dihydrate) 0.25 g CuSO.sub.4*5H.sub.20 (copper sulfate pentahydrate) 0.025 g CoCl.sub.2*6H.sub.20 (cobalt chloride hexahydrate) 0.025 g KI (potassium iodide) 0.8300 g Bring to volume Autoclave

FeEDTA 100.times.--Stock #3 (Per Liter)

TABLE-US-00018 [0416] Na.sub.2EDTA* (sodium EDTA) 3.73 g FeSO.sub.4*7H.sub.20 (iron sulfate heptahydrate) 2.78 g Bring to Volume Solution is photosensitive. Bottle(s) should be wrapped in foil to omit light. Autoclave *EDTA must be completely dissolved before adding iron.

Ca 100.times.--Stock #4 (Per Liter)

TABLE-US-00019 [0417] CaCl.sub.2*2H.sub.20 (calcium chloride dihydrate) 44 g Bring to Volume Autoclave

B5 Vitamin 1000.times.--Stock #5 (Per Liter)

TABLE-US-00020 [0418] Thiamine*HCl 10 g Nicotinic Acid 1 g Pyridoxine*HCl 1 g Myo-Inositol 100 g Bring to Volume Store frozen

4% Glutamine--Stock #6 (Per Liter)

TABLE-US-00021 [0419] DDI water heated to 30.degree. C. 900 mL L-Glutamine 40 g Gradually add while stirring and applying low heat. Do not exceed 35.degree. C. Bring to Volume Filter Sterilize Store frozen* *Note: Warm thawed stock in 31.degree. C. bath to fully dissolve crystals.

[0420] After maturation in SHaM liquid media, individual embryos were removed from the clusters, dried and screened for alterations in their fatty acid compositions as described supra.

[0421] A subset of soybean embryos (i.e., six embryos per event) transformed with either pKR916 (SEQ ID NO:89) and pKR328 (SEQ ID NO:94), or pKR1038 (SEQ ID NO:93) and pKR328 (SEQ ID NO:94), were harvested and picked into glass GC vials and fatty acid methyl esters were prepared by transesterification. For transesterification, 50 .mu.L of trimethylsulfonium hydroxide (TMSH) and 0.5 mL of hexane were added to the embryos in glass vials and incubated for 30 min at room temperature while shaking. Fatty acid methyl esters (5 .mu.L injected from hexane layer) were separated and quantified using a Hewlett-Packard 6890 Gas Chromatograph fitted with an Omegawax 320 fused silica capillary column (Catalog No. 24152, Supelco Inc.). The oven temperature was programmed to hold at 220.degree. C. for 2.6 min, increase to 240.degree. C. at 20.degree. C./min and then hold for an additional 2.4 min. Carrier gas was supplied by a Whatman hydrogen generator. Retention times were compared to those for methyl esters of standards commercially available (Nu-Chek Prep, Inc.).

[0422] In this way, 60 events transformed with pKR916 (SEQ ID NO:89) and pKR328 (SEQ ID NO:94) and 31 events transformed with pKR1038 (SEQ ID NO:93) and pKR328 (SEQ ID NO:94) were analyzed. The average fatty acid profiles for the ten events having the highest delta-5 desaturase activity for each transformation (pKR916 and pKR328, pKR1038 and pKR328) are shown in FIG. 12A and FIG. 12B, respectively.

[0423] In FIGS. 12A and 12B, fatty acids are identified as 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic acid), LA, ALA, EDA, SCI, DGLA, ARA, ERA, JUP, ETA and EPA. Fatty acids listed as "others" include: 18:2 (5,9), GLA, STA, 20:0, 20:1(11), 20:2 (7,11) or 20:2 (8,11) and DPA. Each of these "other" fatty acids is present at a relative abundance of less than 3.0% of the total fatty acids. Fatty acid compositions for an individual embryo were expressed as the weight percent (wt. %) of total fatty acids and the average fatty acid composition is an average of six individual embryos for each event.

[0424] The activity of the delta-5 desaturase for the "correct" substrates (i.e., DGLA and ETA) is expressed as percent delta-5 desaturation ("Correct % delta-5 desat"), calculated according to the following formula: product]/[substrate+product])*100.

[0425] More specifically, the percent delta-5 desaturation for the "correct" substrates was determined as: ([ARA+EPA]/[DGLA+ETA+ARA+EPA])*100.

[0426] The activity of the delta-5 desaturase for the "wrong" substrates (i.e., EDA and ERA) is also expressed as percent delta-5 desaturation ("Wrong % delta-5 desat"), calculated as: ([SCI+JUP]/[EDA+ERA+SCI+JUP])*100.

[0427] The substrate specificities of MaD5 and RD5 for the "correct" substrates (i.e., DGLA and ETA) versus the "wrong" substrates (i.e., EDA and ERA) were compared and the comparison is shown in FIG. 13. In FIG. 13, the activity of the delta-5 desaturase for the "correct" substrates ("Correct % delta-5 desat") is plotted on the x-axis and the activity of the delta-5 desaturase for the "wrong" substrates ("Wrong % delta-5 desat") is plotted on the y-axis for MaD5 (data from FIG. 12A) and RD5 (data from FIG. 12B).

Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 94 <210> SEQ ID NO 1 <211> LENGTH: 1392 <212> TYPE: DNA <213> ORGANISM: Peridinium sp. CCMP626 <220> FEATURE: <221> NAME/KEY: misc_feature <223> OTHER INFORMATION: delta-5 desaturase <400> SEQUENCE: 1 atggctccag atgcggacaa gttgagacag cgcaaggcgc aatcgattca agacacggct 60 gattcgcaag ctaccgaact caagattggc accctgaagg gcttgcaggg gacagaaatc 120 gtcattgatg gagacattta cgatataaaa gactttgatc accccggtgg tgaatccatc 180 atgacttttg ggggaaacga tgtcaccgcc acgtacaaga tgatccaccc ctaccactct 240 aagcaccatt tggagaagat gaagaaagtg ggacgagttc cggactacac ctcggaatac 300 aagtttgata ctccctttga gcgtgaaatc aagcaagagg tcttcaagat tgtgcgacga 360 ggccgcgagt ttggaacacc tggatacttc ttccgggctt tctgctacat tggacttttc 420 ttttacttgc agtatttgtg ggtcacgact cccactacct ttgccttggc gatcttctat 480 ggtgtttcgc aagctttcat tggtttgaac gtacaacatg atgccaacca cggagctgcc 540 tccaagaagc cttggatcaa taacttgcta ggattggggg ctgactttat cggaggttcc 600 aaatggttgt ggatgaacca gcactggacg caccacacat acaccaacca ccatgagaag 660 gatcccgatg ccttgggcgc tgaaccaatg ttgttgttca atgattatcc cttgggtcac 720 ccaaagcgta ctttgattca ccacttccag gccttctatt accttttcgt cttggccgga 780 tactgggtct cttcggtctt caaccctcaa attttggact tgcaacaccg cggtgctcaa 840 gcggttggaa tgaaaatgga gaacgattac attgccaaaa gccgaaagta tgccatcttc 900 ttgcgtctct tgtatattta caccaacatt gtcgctccga tccaaaacca aggcttctcg 960 ttgaccgtgg tcgcccacat tttgaccatg ggcgtcgctt ccagtttgac tttggcgact 1020 ctttttgcct tgtcgcacaa ttttgaaaac gcggatcgcg atcccactta cgaggcccgc 1080 aagggaggag agcctgtttg ttggttcaag tcgcaagtcg aaacctcgtc aacttacgga 1140 ggtttcatct cgggttgctt gacgggcgga ctcaacttcc aagtggaaca ccacttgttc 1200 cctcgtatga gttcggcctg gtacccctac attgccccta ctgttcgaga ggtttgcaaa 1260 aagcacggag tcaagtacgc atactatccc tgggtctggc aaaacttgat ttcaactgtc 1320 aagtatctgc atcaaagcgg aactggatcc aactggaaga atggcgccaa cccctactcg 1380 ggaaaattgt aa 1392 <210> SEQ ID NO 2 <211> LENGTH: 463 <212> TYPE: PRT <213> ORGANISM: Peridinium sp. CCMP626 <400> SEQUENCE: 2 Met Ala Pro Asp Ala Asp Lys Leu Arg Gln Arg Lys Ala Gln Ser Ile 1 5 10 15 Gln Asp Thr Ala Asp Ser Gln Ala Thr Glu Leu Lys Ile Gly Thr Leu 20 25 30 Lys Gly Leu Gln Gly Thr Glu Ile Val Ile Asp Gly Asp Ile Tyr Asp 35 40 45 Ile Lys Asp Phe Asp His Pro Gly Gly Glu Ser Ile Met Thr Phe Gly 50 55 60 Gly Asn Asp Val Thr Ala Thr Tyr Lys Met Ile His Pro Tyr His Ser 65 70 75 80 Lys His His Leu Glu Lys Met Lys Lys Val Gly Arg Val Pro Asp Tyr 85 90 95 Thr Ser Glu Tyr Lys Phe Asp Thr Pro Phe Glu Arg Glu Ile Lys Gln 100 105 110 Glu Val Phe Lys Ile Val Arg Arg Gly Arg Glu Phe Gly Thr Pro Gly 115 120 125 Tyr Phe Phe Arg Ala Phe Cys Tyr Ile Gly Leu Phe Phe Tyr Leu Gln 130 135 140 Tyr Leu Trp Val Thr Thr Pro Thr Thr Phe Ala Leu Ala Ile Phe Tyr 145 150 155 160 Gly Val Ser Gln Ala Phe Ile Gly Leu Asn Val Gln His Asp Ala Asn 165 170 175 His Gly Ala Ala Ser Lys Lys Pro Trp Ile Asn Asn Leu Leu Gly Leu 180 185 190 Gly Ala Asp Phe Ile Gly Gly Ser Lys Trp Leu Trp Met Asn Gln His 195 200 205 Trp Thr His His Thr Tyr Thr Asn His His Glu Lys Asp Pro Asp Ala 210 215 220 Leu Gly Ala Glu Pro Met Leu Leu Phe Asn Asp Tyr Pro Leu Gly His 225 230 235 240 Pro Lys Arg Thr Leu Ile His His Phe Gln Ala Phe Tyr Tyr Leu Phe 245 250 255 Val Leu Ala Gly Tyr Trp Val Ser Ser Val Phe Asn Pro Gln Ile Leu 260 265 270 Asp Leu Gln His Arg Gly Ala Gln Ala Val Gly Met Lys Met Glu Asn 275 280 285 Asp Tyr Ile Ala Lys Ser Arg Lys Tyr Ala Ile Phe Leu Arg Leu Leu 290 295 300 Tyr Ile Tyr Thr Asn Ile Val Ala Pro Ile Gln Asn Gln Gly Phe Ser 305 310 315 320 Leu Thr Val Val Ala His Ile Leu Thr Met Gly Val Ala Ser Ser Leu 325 330 335 Thr Leu Ala Thr Leu Phe Ala Leu Ser His Asn Phe Glu Asn Ala Asp 340 345 350 Arg Asp Pro Thr Tyr Glu Ala Arg Lys Gly Gly Glu Pro Val Cys Trp 355 360 365 Phe Lys Ser Gln Val Glu Thr Ser Ser Thr Tyr Gly Gly Phe Ile Ser 370 375 380 Gly Cys Leu Thr Gly Gly Leu Asn Phe Gln Val Glu His His Leu Phe 385 390 395 400 Pro Arg Met Ser Ser Ala Trp Tyr Pro Tyr Ile Ala Pro Thr Val Arg 405 410 415 Glu Val Cys Lys Lys His Gly Val Lys Tyr Ala Tyr Tyr Pro Trp Val 420 425 430 Trp Gln Asn Leu Ile Ser Thr Val Lys Tyr Leu His Gln Ser Gly Thr 435 440 445 Gly Ser Asn Trp Lys Asn Gly Ala Asn Pro Tyr Ser Gly Lys Leu 450 455 460 <210> SEQ ID NO 3 <211> LENGTH: 1392 <212> TYPE: DNA <213> ORGANISM: Peridinium sp. CCMP626 <220> FEATURE: <221> NAME/KEY: misc_feature <223> OTHER INFORMATION: synthetic delta-5 desaturase (codon-optimized) for Yarrowia lipolytica <400> SEQUENCE: 3 atggctcccg acgccgacaa gctgcgacag cgaaaggctc agtccatcca ggacactgcc 60 gattctcagg ctaccgagct caagattggc accctgaagg gtctccaagg caccgagatc 120 gtcattgatg gcgacatcta cgacatcaaa gacttcgatc accctggagg cgaatccatc 180 atgacctttg gtggcaacga cgttactgcc acctacaaga tgattcatcc ctaccactcg 240 aagcatcacc tggagaagat gaaaaaggtc ggtcgagtgc ccgactacac ctccgagtac 300 aagttcgata ctcccttcga acgagagatc aaacaggagg tcttcaagat tgtgcgaaga 360 ggtcgagagt ttggaacacc tggctacttc tttcgagcct tctgctacat cggtctcttc 420 ttttacctgc agtatctctg ggttaccact cctaccactt tcgcccttgc tatcttctac 480 ggtgtgtctc aggccttcat tggcctgaac gtccagcacg acgccaacca cggagctgcc 540 tccaaaaagc cctggatcaa caatttgctc ggcctgggtg ccgactttat cggaggctcc 600 aagtggctct ggatgaacca gcactggacc catcacactt acaccaacca tcacgagaag 660 gatcccgacg ccctgggtgc agagcctatg ctgctcttca acgactatcc cttgggtcac 720 cccaagcgaa ccctcattca tcacttccaa gccttctact atctgtttgt ccttgctggc 780 tactgggtgt cttcggtgtt caaccctcag atcctggacc tccagcaccg aggtgcccag 840 gctgtcggca tgaagatgga gaacgactac attgccaagt ctcgaaagta cgctatcttc 900 ctgcgactcc tgtacatcta caccaacatt gtggctccca tccagaacca aggcttttcg 960 ctcaccgtcg ttgctcacat tcttactatg ggtgtcgcct ccagcctgac cctcgctact 1020 ctgttcgccc tctcccacaa cttcgagaac gcagatcggg atcccaccta cgaggctcga 1080 aagggaggcg agcctgtctg ttggttcaag tcgcaggtgg aaacctcctc tacttacggt 1140 ggcttcattt ccggttgcct tacaggcgga ctcaactttc aggtcgagca tcacctgttt 1200 cctcgaatgt cctctgcctg gtacccctac atcgctccta ccgttcgaga ggtctgcaaa 1260 aagcacggcg tcaagtacgc ctactatccc tgggtgtggc agaacctcat ctcgaccgtc 1320 aagtacctgc atcagtccgg aactggctcg aactggaaga acggtgccaa tccctactct 1380 ggcaagctgt aa 1392 <210> SEQ ID NO 4 <211> LENGTH: 563 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: 563 bp fragment of pT-12-D5 <400> SEQUENCE: 4 ggtcatcatg tttataccaa ccaccatgag aaggatcccg atgccttggg cgctgaacca 60 atgttgttgt tcaatgatta tcccttgggt cacccaaagc gtactttgat tcaccacttc 120 caggccttct attacctttt cgtcttggcc ggatactggg tctcttcggt cttcaaccct 180 caaattttgg acttgcaaca ccgcggtgct caagcggttg gaatgaaaat ggagaacgat 240 tacattgcca aaagccgaaa gtatgccatc ttcttgcgtc tcttgtatat ttacaccaac 300 attgtcgctc cgatccaaaa ccaaggcttc tcgttgaccg tggtcgccca cattttgacc 360 atgggcgtcg cttccagttt gactttggcg actctttttg cctcgtcgca caattttgaa 420 aacgcggatc gcgatcccac ttacgaggcc cgcaaggggg gagagcctgt ttgttggttc 480 aagtcgcaag tcgaaacctc gtcaacttac ggaggtttca tctcgggttg cttgacgggc 540 ggactcaact tccaagtatc aca 563 <210> SEQ ID NO 5 <211> LENGTH: 187 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: translation of 563 bp fragment of pT-12-D5 <400> SEQUENCE: 5 Gly His His Val Tyr Thr Asn His His Glu Lys Asp Pro Asp Ala Leu 1 5 10 15 Gly Ala Glu Pro Met Leu Leu Phe Asn Asp Tyr Pro Leu Gly His Pro 20 25 30 Lys Arg Thr Leu Ile His His Phe Gln Ala Phe Tyr Tyr Leu Phe Val 35 40 45 Leu Ala Gly Tyr Trp Val Ser Ser Val Phe Asn Pro Gln Ile Leu Asp 50 55 60 Leu Gln His Arg Gly Ala Gln Ala Val Gly Met Lys Met Glu Asn Asp 65 70 75 80 Tyr Ile Ala Lys Ser Arg Lys Tyr Ala Ile Phe Leu Arg Leu Leu Tyr 85 90 95 Ile Tyr Thr Asn Ile Val Ala Pro Ile Gln Asn Gln Gly Phe Ser Leu 100 105 110 Thr Val Val Ala His Ile Leu Thr Met Gly Val Ala Ser Ser Leu Thr 115 120 125 Leu Ala Thr Leu Phe Ala Ser Ser His Asn Phe Glu Asn Ala Asp Arg 130 135 140 Asp Pro Thr Tyr Glu Ala Arg Lys Gly Gly Glu Pro Val Cys Trp Phe 145 150 155 160 Lys Ser Gln Val Glu Thr Ser Ser Thr Tyr Gly Gly Phe Ile Ser Gly 165 170 175 Cys Leu Thr Gly Gly Leu Asn Phe Gln Val Ser 180 185 <210> SEQ ID NO 6 <211> LENGTH: 693 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: 693 bp fragment of pT-RD5-5'C2 <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (10)..(10) <223> OTHER INFORMATION: n is a, c, g, or t <400> SEQUENCE: 6 aatcgtcatn gatggagaca tttacgatat aaaagacttt gatcaccccg gtggtgaatc 60 catcatgact tttgggggaa acgatgtcac cgccacgtac aagatgatcc acccctacca 120 ctctaagcac catttggaga agatgaagaa agtgggacga gttccggact acacctcgga 180 atacaagttt gatactccct ttgagcgtga aatcaagyaa gaggtcttca agattgtgcg 240 acgaggccgc gagtttggaa cacctggata cttcttccgg gctttctgct acattggact 300 tttcttttac ttgcagtatt tgtgggtcac gactcccact acctttgcct tggcgatctt 360 ctatggtgtt tcgcaagctt tcattggttt gaacgtacaa catgatgcca accacggagc 420 tgcctccaag aagccttgga tcaataactt gctaggattg ggggctgact ttatcggagg 480 ttccaaatgg ttgtggatga accagcactg gacgcaccac acatacacca accaccatga 540 gaaggatccc gatgccttgg gcgctgaacc aatgttgttg ttcaatgatt atcccttggg 600 tcacccaaag cgtactttga ttcaccactt ccaggccttc tattaccttt tcgtcttggc 660 cggatactgg gtctcttcgg tcttcaaccc tca 693 <210> SEQ ID NO 7 <211> LENGTH: 511 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: 511 bp 5' extended fragment of pT-RD5-5'C2 <400> SEQUENCE: 7 aatcgtcatt gatggagaca tttacgatat aaaagacttt gatcaccccg gtggtgaatc 60 catcatgact tttgggggaa acgatgtcac cgccacgtac aagatgatcc acccctacca 120 ctctaagcac catttggaga agatgaagaa agtgggacga gttccggact acacctcgga 180 atacaagttt gatactccct ttgagcgtga aatcaagyaa gaggtcttca agattgtgcg 240 acgaggccgc gagtttggaa cacctggata cttcttccgg gctttctgct acattggact 300 tttcttttac ttgcagtatt tgtgggtcac gactcccact acctttgcct tggcgatctt 360 ctatggtgtt tcgcaagctt tcattggttt gaacgtacaa catgatgcca accacggagc 420 tgcctccaag aagccttgga tcaataactt gctaggattg ggggctgact ttatcggagg 480 ttccaaatgg ttgtggatga accagcactg g 511 <210> SEQ ID NO 8 <211> LENGTH: 358 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: 358 bp fragment of pT-RD5-5'2nd <400> SEQUENCE: 8 gatttctttc gttggcattt ttcgttggga aagactcttg caacgatggc tccagatgcg 60 gacaagttga gacagcgcaa ggcgcaatcg attcaagaca cggctgattc gcaagctacc 120 gaactcaaga ttggcaccct gaagggcttg caggggacag aaatcgtcat tgatggagac 180 atttacgata taaaagactt tgatcacccc ggtggtgaat ccatcatgac ttttggagga 240 aacgatgtca ctgccacgta caagatgatc cacccctacc actctaagca ccatttggag 300 aagatgaaga aagtgggacg agttctggac tacacctcgg aatacaagtt tgatactc 358 <210> SEQ ID NO 9 <211> LENGTH: 161 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: 161 bp 5' extended fragment of pT-RD5-5'2nd <400> SEQUENCE: 9 gatttctttc gttggcattt ttcgttggga aagactcttg caacgatggc tccagatgcg 60 gacaagttga gacagcgcaa ggcgcaatcg attcaagaca cggctgattc gcaagctacc 120 gaactcaaga ttggcaccct gaagggcttg caggggacag a 161 <210> SEQ ID NO 10 <211> LENGTH: 299 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: 299 bp fragment of pT-RD5-3' <400> SEQUENCE: 10 gaggtttcat ctcgggttgt ttgacgggcg gactcaactt tcaagtggaa caccacttgt 60 tccctcgtat gagttcggcc tggtacccct acattgcccc tgctgttcga gaggtttgca 120 aaaagcacgg agtcaagtac gcatactatc cctgggtctg gcaaaacttg atttcaactg 180 tcaagtatct gcatcaaagc ggaactggat ccaactggaa gaatggcgcc aacccctact 240 cgggaaaatt gtaaatgaat tctagtcaag atgggtcact gcattcaaaa aaaaaaaaa 299 <210> SEQ ID NO 11 <211> LENGTH: 247 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: 247 bp of 3' downstream sequence of pT-RD5-3' <400> SEQUENCE: 11 ccacttgttc cctcgtatga gttcggcctg gtacccctac attgcccctg ctgttcgaga 60 ggtttgcaaa aagcacggag tcaagtacgc atactatccc tgggtctggc aaaacttgat 120 ttcaactgtc aagtatctgc atcaaagcgg aactggatcc aactggaaga atggcgccaa 180 cccctactcg ggaaaattgt aaatgaattc tagtcaagat gggtcactgc attcaaaaaa 240 aaaaaaa 247 <210> SEQ ID NO 12 <211> LENGTH: 456 <212> TYPE: PRT <213> ORGANISM: Pythium irregulare <220> FEATURE: <221> NAME/KEY: MISC_FEATURE <223> OTHER INFORMATION: delta-5 desaturase <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: GenBank Accession No. AAL13311 <400> SEQUENCE: 12 Met Gly Thr Asp Gln Gly Lys Thr Phe Thr Trp Gln Glu Val Ala Lys 1 5 10 15 His Asn Thr Ala Lys Ser Ala Trp Val Ile Ile Arg Gly Glu Val Tyr 20 25 30 Asp Val Thr Glu Trp Ala Asp Lys His Pro Gly Gly Ser Glu Leu Ile 35 40 45 Val Leu His Ser Gly Arg Glu Cys Thr Asp Thr Phe Tyr Ser Tyr His 50 55 60 Pro Phe Ser Asn Arg Ala Asp Lys Ile Leu Ala Lys Tyr Lys Ile Gly 65 70 75 80 Lys Leu Val Gly Gly Tyr Glu Phe Pro Val Phe Lys Pro Asp Ser Gly 85 90 95 Phe Tyr Lys Glu Cys Ser Glu Arg Val Ala Glu Tyr Phe Lys Thr Asn 100 105 110 Asn Leu Asp Pro Lys Ala Ala Phe Ala Gly Leu Trp Arg Met Val Phe 115 120 125 Val Phe Ala Val Ala Ala Leu Ala Tyr Met Gly Met Asn Glu Leu Ile 130 135 140 Pro Gly Asn Val Tyr Ala Gln Tyr Ala Trp Gly Val Val Phe Gly Val 145 150 155 160 Phe Gln Ala Leu Pro Leu Leu His Val Met His Asp Ser Ser His Ala 165 170 175 Ala Cys Ser Ser Ser Pro Ala Met Trp Gln Ile Ile Gly Arg Gly Val 180 185 190 Met Asp Trp Phe Ala Gly Ala Ser Met Val Ser Trp Leu Asn Gln His 195 200 205 Val Val Gly His His Ile Tyr Thr Asn Val Ala Gly Ala Asp Pro Asp 210 215 220 Leu Pro Val Asp Phe Glu Ser Asp Val Arg Arg Ile Val His Arg Gln 225 230 235 240 Val Leu Leu Pro Ile Tyr Lys Phe Gln His Ile Tyr Leu Pro Pro Leu 245 250 255 Tyr Gly Val Leu Gly Leu Lys Phe Arg Ile Gln Asp Val Phe Glu Thr 260 265 270 Phe Val Ser Leu Thr Asn Gly Pro Val Arg Val Asn Pro His Pro Val 275 280 285 Ser Asp Trp Val Gln Met Ile Phe Ala Lys Ala Phe Trp Thr Phe Tyr 290 295 300 Arg Ile Tyr Ile Pro Leu Val Trp Leu Lys Ile Thr Pro Ser Thr Phe 305 310 315 320 Trp Gly Val Phe Phe Leu Ala Glu Phe Thr Thr Gly Trp Tyr Leu Ala 325 330 335 Phe Asn Phe Gln Val Ser His Val Ser Thr Glu Cys Glu Tyr Pro Cys 340 345 350 Gly Asp Ala Pro Ser Ala Glu Val Gly Asp Glu Trp Ala Ile Ser Gln 355 360 365 Val Lys Ser Ser Val Asp Tyr Ala His Gly Ser Pro Leu Ala Ala Phe 370 375 380 Leu Cys Gly Ala Leu Asn Tyr Gln Val Thr His His Leu Tyr Pro Gly 385 390 395 400 Ile Ser Gln Tyr His Tyr Pro Ala Ile Ala Pro Ile Ile Ile Asp Val 405 410 415 Cys Lys Lys Tyr Asn Ile Lys Tyr Thr Val Leu Pro Thr Phe Thr Glu 420 425 430 Ala Leu Leu Ala His Phe Lys His Leu Lys Asn Met Gly Glu Leu Gly 435 440 445 Lys Pro Val Glu Ile His Met Gly 450 455 <210> SEQ ID NO 13 <211> LENGTH: 477 <212> TYPE: PRT <213> ORGANISM: Phytophthora megasperma <220> FEATURE: <221> NAME/KEY: MISC_FEATURE <223> OTHER INFORMATION: delta-5 desaturase <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: GenBank Accession No. CAD53323 <400> SEQUENCE: 13 Met Ala Pro Ile Glu Thr Val Lys Asp Ala Asn Glu Gly Leu His Gln 1 5 10 15 Arg Lys Gly Ala Ala Ala Ala Ser Lys Asp Thr Thr Thr Phe Thr Trp 20 25 30 Gln Asp Val Ala Lys His Asn Thr Ala Lys Ser Ala Trp Val Thr Ile 35 40 45 Arg Gly Val Val Tyr Asp Val Thr Glu Trp Ala Asp Arg His Pro Gly 50 55 60 Gly Arg Glu Leu Val Leu Leu His Ser Gly Arg Glu Cys Thr Asp Thr 65 70 75 80 Phe Asp Ser Tyr His Pro Phe Ser Asp Arg Ala Asp Lys Ile Leu Ala 85 90 95 Lys Tyr Ala Ile Gly Lys Leu Val Gly Gly Ser Glu Phe Pro Thr Tyr 100 105 110 Lys Pro Asp Thr Gly Phe Tyr Lys Glu Cys Cys Asp Arg Val Asn Gln 115 120 125 Tyr Phe Lys Asp Asn Lys Leu Asp Pro Arg Ser Pro Tyr Ser Gly Leu 130 135 140 Trp Arg Met Ile Leu Val Ala Ile Val Gly Ala Val Ala Tyr Met Gly 145 150 155 160 Met Asn Gln Leu Leu Pro Gly Asn Ile Tyr Ala His Tyr Ala Trp Gly 165 170 175 Ala Leu Phe Gly Val Cys Gln Ala Leu Pro Leu Leu His Val Met His 180 185 190 Asp Ala Ser His Ala Ala Ile Thr Ser Ser Pro Thr Gly Trp Arg Leu 195 200 205 Ile Gly Arg Leu Ala Met Asp Trp Val Ala Gly Ala Asn Met Val Ser 210 215 220 Trp Leu Asn Gln His Val Val Gly His His Ile Tyr Thr Asn Val Ala 225 230 235 240 Gly Ala Asp Pro Asp Leu Pro Val Asp Phe Lys Ser Asp Val Arg Arg 245 250 255 Ile Val Tyr Arg Gln Val Leu Leu Pro Ile Tyr Lys Tyr Gln His Leu 260 265 270 Tyr Leu Pro Pro Leu Tyr Gly Val Leu Gly Leu Lys Phe Arg Val Gln 275 280 285 Asp Val Phe Glu Thr Phe Val Thr Leu Thr Asn Gly Pro Leu Arg Val 290 295 300 Asn Pro Leu Ser Val Gly Asp Trp Ala Glu Met Ile Leu Ser Lys Ala 305 310 315 320 Phe Trp Val Phe Tyr Arg Ile Tyr Leu Pro Leu Ala Val Leu Gln Val 325 330 335 Asp Pro Ala Arg Phe Trp Gly Val Phe Phe Leu Ala Glu Phe Ser Thr 340 345 350 Gly Trp Tyr Leu Ala Phe Asn Phe Gln Val Ser His Val Ser Thr Ala 355 360 365 Cys Glu Tyr Pro Gly Gly Asp Glu Glu Val Thr Ser Ile Asp Asp Glu 370 375 380 Trp Ala Ile Ser Gln Val Lys Ser Ser Val Asp Tyr Gly His Gly Ser 385 390 395 400 Phe Ile Thr Thr Phe Leu Thr Gly Ala Leu Asn Tyr Gln Val Thr His 405 410 415 His Leu Phe Pro Gly Val Ser Gln Tyr His Tyr Pro Ala Ile Ala Pro 420 425 430 Leu Ile Leu Asp Val Cys His Lys Tyr Lys Val Lys Tyr Asn Val Leu 435 440 445 Pro Asp Phe Thr Ala Ala Met Ala Gly His Phe Asp His Leu Val Ile 450 455 460 Met Gly Lys Met Gly Lys Arg Val Thr Ile His Met Gly 465 470 475 <210> SEQ ID NO 14 <211> LENGTH: 469 <212> TYPE: PRT <213> ORGANISM: Phaeodactylum tricornutum <220> FEATURE: <221> NAME/KEY: MISC_FEATURE <223> OTHER INFORMATION: delta-5 desaturase <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: GenBank Accession No. AAL92562 <400> SEQUENCE: 14 Met Ala Pro Asp Ala Asp Lys Leu Arg Gln Arg Gln Thr Thr Ala Val 1 5 10 15 Ala Lys His Asn Ala Ala Thr Ile Ser Thr Gln Glu Arg Leu Cys Ser 20 25 30 Leu Ser Ser Leu Lys Gly Glu Glu Val Cys Ile Asp Gly Ile Ile Tyr 35 40 45 Asp Leu Gln Ser Phe Asp His Pro Gly Gly Glu Thr Ile Lys Met Phe 50 55 60 Gly Gly Asn Asp Val Thr Val Gln Tyr Lys Met Ile His Pro Tyr His 65 70 75 80 Thr Glu Lys His Leu Glu Lys Met Lys Arg Val Gly Lys Val Thr Asp 85 90 95 Phe Val Cys Glu Tyr Lys Phe Asp Thr Glu Phe Glu Arg Glu Ile Lys 100 105 110 Arg Glu Val Phe Lys Ile Val Arg Arg Gly Lys Asp Phe Gly Thr Leu 115 120 125 Gly Trp Phe Phe Arg Ala Phe Cys Tyr Ile Ala Ile Phe Phe Tyr Leu 130 135 140 Gln Tyr His Trp Val Thr Thr Gly Thr Ser Trp Leu Leu Ala Val Ala 145 150 155 160 Tyr Gly Ile Ser Gln Ala Met Ile Gly Met Asn Val Gln His Asp Ala 165 170 175 Asn His Gly Ala Thr Ser Lys Arg Pro Trp Val Asn Asp Met Leu Gly 180 185 190 Leu Gly Ala Asp Phe Ile Gly Gly Ser Lys Trp Leu Trp Gln Glu Gln 195 200 205 His Trp Thr His His Ala Tyr Thr Asn His Ala Glu Met Asp Pro Asp 210 215 220 Ser Phe Gly Ala Glu Pro Met Leu Leu Phe Asn Asp Tyr Pro Leu Asp 225 230 235 240 His Pro Ala Arg Thr Trp Leu His Arg Phe Gln Ala Phe Phe Tyr Met 245 250 255 Pro Val Leu Ala Gly Tyr Trp Leu Ser Ala Val Phe Asn Pro Gln Ile 260 265 270 Leu Asp Leu Gln Gln Arg Gly Ala Leu Ser Val Gly Ile Arg Leu Asp 275 280 285 Asn Ala Phe Ile His Ser Arg Arg Lys Tyr Ala Val Phe Trp Arg Ala 290 295 300 Val Tyr Ile Ala Val Asn Val Ile Ala Pro Phe Tyr Thr Asn Ser Gly 305 310 315 320 Leu Glu Trp Ser Trp Arg Val Phe Gly Asn Ile Met Leu Met Gly Val 325 330 335 Ala Glu Ser Leu Ala Leu Ala Val Leu Phe Ser Leu Ser His Asn Phe 340 345 350 Glu Ser Ala Asp Arg Asp Pro Thr Ala Pro Leu Lys Lys Thr Gly Glu 355 360 365 Pro Val Asp Trp Phe Lys Thr Gln Val Glu Thr Ser Cys Thr Tyr Gly 370 375 380 Gly Phe Leu Ser Gly Cys Phe Thr Gly Gly Leu Asn Phe Gln Val Glu 385 390 395 400 His His Leu Phe Pro Arg Met Ser Ser Ala Trp Tyr Pro Tyr Ile Ala 405 410 415 Pro Lys Val Arg Glu Ile Cys Ala Lys His Gly Val His Tyr Ala Tyr 420 425 430 Tyr Pro Trp Ile His Gln Asn Phe Leu Ser Thr Val Arg Tyr Met His 435 440 445 Ala Ala Gly Thr Gly Ala Asn Trp Arg Gln Met Ala Arg Glu Asn Pro 450 455 460 Leu Thr Gly Arg Ala 465 <210> SEQ ID NO 15 <211> LENGTH: 467 <212> TYPE: PRT <213> ORGANISM: Dictyostelium discoideum <220> FEATURE: <221> NAME/KEY: MISC_FEATURE <223> OTHER INFORMATION: delta-5 desaturase <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: GenBank Accession No. XP_640331 <400> SEQUENCE: 15 Met Met Glu Thr Asn Asn Glu Asn Lys Glu Lys Leu Lys Leu Tyr Thr 1 5 10 15 Trp Asp Glu Val Ser Lys His Asn Gln Lys Asn Asp Leu Trp Ile Ile 20 25 30 Val Asp Gly Lys Val Tyr Asn Ile Thr Lys Trp Val Pro Leu His Pro 35 40 45 Gly Gly Glu Asp Ile Leu Leu Leu Ser Ala Gly Arg Asp Ala Thr Asn 50 55 60 Leu Phe Glu Ser Tyr His Pro Met Thr Asp Lys His Tyr Ser Leu Ile 65 70 75 80 Lys Gln Tyr Glu Ile Gly Tyr Ile Ser Ser Tyr Glu His Pro Lys Tyr 85 90 95 Val Glu Lys Ser Glu Phe Tyr Ser Thr Leu Lys Gln Arg Val Arg Lys 100 105 110 His Phe Gln Thr Ser Ser Gln Asp Pro Lys Val Ser Val Gly Val Phe 115 120 125 Thr Arg Met Val Leu Ile Tyr Leu Phe Leu Phe Val Thr Tyr Tyr Leu 130 135 140 Ser Gln Phe Ser Thr Asp Arg Phe Trp Leu Asn Cys Ile Phe Ala Val 145 150 155 160 Leu Tyr Gly Val Ala Asn Ser Leu Phe Gly Leu His Thr Met His Asp 165 170 175 Ala Cys His Thr Ala Ile Thr His Asn Pro Met Thr Trp Lys Ile Leu 180 185 190 Gly Ala Thr Phe Asp Leu Phe Ala Gly Ala Ser Phe Tyr Ala Trp Cys 195 200 205 His Gln His Val Ile Gly His His Leu Tyr Thr Asn Val Arg Asn Ala 210 215 220 Asp Pro Asp Leu Gly Gln Gly Glu Ile Asp Phe Arg Val Val Thr Pro 225 230 235 240 Tyr Gln Ala Arg Ser Trp Tyr His Lys Tyr Gln His Ile Tyr Ala Pro 245 250 255 Ile Leu Tyr Gly Val Tyr Ala Leu Lys Tyr Arg Ile Gln Asp His Glu 260 265 270 Ile Phe Thr Lys Lys Ser Asn Gly Ala Ile Arg Tyr Ser Pro Ile Ser 275 280 285 Thr Ile Asp Thr Ala Ile Phe Ile Leu Gly Lys Leu Val Phe Ile Ile 290 295 300 Ser Arg Phe Ile Leu Pro Leu Ile Tyr Asn His Ser Phe Ser His Leu 305 310 315 320 Ile Cys Phe Phe Leu Ile Ser Glu Leu Val Leu Gly Trp Tyr Leu Ala 325 330 335 Ile Ser Phe Gln Val Ser His Val Val Glu Asp Leu Gln Phe Met Ala 340 345 350 Thr Pro Glu Ile Phe Asp Gly Ala Asp His Pro Leu Pro Thr Thr Phe 355 360 365 Asn Gln Asp Trp Ala Ile Leu Gln Val Lys Thr Thr Gln Asp Tyr Ala 370 375 380 Gln Asp Ser Val Leu Ser Thr Phe Phe Ser Gly Gly Leu Asn Leu Gln 385 390 395 400 Val Ile His His Cys Phe Pro Thr Ile Ala Gln Asp Tyr Tyr Pro Gln 405 410 415 Ile Val Pro Ile Leu Lys Glu Val Cys Lys Glu Tyr Asn Val Thr Tyr 420 425 430 His Tyr Lys Pro Thr Phe Thr Glu Ala Ile Lys Ser His Ile Asn Tyr 435 440 445 Leu Tyr Lys Met Gly Asn Asp Pro Asp Tyr Val Arg Lys Pro Val Asn 450 455 460 Lys Asn Asp 465 <210> SEQ ID NO 16 <211> LENGTH: 421 <212> TYPE: PRT <213> ORGANISM: Euglena gracilis <220> FEATURE: <221> NAME/KEY: MISC_FEATURE <223> OTHER INFORMATION: delta-8 desaturase <300> PUBLICATION INFORMATION: <302> TITLE: DELTA-8 DESATURASE AND ITS USE IN MAKING POLYUNSATURATED FATTY ACIDS <310> PATENT DOCUMENT NUMBER: WO2006012325 <311> PATENT FILING DATE: 2005-06-24 <312> PUBLICATION DATE: 2006-02-02 <313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(421) <300> PUBLICATION INFORMATION: <302> TITLE: DELTA-8 DESATURASE AND ITS USE IN MAKING POLYUNSATURATED FATTY ACIDS <310> PATENT DOCUMENT NUMBER: WO2006012326 <311> PATENT FILING DATE: 2005-06-24 <312> PUBLICATION DATE: 2006-02-02 <313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(421) <400> SEQUENCE: 16 Met Lys Ser Lys Arg Gln Ala Leu Pro Leu Thr Ile Asp Gly Thr Thr 1 5 10 15 Tyr Asp Val Ser Ala Trp Val Asn Phe His Pro Gly Gly Ala Glu Ile 20 25 30 Ile Glu Asn Tyr Gln Gly Arg Asp Ala Thr Asp Ala Phe Met Val Met 35 40 45 His Ser Gln Glu Ala Phe Asp Lys Leu Lys Arg Met Pro Lys Ile Asn 50 55 60 Pro Ser Ser Glu Leu Pro Pro Gln Ala Ala Val Asn Glu Ala Gln Glu 65 70 75 80 Asp Phe Arg Lys Leu Arg Glu Glu Leu Ile Ala Thr Gly Met Phe Asp 85 90 95 Ala Ser Pro Leu Trp Tyr Ser Tyr Lys Ile Ser Thr Thr Leu Gly Leu 100 105 110 Gly Val Leu Gly Tyr Phe Leu Met Val Gln Tyr Gln Met Tyr Phe Ile 115 120 125 Gly Ala Val Leu Leu Gly Met His Tyr Gln Gln Met Gly Trp Leu Ser 130 135 140 His Asp Ile Cys His His Gln Thr Phe Lys Asn Arg Asn Trp Asn Asn 145 150 155 160 Leu Val Gly Leu Val Phe Gly Asn Gly Leu Gln Gly Phe Ser Val Thr 165 170 175 Trp Trp Lys Asp Arg His Asn Ala His His Ser Ala Thr Asn Val Gln 180 185 190 Gly His Asp Pro Asp Ile Asp Asn Leu Pro Leu Leu Ala Trp Ser Glu 195 200 205 Asp Asp Val Thr Arg Ala Ser Pro Ile Ser Arg Lys Leu Ile Gln Phe 210 215 220 Gln Gln Tyr Tyr Phe Leu Val Ile Cys Ile Leu Leu Arg Phe Ile Trp 225 230 235 240 Cys Phe Gln Ser Val Leu Thr Val Arg Ser Leu Lys Asp Arg Asp Asn 245 250 255 Gln Phe Tyr Arg Ser Gln Tyr Lys Lys Glu Ala Ile Gly Leu Ala Leu 260 265 270 His Trp Thr Leu Lys Thr Leu Phe His Leu Phe Phe Met Pro Ser Ile 275 280 285 Leu Thr Ser Leu Leu Val Phe Phe Val Ser Glu Leu Val Gly Gly Phe 290 295 300 Gly Ile Ala Ile Val Val Phe Met Asn His Tyr Pro Leu Glu Lys Ile 305 310 315 320 Gly Asp Ser Val Trp Asp Gly His Gly Phe Ser Val Gly Gln Ile His 325 330 335 Glu Thr Met Asn Ile Arg Arg Gly Ile Ile Thr Asp Trp Phe Phe Gly 340 345 350 Gly Leu Asn Tyr Gln Ile Glu His His Leu Trp Pro Thr Leu Pro Arg 355 360 365 His Asn Leu Thr Ala Val Ser Tyr Gln Val Glu Gln Leu Cys Gln Lys 370 375 380 His Asn Leu Pro Tyr Arg Asn Pro Leu Pro His Glu Gly Leu Val Ile 385 390 395 400 Leu Leu Arg Tyr Leu Ala Val Phe Ala Arg Met Ala Glu Lys Gln Pro 405 410 415 Ala Gly Lys Ala Leu 420 <210> SEQ ID NO 17 <211> LENGTH: 1269 <212> TYPE: DNA <213> ORGANISM: Pavlova lutheri CCMP459 <220> FEATURE: <221> NAME/KEY: misc_feature <223> OTHER INFORMATION: delta-8 desaturase <400> SEQUENCE: 17 atgggcaagg gtggagacgg cggcgcgcag gcggtgagcg ggaccgacgc gtctctcgct 60 gaggtgagct ccgtcgatag caagagcgtg cacgtcgtgc tctacggcaa gcgcgtggat 120 gtcacaaagt tccagaaggc acacccgggc gggagcaagg tgttccgcat cttccaggag 180 cgcgacgcga cggagcagtt cgagtcttac cactcgccca aggccatcaa gatgatggag 240 ggcatgctca agaagtcgga ggatgcgccc gcttccgtgc ccctgccctc gcggtccacc 300 atgggcacgg agttcaagga gatgattgag cgccacaaga gggctggtct ctacgaccct 360 tgcccgttgg acgagctgtt caagctcacc atcgtccttg cgcccatctt cgtgggcgcc 420 tatctcgtgc ggagcggcgt ctcgcccctc gcgggcgcgc tctccatggg ctttggcttc 480 tacctcgacg gctggcttgc tcacgactac ctgcatcacg cagtcttcaa gggctcggtc 540 aacacgctcg tcaaggcgaa caacgccatg ggatacgccc tcggcttcct ccagggctac 600 gacgtggcct ggtggcgcgc gcgccataac acgcaccacg tgtgcaccaa cgaggatggt 660 tcggacccgg acatcaagac ggcgcccctg ctcatctacg tgcgagagaa cccgtccatt 720 gccaagcggc tcaacttctt ccagcgctgg cagcagtact actatgtgcc gaccatggcc 780 atcctcgacc tctactggcg cctggagtcc atcgcgtacg tggctgtgcg cctgcctaag 840 atgtggatgc aggccgccgc tcttgccgct cactacgcgc tcctgtgctg ggtcttcgca 900 gcgcatctca acctcatccc tctcatgatg gttgcacgcg gcttcgcgac gggcatcgtt 960 gtctttgcaa cccactatgg tgaggacatc ctcgaccgcg agcacgtcga gggcatgacg 1020 ctcgtcgagc agaccgccaa gacctcccgt aacatcacgg gcggctggct agtgaacgtg 1080 ctcacgggct tcatctccct gcagaccgag catcacctct tccccatgat gcccaccggc 1140 aacctaatga ctatccagcc cgaggtacgc gacttcttca agaagcatgg cctcgagtac 1200 cgcgagggca acctcttcca gtgcgtgcac cagaacatca aggctctcgc cttcgagcac 1260 ctcctccac 1269 <210> SEQ ID NO 18 <211> LENGTH: 423 <212> TYPE: PRT <213> ORGANISM: Pavlova lutheri CCMP459 <400> SEQUENCE: 18 Met Gly Lys Gly Gly Asp Gly Gly Ala Gln Ala Val Ser Gly Thr Asp 1 5 10 15 Ala Ser Leu Ala Glu Val Ser Ser Val Asp Ser Lys Ser Val His Val 20 25 30 Val Leu Tyr Gly Lys Arg Val Asp Val Thr Lys Phe Gln Lys Ala His 35 40 45 Pro Gly Gly Ser Lys Val Phe Arg Ile Phe Gln Glu Arg Asp Ala Thr 50 55 60 Glu Gln Phe Glu Ser Tyr His Ser Pro Lys Ala Ile Lys Met Met Glu 65 70 75 80 Gly Met Leu Lys Lys Ser Glu Asp Ala Pro Ala Ser Val Pro Leu Pro 85 90 95 Ser Arg Ser Thr Met Gly Thr Glu Phe Lys Glu Met Ile Glu Arg His 100 105 110 Lys Arg Ala Gly Leu Tyr Asp Pro Cys Pro Leu Asp Glu Leu Phe Lys 115 120 125 Leu Thr Ile Val Leu Ala Pro Ile Phe Val Gly Ala Tyr Leu Val Arg 130 135 140 Ser Gly Val Ser Pro Leu Ala Gly Ala Leu Ser Met Gly Phe Gly Phe 145 150 155 160 Tyr Leu Asp Gly Trp Leu Ala His Asp Tyr Leu His His Ala Val Phe 165 170 175 Lys Gly Ser Val Asn Thr Leu Val Lys Ala Asn Asn Ala Met Gly Tyr 180 185 190 Ala Leu Gly Phe Leu Gln Gly Tyr Asp Val Ala Trp Trp Arg Ala Arg 195 200 205 His Asn Thr His His Val Cys Thr Asn Glu Asp Gly Ser Asp Pro Asp 210 215 220 Ile Lys Thr Ala Pro Leu Leu Ile Tyr Val Arg Glu Asn Pro Ser Ile 225 230 235 240 Ala Lys Arg Leu Asn Phe Phe Gln Arg Trp Gln Gln Tyr Tyr Tyr Val 245 250 255 Pro Thr Met Ala Ile Leu Asp Leu Tyr Trp Arg Leu Glu Ser Ile Ala 260 265 270 Tyr Val Ala Val Arg Leu Pro Lys Met Trp Met Gln Ala Ala Ala Leu 275 280 285 Ala Ala His Tyr Ala Leu Leu Cys Trp Val Phe Ala Ala His Leu Asn 290 295 300 Leu Ile Pro Leu Met Met Val Ala Arg Gly Phe Ala Thr Gly Ile Val 305 310 315 320 Val Phe Ala Thr His Tyr Gly Glu Asp Ile Leu Asp Arg Glu His Val 325 330 335 Glu Gly Met Thr Leu Val Glu Gln Thr Ala Lys Thr Ser Arg Asn Ile 340 345 350 Thr Gly Gly Trp Leu Val Asn Val Leu Thr Gly Phe Ile Ser Leu Gln 355 360 365 Thr Glu His His Leu Phe Pro Met Met Pro Thr Gly Asn Leu Met Thr 370 375 380 Ile Gln Pro Glu Val Arg Asp Phe Phe Lys Lys His Gly Leu Glu Tyr 385 390 395 400 Arg Glu Gly Asn Leu Phe Gln Cys Val His Gln Asn Ile Lys Ala Leu 405 410 415 Ala Phe Glu His Leu Leu His 420 <210> SEQ ID NO 19 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Conserved Region 1 <220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION: (4)..(4) <223> OTHER INFORMATION: Xaa = Ile or Val <400> SEQUENCE: 19 Gly His His Xaa Tyr Thr Asn 1 5 <210> SEQ ID NO 20 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Conserved Region 2 <220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION: (4)..(4) <223> OTHER INFORMATION: Xaa = Val or Ala <220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa = Ser or Asn <400> SEQUENCE: 20 Asn Phe Gln Xaa Xaa His Val 1 5 <210> SEQ ID NO 21 <211> LENGTH: 476 <212> TYPE: PRT <213> ORGANISM: Thalassiosira pseudonana <220> FEATURE: <221> NAME/KEY: MISC_FEATURE <223> OTHER INFORMATION: delta-8 desaturase <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: GenBank Accession No. AAX14502 <400> SEQUENCE: 21 Met Pro Pro Asn Ala Asp Ile Ser Arg Ile Arg Asn Arg Ile Pro Thr 1 5 10 15 Lys Thr Gly Thr Val Ala Ser Ala Asp Asn Asn Asp Pro Ala Thr Gln 20 25 30 Ser Val Arg Thr Leu Lys Ser Leu Lys Gly Asn Glu Val Val Ile Asn 35 40 45 Gly Thr Ile Tyr Asp Ile Ala Asp Phe Val His Pro Gly Gly Glu Val 50 55 60 Val Lys Phe Phe Gly Gly Asn Asp Val Thr Ile Gln Tyr Asn Met Ile 65 70 75 80 His Pro Tyr His Thr Gly Lys His Leu Glu Lys Met Lys Ala Val Gly 85 90 95 Lys Val Val Asp Trp Gln Ser Asp Tyr Lys Phe Asp Thr Pro Phe Glu 100 105 110 Arg Glu Ile Lys Ser Glu Val Phe Lys Ile Val Arg Arg Gly Arg Glu 115 120 125 Phe Gly Thr Thr Gly Tyr Phe Leu Arg Ala Phe Phe Tyr Ile Ala Leu 130 135 140 Phe Phe Thr Met Gln Tyr Thr Phe Ala Thr Cys Thr Thr Phe Thr Thr 145 150 155 160 Tyr Asp His Trp Tyr Gln Ser Gly Val Phe Ile Ala Ile Val Phe Gly 165 170 175 Ile Ser Gln Ala Phe Ile Gly Leu Asn Val Gln His Asp Ala Asn His 180 185 190 Gly Ala Ala Ser Lys Arg Pro Trp Val Asn Asp Leu Leu Gly Phe Gly 195 200 205 Thr Asp Leu Ile Gly Ser Asn Lys Trp Asn Trp Met Ala Gln His Trp 210 215 220 Thr His His Ala Tyr Thr Asn His Ser Glu Lys Asp Pro Asp Ser Phe 225 230 235 240 Ser Ser Glu Pro Met Phe Ala Phe Asn Asp Tyr Pro Ile Gly His Pro 245 250 255 Lys Arg Lys Trp Trp His Arg Phe Gln Gly Gly Tyr Phe Leu Phe Met 260 265 270 Leu Gly Leu Tyr Trp Leu Pro Thr Val Phe Asn Pro Gln Phe Ile Asp 275 280 285 Leu Arg Gln Arg Gly Ala Gln Tyr Val Gly Ile Gln Met Glu Asn Asp 290 295 300 Phe Ile Val Lys Arg Arg Lys Tyr Ala Val Ala Leu Arg Met Met Tyr 305 310 315 320 Ile Tyr Leu Asn Ile Val Ser Pro Phe Met Asn Asn Gly Leu Ser Trp 325 330 335 Ser Thr Phe Gly Ile Ile Met Leu Met Gly Ile Ser Glu Ser Leu Thr 340 345 350 Leu Ser Val Leu Phe Ser Leu Ser His Asn Phe Ile Asn Ser Asp Arg 355 360 365 Asp Pro Thr Ala Asp Phe Lys Lys Thr Gly Glu Gln Val Cys Trp Phe 370 375 380 Lys Ser Gln Val Glu Thr Ser Ser Thr Tyr Gly Gly Phe Ile Ser Gly 385 390 395 400 Cys Leu Thr Gly Gly Leu Asn Phe Gln Val Glu His His Leu Phe Pro 405 410 415 Arg Met Ser Ser Ala Trp Tyr Pro Tyr Ile Ala Pro Thr Val Arg Glu 420 425 430 Val Cys Lys Lys His Gly Met Ser Tyr Ala Tyr Tyr Pro Trp Ile Gly 435 440 445 Gln Asn Leu Val Ser Thr Phe Lys Tyr Met His Arg Ala Gly Ser Gly 450 455 460 Ala Asn Trp Glu Leu Lys Pro Leu Ser Gly Ser Ala 465 470 475 <210> SEQ ID NO 22 <211> LENGTH: 8165 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pZUF17 <400> SEQUENCE: 22 gtacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 60 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 120 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 180 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 240 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 300 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 360 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 420 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 480 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 540 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 600 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 660 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 720 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 780 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 840 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 900 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 960 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 1020 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 1080 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 1140 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 1200 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 1260 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 1320 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 1380 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 1440 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 1500 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 1560 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 1620 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 1680 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 1740 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 1800 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 1860 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 1920 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 1980 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 2040 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 2100 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 2160 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 2220 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 2280 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 2340 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 2400 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 2460 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 2520 aacgcgaatt ttaacaaaat attaacgctt acaatttcca ttcgccattc aggctgcgca 2580 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 2640 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta 2700 aaacgacggc cagtgaattg taatacgact cactataggg cgaattgggt accgggcccc 2760 ccctcgaggt cgatggtgtc gataagcttg atatcgaatt catgtcacac aaaccgatct 2820 tcgcctcaag gaaacctaat tctacatccg agagactgcc gagatccagt ctacactgat 2880 taattttcgg gccaataatt taaaaaaatc gtgttatata atattatatg tattatatat 2940 atacatcatg atgatactga cagtcatgtc ccattgctaa atagacagac tccatctgcc 3000 gcctccaact gatgttctca atatttaagg ggtcatctcg cattgtttaa taataaacag 3060 actccatcta ccgcctccaa atgatgttct caaaatatat tgtatgaact tatttttatt 3120 acttagtatt attagacaac ttacttgctt tatgaaaaac acttcctatt taggaaacaa 3180 tttataatgg cagttcgttc atttaacaat ttatgtagaa taaatgttat aaatgcgtat 3240 gggaaatctt aaatatggat agcataaatg atatctgcat tgcctaattc gaaatcaaca 3300 gcaacgaaaa aaatcccttg tacaacataa atagtcatcg agaaatatca actatcaaag 3360 aacagctatt cacacgttac tattgagatt attattggac gagaatcaca cactcaactg 3420 tctttctctc ttctagaaat acaggtacaa gtatgtacta ttctcattgt tcatacttct 3480 agtcatttca tcccacatat tccttggatt tctctccaat gaatgacatt ctatcttgca 3540 aattcaacaa ttataataag atataccaaa gtagcggtat agtggcaatc aaaaagcttc 3600 tctggtgtgc ttctcgtatt tatttttatt ctaatgatcc attaaaggta tatatttatt 3660 tcttgttata taatcctttt gtttattaca tgggctggat acataaaggt attttgattt 3720 aattttttgc ttaaattcaa tcccccctcg ttcagtgtca actgtaatgg taggaaatta 3780 ccatactttt gaagaagcaa aaaaaatgaa agaaaaaaaa aatcgtattt ccaggttaga 3840 cgttccgcag aatctagaat gcggtatgcg gtacattgtt cttcgaacgt aaaagttgcg 3900 ctccctgaga tattgtacat ttttgctttt acaagtacaa gtacatcgta caactatgta 3960 ctactgttga tgcatccaca acagtttgtt ttgttttttt ttgttttttt tttttctaat 4020 gattcattac cgctatgtat acctacttgt acttgtagta agccgggtta ttggcgttca 4080 attaatcata gacttatgaa tctgcacggt gtgcgctgcg agttactttt agcttatgca 4140 tgctacttgg gtgtaatatt gggatctgtt cggaaatcaa cggatgctca atcgatttcg 4200 acagtaatta attaagtcat acacaagtca gctttcttcg agcctcatat aagtataagt 4260 agttcaacgt attagcactg tacccagcat ctccgtatcg agaaacacaa caacatgccc 4320 cattggacag atcatgcgga tacacaggtt gtgcagtatc atacatactc gatcagacag 4380 gtcgtctgac catcatacaa gctgaacaag cgctccatac ttgcacgctc tctatataca 4440 cagttaaatt acatatccat agtctaacct ctaacagtta atcttctggt aagcctccca 4500 gccagccttc tggtatcgct tggcctcctc aataggatct cggttctggc cgtacagacc 4560 tcggccgaca attatgatat ccgttccggt agacatgaca tcctcaacag ttcggtactg 4620 ctgtccgaga gcgtctccct tgtcgtcaag acccaccccg ggggtcagaa taagccagtc 4680 ctcagagtcg cccttaggtc ggttctgggc aatgaagcca accacaaact cggggtcgga 4740 tcgggcaagc tcaatggtct gcttggagta ctcgccagtg gccagagagc ccttgcaaga 4800 cagctcggcc agcatgagca gacctctggc cagcttctcg ttgggagagg ggactaggaa 4860 ctccttgtac tgggagttct cgtagtcaga gacgtcctcc ttcttctgtt cagagacagt 4920 ttcctcggca ccagctcgca ggccagcaat gattccggtt ccgggtacac cgtgggcgtt 4980 ggtgatatcg gaccactcgg cgattcggtg acaccggtac tggtgcttga cagtgttgcc 5040 aatatctgcg aactttctgt cctcgaacag gaagaaaccg tgcttaagag caagttcctt 5100 gagggggagc acagtgccgg cgtaggtgaa gtcgtcaatg atgtcgatat gggttttgat 5160 catgcacaca taaggtccga ccttatcggc aagctcaatg agctccttgg tggtggtaac 5220 atccagagaa gcacacaggt tggttttctt ggctgccacg agcttgagca ctcgagcggc 5280 aaaggcggac ttgtggacgt tagctcgagc ttcgtaggag ggcattttgg tggtgaagag 5340 gagactgaaa taaatttagt ctgcagaact ttttatcgga accttatctg gggcagtgaa 5400 gtatatgtta tggtaatagt tacgagttag ttgaacttat agatagactg gactatacgg 5460 ctatcggtcc aaattagaaa gaacgtcaat ggctctctgg gcgtcgcctt tgccgacaaa 5520 aatgtgatca tgatgaaagc cagcaatgac gttgcagctg atattgttgt cggccaaccg 5580 cgccgaaaac gcagctgtca gacccacagc ctccaacgaa gaatgtatcg tcaaagtgat 5640 ccaagcacac tcatagttgg agtcgtactc caaaggcggc aatgacgagt cagacagata 5700 ctcgtcgact caggcgacga cggaattcct gcagcccatc tgcagaattc aggagagacc 5760 gggttggcgg cgtatttgtg tcccaaaaaa cagccccaat tgccccggag aagacggcca 5820 ggccgcctag atgacaaatt caacaactca cagctgactt tctgccattg ccactagggg 5880 ggggcctttt tatatggcca agccaagctc tccacgtcgg ttgggctgca cccaacaata 5940 aatgggtagg gttgcaccaa caaagggatg ggatgggggg tagaagatac gaggataacg 6000 gggctcaatg gcacaaataa gaacgaatac tgccattaag actcgtgatc cagcgactga 6060 caccattgca tcatctaagg gcctcaaaac tacctcggaa ctgctgcgct gatctggaca 6120 ccacagaggt tccgagcact ttaggttgca ccaaatgtcc caccaggtgc aggcagaaaa 6180 cgctggaaca gcgtgtacag tttgtcttaa caaaaagtga gggcgctgag gtcgagcagg 6240 gtggtgtgac ttgttatagc ctttagagct gcgaaagcgc gtatggattt ggctcatcag 6300 gccagattga gggtctgtgg acacatgtca tgttagtgta cttcaatcgc cccctggata 6360 tagccccgac aataggccgt ggcctcattt ttttgccttc cgcacatttc cattgctcgg 6420 tacccacacc ttgcttctcc tgcacttgcc aaccttaata ctggtttaca ttgaccaaca 6480 tcttacaagc ggggggcttg tctagggtat atataaacag tggctctccc aatcggttgc 6540 cagtctcttt tttcctttct ttccccacag attcgaaatc taaactacac atcacacaat 6600 gcctgttact gacgtcctta agcgaaagtc cggtgtcatc gtcggcgacg atgtccgagc 6660 cgtgagtatc cacgacaaga tcagtgtcga gacgacgcgt tttgtgtaat gacacaatcc 6720 gaaagtcgct agcaacacac actctctaca caaactaacc cagctctcca tggctgagga 6780 taagaccaag gtcgagttcc ctaccctgac tgagctgaag cactctatcc ctaacgcttg 6840 ctttgagtcc aacctcggac tctcgctcta ctacactgcc cgagcgatct tcaacgcatc 6900 tgcctctgct gctctgctct acgctgcccg atctactccc ttcattgccg ataacgttct 6960 gctccacgct ctggtttgcg ccacctacat ctacgtgcag ggtgtcatct tctggggttt 7020 ctttaccgtc ggtcacgact gtggtcactc tgccttctcc cgataccact ccgtcaactt 7080 catcattggc tgcatcatgc actctgccat tctgactccc ttcgagtcct ggcgagtgac 7140 ccaccgacac catcacaaga acactggcaa cattgataag gacgagatct tctaccctca 7200 tcggtccgtc aaggacctcc aggacgtgcg acaatgggtc tacaccctcg gaggtgcttg 7260 gtttgtctac ctgaaggtcg gatatgctcc tcgaaccatg tcccactttg acccctggga 7320 ccctctcctg cttcgacgag cctccgctgt catcgtgtcc ctcggagtct gggctgcctt 7380 cttcgctgcc tacgcctacc tcacatactc gctcggcttt gccgtcatgg gcctctacta 7440 ctatgctcct ctctttgtct ttgcttcgtt cctcgtcatt actaccttct tgcatcacaa 7500 cgacgaagct actccctggt acggtgactc ggagtggacc tacgtcaagg gcaacctgag 7560 ctccgtcgac cgatcgtacg gagctttcgt ggacaacctg tctcaccaca ttggcaccca 7620 ccaggtccat cacttgttcc ctatcattcc ccactacaag ctcaacgaag ccaccaagca 7680 ctttgctgcc gcttaccctc acctcgtgag acgtaacgac gagcccatca ttactgcctt 7740 cttcaagacc gctcacctct ttgtcaacta cggagctgtg cccgagactg ctcagatttt 7800 caccctcaaa gagtctgccg ctgcagccaa ggccaagagc gactaagcgg ccgcaagtgt 7860 ggatggggaa gtgagtgccc ggttctgtgt gcacaattgg caatccaaga tggatggatt 7920 caacacaggg atatagcgag ctacgtggtg gtgcgaggat atagcaacgg atatttatgt 7980 ttgacacttg agaatgtacg atacaagcac tgtccaagta caatactaaa catactgtac 8040 atactcatac tcgtacccgg gcaacggttt cacttgagtg cagtggctag tgctcttact 8100 cgtacagtgt gcaatactgc gtatcatagt ctttgatgta tatcgtattc attcatgtta 8160 gttgc 8165 <210> SEQ ID NO 23 <211> LENGTH: 8480 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pDMW368 <400> SEQUENCE: 23 ggccgcaagt gtggatgggg aagtgagtgc ccggttctgt gtgcacaatt ggcaatccaa 60 gatggatgga ttcaacacag ggatatagcg agctacgtgg tggtgcgagg atatagcaac 120 ggatatttat gtttgacact tgagaatgta cgatacaagc actgtccaag tacaatacta 180 aacatactgt acatactcat actcgtaccc gggcaacggt ttcacttgag tgcagtggct 240 agtgctctta ctcgtacagt gtgcaatact gcgtatcata gtctttgatg tatatcgtat 300 tcattcatgt tagttgcgta cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 360 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 420 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 480 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 540 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 600 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 660 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 720 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 780 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 840 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 900 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 960 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 1020 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 1080 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 1140 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 1200 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 1260 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 1320 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 1380 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 1440 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 1500 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 1560 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 1620 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 1680 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 1740 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 1800 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 1860 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 1920 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 1980 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 2040 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 2100 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 2160 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 2220 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 2280 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 2340 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 2400 cccgaaaagt gccacctgac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg 2460 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct 2520 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc 2580 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg 2640 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt 2700 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg 2760 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc 2820 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttccattc 2880 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg 2940 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc 3000 ccagtcacga cgttgtaaaa cgacggccag tgaattgtaa tacgactcac tatagggcga 3060 attgggtacc gggccccccc tcgaggtcga tggtgtcgat aagcttgata tcgaattcat 3120 gtcacacaaa ccgatcttcg cctcaaggaa acctaattct acatccgaga gactgccgag 3180 atccagtcta cactgattaa ttttcgggcc aataatttaa aaaaatcgtg ttatataata 3240 ttatatgtat tatatatata catcatgatg atactgacag tcatgtccca ttgctaaata 3300 gacagactcc atctgccgcc tccaactgat gttctcaata tttaaggggt catctcgcat 3360 tgtttaataa taaacagact ccatctaccg cctccaaatg atgttctcaa aatatattgt 3420 atgaacttat ttttattact tagtattatt agacaactta cttgctttat gaaaaacact 3480 tcctatttag gaaacaattt ataatggcag ttcgttcatt taacaattta tgtagaataa 3540 atgttataaa tgcgtatggg aaatcttaaa tatggatagc ataaatgata tctgcattgc 3600 ctaattcgaa atcaacagca acgaaaaaaa tcccttgtac aacataaata gtcatcgaga 3660 aatatcaact atcaaagaac agctattcac acgttactat tgagattatt attggacgag 3720 aatcacacac tcaactgtct ttctctcttc tagaaataca ggtacaagta tgtactattc 3780 tcattgttca tacttctagt catttcatcc cacatattcc ttggatttct ctccaatgaa 3840 tgacattcta tcttgcaaat tcaacaatta taataagata taccaaagta gcggtatagt 3900 ggcaatcaaa aagcttctct ggtgtgcttc tcgtatttat ttttattcta atgatccatt 3960 aaaggtatat atttatttct tgttatataa tccttttgtt tattacatgg gctggataca 4020 taaaggtatt ttgatttaat tttttgctta aattcaatcc cccctcgttc agtgtcaact 4080 gtaatggtag gaaattacca tacttttgaa gaagcaaaaa aaatgaaaga aaaaaaaaat 4140 cgtatttcca ggttagacgt tccgcagaat ctagaatgcg gtatgcggta cattgttctt 4200 cgaacgtaaa agttgcgctc cctgagatat tgtacatttt tgcttttaca agtacaagta 4260 catcgtacaa ctatgtacta ctgttgatgc atccacaaca gtttgttttg tttttttttg 4320 tttttttttt ttctaatgat tcattaccgc tatgtatacc tacttgtact tgtagtaagc 4380 cgggttattg gcgttcaatt aatcatagac ttatgaatct gcacggtgtg cgctgcgagt 4440 tacttttagc ttatgcatgc tacttgggtg taatattggg atctgttcgg aaatcaacgg 4500 atgctcaatc gatttcgaca gtaattaatt aagtcataca caagtcagct ttcttcgagc 4560 ctcatataag tataagtagt tcaacgtatt agcactgtac ccagcatctc cgtatcgaga 4620 aacacaacaa catgccccat tggacagatc atgcggatac acaggttgtg cagtatcata 4680 catactcgat cagacaggtc gtctgaccat catacaagct gaacaagcgc tccatacttg 4740 cacgctctct atatacacag ttaaattaca tatccatagt ctaacctcta acagttaatc 4800 ttctggtaag cctcccagcc agccttctgg tatcgcttgg cctcctcaat aggatctcgg 4860 ttctggccgt acagacctcg gccgacaatt atgatatccg ttccggtaga catgacatcc 4920 tcaacagttc ggtactgctg tccgagagcg tctcccttgt cgtcaagacc caccccgggg 4980 gtcagaataa gccagtcctc agagtcgccc ttaggtcggt tctgggcaat gaagccaacc 5040 acaaactcgg ggtcggatcg ggcaagctca atggtctgct tggagtactc gccagtggcc 5100 agagagccct tgcaagacag ctcggccagc atgagcagac ctctggccag cttctcgttg 5160 ggagagggga ctaggaactc cttgtactgg gagttctcgt agtcagagac gtcctccttc 5220 ttctgttcag agacagtttc ctcggcacca gctcgcaggc cagcaatgat tccggttccg 5280 ggtacaccgt gggcgttggt gatatcggac cactcggcga ttcggtgaca ccggtactgg 5340 tgcttgacag tgttgccaat atctgcgaac tttctgtcct cgaacaggaa gaaaccgtgc 5400 ttaagagcaa gttccttgag ggggagcaca gtgccggcgt aggtgaagtc gtcaatgatg 5460 tcgatatggg ttttgatcat gcacacataa ggtccgacct tatcggcaag ctcaatgagc 5520 tccttggtgg tggtaacatc cagagaagca cacaggttgg ttttcttggc tgccacgagc 5580 ttgagcactc gagcggcaaa ggcggacttg tggacgttag ctcgagcttc gtaggagggc 5640 attttggtgg tgaagaggag actgaaataa atttagtctg cagaactttt tatcggaacc 5700 ttatctgggg cagtgaagta tatgttatgg taatagttac gagttagttg aacttataga 5760 tagactggac tatacggcta tcggtccaaa ttagaaagaa cgtcaatggc tctctgggcg 5820 tcgcctttgc cgacaaaaat gtgatcatga tgaaagccag caatgacgtt gcagctgata 5880 ttgttgtcgg ccaaccgcgc cgaaaacgca gctgtcagac ccacagcctc caacgaagaa 5940 tgtatcgtca aagtgatcca agcacactca tagttggagt cgtactccaa aggcggcaat 6000 gacgagtcag acagatactc gtcgactcag gcgacgacgg aattcctgca gcccatctgc 6060 agaattcagg agagaccggg ttggcggcgt atttgtgtcc caaaaaacag ccccaattgc 6120 cccggagaag acggccaggc cgcctagatg acaaattcaa caactcacag ctgactttct 6180 gccattgcca ctaggggggg gcctttttat atggccaagc caagctctcc acgtcggttg 6240 ggctgcaccc aacaataaat gggtagggtt gcaccaacaa agggatggga tggggggtag 6300 aagatacgag gataacgggg ctcaatggca caaataagaa cgaatactgc cattaagact 6360 cgtgatccag cgactgacac cattgcatca tctaagggcc tcaaaactac ctcggaactg 6420 ctgcgctgat ctggacacca cagaggttcc gagcacttta ggttgcacca aatgtcccac 6480 caggtgcagg cagaaaacgc tggaacagcg tgtacagttt gtcttaacaa aaagtgaggg 6540 cgctgaggtc gagcagggtg gtgtgacttg ttatagcctt tagagctgcg aaagcgcgta 6600 tggatttggc tcatcaggcc agattgaggg tctgtggaca catgtcatgt tagtgtactt 6660 caatcgcccc ctggatatag ccccgacaat aggccgtggc ctcatttttt tgccttccgc 6720 acatttccat tgctcggtac ccacaccttg cttctcctgc acttgccaac cttaatactg 6780 gtttacattg accaacatct tacaagcggg gggcttgtct agggtatata taaacagtgg 6840 ctctcccaat cggttgccag tctctttttt cctttctttc cccacagatt cgaaatctaa 6900 actacacatc acacaatgcc tgttactgac gtccttaagc gaaagtccgg tgtcatcgtc 6960 ggcgacgatg tccgagccgt gagtatccac gacaagatca gtgtcgagac gacgcgtttt 7020 gtgtaatgac acaatccgaa agtcgctagc aacacacact ctctacacaa actaacccag 7080 ctctccatgg ctccagatgc ggacaagttg agacagcgca aggcgcaatc gattcaagac 7140 acggctgatt cgcaagctac cgaactcaag attggcaccc tgaagggctt gcaggggaca 7200 gaaatcgtca ttgatggaga catttacgat ataaaagact ttgatcaccc cggtggtgaa 7260 tccatcatga cttttggggg aaacgatgtc accgccacgt acaagatgat ccacccctac 7320 cactctaagc accatttgga gaagatgaag aaagtgggac gagttccgga ctacacctcg 7380 gaatacaagt ttgatactcc ctttgagcgt gaaatcaagc aagaggtctt caagattgtg 7440 cgacgaggcc gcgagtttgg aacacctgga tacttcttcc gggctttctg ctacattgga 7500 cttttctttt acttgcagta tttgtgggtc acgactccca ctacctttgc cttggcgatc 7560 ttctatggtg tttcgcaagc tttcattggt ttgaacgtac aacatgatgc caaccacgga 7620 gctgcctcca agaagccttg gatcaataac ttgctaggat tgggggctga ctttatcgga 7680 ggttccaaat ggttgtggat gaaccagcac tggacgcacc acacatacac caaccaccat 7740 gagaaggatc ccgatgcctt gggcgctgaa ccaatgttgt tgttcaatga ttatcccttg 7800 ggtcacccaa agcgtacttt gattcaccac ttccaggcct tctattacct tttcgtcttg 7860 gccggatact gggtctcttc ggtcttcaac cctcaaattt tggacttgca acaccgcggt 7920 gctcaagcgg ttggaatgaa aatggagaac gattacattg ccaaaagccg aaagtatgcc 7980 atcttcttgc gtctcttgta tatttacacc aacattgtcg ctccgatcca aaaccaaggc 8040 ttctcgttga ccgtggtcgc ccacattttg accatgggcg tcgcttccag tttgactttg 8100 gcgactcttt ttgccttgtc gcacaatttt gaaaacgcgg atcgcgatcc cacttacgag 8160 gcccgcaagg gaggagagcc tgtttgttgg ttcaagtcgc aagtcgaaac ctcgtcaact 8220 tacggaggtt tcatctcggg ttgcttgacg ggcggactca acttccaagt ggaacaccac 8280 ttgttccctc gtatgagttc ggcctggtac ccctacattg cccctactgt tcgagaggtt 8340 tgcaaaaagc acggagtcaa gtacgcatac tatccctggg tctggcaaaa cttgatttca 8400 actgtcaagt atctgcatca aagcggaact ggatccaact ggaagaatgg cgccaacccc 8460 tactcgggaa aattgtaagc 8480 <210> SEQ ID NO 24 <211> LENGTH: 12649 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKUNF12T6E <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (2507)..(2507) <223> OTHER INFORMATION: n is a, c, g, or t <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (2512)..(2515) <223> OTHER INFORMATION: n is a, c, g, or t <400> SEQUENCE: 24 taaccctcac taaagggaac aaaagctgga gctccaccgc ggacacaata tctggtcaaa 60 tttcagtttc gttacataaa tcgttatgtc aaaggagtgt gggaggttaa gagaattatc 120 accggcaaac tatctgttaa ttgctaggta cctctagacg tccacccggg tcgcttggcg 180 gccgaagagg ccggaatctc gggccgcggt ggcggccgct tagttggtct tggacttctt 240 gggcttcttc aggtaggact ggacaaagaa gttgccgaac agagcgagca gggtgatcat 300 gtacacgccg agcagctgga ccagagcctg agggtagtcg caggggaaga ggtagtcgta 360 cagggactgc accagcatag ccatgaactg ggtcatctgc agagtggtga tgtagggctt 420 gatgggcttg acgaagccga agccctgaga ggaaaagaag tagtaggcgt acatgacggt 480 gtggacgaag gagttgagga tgacggagaa gtaggcgtcg ccaccaggag cgtacttggc 540 aatagcccac cagatggcga agatggtggc atggtggtac acgtgcagga aggagacctg 600 gttgaacttc ttgcacagga tcatgatagc ggtgtccagg aactcgtagg ccttggagac 660 gtagaacacg tagacgattc gggacatgcc ctgagcgtgg gactcgttgc ccttctccat 720 gtcgttgccg aagaccttgt agccacccag gatagcctgt cggatggtct cgacgcacat 780 gtagagggac agtccgaaga ggaacaggtt gtggagcagc ttgatggtct tcagctcgaa 840 gggcttctcc atctgcttca tgatgggaat gccgaagagc agcatggcca tgtagccgac 900 ctcgaaggcg agcatggtgg agacgtccat catgggcaga ccgtcggtca gagcgtaggg 960 cttagctccg tccatccact ggtcgacacc ggtctcgact cgtccgacca cgtcgtccca 1020 gacagaggag ttggccatgg tgaatgattc ttatactcag aaggaaatgc ttaacgattt 1080 cgggtgtgag ttgacaagga gagagagaaa agaagaggaa aggtaattcg gggacggtgg 1140 tcttttatac ccttggctaa agtcccaacc acaaagcaaa aaaattttca gtagtctatt 1200 ttgcgtccgg catgggttac ccggatggcc agacaaagaa actagtacaa agtctgaaca 1260 agcgtagatt ccagactgca gtaccctacg cccttaacgg caagtgtggg aaccggggga 1320 ggtttgatat gtggggtgaa gggggctctc gccggggttg ggcccgctac tgggtcaatt 1380 tggggtcaat tggggcaatt ggggctgttt tttgggacac aaatacgccg ccaacccggt 1440 ctctcctgaa ttctgcatcg atcgaggaag aggacaagcg gctgcttctt aagtttgtga 1500 catcagtatc caaggcacca ttgcaaggat tcaaggcttt gaacccgtca tttgccattc 1560 gtaacgctgg tagacaggtt gatcggttcc ctacggcctc cacctgtgtc aatcttctca 1620 agctgcctga ctatcaggac attgatcaac ttcggaagaa acttttgtat gccattcgat 1680 cacatgctgg tttcgatttg tcttagagga acgcatatac agtaatcata gagaataaac 1740 gatattcatt tattaaagta gatagttgag gtagaagttg taaagagtga taaatagcgg 1800 ccgcgcctac ttaagcaacg ggcttgataa cagcgggggg ggtgcccacg ttgttgcggt 1860 tgcggaagaa cagaacaccc ttaccagcac cctcggcacc agcgctgggc tcaacccact 1920 ggcacatacg cgcactgcgg tacatggcgc ggatgaagcc acgaggacca tcctggacat 1980 cagcccggta gtgcttgccc atgatgggct taatggcctc ggtggcctcg tccgcgttgt 2040 agaaggggat gctgctgacg tagtggtgga ggacatgagt ctcgatgatg ccgtggagaa 2100 ggtggcggcc gatgaagccc atctcacggt caatggtagc agcggcacca cggacgaagt 2160 tccactcgtc gttggtgtag tggggaaggg tagggtcggt gtgctggagg aaggtgatgg 2220 caacgagcca gtggttaacc cagaggtagg gaacaaagta ccagatggcc atgttgtaga 2280 aaccgaactt ctgaacgagg aagtacagag cagtggccat cagaccgata ccaatatcgc 2340 tgaggacgat gagcttagcg tcactgttct cgtacagagg gctgcgggga tcgaagtggt 2400 taacaccacc gccgaggccg ttatgcttgc ccttgccgcg accctcacgc tggcgctcgt 2460 ggtagttgtg gccggtaaca ttggtgatga ggtagttggg ccagccnacg annnnctcag 2520 taagatgagc gagctcgtgg gtcatctttc cgagacgagt agcctgctgc tcgcgggttc 2580 ggggaacgaa gaccatgtca cgctccatgt tgccagtggc cttgtggtgc tttcggtggg 2640 agatttgcca gctgaagtag gggacaagga gggaagagtg aagaacccag ccagtaatgt 2700 cgttgatgat gcgagaatcg gagaaagcac cgtgaccgca ctcatgggca ataacccaga 2760 gaccagtacc gaaaagaccc tgaagaacgg tgtacacggc ccacagacca gcgcgggcgg 2820 gggtggaggg gatatattcg ggggtcacaa agttgtacca gatgctgaaa gtggtagtca 2880 ggaggacaat gtcgcggagg atataaccgt atcccttgag agcggagcgc ttgaagcagt 2940 gcttagggat ggcattgtag atgtccttga tggtaaagtc gggaacctcg aactggttgc 3000 cgtaggtgtc gagcatgaca ccatactcgg acttgggctt ggcgatatca acctcggaca 3060 tggacgagag cgatgtggaa gaggccgagt ggcggggaga gtctgaagga gagacggcgg 3120 cagactcaga atccgtcaca gtagttgagg tgacggtgcg tctaagcgca gggttctgct 3180 tgggcagagc cgaagtggac gccatggaga gctgggttag tttgtgtaga gagtgtgtgt 3240 tgctagcgac tttcggattg tgtcattaca caaaacgcgt cgtctcgaca ctgatcttgt 3300 cgtggatact cacggctcgg acatcgtcgc cgacgatgac accggacttt cgcttaagga 3360 cgtcagtaac aggcattgtg tgatgtgtag tttagatttc gaatctgtgg ggaaagaaag 3420 gaaaaaagag actggcaacc gattgggaga gccactgttt atatataccc tagacaagcc 3480 ccccgcttgt aagatgttgg tcaatgtaaa ccagtattaa ggttggcaag tgcaggagaa 3540 gcaaggtgtg ggtaccgagc aatggaaatg tgcggaaggc aaaaaaatga ggccacggcc 3600 tattgtcggg gctatatcca gggggcgatt gaagtacact aacatgacat gtgtccacag 3660 accctcaatc tggcctgatg agccaaatcc atacgcgctt tcgcagctct aaaggctata 3720 acaagtcaca ccaccctgct cgacctcagc gccctcactt tttgttaaga caaactgtac 3780 acgctgttcc agcgttttct gcctgcacct ggtgggacat ttggtgcaac ctaaagtgct 3840 cggaacctct gtggtgtcca gatcagcgca gcagttccga ggtagttttg aggcccttag 3900 atgatgcaat ggtgtcagtc gctggatcac gagtcttaat ggcagtattc gttcttattt 3960 gtgccattga gccccgttat cctcgtatct tctacccccc atcccatccc tttgttggtg 4020 caaccctacc catttattgt tgggtgcagc ccaaccgacg tggagagctt ggcttggcca 4080 tataaaaagg ccccccccta gtggcaatgg cagaaagtca gctgtgagtt gttgaatttg 4140 tcatctaggc ggcctggccg tcttctccgg ggcaattgtt cctctatagt actgcgtaca 4200 ctgtttaaac agtgtacgca gatctgcgac gacggaattc ctgcagccca tctgcagaat 4260 tcaggagaga ccgggttggc ggcgtatttg tgtcccaaaa aacagcccca attgccccaa 4320 ttgaccccaa attgacccag tagcgggccc aaccccggcg agagccccct tcaccccaca 4380 tatcaaacct cccccggttc ccacacttgc cgttaagggc gtagggtact gcagtctgga 4440 atctacgctt gttcagactt tgtactagtt tctttgtctg gccatccggg taacccatgc 4500 cggacgcaaa atagactact gaaaattttt ttgctttgtg gttgggactt tagccaaggg 4560 tataaaagac caccgtcccc gaattacctt tcctcttctt ttctctctct ccttgtcaac 4620 tcacacccga aatcgttaag catttccttc tgagtataag aatcattcac catggctgcc 4680 gctccctctg tgcgaacctt tacccgagcc gaggttctga acgctgaggc tctgaacgag 4740 ggcaagaagg acgctgaggc tcccttcctg atgatcatcg acaacaaggt gtacgacgtc 4800 cgagagttcg tccctgacca tcctggaggc tccgtgattc tcacccacgt tggcaaggac 4860 ggcaccgacg tctttgacac ctttcatccc gaggctgctt gggagactct cgccaacttc 4920 tacgttggag acattgacga gtccgaccga gacatcaaga acgatgactt tgccgctgag 4980 gtccgaaagc tgcgaaccct gttccagtct ctcggctact acgactcctc taaggcctac 5040 tacgccttca aggtctcctt caacctctgc atctggggac tgtccaccgt cattgtggcc 5100 aagtggggtc agacctccac cctcgccaac gtgctctctg ctgccctgct cggcctgttc 5160 tggcagcagt gcggatggct ggctcacgac tttctgcacc accaggtctt ccaggaccga 5220 ttctggggtg atctcttcgg agccttcctg ggaggtgtct gccagggctt ctcctcttcc 5280 tggtggaagg acaagcacaa cactcaccat gccgctccca acgtgcatgg cgaggatcct 5340 gacattgaca cccaccctct cctgacctgg tccgagcacg ctctggagat gttctccgac 5400 gtccccgatg aggagctgac ccgaatgtgg tctcgattca tggtcctgaa ccagacctgg 5460 ttctacttcc ccattctctc cttcgctcga ctgtcttggt gcctccagtc cattctcttt 5520 gtgctgccca acggtcaggc tcacaagccc tccggagctc gagtgcccat ctccctggtc 5580 gagcagctgt ccctcgccat gcactggacc tggtacctcg ctaccatgtt cctgttcatc 5640 aaggatcctg tcaacatgct cgtgtacttc ctggtgtctc aggctgtgtg cggaaacctg 5700 ctcgccatcg tgttctccct caaccacaac ggtatgcctg tgatctccaa ggaggaggct 5760 gtcgacatgg atttctttac caagcagatc atcactggtc gagatgtcca tcctggactg 5820 ttcgccaact ggttcaccgg tggcctgaac taccagatcg agcatcacct gttcccttcc 5880 atgcctcgac acaacttctc caagatccag cctgccgtcg agaccctgtg caagaagtac 5940 aacgtccgat accacaccac tggtatgatc gagggaactg ccgaggtctt ctcccgactg 6000 aacgaggtct ccaaggccac ctccaagatg ggcaaggctc agtaagcggc cgcatgagaa 6060 gataaatata taaatacatt gagatattaa atgcgctaga ttagagagcc tcatactgct 6120 cggagagaag ccaagacgag tactcaaagg ggattacacc atccatatcc acagacacaa 6180 gctggggaaa ggttctatat acactttccg gaataccgta gtttccgatg ttatcaatgg 6240 gggcagccag gatttcaggc acttcggtgt ctcggggtga aatggcgttc ttggcctcca 6300 tcaagtcgta ccatgtcttc atttgcctgt caaagtaaaa cagaagcaga tgaagaatga 6360 acttgaagtg aaggaattta aattgccccg gagaagacgg ccaggccgcc tagatgacaa 6420 attcaacaac tcacagctga ctttctgcca ttgccactag gggggggcct ttttatatgg 6480 ccaagccaag ctctccacgt cggttgggct gcacccaaca ataaatgggt agggttgcac 6540 caacaaaggg atgggatggg gggtagaaga tacgaggata acggggctca atggcacaaa 6600 taagaacgaa tactgccatt aagactcgtg atccagcgac tgacaccatt gcatcatcta 6660 agggcctcaa aactacctcg gaactgctgc gctgatctgg acaccacaga ggttccgagc 6720 actttaggtt gcaccaaatg tcccaccagg tgcaggcaga aaacgctgga acagcgtgta 6780 cagtttgtct taacaaaaag tgagggcgct gaggtcgagc agggtggtgt gacttgttat 6840 agcctttaga gctgcgaaag cgcgtatgga tttggctcat caggccagat tgagggtctg 6900 tggacacatg tcatgttagt gtacttcaat cgccccctgg atatagcccc gacaataggc 6960 cgtggcctca tttttttgcc ttccgcacat ttccattgct cggtacccac accttgcttc 7020 tcctgcactt gccaacctta atactggttt acattgacca acatcttaca agcggggggc 7080 ttgtctaggg tatatataaa cagtggctct cccaatcggt tgccagtctc ttttttcctt 7140 tctttcccca cagattcgaa atctaaacta cacatcacac aatgcctgtt actgacgtcc 7200 ttaagcgaaa gtccggtgtc atcgtcggcg acgatgtccg agccgtgagt atccacgaca 7260 agatcagtgt cgagacgacg cgttttgtgt aatgacacaa tccgaaagtc gctagcaaca 7320 cacactctct acacaaacta acccagctct ccatggagtc cattgctccc ttcctgccct 7380 ccaagatgcc tcaggacctg ttcatggacc tcgccagcgc tatcggtgtc cgagctgctc 7440 cctacgtcga tcccctggag gctgccctgg ttgcccaggc cgagaagtac attcccacca 7500 ttgtccatca cactcgaggc ttcctggttg ccgtggagtc tcccctggct cgagagctgc 7560 ctctgatgaa ccccttccac gtgctcctga tcgtgctcgc ctacctggtc accgtgtttg 7620 tgggtatgca gatcatgaag aactttgaac gattcgaggt caagaccttc tccctcctgc 7680 acaacttctg tctggtctcc atctccgcct acatgtgcgg tggcatcctg tacgaggctt 7740 atcaggccaa ctatggactg tttgagaacg ctgccgatca caccttcaag ggtctcccta 7800 tggctaagat gatctggctc ttctacttct ccaagatcat ggagtttgtc gacaccatga 7860 tcatggtcct caagaagaac aaccgacaga tttcctttct gcacgtgtac caccactctt 7920 ccatcttcac catctggtgg ctggtcacct tcgttgctcc caacggtgaa gcctacttct 7980 ctgctgccct gaactccttc atccacgtca tcatgtacgg ctactacttt ctgtctgccc 8040 tgggcttcaa gcaggtgtcg ttcatcaagt tctacatcac tcgatcccag atgacccagt 8100 tctgcatgat gtctgtccag tcttcctggg acatgtacgc catgaaggtc cttggccgac 8160 ctggataccc cttcttcatc accgctctgc tctggttcta catgtggacc atgctcggtc 8220 tcttctacaa cttttaccga aagaacgcca agctcgccaa gcaggccaag gctgacgctg 8280 ccaaggagaa ggccagaaag ctccagtaag cggccgcaag tgtggatggg gaagtgagtg 8340 cccggttctg tgtgcacaat tggcaatcca agatggatgg attcaacaca gggatatagc 8400 gagctacgtg gtggtgcgag gatatagcaa cggatattta tgtttgacac ttgagaatgt 8460 acgatacaag cactgtccaa gtacaatact aaacatactg tacatactca tactcgtacc 8520 cgggcaacgg tttcacttga gtgcagtggc tagtgctctt actcgtacag tgtgcaatac 8580 tgcgtatcat agtctttgat gtatatcgta ttcattcatg ttagttgcgt acgaagtcgt 8640 caatgatgtc gatatgggtt ttgatcatgc acacataagg tccgacctta tcggcaagct 8700 caatgagctc cttggtggtg gtaacatcca gagaagcaca caggttggtt ttcttggctg 8760 ccacgagctt gagcactcga gcggcaaagg cggacttgtg gacgttagct cgagcttcgt 8820 aggagggcat tttggtggtg aagaggagac tgaaataaat ttagtctgca gaacttttta 8880 tcggaacctt atctggggca gtgaagtata tgttatggta atagttacga gttagttgaa 8940 cttatagata gactggacta tacggctatc ggtccaaatt agaaagaacg tcaatggctc 9000 tctgggcgtc gcctttgccg acaaaaatgt gatcatgatg aaagccagca atgacgttgc 9060 agctgatatt gttgtcggcc aaccgcgccg aaaacgcagc tgtcagaccc acagcctcca 9120 acgaagaatg tatcgtcaaa gtgatccaag cacactcata gttggagtcg tactccaaag 9180 gcggcaatga cgagtcagac agatactcgt cgaccttttc cttgggaacc accaccgtca 9240 gcccttctga ctcacgtatt gtagccaccg acacaggcaa cagtccgtgg atagcagaat 9300 atgtcttgtc ggtccatttc tcaccaactt taggcgtcaa gtgaatgttg cagaagaagt 9360 atgtgccttc attgagaatc ggtgttgctg atttcaataa agtcttgaga tcagtttggc 9420 gcgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg 9480 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 9540 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 9600 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 9660 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 9720 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 9780 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 9840 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 9900 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 9960 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 10020 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 10080 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt 10140 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 10200 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 10260 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 10320 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 10380 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 10440 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 10500 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 10560 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 10620 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 10680 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac 10740 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 10800 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 10860 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 10920 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 10980 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat 11040 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 11100 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 11160 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 11220 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 11280 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 11340 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 11400 aaaagtgcca cctgatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca 11460 tcaggaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag 11520 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac 11580 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga 11640 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc 11700 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg 11760 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa 11820 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac 11880 caccacaccc gccgcgctta atgcgccgct acagggcgcg tccattcgcc attcaggctg 11940 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa 12000 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt 12060 tgtaaaacga cggccagtga attgtaatac gactcactat agggcgaatt gggcccgacg 12120 tcgcatgcag tggtggtatt gtgactgggg atgtagttga gaataagtca tacacaagtc 12180 agctttcttc gagcctcata taagtataag tagttcaacg tattagcact gtacccagca 12240 tctccgtatc gagaaacaca acaacatgcc ccattggaca gatcatgcgg atacacaggt 12300 tgtgcagtat catacatact cgatcagaca ggtcgtctga ccatcataca agctgaacaa 12360 gcgctccata cttgcacgct ctctatatac acagttaaat tacatatcca tagtctaacc 12420 tctaacagtt aatcttctgg taagcctccc agccagcctt ctggtatcgc ttggcctcct 12480 caataggatc tcggttctgg ccgtacagac ctcggccgac aattatgata tccgttccgg 12540 tagacatgac atcctcaaca gttcggtact gctgtccgag agcgtctccc ttgtcgtcaa 12600 gacccacccc gggggtcaga ataagccagt cctcagagtc gcccttaat 12649 <210> SEQ ID NO 25 <211> LENGTH: 819 <212> TYPE: DNA <213> ORGANISM: Thraustochytrium aureum <220> FEATURE: <221> NAME/KEY: misc_feature <223> OTHER INFORMATION: synthetic elongase (codon-optimized) for Yarrowia lipolytica <400> SEQUENCE: 25 atggccaact cctctgtctg ggacgacgtg gtcggacgag tcgagaccgg tgtcgaccag 60 tggatggacg gagctaagcc ctacgctctg accgacggtc tgcccatgat ggacgtctcc 120 accatgctcg ccttcgaggt cggctacatg gccatgctgc tcttcggcat tcccatcatg 180 aagcagatgg agaagccctt cgagctgaag accatcaagc tgctccacaa cctgttcctc 240 ttcggactgt ccctctacat gtgcgtcgag accatccgac aggctatcct gggtggctac 300 aaggtcttcg gcaacgacat ggagaagggc aacgagtccc acgctcaggg catgtcccga 360 atcgtctacg tgttctacgt ctccaaggcc tacgagttcc tggacaccgc tatcatgatc 420 ctgtgcaaga agttcaacca ggtctccttc ctgcacgtgt accaccatgc caccatcttc 480 gccatctggt gggctattgc caagtacgct cctggtggcg acgcctactt ctccgtcatc 540 ctcaactcct tcgtccacac cgtcatgtac gcctactact tcttttcctc tcagggcttc 600 ggcttcgtca agcccatcaa gccctacatc accactctgc agatgaccca gttcatggct 660 atgctggtgc agtccctgta cgactacctc ttcccctgcg actaccctca ggctctggtc 720 cagctgctcg gcgtgtacat gatcaccctg ctcgctctgt tcggcaactt ctttgtccag 780 tcctacctga agaagcccaa gaagtccaag accaactaa 819 <210> SEQ ID NO 26 <211> LENGTH: 272 <212> TYPE: PRT <213> ORGANISM: Thraustochytrium aureum <400> SEQUENCE: 26 Met Ala Asn Ser Ser Val Trp Asp Asp Val Val Gly Arg Val Glu Thr 1 5 10 15 Gly Val Asp Gln Trp Met Asp Gly Ala Lys Pro Tyr Ala Leu Thr Asp 20 25 30 Gly Leu Pro Met Met Asp Val Ser Thr Met Leu Ala Phe Glu Val Gly 35 40 45 Tyr Met Ala Met Leu Leu Phe Gly Ile Pro Ile Met Lys Gln Met Glu 50 55 60 Lys Pro Phe Glu Leu Lys Thr Ile Lys Leu Leu His Asn Leu Phe Leu 65 70 75 80 Phe Gly Leu Ser Leu Tyr Met Cys Val Glu Thr Ile Arg Gln Ala Ile 85 90 95 Leu Gly Gly Tyr Lys Val Phe Gly Asn Asp Met Glu Lys Gly Asn Glu 100 105 110 Ser His Ala Gln Gly Met Ser Arg Ile Val Tyr Val Phe Tyr Val Ser 115 120 125 Lys Ala Tyr Glu Phe Leu Asp Thr Ala Ile Met Ile Leu Cys Lys Lys 130 135 140 Phe Asn Gln Val Ser Phe Leu His Val Tyr His His Ala Thr Ile Phe 145 150 155 160 Ala Ile Trp Trp Ala Ile Ala Lys Tyr Ala Pro Gly Gly Asp Ala Tyr 165 170 175 Phe Ser Val Ile Leu Asn Ser Phe Val His Thr Val Met Tyr Ala Tyr 180 185 190 Tyr Phe Phe Ser Ser Gln Gly Phe Gly Phe Val Lys Pro Ile Lys Pro 195 200 205 Tyr Ile Thr Thr Leu Gln Met Thr Gln Phe Met Ala Met Leu Val Gln 210 215 220 Ser Leu Tyr Asp Tyr Leu Phe Pro Cys Asp Tyr Pro Gln Ala Leu Val 225 230 235 240 Gln Leu Leu Gly Val Tyr Met Ile Thr Leu Leu Ala Leu Phe Gly Asn 245 250 255 Phe Phe Val Gln Ser Tyr Leu Lys Lys Pro Lys Lys Ser Lys Thr Asn 260 265 270 <210> SEQ ID NO 27 <211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer AP <400> SEQUENCE: 27 ggccacgcgt cgactagtac tttttttttt ttttttt 37 <210> SEQ ID NO 28 <211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Smart IV oligonucleotide primer <400> SEQUENCE: 28 aagcagtggt atcaacgcag agtggccatt acggccggg 39 <210> SEQ ID NO 29 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer CDSIII 5' <400> SEQUENCE: 29 aagcagtggt atcaacgcag agt 23 <210> SEQ ID NO 30 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Degenerate primer 5-1A <400> SEQUENCE: 30 gghcaycayr tbtayacaaa 20 <210> SEQ ID NO 31 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Degenerate primer 5-1B <400> SEQUENCE: 31 gghcaycayr tbtayaccaa 20 <210> SEQ ID NO 32 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Degenerate primer 5-1C <400> SEQUENCE: 32 gghcaycayr tbtayacgaa 20 <210> SEQ ID NO 33 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Degenerate primer 5-1D <400> SEQUENCE: 33 gghcaycayr tbtayactaa 20 <210> SEQ ID NO 34 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Degenerate primer 5-4AR <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (9)..(9) <223> OTHER INFORMATION: n is a, c, g, or t <400> SEQUENCE: 34 acrtgrytna cytgraagtt 20 <210> SEQ ID NO 35 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Degenerate primer 5-4BR <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (9)..(9) <223> OTHER INFORMATION: n is a, c, g, or t <400> SEQUENCE: 35 acrtgrytna cytgraaatt 20 <210> SEQ ID NO 36 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Degenerate primer 5-4CR <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (6)..(6) <223> OTHER INFORMATION: n is a, c, g, or t <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (9)..(9) <223> OTHER INFORMATION: n is a, c, g, or t <400> SEQUENCE: 36 acrtgngana cytgraagtt 20 <210> SEQ ID NO 37 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Degenerate primer 5-4DR <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (6)..(6) <223> OTHER INFORMATION: n is a, c, g, or t <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (9)..(9) <223> OTHER INFORMATION: n is a, c, g, or t <400> SEQUENCE: 37 acrtgngana cytgraaatt 20 <210> SEQ ID NO 38 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer ODMW520 <400> SEQUENCE: 38 cgttctccat tttcattcca accgc 25 <210> SEQ ID NO 39 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer ODMW521 <400> SEQUENCE: 39 ggttgaagac cgaagagacc cagtatcc 28 <210> SEQ ID NO 40 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer DNR CDS 5' <400> SEQUENCE: 40 caacgcagag tggccattac gg 22 <210> SEQ ID NO 41 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer ODMW541 <400> SEQUENCE: 41 ctcgcggcct cgtcgcacaa tcttgaag 28 <210> SEQ ID NO 42 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer ODMW542 <400> SEQUENCE: 42 gagtatcaaa cttgtattcc gaggtgtag 29 <210> SEQ ID NO 43 <211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer ODMW523 <400> SEQUENCE: 43 ggttcaagtc gcaagtcgaa acctcg 26 <210> SEQ ID NO 44 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer AUAP <400> SEQUENCE: 44 ggccacgcgt cgactagtac 20 <210> SEQ ID NO 45 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer ODMW524 <400> SEQUENCE: 45 caacttacgg aggtttcatc tcggg 25 <210> SEQ ID NO 46 <211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer YL807 <400> SEQUENCE: 46 tttccatggc tccagatgcg gacaagttga g 31 <210> SEQ ID NO 47 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer YL810 <400> SEQUENCE: 47 caaggcatcg ggatccttct catggtggt 29 <210> SEQ ID NO 48 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer YL808 <400> SEQUENCE: 48 tttgcggccg cttacaattt tcccgagtag gggttggcgc ca 42 <210> SEQ ID NO 49 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer YL809 <400> SEQUENCE: 49 accaccatga gaaggatccc gatgccttg 29 <210> SEQ ID NO 50 <211> LENGTH: 4112 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pRD5S <400> SEQUENCE: 50 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa 420 tgcatctaga tccatggctc ccgacgccga caagctgcga cagcgaaagg ctcagtccat 480 ccaggacact gccgattctc aggctaccga gctcaagatt ggcaccctga agggtctcca 540 aggcaccgag atcgtcattg atggcgacat ctacgacatc aaagacttcg atcaccctgg 600 aggcgaatcc atcatgacct ttggtggcaa cgacgttact gccacctaca agatgattca 660 tccctaccac tcgaagcatc acctggagaa gatgaaaaag gtcggtcgag tgcccgacta 720 cacctccgag tacaagttcg atactccctt cgaacgagag atcaaacagg aggtcttcaa 780 gattgtgcga agaggtcgag agtttggaac acctggctac ttctttcgag ccttctgcta 840 catcggtctc ttcttttacc tgcagtatct ctgggttacc actcctacca ctttcgccct 900 tgctatcttc tacggtgtgt ctcaggcctt cattggcctg aacgtccagc acgacgccaa 960 ccacggagct gcctccaaaa agccctggat caacaatttg ctcggcctgg gtgccgactt 1020 tatcggaggc tccaagtggc tctggatgaa ccagcactgg acccatcaca cttacaccaa 1080 ccatcacgag aaggatcccg acgccctggg tgcagagcct atgctgctct tcaacgacta 1140 tcccttgggt caccccaagc gaaccctcat tcatcacttc caagccttct actatctgtt 1200 tgtccttgct ggctactggg tgtcttcggt gttcaaccct cagatcctgg acctccagca 1260 ccgaggtgcc caggctgtcg gcatgaagat ggagaacgac tacattgcca agtctcgaaa 1320 gtacgctatc ttcctgcgac tcctgtacat ctacaccaac attgtggctc ccatccagaa 1380 ccaaggcttt tcgctcaccg tcgttgctca cattcttact atgggtgtcg cctccagcct 1440 gaccctcgct actctgttcg ccctctccca caacttcgag aacgcagatc gggatcccac 1500 ctacgaggct cgaaagggag gcgagcctgt ctgttggttc aagtcgcagg tggaaacctc 1560 ctctacttac ggtggcttca tttccggttg ccttacaggc ggactcaact ttcaggtcga 1620 gcatcacctg tttcctcgaa tgtcctctgc ctggtacccc tacatcgctc ctaccgttcg 1680 agaggtctgc aaaaagcacg gcgtcaagta cgcctactat ccctgggtgt ggcagaacct 1740 catctcgacc gtcaagtacc tgcatcagtc cggaactggc tcgaactgga agaacggtgc 1800 caatccctac tctggcaagc tgtaagcggc cgcatcggat cccgggcccg tcgactgcag 1860 aggcctgcat gcaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta 1920 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc 1980 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 2040 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 2100 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 2160 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 2220 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 2280 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 2340 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 2400 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 2460 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 2520 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 2580 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 2640 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 2700 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct 2760 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 2820 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 2880 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 2940 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 3000 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 3060 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 3120 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 3180 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 3240 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 3300 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 3360 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 3420 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 3480 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 3540 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 3600 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 3660 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 3720 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 3780 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 3840 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 3900 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 3960 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 4020 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta 4080 taaaaatagg cgtatcacga ggccctttcg tc 4112 <210> SEQ ID NO 51 <211> LENGTH: 8480 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pZURD5S <400> SEQUENCE: 51 catggctccc gacgccgaca agctgcgaca gcgaaaggct cagtccatcc aggacactgc 60 cgattctcag gctaccgagc tcaagattgg caccctgaag ggtctccaag gcaccgagat 120 cgtcattgat ggcgacatct acgacatcaa agacttcgat caccctggag gcgaatccat 180 catgaccttt ggtggcaacg acgttactgc cacctacaag atgattcatc cctaccactc 240 gaagcatcac ctggagaaga tgaaaaaggt cggtcgagtg cccgactaca cctccgagta 300 caagttcgat actcccttcg aacgagagat caaacaggag gtcttcaaga ttgtgcgaag 360 aggtcgagag tttggaacac ctggctactt ctttcgagcc ttctgctaca tcggtctctt 420 cttttacctg cagtatctct gggttaccac tcctaccact ttcgcccttg ctatcttcta 480 cggtgtgtct caggccttca ttggcctgaa cgtccagcac gacgccaacc acggagctgc 540 ctccaaaaag ccctggatca acaatttgct cggcctgggt gccgacttta tcggaggctc 600 caagtggctc tggatgaacc agcactggac ccatcacact tacaccaacc atcacgagaa 660 ggatcccgac gccctgggtg cagagcctat gctgctcttc aacgactatc ccttgggtca 720 ccccaagcga accctcattc atcacttcca agccttctac tatctgtttg tccttgctgg 780 ctactgggtg tcttcggtgt tcaaccctca gatcctggac ctccagcacc gaggtgccca 840 ggctgtcggc atgaagatgg agaacgacta cattgccaag tctcgaaagt acgctatctt 900 cctgcgactc ctgtacatct acaccaacat tgtggctccc atccagaacc aaggcttttc 960 gctcaccgtc gttgctcaca ttcttactat gggtgtcgcc tccagcctga ccctcgctac 1020 tctgttcgcc ctctcccaca acttcgagaa cgcagatcgg gatcccacct acgaggctcg 1080 aaagggaggc gagcctgtct gttggttcaa gtcgcaggtg gaaacctcct ctacttacgg 1140 tggcttcatt tccggttgcc ttacaggcgg actcaacttt caggtcgagc atcacctgtt 1200 tcctcgaatg tcctctgcct ggtaccccta catcgctcct accgttcgag aggtctgcaa 1260 aaagcacggc gtcaagtacg cctactatcc ctgggtgtgg cagaacctca tctcgaccgt 1320 caagtacctg catcagtccg gaactggctc gaactggaag aacggtgcca atccctactc 1380 tggcaagctg taagcggccg caagtgtgga tggggaagtg agtgcccggt tctgtgtgca 1440 caattggcaa tccaagatgg atggattcaa cacagggata tagcgagcta cgtggtggtg 1500 cgaggatata gcaacggata tttatgtttg acacttgaga atgtacgata caagcactgt 1560 ccaagtacaa tactaaacat actgtacata ctcatactcg tacccgggca acggtttcac 1620 ttgagtgcag tggctagtgc tcttactcgt acagtgtgca atactgcgta tcatagtctt 1680 tgatgtatat cgtattcatt catgttagtt gcgtacgagc cggaagcata aagtgtaaag 1740 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt 1800 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 1860 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 1920 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 1980 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 2040 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 2100 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 2160 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 2220 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 2280 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 2340 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 2400 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 2460 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct 2520 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 2580 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 2640 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 2700 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 2760 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 2820 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 2880 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 2940 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 3000 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 3060 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 3120 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 3180 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 3240 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 3300 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 3360 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 3420 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 3480 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 3540 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 3600 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 3660 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg 3720 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 3780 ttccgcgcac atttccccga aaagtgccac ctgacgcgcc ctgtagcggc gcattaagcg 3840 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg 3900 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 3960 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 4020 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 4080 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 4140 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt 4200 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc 4260 ttacaatttc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc 4320 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt 4380 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgaat tgtaatacga 4440 ctcactatag ggcgaattgg gtaccgggcc ccccctcgag gtcgatggtg tcgataagct 4500 tgatatcgaa ttcatgtcac acaaaccgat cttcgcctca aggaaaccta attctacatc 4560 cgagagactg ccgagatcca gtctacactg attaattttc gggccaataa tttaaaaaaa 4620 tcgtgttata taatattata tgtattatat atatacatca tgatgatact gacagtcatg 4680 tcccattgct aaatagacag actccatctg ccgcctccaa ctgatgttct caatatttaa 4740 ggggtcatct cgcattgttt aataataaac agactccatc taccgcctcc aaatgatgtt 4800 ctcaaaatat attgtatgaa cttattttta ttacttagta ttattagaca acttacttgc 4860 tttatgaaaa acacttccta tttaggaaac aatttataat ggcagttcgt tcatttaaca 4920 atttatgtag aataaatgtt ataaatgcgt atgggaaatc ttaaatatgg atagcataaa 4980 tgatatctgc attgcctaat tcgaaatcaa cagcaacgaa aaaaatccct tgtacaacat 5040 aaatagtcat cgagaaatat caactatcaa agaacagcta ttcacacgtt actattgaga 5100 ttattattgg acgagaatca cacactcaac tgtctttctc tcttctagaa atacaggtac 5160 aagtatgtac tattctcatt gttcatactt ctagtcattt catcccacat attccttgga 5220 tttctctcca atgaatgaca ttctatcttg caaattcaac aattataata agatatacca 5280 aagtagcggt atagtggcaa tcaaaaagct tctctggtgt gcttctcgta tttattttta 5340 ttctaatgat ccattaaagg tatatattta tttcttgtta tataatcctt ttgtttatta 5400 catgggctgg atacataaag gtattttgat ttaatttttt gcttaaattc aatcccccct 5460 cgttcagtgt caactgtaat ggtaggaaat taccatactt ttgaagaagc aaaaaaaatg 5520 aaagaaaaaa aaaatcgtat ttccaggtta gacgttccgc agaatctaga atgcggtatg 5580 cggtacattg ttcttcgaac gtaaaagttg cgctccctga gatattgtac atttttgctt 5640 ttacaagtac aagtacatcg tacaactatg tactactgtt gatgcatcca caacagtttg 5700 ttttgttttt ttttgttttt tttttttcta atgattcatt accgctatgt atacctactt 5760 gtacttgtag taagccgggt tattggcgtt caattaatca tagacttatg aatctgcacg 5820 gtgtgcgctg cgagttactt ttagcttatg catgctactt gggtgtaata ttgggatctg 5880 ttcggaaatc aacggatgct caatcgattt cgacagtaat taattaagtc atacacaagt 5940 cagctttctt cgagcctcat ataagtataa gtagttcaac gtattagcac tgtacccagc 6000 atctccgtat cgagaaacac aacaacatgc cccattggac agatcatgcg gatacacagg 6060 ttgtgcagta tcatacatac tcgatcagac aggtcgtctg accatcatac aagctgaaca 6120 agcgctccat acttgcacgc tctctatata cacagttaaa ttacatatcc atagtctaac 6180 ctctaacagt taatcttctg gtaagcctcc cagccagcct tctggtatcg cttggcctcc 6240 tcaataggat ctcggttctg gccgtacaga cctcggccga caattatgat atccgttccg 6300 gtagacatga catcctcaac agttcggtac tgctgtccga gagcgtctcc cttgtcgtca 6360 agacccaccc cgggggtcag aataagccag tcctcagagt cgcccttagg tcggttctgg 6420 gcaatgaagc caaccacaaa ctcggggtcg gatcgggcaa gctcaatggt ctgcttggag 6480 tactcgccag tggccagaga gcccttgcaa gacagctcgg ccagcatgag cagacctctg 6540 gccagcttct cgttgggaga ggggactagg aactccttgt actgggagtt ctcgtagtca 6600 gagacgtcct ccttcttctg ttcagagaca gtttcctcgg caccagctcg caggccagca 6660 atgattccgg ttccgggtac accgtgggcg ttggtgatat cggaccactc ggcgattcgg 6720 tgacaccggt actggtgctt gacagtgttg ccaatatctg cgaactttct gtcctcgaac 6780 aggaagaaac cgtgcttaag agcaagttcc ttgaggggga gcacagtgcc ggcgtaggtg 6840 aagtcgtcaa tgatgtcgat atgggttttg atcatgcaca cataaggtcc gaccttatcg 6900 gcaagctcaa tgagctcctt ggtggtggta acatccagag aagcacacag gttggttttc 6960 ttggctgcca cgagcttgag cactcgagcg gcaaaggcgg acttgtggac gttagctcga 7020 gcttcgtagg agggcatttt ggtggtgaag aggagactga aataaattta gtctgcagaa 7080 ctttttatcg gaaccttatc tggggcagtg aagtatatgt tatggtaata gttacgagtt 7140 agttgaactt atagatagac tggactatac ggctatcggt ccaaattaga aagaacgtca 7200 atggctctct gggcgtcgcc tttgccgaca aaaatgtgat catgatgaaa gccagcaatg 7260 acgttgcagc tgatattgtt gtcggccaac cgcgccgaaa acgcagctgt cagacccaca 7320 gcctccaacg aagaatgtat cgtcaaagtg atccaagcac actcatagtt ggagtcgtac 7380 tccaaaggcg gcaatgacga gtcagacaga tactcgtcga ctcaggcgac gacggaattc 7440 ctgcagccca tctgcagaat tcaggagaga ccgggttggc ggcgtatttg tgtcccaaaa 7500 aacagcccca attgccccgg agaagacggc caggccgcct agatgacaaa ttcaacaact 7560 cacagctgac tttctgccat tgccactagg ggggggcctt tttatatggc caagccaagc 7620 tctccacgtc ggttgggctg cacccaacaa taaatgggta gggttgcacc aacaaaggga 7680 tgggatgggg ggtagaagat acgaggataa cggggctcaa tggcacaaat aagaacgaat 7740 actgccatta agactcgtga tccagcgact gacaccattg catcatctaa gggcctcaaa 7800 actacctcgg aactgctgcg ctgatctgga caccacagag gttccgagca ctttaggttg 7860 caccaaatgt cccaccaggt gcaggcagaa aacgctggaa cagcgtgtac agtttgtctt 7920 aacaaaaagt gagggcgctg aggtcgagca gggtggtgtg acttgttata gcctttagag 7980 ctgcgaaagc gcgtatggat ttggctcatc aggccagatt gagggtctgt ggacacatgt 8040 catgttagtg tacttcaatc gccccctgga tatagccccg acaataggcc gtggcctcat 8100 ttttttgcct tccgcacatt tccattgctc ggtacccaca ccttgcttct cctgcacttg 8160 ccaaccttaa tactggttta cattgaccaa catcttacaa gcggggggct tgtctagggt 8220 atatataaac agtggctctc ccaatcggtt gccagtctct tttttccttt ctttccccac 8280 agattcgaaa tctaaactac acatcacaca atgcctgtta ctgacgtcct taagcgaaag 8340 tccggtgtca tcgtcggcga cgatgtccga gccgtgagta tccacgacaa gatcagtgtc 8400 gagacgacgc gttttgtgta atgacacaat ccgaaagtcg ctagcaacac acactctcta 8460 cacaaactaa cccagctctc 8480 <210> SEQ ID NO 52 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer T7 <400> SEQUENCE: 52 ggaaacagct atgaccatg 19 <210> SEQ ID NO 53 <211> LENGTH: 459 <212> TYPE: PRT <213> ORGANISM: Rhizopus stolonifer <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: GenBank Accession No. AAX22052 <400> SEQUENCE: 53 Met Ser Thr Leu Asp Arg Gln Ser Ile Phe Thr Ile Lys Glu Leu Glu 1 5 10 15 Ser Ile Ser Gln Arg Ile His Asp Gly Asp Glu Glu Ala Met Lys Phe 20 25 30 Ile Ile Ile Asp Lys Lys Val Tyr Asp Val Thr Glu Phe Ile Glu Asp 35 40 45 His Pro Gly Gly Ala Gln Val Leu Leu Thr His Val Gly Lys Asp Ala 50 55 60 Ser Asp Val Phe His Ala Met His Pro Glu Ser Ala Tyr Glu Val Leu 65 70 75 80 Asn Asn Tyr Phe Val Gly Asp Val Gln Glu Thr Val Val Thr Glu Lys 85 90 95 Ser Ser Ser Ala Gln Phe Ala Val Glu Met Arg Gln Leu Arg Asp Gln 100 105 110 Leu Lys Lys Glu Gly Tyr Phe His Ser Ser Lys Leu Phe Tyr Ala Tyr 115 120 125 Lys Val Leu Ser Thr Leu Ala Ile Cys Ile Ala Gly Leu Ser Leu Leu 130 135 140 Tyr Ala Tyr Gly Arg Thr Ser Thr Leu Ala Val Val Ala Ser Ala Ile 145 150 155 160 Thr Val Gly Ile Phe Trp Gln Gln Cys Gly Trp Leu Ala His Asp Phe 165 170 175 Gly His His Gln Cys Phe Glu Asp Arg Thr Trp Asn Asp Val Leu Val 180 185 190 Val Phe Leu Gly Asn Phe Cys Gln Gly Phe Ser Leu Ser Trp Trp Lys 195 200 205 Asn Lys His Asn Thr His His Ala Ser Thr Asn Val His Gly Gln Asp 210 215 220 Pro Asp Ile Asp Thr Ala Pro Val Leu Leu Trp Asp Glu Tyr Ala Ser 225 230 235 240 Ala Ala Tyr Tyr Ala Ser Leu Asp Gln Glu Pro Thr Met Val Ser Arg 245 250 255 Phe Leu Ala Glu Gln Val Leu Pro His Gln Thr Arg Tyr Phe Phe Phe 260 265 270 Ile Leu Ala Phe Ala Arg Leu Ser Trp Ala Leu Gln Ser Leu Ser Tyr 275 280 285 Ser Phe Lys Lys Glu Ser Ile Asn Lys Ser Arg Gln Leu Asn Leu Phe 290 295 300 Glu Arg Val Cys Ile Val Gly His Trp Ala Leu Phe Ala Phe Cys Ile 305 310 315 320 Tyr Ser Trp Cys Ser Asn Val Tyr His Met Val Leu Phe Phe Leu Val 325 330 335 Ser Gln Ala Thr Thr Gly Tyr Thr Leu Ala Leu Val Phe Ala Leu Asn 340 345 350 His Asn Gly Met Pro Val Ile Thr Glu Glu Lys Ala Glu Ser Met Glu 355 360 365 Phe Phe Glu Ile Gln Val Ile Thr Gly Arg Asp Val Thr Leu Ser Pro 370 375 380 Leu Gly Asp Trp Phe Met Gly Gly Leu Asn Tyr Gln Ile Glu His His 385 390 395 400 Val Phe Pro Asn Met Pro Arg His Asn Leu Pro Thr Val Lys Pro Met 405 410 415 Val Lys Ser Leu Cys Gln Lys Tyr Asp Ile Asn Tyr His Asp Thr Gly 420 425 430 Phe Leu Lys Gly Thr Leu Glu Val Leu Gln Thr Leu Asp Ile Thr Ser 435 440 445 Lys Leu Ser Leu Gln Leu Ser Lys Lys Ser Phe 450 455 <210> SEQ ID NO 54 <211> LENGTH: 695 <212> TYPE: DNA <213> ORGANISM: Pavlova lutheri <400> SEQUENCE: 54 agggccaagg gtgccaacca ccttccacgt gagactacac accgtaggcc gatgggcaag 60 ggtggagacg gcggcgcgca ggcggtgagc gggaccgacg cgtctctcgc tgaggtgagc 120 tccgtcgata gcaagagcgt gcacgtcgtg ctctacggca agcgcgtgga tgtcacaaag 180 ttccagggct acgacgtggc ctggtggcgc gcgcgccata acacgcacca cgtgtgcacc 240 aacgaggatg gttcggaccc ggacatcaag acggcgcccc tgctcatcta cgtgcgagag 300 aacccgtcca ttgccaagcg gctcaacttc ttccagcgct ggcagcagta ctactatgtg 360 ccgaccatgg ccatcctcga cctctactgg cgcctggagt ccatcgcgta cgtggctgtg 420 cgcctgccta agatgtggat gcaggccgcc gctcttgccg ctcactacgc gctcctgtgc 480 tgggtcttcg cagcgcatct caacctcatc cctctcatga tggttgcacg cggcttcgcg 540 acgggcatcg ttgtctttgc aacccactat ggtgaggaca tcctcgaccg cgagcacgtc 600 gagggcatga cgctcgtcga gcagaccgcc aagacctccc gtaacatcac gggcggctgg 660 ctagtgaacg tgctcacggg cttcatctcc ctgca 695 <210> SEQ ID NO 55 <211> LENGTH: 1106 <212> TYPE: DNA <213> ORGANISM: Pavlova lutheri <400> SEQUENCE: 55 agggccaagg gtgccaacca ccttccacgt gagactacac accgtaggcc gatgggcaag 60 ggtggagacg gcggcgcgca ggcggtgagc gggaccgacg cgtctctcgc tgaggtgagc 120 tccgtcgata gcaagagcgt gcacgtcgtg ctctacggca agcgcgtgga tgtcacaaag 180 ttccagggct acgacgtggc ctggtggcgc gcgcgccata acacgcacca cgtgtgcacc 240 aacgaggatg gttcggaccc ggacatcaag acggcgcccc tgctcatcta cgtgcgagag 300 aacccgtcca ttgccaagcg gctcaacttc ttccagcgct ggcagcagta ctactatgtg 360 ccgaccatgg ccatcctcga cctctactgg cgcctggagt ccatcgcgta cgtggctgtg 420 cgcctgccta agatgtggat gcaggccgcc gctcttgccg ctcactacgc gctcctgtgc 480 tgggtcttcg cagcgcatct caacctcatc cctctcatga tggttgcacg cggcttcgcg 540 acgggcatcg ttgtctttgc aacccactat ggtgaggaca tcctcgaccg cgagcacgtc 600 gagggcatga cgctcgtcga gcagaccgcc aagacctccc gtaacatcac gggcggctgg 660 ctagtgaacg tgctcacggg cttcatctcc ctgcagaccg agcatcacct cttccccatg 720 atgcccaccg gcaacctaat gactatccag cccgaggtac gcgacttctt caagaagcat 780 ggcctcgagt accgcgaggg caacctcttc cagtgcgtgc accagaacat caaggctctc 840 gccttcgagc acctcctcca ctgagcgtca ccactcaagc gtcctaagtg cacaggtact 900 gtcttctgac cgatggccgc gcggctccct cggctggcag tggggccaac gagtggcctc 960 gcgggatcgg gcacgatcgg gcctccatga aacttcagtg ttcagagaca agccgacaac 1020 ctccgcatcg tgagaaatct tttaaagcag tatgttccat cacgccgctt ttgcagtcaa 1080 taacattacc caaaaaaaaa aaaaaa 1106 <210> SEQ ID NO 56 <211> LENGTH: 287 <212> TYPE: PRT <213> ORGANISM: Pavlova lutheri <400> SEQUENCE: 56 Arg Ala Lys Gly Ala Asn His Leu Pro Arg Glu Thr Thr His Arg Arg 1 5 10 15 Pro Met Gly Lys Gly Gly Asp Gly Gly Ala Gln Ala Val Ser Gly Thr 20 25 30 Asp Ala Ser Leu Ala Glu Val Ser Ser Val Asp Ser Lys Ser Val His 35 40 45 Val Val Leu Tyr Gly Lys Arg Val Asp Val Thr Lys Phe Gln Gly Tyr 50 55 60 Asp Val Ala Trp Trp Arg Ala Arg His Asn Thr His His Val Cys Thr 65 70 75 80 Asn Glu Asp Gly Ser Asp Pro Asp Ile Lys Thr Ala Pro Leu Leu Ile 85 90 95 Tyr Val Arg Glu Asn Pro Ser Ile Ala Lys Arg Leu Asn Phe Phe Gln 100 105 110 Arg Trp Gln Gln Tyr Tyr Tyr Val Pro Thr Met Ala Ile Leu Asp Leu 115 120 125 Tyr Trp Arg Leu Glu Ser Ile Ala Tyr Val Ala Val Arg Leu Pro Lys 130 135 140 Met Trp Met Gln Ala Ala Ala Leu Ala Ala His Tyr Ala Leu Leu Cys 145 150 155 160 Trp Val Phe Ala Ala His Leu Asn Leu Ile Pro Leu Met Met Val Ala 165 170 175 Arg Gly Phe Ala Thr Gly Ile Val Val Phe Ala Thr His Tyr Gly Glu 180 185 190 Asp Ile Leu Asp Arg Glu His Val Glu Gly Met Thr Leu Val Glu Gln 195 200 205 Thr Ala Lys Thr Ser Arg Asn Ile Thr Gly Gly Trp Leu Val Asn Val 210 215 220 Leu Thr Gly Phe Ile Ser Leu Gln Thr Glu His His Leu Phe Pro Met 225 230 235 240 Met Pro Thr Gly Asn Leu Met Thr Ile Gln Pro Glu Val Arg Asp Phe 245 250 255 Phe Lys Lys His Gly Leu Glu Tyr Arg Glu Gly Asn Leu Phe Gln Cys 260 265 270 Val His Gln Asn Ile Lys Ala Leu Ala Phe Glu His Leu Leu His 275 280 285 <210> SEQ ID NO 57 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer SeqE <400> SEQUENCE: 57 cgacacactc caatctttcc 20 <210> SEQ ID NO 58 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer SeqW <400> SEQUENCE: 58 ggtggctgga gttagacatc 20 <210> SEQ ID NO 59 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer AP1 <400> SEQUENCE: 59 gtaatacgac tcactatagg gc 22 <210> SEQ ID NO 60 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer GSP PvDES <400> SEQUENCE: 60 ctgcgaagac ccagcacagg 20 <210> SEQ ID NO 61 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer M13-28Rev <400> SEQUENCE: 61 gtaatacgac tcactatagg gc 22 <210> SEQ ID NO 62 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer PavDes seq <400> SEQUENCE: 62 ttgtggcgct caatcatctc c 21 <210> SEQ ID NO 63 <211> LENGTH: 1294 <212> TYPE: DNA <213> ORGANISM: Pavlova lutheri <400> SEQUENCE: 63 ctttgcgagc gcggcgcaga cgattgcggc ccgtagtgat cgcggtgcgc attgctgtgt 60 ttctagtttt gctgacgccc ggcccgataa tgacaccttc tcccgtttga aatactaata 120 agtaactata ttataatatt caaaggtggc gactatggat ctccttttct aaagttcagc 180 ggaattggga atcggagaaa tttcgagata tgtcataatc acgtgctcta tctcgaatga 240 accgcggccg gtgagcgatt actcgggaag ccaattccta ttaacgagtc aggggggatc 300 tttgaggtga gtcggccacg cagagagagc aaggaatcat cctcatccgc cgttctcgag 360 aaagagccaa gggtgccaac caccttccac gtgagactac acaccgtagg ccgatgggca 420 agggtggaga cggcggcgcg caggcggcga gcgggaccga cgcatctctc gctgaggtga 480 gctccgtcga tagcaagagc gtgcgcgtcg tgctctacgg caagcgcgtg gatgtcacaa 540 agttccagag ggcacacccg ggcgggagca aggtgttccg catcttccag gagcgcgacg 600 cgacggagca gttcgagtct taccactcgc ccaaggccat caagatgatg gagggcatgc 660 tcaagaagtc ggaggatgcg cccgcttccg tgcccctgcc ctcgcggtcc accatgggca 720 cggagttcaa ggagatgatt gagcgccaca agagggctgg tctctacgac ccttgcccgt 780 tggacgagct gttcaagctc accatcgtcc ttgcgcccat cttcgtgggc gcctatctcg 840 tgcggagcgg cgtctcgccc ctcgcgggcg cgctctccat gggctttggc ttctacctcg 900 acggctggct tgctcacrac tacctgcatc acgcagtctt caagggctcg gtcaacacgc 960 tcgtcaaggc gaacaacgcc atgggatacg ccctcggctt cctccagggc tacgacgtgg 1020 cctggtggcg cgcgcgccat aacacgcacc acgtgtgcac caacgaggat ggttcggacc 1080 cggacatcaa gacggcgccc ctgctcatct acgtgcgaga gaacccgtcc attgccaagc 1140 ggctcaactt cttccagcgc tggcagcagt actactatgt gccgaccatg gccatcctcg 1200 acctctactg gcgcctggag tccatcgcgt acgtggctgt gcgcctgcct aagatgtgga 1260 tgcaggccgc cgctcttgcc gctcactacg cgct 1294 <210> SEQ ID NO 64 <211> LENGTH: 1927 <212> TYPE: DNA <213> ORGANISM: Pavlova lutheri <400> SEQUENCE: 64 ctttgcgagc gcggcgcaga cgattgcggc ccgtagtgat cgcggtgcgc attgctgtgt 60 ttctagtttt gctgacgccc ggcccgataa tgacaccttc tcccgtttga aatactaata 120 agtaactata ttataatatt caaaggtggc gactatggat ctccttttct aaagttcagc 180 ggaattggga atcggagaaa tttcgagata tgtcataatc acgtgctcta tctcgaatga 240 accgcggccg gtgagcgatt actcgggaag ccaattccta ttaacgagtc aggggggatc 300 tttgaggtga gtcggccacg cagagagagc aaggaatcat cctcatccgc cgttctcgag 360 aaagagccaa gggtgccaac caccttccac gtgagactac acaccgtagg ccgatgggca 420 agggtggaga cggcggcgcg caggcggtga gcgggaccga cgcgtctctc gctgaggtga 480 gctccgtcga tagcaagagc gtgcacgtcg tgctctacgg caagcgcgtg gatgtcacaa 540 agttccagaa ggcacacccg ggcgggagca aggtgttccg catcttccag gagcgcgacg 600 cgacggagca gttcgagtct taccactcgc ccaaggccat caagatgatg gagggcatgc 660 tcaagaagtc ggaggatgcg cccgcttccg tgcccctgcc ctcgcggtcc accatgggca 720 cggagttcaa ggagatgatt gagcgccaca agagggctgg tctctacgac ccttgcccgt 780 tggacgagct gttcaagctc accatcgtcc ttgcgcccat cttcgtgggc gcctatctcg 840 tgcggagcgg cgtctcgccc ctcgcgggcg cgctctccat gggctttggc ttctacctcg 900 acggctggct tgctcacgac tacctgcatc acgcagtctt caagggctcg gtcaacacgc 960 tcgtcaaggc gaacaacgcc atgggatacg ccctcggctt cctccagggc tacgacgtgg 1020 cctggtggcg cgcgcgccat aacacgcacc acgtgtgcac caacgaggat ggttcggacc 1080 cggacatcaa gacggcgccc ctgctcatct acgtgcgaga gaacccgtcc attgccaagc 1140 ggctcaactt cttccagcgc tggcagcagt actactatgt gccgaccatg gccatcctcg 1200 acctctactg gcgcctggag tccatcgcgt acgtggctgt gcgcctgcct aagatgtgga 1260 tgcaggccgc cgctcttgcc gctcactacg cgctcctgtg ctgggtcttc gcagcgcatc 1320 tcaacctcat ccctctcatg atggttgcac gcggcttcgc gacgggcatc gttgtctttg 1380 caacccacta tggtgaggac atcctcgacc gcgagcacgt cgagggcatg acgctcgtcg 1440 agcagaccgc caagacctcc cgtaacatca cgggcggctg gctagtgaac gtgctcacgg 1500 gcttcatctc cctgcagacc gagcatcacc tcttccccat gatgcccacc ggcaacctaa 1560 tgactatcca gcccgaggta cgcgacttct tcaagaagca tggcctcgag taccgcgagg 1620 gcaacctctt ccagtgcgtg caccagaaca tcaaggctct cgccttcgag cacctcctcc 1680 actgagcgtc accactcaag cgtcctaagt gcacaggtac tgtcttctga ccgatggccg 1740 cgcggctccc tcggctggca gtggggccaa cgagtggcct cgcgggatcg ggcacgatcg 1800 ggcctccatg aaacttcagt gttcagagac aagccgacaa cctccgcatc gtgagaaatc 1860 ttttaaagca gtatgttcca tcacgccgct tttgcagtca ataacattac ccaaaaaaaa 1920 aaaaaaa 1927 <210> SEQ ID NO 65 <211> LENGTH: 459 <212> TYPE: PRT <213> ORGANISM: Rhizopus stolonifer <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: GenBank Accession No. ABB96724 <400> SEQUENCE: 65 Met Ser Thr Leu Asp Arg Gln Ser Ile Phe Thr Ile Lys Glu Leu Glu 1 5 10 15 Ser Ile Ser Gln Arg Ile His Asp Gly Asp Glu Glu Ala Met Lys Phe 20 25 30 Ile Ile Ile Asp Lys Lys Val Tyr Asp Val Thr Glu Phe Ile Glu Asp 35 40 45 His Pro Gly Gly Ala Gln Val Leu Leu Thr His Val Gly Lys Asp Ala 50 55 60 Ser Asp Val Phe His Ala Met His Pro Glu Ser Ala Tyr Glu Val Leu 65 70 75 80 Asn Asn Tyr Phe Val Gly Asp Val Gln Glu Thr Val Val Thr Glu Lys 85 90 95 Ser Ser Ser Ala Gln Phe Ala Val Glu Met Arg Gln Leu Arg Asp Gln 100 105 110 Leu Lys Lys Glu Gly Tyr Phe His Ser Ser Lys Leu Phe Tyr Ala Tyr 115 120 125 Lys Val Leu Ser Thr Leu Ala Ile Cys Ile Ala Gly Leu Ser Pro Leu 130 135 140 Tyr Ala Tyr Gly Arg Thr Ser Thr Leu Ala Val Val Ala Ser Ala Ile 145 150 155 160 Thr Val Gly Ile Phe Trp Gln Gln Cys Gly Trp Leu Ala His Asp Phe 165 170 175 Gly His His Gln Cys Phe Glu Asp Arg Thr Trp Asn Asp Val Leu Val 180 185 190 Val Phe Leu Gly Asn Phe Cys Gln Gly Phe Ser Leu Ser Trp Trp Lys 195 200 205 Asn Lys His Asn Thr His His Ala Ser Thr Asn Val His Gly Gln Asp 210 215 220 Pro Asp Ile Asp Thr Ala Pro Val Leu Leu Trp Asp Glu Tyr Ala Ser 225 230 235 240 Ala Ala Tyr Tyr Ala Ser Leu Asp Gln Glu Pro Thr Met Val Ser Arg 245 250 255 Phe Leu Ala Glu Gln Val Leu Pro His Gln Thr Arg Tyr Phe Phe Phe 260 265 270 Ile Leu Ala Phe Ala Arg Leu Ser Trp Ala Leu Gln Ser Leu Ser Tyr 275 280 285 Ser Phe Lys Lys Glu Ser Ile Asn Lys Ser Arg Gln Leu Asn Leu Phe 290 295 300 Glu Arg Val Cys Ile Val Gly His Trp Ala Leu Ser Ala Phe Cys Ile 305 310 315 320 Tyr Ser Trp Cys Ser Asn Val Tyr His Met Val Leu Phe Phe Leu Val 325 330 335 Ser Gln Ala Thr Thr Gly Tyr Thr Leu Ala Leu Val Phe Ala Leu Asn 340 345 350 His Asn Gly Met Pro Val Ile Thr Glu Glu Lys Ala Glu Ser Met Glu 355 360 365 Phe Phe Glu Ile Gln Val Ile Thr Gly Arg Asp Val Thr Leu Ser Pro 370 375 380 Leu Gly Asp Trp Phe Met Gly Gly Leu Asn Tyr Gln Ile Glu His His 385 390 395 400 Val Phe Pro Asn Met Pro Arg His Asn Leu Pro Thr Val Lys Pro Met 405 410 415 Val Lys Ser Leu Cys Gln Lys Tyr Asp Ile Asn Tyr His Asp Thr Gly 420 425 430 Phe Leu Lys Gly Thr Leu Glu Val Leu Gln Thr Leu Asp Ile Thr Ser 435 440 445 Lys Leu Ser Leu Gln Leu Ser Lys Lys Ser Phe 450 455 <210> SEQ ID NO 66 <211> LENGTH: 427 <212> TYPE: PRT <213> ORGANISM: Pavlova salina <300> PUBLICATION INFORMATION: <302> TITLE: SYNTHESIS OF LONG-CHAIN POLYUNSATURATED FATTY ACIDS BY RECOMBINANT CELLS <310> PATENT DOCUMENT NUMBER: WO 2005/103253 <311> PATENT FILING DATE: 2005-04-22 <312> PUBLICATION DATE: 2005-11-03 <313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(427) <400> SEQUENCE: 66 Met Gly Arg Gly Gly Asp Ser Ser Gly Gln Ala His Pro Ala Ala Glu 1 5 10 15 Leu Ala Val Pro Ser Asp Arg Ala Glu Val Ser Asn Ala Asp Ser Lys 20 25 30 Ala Leu His Ile Val Leu Tyr Gly Lys Arg Val Asp Val Thr Lys Phe 35 40 45 Gln Arg Thr His Pro Gly Gly Ser Lys Val Phe Arg Ile Phe Gln Asp 50 55 60 Arg Asp Ala Thr Glu Gln Phe Glu Ser Tyr His Ser Lys Arg Ala Ile 65 70 75 80 Lys Met Met Glu Gly Met Leu Lys Lys Ser Glu Asp Ala Pro Ala Asp 85 90 95 Thr Pro Leu Pro Ser Gln Ser Pro Met Gly Lys Asp Phe Lys Ala Met 100 105 110 Ile Glu Arg His Val Ala Ala Gly Tyr Tyr Asp Pro Cys Pro Leu Asp 115 120 125 Glu Leu Phe Lys Leu Ser Leu Val Leu Leu Pro Thr Phe Ala Gly Met 130 135 140 Tyr Met Leu Lys Ala Gly Val Gly Ser Pro Leu Cys Gly Ala Leu Met 145 150 155 160 Val Ser Phe Gly Trp Tyr Leu Asp Gly Trp Leu Ala His Asp Tyr Leu 165 170 175 His His Ser Val Phe Lys Gly Ser Val Ala Arg Thr Val Gly Trp Asn 180 185 190 Asn Ala Ala Gly Tyr Phe Leu Gly Phe Val Gln Gly Tyr Ala Val Glu 195 200 205 Trp Trp Arg Ala Arg His Asn Thr His His Val Cys Thr Asn Glu Asp 210 215 220 Gly Ser Asp Pro Asp Ile Lys Thr Ala Pro Leu Leu Ile Tyr Val Arg 225 230 235 240 Asn Lys Pro Ser Ile Ala Lys Arg Leu Asn Ala Phe Gln Arg Tyr Gln 245 250 255 Gln Tyr Tyr Tyr Val Pro Val Met Ala Ile Leu Asp Leu Tyr Trp Arg 260 265 270 Leu Glu Ser Ile Ala Tyr Val Ala Met Arg Leu Pro Lys Met Leu Pro 275 280 285 Gln Ala Leu Ala Leu Val Ala His Tyr Ala Ile Val Ala Trp Val Phe 290 295 300 Ala Gly Asn Tyr His Leu Leu Pro Leu Val Thr Val Leu Arg Gly Phe 305 310 315 320 Gly Thr Gly Ile Thr Val Phe Ala Thr His Tyr Gly Glu Asp Ile Leu 325 330 335 Asp Ala Asp Gln Val Arg His Met Thr Leu Val Glu Gln Thr Ala Leu 340 345 350 Thr Ser Arg Asn Ile Ser Gly Gly Trp Leu Val Asn Val Leu Thr Gly 355 360 365 Phe Ile Ser Leu Gln Thr Glu His His Leu Phe Pro Met Met Pro Thr 370 375 380 Gly Asn Leu Met Thr Ile Gln Pro Glu Val Arg Ala Phe Phe Lys Lys 385 390 395 400 His Gly Leu Glu Tyr Arg Glu Gly Asn Leu Ile Glu Cys Val Arg Gln 405 410 415 Asn Ile Arg Ala Leu Ala Phe Glu His Leu Leu 420 425 <210> SEQ ID NO 67 <211> LENGTH: 1338 <212> TYPE: DNA <213> ORGANISM: Mortierella alpina <400> SEQUENCE: 67 atgggaacgg accaaggaaa aaccttcacc tgggaagagc tggcggccca taacaccaag 60 gacgacctac tcttggccat ccgcggcagg gtgtacgatg tcacaaagtt cttgagccgc 120 catcctggtg gagtggacac tctcctgctc ggagctggcc gagatgttac tccggtcttt 180 gagatgtatc acgcgtttgg ggctgcagat gccattatga agaagtacta tgtcggtaca 240 ctggtctcga atgagctgcc catcttcccg gagccaacgg tgttccacaa aaccatcaag 300 acgagagtcg agggctactt tacggatcgg aacattgatc ccaagaatag accagagatc 360 tggggacgat acgctcttat ctttggatcc ttgatcgctt cctactacgc gcagctcttt 420 gtgcctttcg ttgtcgaacg cacatggctt caggtggtgt ttgcaatcat catgggattt 480 gcgtgcgcac aagtcggact caaccctctt catgatgcgt ctcacttttc agtgacccac 540 aaccccactg tctggaagat tctgggagcc acgcacgact ttttcaacgg agcatcgtac 600 ctggtgtgga tgtaccaaca tatgctcggc catcacccct acaccaacat tgctggagca 660 gatcccgacg tgtcgacgtc tgagcccgat gttcgtcgta tcaagcccaa ccaaaagtgg 720 tttgtcaacc acatcaacca gcacatgttt gttcctttcc tgtacggact gctggcgttc 780 aaggtgcgca ttcaggacat caacattttg tactttgtca agaccaatga cgctattcgt 840 gtcaatccca tctcgacatg gcacactgtg atgttctggg gcggcaaggc tttctttgtc 900 tggtatcgcc tgattgttcc cctgcagtat ctgcccctgg gcaaggtgct gctcttgttc 960 acggtcgcgg acatggtgtc gtcttactgg ctggcgctga ccttccaggc gaaccacgtt 1020 gttgaggaag ttcagtggcc gttgcctgac gagaacggga tcatccaaaa ggactgggca 1080 gctatgcagg tcgagactac gcaggattac gcacacgatt cgcacctctg gaccagcatc 1140 actggcagct tgaactacca ggctgtgcac catctgttcc ccaacgtgtc gcagcaccat 1200 tatcccgata ttctggccat catcaagaac acctgcagcg agtacaaggt tccatacctt 1260 gtcaaggata cgttttggca agcatttgct tcacatttgg agcacttgcg tgttcttgga 1320 ctccgtccca aggaagag 1338 <210> SEQ ID NO 68 <211> LENGTH: 446 <212> TYPE: PRT <213> ORGANISM: Mortierella alpina <400> SEQUENCE: 68 Met Gly Thr Asp Gln Gly Lys Thr Phe Thr Trp Glu Glu Leu Ala Ala 1 5 10 15 His Asn Thr Lys Asp Asp Leu Leu Leu Ala Ile Arg Gly Arg Val Tyr 20 25 30 Asp Val Thr Lys Phe Leu Ser Arg His Pro Gly Gly Val Asp Thr Leu 35 40 45 Leu Leu Gly Ala Gly Arg Asp Val Thr Pro Val Phe Glu Met Tyr His 50 55 60 Ala Phe Gly Ala Ala Asp Ala Ile Met Lys Lys Tyr Tyr Val Gly Thr 65 70 75 80 Leu Val Ser Asn Glu Leu Pro Ile Phe Pro Glu Pro Thr Val Phe His 85 90 95 Lys Thr Ile Lys Thr Arg Val Glu Gly Tyr Phe Thr Asp Arg Asn Ile 100 105 110 Asp Pro Lys Asn Arg Pro Glu Ile Trp Gly Arg Tyr Ala Leu Ile Phe 115 120 125 Gly Ser Leu Ile Ala Ser Tyr Tyr Ala Gln Leu Phe Val Pro Phe Val 130 135 140 Val Glu Arg Thr Trp Leu Gln Val Val Phe Ala Ile Ile Met Gly Phe 145 150 155 160 Ala Cys Ala Gln Val Gly Leu Asn Pro Leu His Asp Ala Ser His Phe 165 170 175 Ser Val Thr His Asn Pro Thr Val Trp Lys Ile Leu Gly Ala Thr His 180 185 190 Asp Phe Phe Asn Gly Ala Ser Tyr Leu Val Trp Met Tyr Gln His Met 195 200 205 Leu Gly His His Pro Tyr Thr Asn Ile Ala Gly Ala Asp Pro Asp Val 210 215 220 Ser Thr Ser Glu Pro Asp Val Arg Arg Ile Lys Pro Asn Gln Lys Trp 225 230 235 240 Phe Val Asn His Ile Asn Gln His Met Phe Val Pro Phe Leu Tyr Gly 245 250 255 Leu Leu Ala Phe Lys Val Arg Ile Gln Asp Ile Asn Ile Leu Tyr Phe 260 265 270 Val Lys Thr Asn Asp Ala Ile Arg Val Asn Pro Ile Ser Thr Trp His 275 280 285 Thr Val Met Phe Trp Gly Gly Lys Ala Phe Phe Val Trp Tyr Arg Leu 290 295 300 Ile Val Pro Leu Gln Tyr Leu Pro Leu Gly Lys Val Leu Leu Leu Phe 305 310 315 320 Thr Val Ala Asp Met Val Ser Ser Tyr Trp Leu Ala Leu Thr Phe Gln 325 330 335 Ala Asn His Val Val Glu Glu Val Gln Trp Pro Leu Pro Asp Glu Asn 340 345 350 Gly Ile Ile Gln Lys Asp Trp Ala Ala Met Gln Val Glu Thr Thr Gln 355 360 365 Asp Tyr Ala His Asp Ser His Leu Trp Thr Ser Ile Thr Gly Ser Leu 370 375 380 Asn Tyr Gln Ala Val His His Leu Phe Pro Asn Val Ser Gln His His 385 390 395 400 Tyr Pro Asp Ile Leu Ala Ile Ile Lys Asn Thr Cys Ser Glu Tyr Lys 405 410 415 Val Pro Tyr Leu Val Lys Asp Thr Phe Trp Gln Ala Phe Ala Ser His 420 425 430 Leu Glu His Leu Arg Val Leu Gly Leu Arg Pro Lys Glu Glu 435 440 445 <210> SEQ ID NO 69 <211> LENGTH: 6473 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pY5-22 <400> SEQUENCE: 69 ggtggagctc cagcttttgt tccctttagt gagggttaat ttcgagcttg gcgtaatcat 60 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacgtacgag 120 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg 180 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 240 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca 300 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 360 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 420 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 480 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 540 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 600 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 660 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 720 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 780 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 840 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 900 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 960 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 1020 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 1080 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 1140 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 1200 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 1260 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 1320 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 1380 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 1440 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 1500 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 1560 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 1620 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 1680 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 1740 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 1800 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 1860 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 1920 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 1980 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 2040 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat 2100 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 2160 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgcgc 2220 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 2280 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 2340 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 2400 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc 2460 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 2520 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga 2580 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 2640 attttaacaa aatattaacg cttacaattt ccattcgcca ttcaggctgc gcaactgttg 2700 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc 2760 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac 2820 ggccagtgaa ttgtaatacg actcactata gggcgaattg ggtaccgggc cccccctcga 2880 ggtcgatggt gtcgataagc ttgatatcga attcatgtca cacaaaccga tcttcgcctc 2940 aaggaaacct aattctacat ccgagagact gccgagatcc agtctacact gattaatttt 3000 cgggccaata atttaaaaaa atcgtgttat ataatattat atgtattata tatatacatc 3060 atgatgatac tgacagtcat gtcccattgc taaatagaca gactccatct gccgcctcca 3120 actgatgttc tcaatattta aggggtcatc tcgcattgtt taataataaa cagactccat 3180 ctaccgcctc caaatgatgt tctcaaaata tattgtatga acttattttt attacttagt 3240 attattagac aacttacttg ctttatgaaa aacacttcct atttaggaaa caatttataa 3300 tggcagttcg ttcatttaac aatttatgta gaataaatgt tataaatgcg tatgggaaat 3360 cttaaatatg gatagcataa atgatatctg cattgcctaa ttcgaaatca acagcaacga 3420 aaaaaatccc ttgtacaaca taaatagtca tcgagaaata tcaactatca aagaacagct 3480 attcacacgt tactattgag attattattg gacgagaatc acacactcaa ctgtctttct 3540 ctcttctaga aatacaggta caagtatgta ctattctcat tgttcatact tctagtcatt 3600 tcatcccaca tattccttgg atttctctcc aatgaatgac attctatctt gcaaattcaa 3660 caattataat aagatatacc aaagtagcgg tatagtggca atcaaaaagc ttctctggtg 3720 tgcttctcgt atttattttt attctaatga tccattaaag gtatatattt atttcttgtt 3780 atataatcct tttgtttatt acatgggctg gatacataaa ggtattttga tttaattttt 3840 tgcttaaatt caatcccccc tcgttcagtg tcaactgtaa tggtaggaaa ttaccatact 3900 tttgaagaag caaaaaaaat gaaagaaaaa aaaaatcgta tttccaggtt agacgttccg 3960 cagaatctag aatgcggtat gcggtacatt gttcttcgaa cgtaaaagtt gcgctccctg 4020 agatattgta catttttgct tttacaagta caagtacatc gtacaactat gtactactgt 4080 tgatgcatcc acaacagttt gttttgtttt tttttgtttt ttttttttct aatgattcat 4140 taccgctatg tatacctact tgtacttgta gtaagccggg ttattggcgt tcaattaatc 4200 atagacttat gaatctgcac ggtgtgcgct gcgagttact tttagcttat gcatgctact 4260 tgggtgtaat attgggatct gttcggaaat caacggatgc tcaaccgatt tcgacagtaa 4320 ttaattaagt catacacaag tcagctttct tcgagcctca tataagtata agtagttcaa 4380 cgtattagca ctgtacccag catctccgta tcgagaaaca caacaacatg ccccattgga 4440 cagatcatgc ggatacacag gttgtgcagt atcatacata ctcgatcaga caggtcgtct 4500 gaccatcata caagctgaac aagcgctcca tacttgcacg ctctctatat acacagttaa 4560 attacatatc catagtctaa cctctaacag ttaatcttct ggtaagcctc ccagccagcc 4620 ttctggtatc gcttggcctc ctcaatagga tctcggttct ggccgtacag acctcggccg 4680 acaattatga tatccgttcc ggtagacatg acatcctcaa cagttcggta ctgctgtccg 4740 agagcgtctc ccttgtcgtc aagacccacc ccgggggtca gaataagcca gtcctcagag 4800 tcgcccttag gtcggttctg ggcaatgaag ccaaccacaa actcggggtc ggatcgggca 4860 agctcaatgg tctgcttgga gtactcgcca gtggccagag agcccttgca agacagctcg 4920 gccagcatga gcagacctct ggccagcttc tcgttgggag aggggactag gaactccttg 4980 tactgggagt tctcgtagtc agagacgtcc tccttcttct gttcagagac agtttcctcg 5040 gcaccagctc gcaggccagc aatgattccg gttccgggta caccgtgggc gttggtgata 5100 tcggaccact cggcgattcg gtgacaccgg tactggtgct tgacagtgtt gccaatatct 5160 gcgaactttc tgtcctcgaa caggaagaaa ccgtgcttaa gagcaagttc cttgaggggg 5220 agcacagtgc cggcgtaggt gaagtcgtca atgatgtcga tatgggtttt gatcatgcac 5280 acataaggtc cgaccttatc ggcaagctca atgagctcct tggtggtggt aacatccaga 5340 gaagcacaca ggttggtttt cttggctgcc acgagcttga gcactcgagc ggcaaaggcg 5400 gacttgtgga cgttagctcg agcttcgtag gagggcattt tggtggtgaa gaggagactg 5460 aaataaattt agtctgcaga actttttatc ggaaccttat ctggggcagt gaagtatatg 5520 ttatggtaat agttacgagt tagttgaact tatagataga ctggactata cggctatcgg 5580 tccaaattag aaagaacgtc aatggctctc tgggcgtcgc ctttgccgac aaaaatgtga 5640 tcatgatgaa agccagcaat gacgttgcag ctgatattgt tgtcggccaa ccgcgccgaa 5700 aacgcagctg tcagacccac agcctccaac gaagaatgta tcgtcaaagt gatccaagca 5760 cactcatagt tggagtcgta ctccaaaggc ggcaatgacg agtcagacag atactcgtcg 5820 actcaggcga cgacggaatt cctgcagccc atctgcagaa ttcaggagag accgggttgg 5880 cggcgtattt gtgtcccaaa aaacagcccc aattgcccca attgacccca aattgaccca 5940 gtagcgggcc caaccccggc gagagccccc ttcaccccac atatcaaacc tcccccggtt 6000 cccacacttg ccgttaaggg cgtagggtac tgcagtctgg aatctacgct tgttcagact 6060 ttgtactagt ttctttgtct ggccatccgg gtaacccatg ccggacgcaa aatagactac 6120 tgaaaatttt tttgctttgt ggttgggact ttagccaagg gtataaaaga ccaccgtccc 6180 cgaattacct ttcctcttct tttctctctc tccttgtcaa ctcacacccg aaatcgttaa 6240 gcatttcctt ctgagtataa gaatcattca ccatggatcc actagttcta gagcggccgc 6300 caccgcggcc cgagattccg gcctcttcgg ccgccaagcg acccgggtgg acgtctagag 6360 gtacctagca attaacagat agtttgccgg tgataattct cttaacctcc cacactcctt 6420 tgacataacg atttatgtaa cgaaactgaa atttgaccag atattgtgtc cgc 6473 <210> SEQ ID NO 70 <211> LENGTH: 6970 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pY5-22GPD <400> SEQUENCE: 70 tcgacgcagt aggatgtcct gcacgggtct ttttgtgggg tgtggagaaa ggggtgcttg 60 gagatggaag ccggtagaac cgggctgctt gtgcttggag atggaagccg gtagaaccgg 120 gctgcttggg gggatttggg gccgctgggc tccaaagagg ggtaggcatt tcgttggggt 180 tacgtaattg cggcatttgg gtcctgcgcg catgtcccat tggtcagaat tagtccggat 240 aggagactta tcagccaatc acagcgccgg atccacctgt aggttgggtt gggtgggagc 300 acccctccac agagtagagt caaacagcag cagcaacatg atagttgggg gtgtgcgtgt 360 taaaggaaaa aaaagaagct tgggttatat tcccgctcta tttagaggtt gcgggataga 420 cgccgacgga gggcaatggc gccatggaac cttgcggata tcgatacgcc gcggcggact 480 gcgtccgaac cagctccagc agcgtttttt ccgggccatt gagccgactg cgaccccgcc 540 aacgtgtctt ggcccacgca ctcatgtcat gttggtgttg ggaggccact ttttaagtag 600 cacaaggcac ctagctcgca gcaaggtgtc cgaaccaaag aagcggctgc agtggtgcaa 660 acggggcgga aacggcggga aaaagccacg ggggcacgaa ttgaggcacg ccctcgaatt 720 tgagacgagt cacggcccca ttcgcccgcg caatggctcg ccaacgcccg gtcttttgca 780 ccacatcagg ttaccccaag ccaaaccttt gtgttaaaaa gcttaacata ttataccgaa 840 cgtaggtttg ggcgggcttg ctccgtctgt ccaaggcaac atttatataa gggtctgcat 900 cgccggctca attgaatctt ttttcttctt ctcttctcta tattcattct tgaattaaac 960 acacatcaat ccgcggccgc caccgcggcc cgagattccg gcctcttcgg ccgccaagcg 1020 acccgggtgg acgtctagag gtacctagca attaacagat agtttgccgg tgataattct 1080 cttaacctcc cacactcctt tgacataacg atttatgtaa cgaaactgaa atttgaccag 1140 atattgtgtc cgcggtggag ctccagcttt tgttcccttt agtgagggtt aatttcgagc 1200 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 1260 cacaacgtac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 1320 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 1380 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 1440 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 1500 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 1560 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 1620 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 1680 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 1740 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 1800 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 1860 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 1920 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 1980 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 2040 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc 2100 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 2160 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 2220 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 2280 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 2340 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 2400 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 2460 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 2520 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 2580 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 2640 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 2700 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 2760 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 2820 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 2880 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 2940 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 3000 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 3060 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 3120 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 3180 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 3240 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 3300 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 3360 ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc 3420 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt 3480 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc 3540 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt 3600 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt 3660 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt 3720 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa 3780 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttccattcg ccattcaggc 3840 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga 3900 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac 3960 gttgtaaaac gacggccagt gaattgtaat acgactcact atagggcgaa ttgggtaccg 4020 ggccccccct cgaggtcgat ggtgtcgata agcttgatat cgaattcatg tcacacaaac 4080 cgatcttcgc ctcaaggaaa cctaattcta catccgagag actgccgaga tccagtctac 4140 actgattaat tttcgggcca ataatttaaa aaaatcgtgt tatataatat tatatgtatt 4200 atatatatac atcatgatga tactgacagt catgtcccat tgctaaatag acagactcca 4260 tctgccgcct ccaactgatg ttctcaatat ttaaggggtc atctcgcatt gtttaataat 4320 aaacagactc catctaccgc ctccaaatga tgttctcaaa atatattgta tgaacttatt 4380 tttattactt agtattatta gacaacttac ttgctttatg aaaaacactt cctatttagg 4440 aaacaattta taatggcagt tcgttcattt aacaatttat gtagaataaa tgttataaat 4500 gcgtatggga aatcttaaat atggatagca taaatgatat ctgcattgcc taattcgaaa 4560 tcaacagcaa cgaaaaaaat cccttgtaca acataaatag tcatcgagaa atatcaacta 4620 tcaaagaaca gctattcaca cgttactatt gagattatta ttggacgaga atcacacact 4680 caactgtctt tctctcttct agaaatacag gtacaagtat gtactattct cattgttcat 4740 acttctagtc atttcatccc acatattcct tggatttctc tccaatgaat gacattctat 4800 cttgcaaatt caacaattat aataagatat accaaagtag cggtatagtg gcaatcaaaa 4860 agcttctctg gtgtgcttct cgtatttatt tttattctaa tgatccatta aaggtatata 4920 tttatttctt gttatataat ccttttgttt attacatggg ctggatacat aaaggtattt 4980 tgatttaatt ttttgcttaa attcaatccc ccctcgttca gtgtcaactg taatggtagg 5040 aaattaccat acttttgaag aagcaaaaaa aatgaaagaa aaaaaaaatc gtatttccag 5100 gttagacgtt ccgcagaatc tagaatgcgg tatgcggtac attgttcttc gaacgtaaaa 5160 gttgcgctcc ctgagatatt gtacattttt gcttttacaa gtacaagtac atcgtacaac 5220 tatgtactac tgttgatgca tccacaacag tttgttttgt ttttttttgt tttttttttt 5280 tctaatgatt cattaccgct atgtatacct acttgtactt gtagtaagcc gggttattgg 5340 cgttcaatta atcatagact tatgaatctg cacggtgtgc gctgcgagtt acttttagct 5400 tatgcatgct acttgggtgt aatattggga tctgttcgga aatcaacgga tgctcaaccg 5460 atttcgacag taattaatta agtcatacac aagtcagctt tcttcgagcc tcatataagt 5520 ataagtagtt caacgtatta gcactgtacc cagcatctcc gtatcgagaa acacaacaac 5580 atgccccatt ggacagatca tgcggataca caggttgtgc agtatcatac atactcgatc 5640 agacaggtcg tctgaccatc atacaagctg aacaagcgct ccatacttgc acgctctcta 5700 tatacacagt taaattacat atccatagtc taacctctaa cagttaatct tctggtaagc 5760 ctcccagcca gccttctggt atcgcttggc ctcctcaata ggatctcggt tctggccgta 5820 cagacctcgg ccgacaatta tgatatccgt tccggtagac atgacatcct caacagttcg 5880 gtactgctgt ccgagagcgt ctcccttgtc gtcaagaccc accccggggg tcagaataag 5940 ccagtcctca gagtcgccct taggtcggtt ctgggcaatg aagccaacca caaactcggg 6000 gtcggatcgg gcaagctcaa tggtctgctt ggagtactcg ccagtggcca gagagccctt 6060 gcaagacagc tcggccagca tgagcagacc tctggccagc ttctcgttgg gagaggggac 6120 taggaactcc ttgtactggg agttctcgta gtcagagacg tcctccttct tctgttcaga 6180 gacagtttcc tcggcaccag ctcgcaggcc agcaatgatt ccggttccgg gtacaccgtg 6240 ggcgttggtg atatcggacc actcggcgat tcggtgacac cggtactggt gcttgacagt 6300 gttgccaata tctgcgaact ttctgtcctc gaacaggaag aaaccgtgct taagagcaag 6360 ttccttgagg gggagcacag tgccggcgta ggtgaagtcg tcaatgatgt cgatatgggt 6420 tttgatcatg cacacataag gtccgacctt atcggcaagc tcaatgagct ccttggtggt 6480 ggtaacatcc agagaagcac acaggttggt tttcttggct gccacgagct tgagcactcg 6540 agcggcaaag gcggacttgt ggacgttagc tcgagcttcg taggagggca ttttggtggt 6600 gaagaggaga ctgaaataaa tttagtctgc agaacttttt atcggaacct tatctggggc 6660 agtgaagtat atgttatggt aatagttacg agttagttga acttatagat agactggact 6720 atacggctat cggtccaaat tagaaagaac gtcaatggct ctctgggcgt cgcctttgcc 6780 gacaaaaatg tgatcatgat gaaagccagc aatgacgttg cagctgatat tgttgtcggc 6840 caaccgcgcc gaaaacgcag ctgtcagacc cacagcctcc aacgaagaat gtatcgtcaa 6900 agtgatccaa gcacactcat agttggagtc gtactccaaa ggcggcaatg acgagtcaga 6960 cagatactcg 6970 <210> SEQ ID NO 71 <211> LENGTH: 968 <212> TYPE: DNA <213> ORGANISM: Yarrowia lipolytica <300> PUBLICATION INFORMATION: <302> TITLE: GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE AND PHOSPHOGLYCERATE MUTASE PROMOTERS FOR GENE EXPRESSION IN OLEAGINOUS YEAST <310> PATENT DOCUMENT NUMBER: US-2005-0014270-A1 <311> PATENT FILING DATE: 2004-06-16 <312> PUBLICATION DATE: 2005-01-20 <313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(968) <400> SEQUENCE: 71 tcgacgcagt aggatgtcct gcacgggtct ttttgtgggg tgtggagaaa ggggtgcttg 60 gagatggaag ccggtagaac cgggctgctt gtgcttggag atggaagccg gtagaaccgg 120 gctgcttggg gggatttggg gccgctgggc tccaaagagg ggtaggcatt tcgttggggt 180 tacgtaattg cggcatttgg gtcctgcgcg catgtcccat tggtcagaat tagtccggat 240 aggagactta tcagccaatc acagcgccgg atccacctgt aggttgggtt gggtgggagc 300 acccctccac agagtagagt caaacagcag cagcaacatg atagttgggg gtgtgcgtgt 360 taaaggaaaa aaaagaagct tgggttatat tcccgctcta tttagaggtt gcgggataga 420 cgccgacgga gggcaatggc gccatggaac cttgcggata tcgatacgcc gcggcggact 480 gcgtccgaac cagctccagc agcgtttttt ccgggccatt gagccgactg cgaccccgcc 540 aacgtgtctt ggcccacgca ctcatgtcat gttggtgttg ggaggccact ttttaagtag 600 cacaaggcac ctagctcgca gcaaggtgtc cgaaccaaag aagcggctgc agtggtgcaa 660 acggggcgga aacggcggga aaaagccacg ggggcacgaa ttgaggcacg ccctcgaatt 720 tgagacgagt cacggcccca ttcgcccgcg caatggctcg ccaacgcccg gtcttttgca 780 ccacatcagg ttaccccaag ccaaaccttt gtgttaaaaa gcttaacata ttataccgaa 840 cgtaggtttg ggcgggcttg ctccgtctgt ccaaggcaac atttatataa gggtctgcat 900 cgccggctca attgaatctt ttttcttctt ctcttctcta tattcattct tgaattaaac 960 acacatca 968 <210> SEQ ID NO 72 <211> LENGTH: 8630 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pYZDE2-S <400> SEQUENCE: 72 ggtggagctc cagcttttgt tccctttagt gagggttaat ttcgagcttg gcgtaatcat 60 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacgtacgag 120 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg 180 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 240 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca 300 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 360 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 420 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 480 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 540 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 600 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 660 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 720 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 780 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 840 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 900 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 960 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 1020 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 1080 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 1140 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 1200 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 1260 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 1320 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 1380 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 1440 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 1500 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 1560 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 1620 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 1680 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 1740 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 1800 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 1860 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 1920 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 1980 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 2040 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat 2100 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 2160 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgcgc 2220 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 2280 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 2340 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 2400 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc 2460 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 2520 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga 2580 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 2640 attttaacaa aatattaacg cttacaattt ccattcgcca ttcaggctgc gcaactgttg 2700 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc 2760 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac 2820 ggccagtgaa ttgtaatacg actcactata gggcgaattg ggtaccgggc cccccctcga 2880 ggtcgatggt gtcgataagc ttgatatcga attcatgtca cacaaaccga tcttcgcctc 2940 aaggaaacct aattctacat ccgagagact gccgagatcc agtctacact gattaatttt 3000 cgggccaata atttaaaaaa atcgtgttat ataatattat atgtattata tatatacatc 3060 atgatgatac tgacagtcat gtcccattgc taaatagaca gactccatct gccgcctcca 3120 actgatgttc tcaatattta aggggtcatc tcgcattgtt taataataaa cagactccat 3180 ctaccgcctc caaatgatgt tctcaaaata tattgtatga acttattttt attacttagt 3240 attattagac aacttacttg ctttatgaaa aacacttcct atttaggaaa caatttataa 3300 tggcagttcg ttcatttaac aatttatgta gaataaatgt tataaatgcg tatgggaaat 3360 cttaaatatg gatagcataa atgatatctg cattgcctaa ttcgaaatca acagcaacga 3420 aaaaaatccc ttgtacaaca taaatagtca tcgagaaata tcaactatca aagaacagct 3480 attcacacgt tactattgag attattattg gacgagaatc acacactcaa ctgtctttct 3540 ctcttctaga aatacaggta caagtatgta ctattctcat tgttcatact tctagtcatt 3600 tcatcccaca tattccttgg atttctctcc aatgaatgac attctatctt gcaaattcaa 3660 caattataat aagatatacc aaagtagcgg tatagtggca atcaaaaagc ttctctggtg 3720 tgcttctcgt atttattttt attctaatga tccattaaag gtatatattt atttcttgtt 3780 atataatcct tttgtttatt acatgggctg gatacataaa ggtattttga tttaattttt 3840 tgcttaaatt caatcccccc tcgttcagtg tcaactgtaa tggtaggaaa ttaccatact 3900 tttgaagaag caaaaaaaat gaaagaaaaa aaaaatcgta tttccaggtt agacgttccg 3960 cagaatctag aatgcggtat gcggtacatt gttcttcgaa cgtaaaagtt gcgctccctg 4020 agatattgta catttttgct tttacaagta caagtacatc gtacaactat gtactactgt 4080 tgatgcatcc acaacagttt gttttgtttt tttttgtttt ttttttttct aatgattcat 4140 taccgctatg tatacctact tgtacttgta gtaagccggg ttattggcgt tcaattaatc 4200 atagacttat gaatctgcac ggtgtgcgct gcgagttact tttagcttat gcatgctact 4260 tgggtgtaat attgggatct gttcggaaat caacggatgc tcaaccgatt tcgacagtaa 4320 taatttgaat cgaatcggag cctaaaatga acccgagtat atctcataaa attctcggtg 4380 agaggtctgt gactgtcagt acaaggtgcc ttcattatgc cctcaacctt accatacctc 4440 actgaatgta gtgtacctct aaaaatgaaa tacagtgcca aaagccaagg cactgagctc 4500 gtctaacgga cttgatatac aaccaattaa aacaaatgaa aagaaataca gttctttgta 4560 tcatttgtaa caattaccct gtacaaacta aggtattgaa atcccacaat attcccaaag 4620 tccacccctt tccaaattgt catgcctaca actcatatac caagcactaa cctaccaaac 4680 accactaaaa ccccacaaaa tatatcttac cgaatataca gtaacaagct accaccacac 4740 tcgttgggtg cagtcgccag cttaaagata tctatccaca tcagccacaa ctcccttcct 4800 ttaataaacc gactacaccc ttggctattg aggttatgag tgaatatact gtagacaaga 4860 cactttcaag aagactgttt ccaaaacgta ccactgtcct ccactacaaa cacacccaat 4920 ctgcttcttc tagtcaaggt tgctacaccg gtaaattata aatcatcatt tcattagcag 4980 ggcagggccc tttttataga gtcttataca ctagcggacc ctgccggtag accaacccgc 5040 aggcgcgtca gtttgctcct tccatcaatg cgtcgtagaa acgacttact ccttcttgag 5100 cagctccttg accttgttgg caacaagtct ccgacctcgg aggtggagga agagcctccg 5160 atatcggcgg tagtgatacc agcctcgacg gactccttga cggcagcctc aacagcgtca 5220 ccggcgggct tcatgttaag agagaacttg agcatcatgg cggcagacag aatggtggca 5280 atggggttga ccttctgctt gccgagatcg ggggcagatc cgtgacaggg ctcgtacaga 5340 ccgaacgcct cgttggtgtc gggcagagaa gccagagagg cggagggcag cagacccaga 5400 gaaccgggga tgacggaggc ctcgtcggag atgatatcgc caaacatgtt ggtggtgatg 5460 atgataccat tcatcttgga gggctgcttg atgaggatca tggcggccga gtcgatcagc 5520 tggtggttga gctcgagctg ggggaattcg tccttgagga ctcgagtgac agtctttcgc 5580 caaagtcgag aggaggccag cacgttggcc ttgtcaagag accacacggg aagagggggg 5640 ttgtgctgaa gggccaggaa ggcggccatt cgggcaattc gctcaacctc aggaacggag 5700 taggtctcgg tgtcggaagc gacgccagat ccgtcatcct cctttcgctc tccaaagtag 5760 atacctccga cgagctctcg gacaatgatg aagtcggtgc cctcaacgtt tcggatgggg 5820 gagagatcgg cgagcttggg cgacagcagc tggcagggtc gcaggttggc gtacaggttc 5880 aggtcctttc gcagcttgag gagaccctgc tcgggtcgca cgtcggttcg tccgtcggga 5940 gtggtccata cggtgttggc agcgcctccg acagcaccga gcataataga gtcagccttt 6000 cggcagatgt cgagagtagc gtcggtgatg ggctcgccct ccttctcaat ggcagctcct 6060 ccaatgagtc ggtcctcaaa cacaaactcg gtgccggagg cctcagcaac agacttgagc 6120 accttgacgg cctcggcaat cacctcgggg ccacagaagt cgccgccgag aagaacaatc 6180 ttcttggagt cagtcttggt cttcttagtt tcgggttcca ttgtggatgt gtgtggttgt 6240 atgtgtgatg tggtgtgtgg agtgaaaatc tgtggctggc aaacgctctt gtatatatac 6300 gcacttttgc ccgtgctatg tggaagacta aacctccgaa gattgtgact caggtagtgc 6360 ggtatcggct agggacccaa accttgtcga tgccgatagc gctatcgaac gtaccccagc 6420 cggccgggag tatgtcggag gggacatacg agatcgtcaa gggtttgtgg ccaactggta 6480 aatttaaatg atgtcgacgc agtaggatgt cctgcacggg tctttttgtg gggtgtggag 6540 aaaggggtgc ttggagatgg aagccggtag aaccgggctg cttgtgcttg gagatggaag 6600 ccggtagaac cgggctgctt ggggggattt ggggccgctg ggctccaaag aggggtaggc 6660 atttcgttgg ggttacgtaa ttgcggcatt tgggtcctgc gcgcatgtcc cattggtcag 6720 aattagtccg gataggagac ttatcagcca atcacagcgc cggatccacc tgtaggttgg 6780 gttgggtggg agcacccctc cacagagtag agtcaaacag cagcagcaac atgatagttg 6840 ggggtgtgcg tgttaaagga aaaaaaagaa gcttgggtta tattcccgct ctatttagag 6900 gttgcgggat agacgccgac ggagggcaat ggcgccatgg aaccttgcgg atatcgatac 6960 gccgcggcgg actgcgtccg aaccagctcc agcagcgttt tttccgggcc attgagccga 7020 ctgcgacccc gccaacgtgt cttggcccac gcactcatgt catgttggtg ttgggaggcc 7080 actttttaag tagcacaagg cacctagctc gcagcaaggt gtccgaacca aagaagcggc 7140 tgcagtggtg caaacggggc ggaaacggcg ggaaaaagcc acgggggcac gaattgaggc 7200 acgccctcga atttgagacg agtcacggcc ccattcgccc gcgcaatggc tcgccaacgc 7260 ccggtctttt gcaccacatc aggttacccc aagccaaacc tttgtgttaa aaagcttaac 7320 atattatacc gaacgtaggt ttgggcgggc ttgctccgtc tgtccaaggc aacatttata 7380 taagggtctg catcgccggc tcaattgaat cttttttctt cttctcttct ctatattcat 7440 tcttgaatta aacacacatc aatccatggc aaacagcagc gtgtgggatg atgtggtggg 7500 ccgcgtggag accggcgtgg accagtggat ggatggcgcc aagccgtacg cactcaccga 7560 tgggctcccg atgatggacg tgtccaccat gctggcattc gaggtgggat acatggccat 7620 gctgctcttc ggcatcccga tcatgaagca gatggagaag ccttttgagc tcaagaccat 7680 caagctcttg cacaacttgt ttctcttcgg actttccttg tacatgtgcg tggagaccat 7740 ccgccaggct atcctcggag gctacaaagt gtttggaaac gacatggaga agggcaacga 7800 gtctcatgct cagggcatgt ctcgcatcgt gtacgtgttc tacgtgtcca aggcatacga 7860 gttcttggat accgccatca tgatcctttg caagaagttc aaccaggttt ccttcttgca 7920 tgtgtaccac catgccactc atttttgcca tctggtgggc tatccgccaa gtacgctcca 7980 ggaggtgatg cgtacttttt cagtgatcct caactctttc gtgcacaccg tcatgtacgg 8040 catactactt cttctcctcc caagggttcg ggttcgtgaa gccaatcaag ccgtacatca 8100 ccacccttca gatgacccag ttcatggcaa tgcttgtgca gtccttgtac gactacctct 8160 tcccatgcga ctacccacag gctcttgtgc agctccttgg agtgtacatg atcaccttgc 8220 ttgccctctt cggcaacttt tttgtgcaga gctatcttaa aaagccaaaa aagagcaaga 8280 ccaactaaaa ctgcctgcat gatatgccgc tcgccggcgt tcgaattgac tcagaaagcg 8340 agttaaggcg acacgcaaac tctatatttt ttcaaacgtg ttgccgtcac tcattcgcca 8400 tctgtttact acgtgtctgt tcaatgagca tgttcttgaa tctaaagaat ctcgaatgtt 8460 ttttaaaaaa agaattcgat atcaagctta cgcgtcgacc cgggtggacg tctagaggta 8520 cctagcaatt aacagatagt ttgccggtga taattctctt aacctcccac actcctttga 8580 cataacgatt tatgtaacga aactgaaatt tgaccagata ttgtgtccgc 8630 <210> SEQ ID NO 73 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer GPDsense <400> SEQUENCE: 73 atacgagatc gtcaaggg 18 <210> SEQ ID NO 74 <211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer GPDantisense <400> SEQUENCE: 74 gcggccgcgg attgatgtgt gtttaa 26 <210> SEQ ID NO 75 <211> LENGTH: 6339 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKR136 <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (4078)..(4078) <223> OTHER INFORMATION: n is a, c, g, or t <400> SEQUENCE: 75 ggccgcgaca caagtgtgag agtactaaat aaatgctttg gttgtacgaa atcattacac 60 taaataaaat aatcaaagct tatatatgcc ttccgctaag gccgaatgca aagaaattgg 120 ttctttctcg ttatcttttg ccacttttac tagtacgtat taattactac ttaatcatct 180 ttgtttacgg ctcattatat ccgtacgtcg agtcgacctg caggcatgca agcttggcgt 240 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca 300 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat 360 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt 420 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct 480 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 540 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 600 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 660 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 720 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 780 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 840 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 900 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 960 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 1020 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 1080 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 1140 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 1200 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 1260 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 1320 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 1380 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 1440 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 1500 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 1560 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 1620 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 1680 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 1740 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 1800 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 1860 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 1920 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 1980 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 2040 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 2100 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 2160 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 2220 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 2280 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 2340 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 2400 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc 2460 cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg 2520 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt 2580 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac 2640 tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca 2700 tcaggcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct 2760 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa 2820 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattc gagctcggta 2880 cccggggatc ctctagacgt acgtcctcga agagaagggt taataacaca ttttttaaca 2940 tttttaacac aaattttagt tatttaaaaa tttattaaaa aatttaaaat aagaagagga 3000 actctttaaa taaatctaac ttacaaaatt tatgattttt aataagtttt caccaataaa 3060 aaatgtcata aaaatatgtt aaaaagtata ttatcaatat tctctttatg ataaataaaa 3120 agaaaaaaaa aataaaagtt aagtgaaaat gagattgaag tgactttagg tgtgtataaa 3180 tatatcaacc ccgccaacaa tttatttaat ccaaatatat tgaagtatat tattccatag 3240 cctttattta tttatatatt tattatataa aagctttatt tgttctaggt tgttcatgaa 3300 atattttttt ggttttatct ccgttgtaag aaaatcatgt gctttgtgtc gccactcact 3360 attgcagctt tttcatgcat tggtcagatt gacggttgat tgtatttttg ttttttatgg 3420 ttttgtgtta tgacttaagt cttcatctct ttatctcttc atcaggtttg atggttacct 3480 aatatggtcc atgggtacat gcatggttaa attaggtggc caactttgtt gtgaacgata 3540 gaattttttt tatattaagt aaactatttt tatattatga aataataata aaaaaaatat 3600 tttatcatta ttaacaaaat catattagtt aatttgttaa ctctataata aaagaaatac 3660 tgtaacattc acattacatg gtaacatctt tccacccttt catttgtttt ttgtttgatg 3720 actttttttc ttgtttaaat ttatttccct tcttttaaat ttggaataca ttatcatcat 3780 atataaacta aaatactaaa aacaggatta cacaaatgat aaataataac acaaatattt 3840 ataaatctag ctgcaatata tttaaactag ctatatcgat attgtaaaat aaaactagct 3900 gcattgatac tgataaaaaa atatcatgtg ctttctggac tgatgatgca gtatactttt 3960 gacattgcct ttattttatt tttcagaaaa gctttcttag ttctgggttc ttcattattt 4020 gtttcccatc tccattgtga attgaatcat ttgcttcgtg tcacaaatac aatttagnta 4080 ggtacatgca ttggtcagat tcacggttta ttatgtcatg acttaagttc atggtagtac 4140 attacctgcc acgcatgcat tatattggtt agatttgata ggcaaatttg gttgtcaaca 4200 atataaatat aaataatgtt tttatattac gaaataacag tgatcaaaac aaacagtttt 4260 atctttatta acaagatttt gtttttgttt gatgacgttt tttaatgttt acgctttccc 4320 ccttcttttg aatttagaac actttatcat cataaaatca aatactaaaa aaattacata 4380 tttcataaat aataacacaa atatttttaa aaaatctgaa ataataatga acaatattac 4440 atattatcac gaaaattcat taataaaaat attatataaa taaaatgtaa tagtagttat 4500 atgtaggaaa aaagtactgc acgcataata tatacaaaaa gattaaaatg aactattata 4560 aataataaca ctaaattaat ggtgaatcat atcaaaataa tgaaaaagta aataaaattt 4620 gtaattaact tctatatgta ttacacacac aaataataaa taatagtaaa aaaaattatg 4680 ataaatattt accatctcat aagatattta aaataatgat aaaaatatag attatttttt 4740 atgcaactag ctagccaaaa agagaacacg ggtatatata aaaagagtac ctttaaattc 4800 tactgtactt cctttattcc tgacgttttt atatcaagtg gacatacgtg aagattttaa 4860 ttatcagtct aaatatttca ttagcactta atacttttct gttttattcc tatcctataa 4920 gtagtcccga ttctcccaac attgcttatt cacacaacta actaagaaag tcttccatag 4980 ccccccaagc ggccgcatgg gaacggacca aggaaaaacc ttcacctggg aagagctggc 5040 ggcccataac accaaggacg acctactctt ggccatccgc ggcagggtgt acgatgtcac 5100 aaagttcttg agccgccatc ctggtggagt ggacactctc ctgctcggag ctggccgaga 5160 tgttactccg gtctttgaga tgtatcacgc gtttggggct gcagatgcca ttatgaagaa 5220 gtactatgtc ggtacactgg tctcgaatga gctgcccatc ttcccggagc caacggtgtt 5280 ccacaaaacc atcaagacga gagtcgaggg ctactttacg gatcggaaca ttgatcccaa 5340 gaatagacca gagatctggg gacgatacgc tcttatcttt ggatccttga tcgcttccta 5400 ctacgcgcag ctctttgtgc ctttcgttgt cgaacgcaca tggcttcagg tggtgtttgc 5460 aatcatcatg ggatttgcgt gcgcacaagt cggactcaac cctcttcatg atgcgtctca 5520 cttttcagtg acccacaacc ccactgtctg gaagattctg ggagccacgc acgacttttt 5580 caacggagca tcgtacctgg tgtggatgta ccaacatatg ctcggccatc acccctacac 5640 caacattgct ggagcagatc ccgacgtgtc gacgtctgag cccgatgttc gtcgtatcaa 5700 gcccaaccaa aagtggtttg tcaaccacat caaccagcac atgtttgttc ctttcctgta 5760 cggactgctg gcgttcaagg tgcgcattca ggacatcaac attttgtact ttgtcaagac 5820 caatgacgct attcgtgtca atcccatctc gacatggcac actgtgatgt tctggggcgg 5880 caaggctttc tttgtctggt atcgcctgat tgttcccctg cagtatctgc ccctgggcaa 5940 ggtgctgctc ttgttcacgg tcgcggacat ggtgtcgtct tactggctgg cgctgacctt 6000 ccaggcgaac cacgttgttg aggaagttca gtggccgttg cctgacgaga acgggatcat 6060 ccaaaaggac tgggcagcta tgcaggtcga gactacgcag gattacgcac acgattcgca 6120 cctctggacc agcatcactg gcagcttgaa ctaccaggct gtgcaccatc tgttccccaa 6180 cgtgtcgcag caccattatc ccgatattct ggccatcatc aagaacacct gcagcgagta 6240 caaggttcca taccttgtca aggatacgtt ttggcaagca tttgcttcac atttggagca 6300 cttgcgtgtt cttggactcc gtcccaagga agagtaggc 6339 <210> SEQ ID NO 76 <211> LENGTH: 8319 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pY98 <400> SEQUENCE: 76 ggccgccacc gcggcccgag attccggcct cttcggccgc caagcgaccc gggtggacgt 60 ctagaggtac ctagcaatta acagatagtt tgccggtgat aattctctta acctcccaca 120 ctcctttgac ataacgattt atgtaacgaa actgaaattt gaccagatat tgtgtccgcg 180 gtggagctcc agcttttgtt ccctttagtg agggttaatt tcgagcttgg cgtaatcatg 240 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acgtacgagc 300 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc 360 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat 420 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac 480 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 540 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca 600 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 660 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 720 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 780 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag 840 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 900 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 960 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 1020 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 1080 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 1140 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 1200 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc 1260 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag 1320 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata 1380 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat 1440 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg 1500 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc 1560 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc 1620 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc 1680 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc 1740 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc 1800 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa 1860 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat 1920 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata 1980 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca 2040 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag 2100 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc 2160 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc 2220 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata 2280 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta 2340 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgcgcc 2400 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact 2460 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc 2520 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt 2580 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc 2640 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt 2700 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat 2760 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa 2820 ttttaacaaa atattaacgc ttacaatttc cattcgccat tcaggctgcg caactgttgg 2880 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct 2940 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg 3000 gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtaccgggcc ccccctcgag 3060 gtcgatggtg tcgataagct tgatatcgaa ttcatgtcac acaaaccgat cttcgcctca 3120 aggaaaccta attctacatc cgagagactg ccgagatcca gtctacactg attaattttc 3180 gggccaataa tttaaaaaaa tcgtgttata taatattata tgtattatat atatacatca 3240 tgatgatact gacagtcatg tcccattgct aaatagacag actccatctg ccgcctccaa 3300 ctgatgttct caatatttaa ggggtcatct cgcattgttt aataataaac agactccatc 3360 taccgcctcc aaatgatgtt ctcaaaatat attgtatgaa cttattttta ttacttagta 3420 ttattagaca acttacttgc tttatgaaaa acacttccta tttaggaaac aatttataat 3480 ggcagttcgt tcatttaaca atttatgtag aataaatgtt ataaatgcgt atgggaaatc 3540 ttaaatatgg atagcataaa tgatatctgc attgcctaat tcgaaatcaa cagcaacgaa 3600 aaaaatccct tgtacaacat aaatagtcat cgagaaatat caactatcaa agaacagcta 3660 ttcacacgtt actattgaga ttattattgg acgagaatca cacactcaac tgtctttctc 3720 tcttctagaa atacaggtac aagtatgtac tattctcatt gttcatactt ctagtcattt 3780 catcccacat attccttgga tttctctcca atgaatgaca ttctatcttg caaattcaac 3840 aattataata agatatacca aagtagcggt atagtggcaa tcaaaaagct tctctggtgt 3900 gcttctcgta tttattttta ttctaatgat ccattaaagg tatatattta tttcttgtta 3960 tataatcctt ttgtttatta catgggctgg atacataaag gtattttgat ttaatttttt 4020 gcttaaattc aatcccccct cgttcagtgt caactgtaat ggtaggaaat taccatactt 4080 ttgaagaagc aaaaaaaatg aaagaaaaaa aaaatcgtat ttccaggtta gacgttccgc 4140 agaatctaga atgcggtatg cggtacattg ttcttcgaac gtaaaagttg cgctccctga 4200 gatattgtac atttttgctt ttacaagtac aagtacatcg tacaactatg tactactgtt 4260 gatgcatcca caacagtttg ttttgttttt ttttgttttt tttttttcta atgattcatt 4320 accgctatgt atacctactt gtacttgtag taagccgggt tattggcgtt caattaatca 4380 tagacttatg aatctgcacg gtgtgcgctg cgagttactt ttagcttatg catgctactt 4440 gggtgtaata ttgggatctg ttcggaaatc aacggatgct caaccgattt cgacagtaat 4500 taattaagtc atacacaagt cagctttctt cgagcctcat ataagtataa gtagttcaac 4560 gtattagcac tgtacccagc atctccgtat cgagaaacac aacaacatgc cccattggac 4620 agatcatgcg gatacacagg ttgtgcagta tcatacatac tcgatcagac aggtcgtctg 4680 accatcatac aagctgaaca agcgctccat acttgcacgc tctctatata cacagttaaa 4740 ttacatatcc atagtctaac ctctaacagt taatcttctg gtaagcctcc cagccagcct 4800 tctggtatcg cttggcctcc tcaataggat ctcggttctg gccgtacaga cctcggccga 4860 caattatgat atccgttccg gtagacatga catcctcaac agttcggtac tgctgtccga 4920 gagcgtctcc cttgtcgtca agacccaccc cgggggtcag aataagccag tcctcagagt 4980 cgcccttagg tcggttctgg gcaatgaagc caaccacaaa ctcggggtcg gatcgggcaa 5040 gctcaatggt ctgcttggag tactcgccag tggccagaga gcccttgcaa gacagctcgg 5100 ccagcatgag cagacctctg gccagcttct cgttgggaga ggggactagg aactccttgt 5160 actgggagtt ctcgtagtca gagacgtcct ccttcttctg ttcagagaca gtttcctcgg 5220 caccagctcg caggccagca atgattccgg ttccgggtac accgtgggcg ttggtgatat 5280 cggaccactc ggcgattcgg tgacaccggt actggtgctt gacagtgttg ccaatatctg 5340 cgaactttct gtcctcgaac aggaagaaac cgtgcttaag agcaagttcc ttgaggggga 5400 gcacagtgcc ggcgtaggtg aagtcgtcaa tgatgtcgat atgggttttg atcatgcaca 5460 cataaggtcc gaccttatcg gcaagctcaa tgagctcctt ggtggtggta acatccagag 5520 aagcacacag gttggttttc ttggctgcca cgagcttgag cactcgagcg gcaaaggcgg 5580 acttgtggac gttagctcga gcttcgtagg agggcatttt ggtggtgaag aggagactga 5640 aataaattta gtctgcagaa ctttttatcg gaaccttatc tggggcagtg aagtatatgt 5700 tatggtaata gttacgagtt agttgaactt atagatagac tggactatac ggctatcggt 5760 ccaaattaga aagaacgtca atggctctct gggcgtcgcc tttgccgaca aaaatgtgat 5820 catgatgaaa gccagcaatg acgttgcagc tgatattgtt gtcggccaac cgcgccgaaa 5880 acgcagctgt cagacccaca gcctccaacg aagaatgtat cgtcaaagtg atccaagcac 5940 actcatagtt ggagtcgtac tccaaaggcg gcaatgacga gtcagacaga tactcgtcga 6000 cgcagtagga tgtcctgcac gggtcttttt gtggggtgtg gagaaagggg tgcttggaga 6060 tggaagccgg tagaaccggg ctgcttgtgc ttggagatgg aagccggtag aaccgggctg 6120 cttgggggga tttggggccg ctgggctcca aagaggggta ggcatttcgt tggggttacg 6180 taattgcggc atttgggtcc tgcgcgcatg tcccattggt cagaattagt ccggatagga 6240 gacttatcag ccaatcacag cgccggatcc acctgtaggt tgggttgggt gggagcaccc 6300 ctccacagag tagagtcaaa cagcagcagc aacatgatag ttgggggtgt gcgtgttaaa 6360 ggaaaaaaaa gaagcttggg ttatattccc gctctattta gaggttgcgg gatagacgcc 6420 gacggagggc aatggcgcca tggaaccttg cggatatcga tacgccgcgg cggactgcgt 6480 ccgaaccagc tccagcagcg ttttttccgg gccattgagc cgactgcgac cccgccaacg 6540 tgtcttggcc cacgcactca tgtcatgttg gtgttgggag gccacttttt aagtagcaca 6600 aggcacctag ctcgcagcaa ggtgtccgaa ccaaagaagc ggctgcagtg gtgcaaacgg 6660 ggcggaaacg gcgggaaaaa gccacggggg cacgaattga ggcacgccct cgaatttgag 6720 acgagtcacg gccccattcg cccgcgcaat ggctcgccaa cgcccggtct tttgcaccac 6780 atcaggttac cccaagccaa acctttgtgt taaaaagctt aacatattat accgaacgta 6840 ggtttgggcg ggcttgctcc gtctgtccaa ggcaacattt atataagggt ctgcatcgcc 6900 ggctcaattg aatctttttt cttcttctct tctctatatt cattcttgaa ttaaacacac 6960 atcaatccgc ggccgcatgg gaacggacca aggaaaaacc ttcacctggg aagagctggc 7020 ggcccataac accaaggacg acctactctt ggccatccgc ggcagggtgt acgatgtcac 7080 aaagttcttg agccgccatc ctggtggagt ggacactctc ctgctcggag ctggccgaga 7140 tgttactccg gtctttgaga tgtatcacgc gtttggggct gcagatgcca ttatgaagaa 7200 gtactatgtc ggtacactgg tctcgaatga gctgcccatc ttcccggagc caacggtgtt 7260 ccacaaaacc atcaagacga gagtcgaggg ctactttacg gatcggaaca ttgatcccaa 7320 gaatagacca gagatctggg gacgatacgc tcttatcttt ggatccttga tcgcttccta 7380 ctacgcgcag ctctttgtgc ctttcgttgt cgaacgcaca tggcttcagg tggtgtttgc 7440 aatcatcatg ggatttgcgt gcgcacaagt cggactcaac cctcttcatg atgcgtctca 7500 cttttcagtg acccacaacc ccactgtctg gaagattctg ggagccacgc acgacttttt 7560 caacggagca tcgtacctgg tgtggatgta ccaacatatg ctcggccatc acccctacac 7620 caacattgct ggagcagatc ccgacgtgtc gacgtctgag cccgatgttc gtcgtatcaa 7680 gcccaaccaa aagtggtttg tcaaccacat caaccagcac atgtttgttc ctttcctgta 7740 cggactgctg gcgttcaagg tgcgcattca ggacatcaac attttgtact ttgtcaagac 7800 caatgacgct attcgtgtca atcccatctc gacatggcac actgtgatgt tctggggcgg 7860 caaggctttc tttgtctggt atcgcctgat tgttcccctg cagtatctgc ccctgggcaa 7920 ggtgctgctc ttgttcacgg tcgcggacat ggtgtcgtct tactggctgg cgctgacctt 7980 ccaggcgaac cacgttgttg aggaagttca gtggccgttg cctgacgaga acgggatcat 8040 ccaaaaggac tgggcagcta tgcaggtcga gactacgcag gattacgcac acgattcgca 8100 cctctggacc agcatcactg gcagcttgaa ctaccaggct gtgcaccatc tgttccccaa 8160 cgtgtcgcag caccattatc ccgatattct ggccatcatc aagaacacct gcagcgagta 8220 caaggttcca taccttgtca aggatacgtt ttggcaagca tttgcttcac atttggagca 8280 cttgcgtgtt cttggactcc gtcccaagga agagtaggc 8319 <210> SEQ ID NO 77 <211> LENGTH: 774 <212> TYPE: DNA <213> ORGANISM: Euglena gracilis <400> SEQUENCE: 77 atggaggtgg tgaatgaaat agtctcaatt gggcaggaag ttttacccaa agttgattat 60 gcccaactct ggagtgatgc cagtcactgt gaggtgcttt acttgtccat cgcatttgtc 120 atcttgaagt tcactcttgg cccccttggt ccaaaaggtc agtctcgtat gaagtttgtt 180 ttcaccaatt acaaccttct catgtccatt tattcgttgg gatcattcct ctcaatggca 240 tatgccatgt acaccatcgg tgttatgtct gacaactgcg agaaggcttt tgacaacaac 300 gtcttcagga tcaccacgca gttgttctat ttgagcaagt tcctggagta tattgactcc 360 ttctatttgc cactgatggg caagcctctg acctggttgc aattcttcca tcatttgggg 420 gcaccgatgg atatgtggct gttctataat taccgaaatg aagctgtttg gatttttgtg 480 ctgttgaatg gtttcatcca ctggatcatg tacggttatt attggaccag attgatcaag 540 ctgaagttcc ccatgccaaa atccctgatt acatcaatgc agatcattca attcaatgtt 600 ggtttctaca ttgtctggaa gtacaggaac attccctgtt atcgccaaga tgggatgagg 660 atgtttggct ggttcttcaa ttacttttat gttggcacag tcttgtgttt gttcttgaat 720 ttctatgtgc aaacgtatat cgtcaggaag cacaagggag ccaaaaagat tcag 774 <210> SEQ ID NO 78 <211> LENGTH: 1263 <212> TYPE: DNA <213> ORGANISM: Euglena gracilis <400> SEQUENCE: 78 atgaagtcaa agcgccaagc gcttcccctt acaattgatg gaacaacata tgatgtgtct 60 gcctgggtca atttccaccc tggtggtgcg gaaattatag agaattacca aggaagggat 120 gccactgatg ccttcatggt tatgcactct caagaagcct tcgacaagct caagcgcatg 180 cccaaaatca atcccagttc tgagttgcca ccccaggctg cagtgaatga agctcaagag 240 gatttccgga agctccgaga agagttgatc gcaactggca tgtttgatgc ctcccccctc 300 tggtactcat acaaaatcag caccacactg ggccttggag tgctgggtta tttcctgatg 360 gttcagtatc agatgtattt cattggggca gtgttgcttg ggatgcacta tcaacagatg 420 ggctggcttt ctcatgacat ttgccaccac cagactttca agaaccggaa ctggaacaac 480 ctcgtgggac tggtatttgg caatggtctg caaggttttt ccgtgacatg gtggaaggac 540 agacacaatg cacatcattc ggcaaccaat gttcaagggc acgaccctga tattgacaac 600 ctccccctct tagcctggtc tgaggatgac gtcacacggg cgtcaccgat ttcccgcaag 660 ctcattcagt tccagcagta ctatttcttg gtcatctgta tcttgttgcg gttcatttgg 720 tgtttccaga gcgtgttgac cgtgcgcagt ttgaaggaca gagataacca attctatcgc 780 tctcagtata agaaggaggc cattggcctc gccctgcact ggaccttgaa gaccctgttc 840 cacttattct ttatgcccag catcctcaca tcgctgttgg tgtttttcgt ttcggagctg 900 gttggcggct tcggcattgc gatcgtggtg ttcatgaacc actacccact ggagaagatc 960 ggggactcag tctgggatgg ccatggattc tcggttggcc agatccatga gaccatgaac 1020 attcggcgag ggattatcac agattggttt ttcggaggct tgaattacca gattgagcac 1080 catttgtggc cgaccctccc tcgccacaac ctgacagcgg ttagctacca ggtggaacag 1140 ctgtgccaga agcacaacct gccgtatcgg aacccgctgc cccatgaagg gttggtcatc 1200 ctgctgcgct atctggcggt gttcgcccgg atggcggaga agcaacccgc ggggaaggct 1260 cta 1263 <210> SEQ ID NO 79 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer oEugEL1-1 <400> SEQUENCE: 79 agcggccgca ccatggaggt ggtgaatgaa 30 <210> SEQ ID NO 80 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer oEugEL1-2 <400> SEQUENCE: 80 tgcggccgct cactgaatct ttttggctcc 30 <210> SEQ ID NO 81 <211> LENGTH: 4311 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKR906 <400> SEQUENCE: 81 agcggccgca ccatggaggt ggtgaatgaa atagtctcaa ttgggcagga agttttaccc 60 aaagttgatt atgcccaact ctggagtgat gccagtcact gtgaggtgct ttacttgtcc 120 atcgcatttg tcatcttgaa gttcactctt ggcccccttg gtccaaaagg tcagtctcgt 180 atgaagtttg ttttcaccaa ttacaacctt ctcatgtcca tttattcgtt gggatcattc 240 ctctcaatgg catatgccat gtacaccatc ggtgttatgt ctgacaactg cgagaaggct 300 tttgacaaca acgtcttcag gatcaccacg cagttgttct atttgagcaa gttcctggag 360 tatattgact ccttctattt gccactgatg ggcaagcctc tgacctggtt gcaattcttc 420 catcatttgg gggcaccgat ggatatgtgg ctgttctata attaccgaaa tgaagctgtt 480 tggatttttg tgctgttgaa tggtttcatc cactggatca tgtacggtta ttattggacc 540 agattgatca agctgaagtt ccccatgcca aaatccctga ttacatcaat gcagatcatt 600 caattcaatg ttggtttcta cattgtctgg aagtacagga acattccctg ttatcgccaa 660 gatgggatga ggatgtttgg ctggttcttc aattactttt atgttggcac agtcttgtgt 720 ttgttcttga atttctatgt gcaaacgtat atcgtcagga agcacaaggg agccaaaaag 780 attcagtgag cggccgcacc tgaattccag cacactggcg gccgttacta gtggatccga 840 gctcggtacc aagcttgatg catagcttga gtattctaac gcgtcaccta aatagcttgg 900 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca 960 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca 1020 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 1080 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt 1140 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 1200 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 1260 caaaaggcca gcaaaagccc aggaaccgta aaaaggccgc gttgctggcg tttttccata 1320 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 1380 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 1440 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 1500 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 1560 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 1620 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 1680 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 1740 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 1800 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 1860 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 1920 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 1980 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt agcacgtgtc 2040 agtcctgctc ctcggccacg aagtgcacgc agttgccggc cgggtcgcgc agggcgaact 2100 cccgccccca cggctgctcg ccgatctcgg tcatggccgg cccggaggcg tcccggaagt 2160 tcgtggacac gacctccgac cactcggcgt acagctcgtc caggccgcgc acccacaccc 2220 aggccagggt gttgtccggc accacctggt cctggaccgc gctgatgaac agggtcacgt 2280 cgtcccggac cacaccggcg aagtcgtcct ccacgaagtc ccgggagaac ccgagccggt 2340 cggtccagaa ctcgaccgct ccggcgacgt cgcgcgcggt gagcaccgga acggcactgg 2400 tcaacttggc catggtggcc ctcctcacgt gctattattg aagcatttat cagggttatt 2460 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 2520 gcacatttcc ccgaaaagtg ccacctgtat gcggtgtgaa ataccgcaca gatgcgtaag 2580 gagaaaatac cgcatcagga aattgtaagc gttaataatt cagaagaact cgtcaagaag 2640 gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg 2700 gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg ctatgtcctg 2760 atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc ggccattttc 2820 caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct cgccgtcggg 2880 catgctcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat gctcttcgtc 2940 cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct cgatgcgatg 3000 tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc 3060 atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga gatcctgccc 3120 cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt cgagcacagc 3180 tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt cttgcagttc 3240 attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct gcgctgacag 3300 ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat agccgaatag 3360 cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa 3420 cgatcctcat cctgtctctt gatcagagct tgatcccctg cgccatcaga tccttggcgg 3480 cgagaaagcc atccagttta ctttgcaggg cttcccaacc ttaccagagg gcgccccagc 3540 tggcaattcc ggttcgcttg ctgtccataa aaccgcccag tctagctatc gccatgtaag 3600 cccactgcaa gctacctgct ttctctttgc gcttgcgttt tcccttgtcc agatagccca 3660 gtagctgaca ttcatccggg gtcagcaccg tttctgcgga ctggctttct acgtgaaaag 3720 gatctaggtg aagatccttt ttgataatct catgcctgac atttatattc cccagaacat 3780 caggttaatg gcgtttttga tgtcattttc gcggtggctg agatcagcca cttcttcccc 3840 gataacggag accggcacac tggccatatc ggtggtcatc atgcgccagc tttcatcccc 3900 gatatgcacc accgggtaaa gttcacggga gactttatct gacagcagac gtgcactggc 3960 cagggggatc accatccgtc gccccggcgt gtcaataata tcactctgta catccacaaa 4020 cagacgataa cggctctctc ttttataggt gtaaacctta aactgccgta cgtataggct 4080 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 4140 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg 4200 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tgggccctct 4260 agatgcatgc tcgagcggcc gccagtgtga tggatatctg cagaattcag g 4311 <210> SEQ ID NO 82 <211> LENGTH: 7085 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKR72 <400> SEQUENCE: 82 gtacggatcc gtcgacggcg cgcccgatca tccggatata gttcctcctt tcagcaaaaa 60 acccctcaag acccgtttag aggccccaag gggttatgct agttattgct cagcggtggc 120 agcagccaac tcagcttcct ttcgggcttt gttagcagcc ggatcgatcc aagctgtacc 180 tcactattcc tttgccctcg gacgagtgct ggggcgtcgg tttccactat cggcgagtac 240 ttctacacag ccatcggtcc agacggccgc gcttctgcgg gcgatttgtg tacgcccgac 300 agtcccggct ccggatcgga cgattgcgtc gcatcgaccc tgcgcccaag ctgcatcatc 360 gaaattgccg tcaaccaagc tctgatagag ttggtcaaga ccaatgcgga gcatatacgc 420 ccggagccgc ggcgatcctg caagctccgg atgcctccgc tcgaagtagc gcgtctgctg 480 ctccatacaa gccaaccacg gcctccagaa gaagatgttg gcgacctcgt attgggaatc 540 cccgaacatc gcctcgctcc agtcaatgac cgctgttatg cggccattgt ccgtcaggac 600 attgttggag ccgaaatccg cgtgcacgag gtgccggact tcggggcagt cctcggccca 660 aagcatcagc tcatcgagag cctgcgcgac ggacgcactg acggtgtcgt ccatcacagt 720 ttgccagtga tacacatggg gatcagcaat cgcgcatatg aaatcacgcc atgtagtgta 780 ttgaccgatt ccttgcggtc cgaatgggcc gaacccgctc gtctggctaa gatcggccgc 840 agcgatcgca tccatagcct ccgcgaccgg ctgcagaaca gcgggcagtt cggtttcagg 900 caggtcttgc aacgtgacac cctgtgcacg gcgggagatg caataggtca ggctctcgct 960 gaattcccca atgtcaagca cttccggaat cgggagcgcg gccgatgcaa agtgccgata 1020 aacataacga tctttgtaga aaccatcggc gcagctattt acccgcagga catatccacg 1080 ccctcctaca tcgaagctga aagcacgaga ttcttcgccc tccgagagct gcatcaggtc 1140 ggagacgctg tcgaactttt cgatcagaaa cttctcgaca gacgtcgcgg tgagttcagg 1200 cttttccatg ggtatatctc cttcttaaag ttaaacaaaa ttatttctag agggaaaccg 1260 ttgtggtctc cctatagtga gtcgtattaa tttcgcggga tcgagatcga tccaattcca 1320 atcccacaaa aatctgagct taacagcaca gttgctcctc tcagagcaga atcgggtatt 1380 caacaccctc atatcaacta ctacgttgtg tataacggtc cacatgccgg tatatacgat 1440 gactggggtt gtacaaaggc ggcaacaaac ggcgttcccg gagttgcaca caagaaattt 1500 gccactatta cagaggcaag agcagcagct gacgcgtaca caacaagtca gcaaacagac 1560 aggttgaact tcatccccaa aggagaagct caactcaagc ccaagagctt tgctaaggcc 1620 ctaacaagcc caccaaagca aaaagcccac tggctcacgc taggaaccaa aaggcccagc 1680 agtgatccag ccccaaaaga gatctccttt gccccggaga ttacaatgga cgatttcctc 1740 tatctttacg atctaggaag gaagttcgaa ggtgaaggtg acgacactat gttcaccact 1800 gataatgaga aggttagcct cttcaatttc agaaagaatg ctgacccaca gatggttaga 1860 gaggcctacg cagcaggtct catcaagacg atctacccga gtaacaatct ccaggagatc 1920 aaataccttc ccaagaaggt taaagatgca gtcaaaagat tcaggactaa ttgcatcaag 1980 aacacagaga aagacatatt tctcaagatc agaagtacta ttccagtatg gacgattcaa 2040 ggcttgcttc ataaaccaag gcaagtaata gagattggag tctctaaaaa ggtagttcct 2100 actgaatcta aggccatgca tggagtctaa gattcaaatc gaggatctaa cagaactcgc 2160 cgtgaagact ggcgaacagt tcatacagag tcttttacga ctcaatgaca agaagaaaat 2220 cttcgtcaac atggtggagc acgacactct ggtctactcc aaaaatgtca aagatacagt 2280 ctcagaagac caaagggcta ttgagacttt tcaacaaagg ataatttcgg gaaacctcct 2340 cggattccat tgcccagcta tctgtcactt catcgaaagg acagtagaaa aggaaggtgg 2400 ctcctacaaa tgccatcatt gcgataaagg aaaggctatc attcaagatg cctctgccga 2460 cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag aagacgttcc 2520 aaccacgtct tcaaagcaag tggattgatg tgacatctcc actgacgtaa gggatgacgc 2580 acaatcccac tatccttcgc aagacccttc ctctatataa ggaagttcat ttcatttgga 2640 gaggacacgc tcgagctcat ttctctatta cttcagccat aacaaaagaa ctcttttctc 2700 ttcttattaa accatgaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat 2760 cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc 2820 tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg 2880 tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga 2940 agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca 3000 gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc 3060 ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt 3120 cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga 3180 tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca 3240 ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca 3300 cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga 3360 ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag 3420 gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct 3480 tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca 3540 gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat 3600 cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt 3660 ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg 3720 tccgagggca aaggaatagt gaggtaccta aagaaggagt gcgtcgaagc agatcgttca 3780 aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc 3840 atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta 3900 tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa 3960 aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta 4020 gatcgatgtc gaatcgatca acctgcatta atgaatcggc caacgcgcgg ggagaggcgg 4080 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 4140 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 4200 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4260 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 4320 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 4380 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4440 ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc 4500 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4560 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4620 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 4680 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 4740 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4800 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 4860 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 4920 acgttaaggg attttggtca tgacattaac ctataaaaat aggcgtatca cgaggccctt 4980 tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac 5040 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc 5100 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag 5160 agtgcaccat atggacatat tgtcgttaga acgcggctac aattaataca taaccttatg 5220 tatcatacac atacgattta ggtgacacta tagaacggcg cgccaagctt gttgaaacat 5280 ccctgaagtg tctcatttta ttttatttat tctttgctga taaaaaaata aaataaaaga 5340 agctaagcac acggtcaacc attgctctac tgctaaaagg gttatgtgta gtgttttact 5400 gcataaatta tgcagcaaac aagacaactc aaattaaaaa atttcctttg cttgtttttt 5460 tgttgtctct gacttgactt tcttgtggaa gttggttgta taaggattgg gacaccattg 5520 tccttcttaa tttaatttta ttctttgctg ataaaaaaaa aaatttcata tagtgttaaa 5580 taataatttg ttaaataacc aaaaagtcaa atatgtttac tctcgtttaa ataattgaga 5640 ttcgtccagc aaggctaaac gattgtatag atttatgaca atatttactt ttttatagat 5700 aaatgttata ttataataaa tttatataca tatattatat gttatttatt attattttaa 5760 atccttcaat attttatcaa accaactcat aatttttttt ttatctgtaa gaagcaataa 5820 aattaaatag acccacttta aggatgatcc aacctttata cagagtaaga gagttcaaat 5880 agtacccttt catatacata tcaactaaaa tattagaaat atcatggatc aaaccttata 5940 aagacattaa ataagtggat aagtataata tataaatggg tagtatataa tatataaatg 6000 gatacaaact tctctcttta taattgttat gtctccttaa catcctaata taatacataa 6060 gtgggtaata tataatatat aaatggagac aaacttcttc cattataatt gttatgtctt 6120 cttaacactt atgtctcgtt cacaatgcta aggttagaat tgtttagaaa gtcttatagt 6180 acacatttgt ttttgtacta tttgaagcat tccataagcc gtcacgattc agatgattta 6240 taataataag aggaaattta tcatagaaca ataaggtgca tagatagagt gttaatatat 6300 cataacatcc tttgtttatt catagaagaa gtgagatgga gctcagttat tatactgtta 6360 catggtcgga tacaatattc catgctctcc atgagctctt acacctacat gcattttagt 6420 tcatacttgc ggccgcagta tatcttaaat tctttaatac ggtgtactag gatattgaac 6480 tggttcttga tgatgaaaac ctgggccgag attgcagcta tttatagtca taggtcttgt 6540 taacatgcat ggacatttgg ccacggggtg gcatgcagtt tgacgggtgt tgaaataaac 6600 aaaaatgagg tggcggaaga gaatacgagt ttgaggttgg gttagaaaca acaaatgtga 6660 gggctcatga tgggttgagt tggtgaatgt tttgggctgc tcgattgaca cctttgtgag 6720 tacgtgttgt tgtgcatggc ttttggggtc cagttttttt ttcttgacgc ggcgatcctg 6780 atcagctagt ggataagtga tgtccactgt gtgtgattgc gtttttgttt gaattttatg 6840 aacttagaca ttgctatgca aaggatactc tcattgtgtt ttgtcttctt ttgttccttg 6900 gctttttctt atgatccaag agactagtca gtgttgtggc attcgagact accaagatta 6960 attatgatgg gggaaggata agtaactgat tagtacggac tgttaccaaa ttaattaata 7020 agcggcaaat gaagggcatg gatcaaaagc ttggatctcc tgcaggatct ggccggccgg 7080 atctc 7085 <210> SEQ ID NO 83 <211> LENGTH: 2540 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKS102 <400> SEQUENCE: 83 cgatcatccg gatatagttc ctcctttcag caaaaaaccc ctcaagaccc gtttagaggc 60 cccaaggggt tatgctagtt attgctcagc ggtggcagca gccaactcag cttcctttcg 120 ggctttgtta gcagccggat cgatccaagc tgtacctcac tattcctttg ccctcggacg 180 agtgctgggg cgtcggtttc cactatcggc gagtacttct acacagccat cggtccagac 240 ggccgcgctt ctgcgggcga tttgtgtacg cccgacagtc ccggctccgg atcggacgat 300 tgcgtcgcat cgaccctgcg cccaagctgc atcatcgaaa ttgccgtcaa ccaagctctg 360 atagagttgg tcaagaccaa tgcggagcat atacgcccgg agccgcggcg atcctgcaag 420 ctccggatgc ctccgctcga agtagcgcgt ctgctgctcc atacaagcca accacggcct 480 ccagaagaag atgttggcga cctcgtattg ggaatccccg aacatcgcct cgctccagtc 540 aatgaccgct gttatgcggc cattgtccgt caggacattg ttggagccga aatccgcgtg 600 cacgaggtgc cggacttcgg ggcagtcctc ggcccaaagc atcagctcat cgagagcctg 660 cgcgacggac gcactgacgg tgtcgtccat cacagtttgc cagtgataca catggggatc 720 agcaatcgcg catatgaaat cacgccatgt agtgtattga ccgattcctt gcggtccgaa 780 tgggccgaac ccgctcgtct ggctaagatc ggccgcagcg atcgcatcca tagcctccgc 840 gaccggctgc agaacagcgg gcagttcggt ttcaggcagg tcttgcaacg tgacaccctg 900 tgcacggcgg gagatgcaat aggtcaggct ctcgctgaat tccccaatgt caagcacttc 960 cggaatcggg agcgcggccg atgcaaagtg ccgataaaca taacgatctt tgtagaaacc 1020 atcggcgcag ctatttaccc gcaggacata tccacgccct cctacatcga agctgaaagc 1080 acgagattct tcgccctccg agagctgcat caggtcggag acgctgtcga acttttcgat 1140 cagaaacttc tcgacagacg tcgcggtgag ttcaggcttt tccatgggta tatctccttc 1200 ttaaagttaa acaaaattat ttctagaggg aaaccgttgt ggtctcccta tagtgagtcg 1260 tattaatttc gcgggatcga gatctgatca acctgcatta atgaatcggc caacgcgcgg 1320 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 1380 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 1440 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 1500 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 1560 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 1620 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 1680 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt 1740 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 1800 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 1860 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 1920 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg 1980 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 2040 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 2100 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 2160 acgaaaactc acgttaaggg attttggtca tgacattaac ctataaaaat aggcgtatca 2220 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc 2280 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg 2340 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga 2400 ttgtactgag agtgcaccat atggacatat tgtcgttaga acgcggctac aattaataca 2460 taaccttatg tatcatacac atacgattta ggtgacacta tagaacggcg cgccaagctt 2520 ggatccgtcg acggcgcgcc 2540 <210> SEQ ID NO 84 <211> LENGTH: 4359 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKR197 <400> SEQUENCE: 84 cgcgcccgat catccggata tagttcctcc tttcagcaaa aaacccctca agacccgttt 60 agaggcccca aggggttatg ctagttattg ctcagcggtg gcagcagcca actcagcttc 120 ctttcgggct ttgttagcag ccggatcgat ccaagctgta cctcactatt cctttgccct 180 cggacgagtg ctggggcgtc ggtttccact atcggcgagt acttctacac agccatcggt 240 ccagacggcc gcgcttctgc gggcgatttg tgtacgcccg acagtcccgg ctccggatcg 300 gacgattgcg tcgcatcgac cctgcgccca agctgcatca tcgaaattgc cgtcaaccaa 360 gctctgatag agttggtcaa gaccaatgcg gagcatatac gcccggagcc gcggcgatcc 420 tgcaagctcc ggatgcctcc gctcgaagta gcgcgtctgc tgctccatac aagccaacca 480 cggcctccag aagaagatgt tggcgacctc gtattgggaa tccccgaaca tcgcctcgct 540 ccagtcaatg accgctgtta tgcggccatt gtccgtcagg acattgttgg agccgaaatc 600 cgcgtgcacg aggtgccgga cttcggggca gtcctcggcc caaagcatca gctcatcgag 660 agcctgcgcg acggacgcac tgacggtgtc gtccatcaca gtttgccagt gatacacatg 720 gggatcagca atcgcgcata tgaaatcacg ccatgtagtg tattgaccga ttccttgcgg 780 tccgaatggg ccgaacccgc tcgtctggct aagatcggcc gcagcgatcg catccatagc 840 ctccgcgacc ggctgcagaa cagcgggcag ttcggtttca ggcaggtctt gcaacgtgac 900 accctgtgca cggcgggaga tgcaataggt caggctctcg ctgaattccc caatgtcaag 960 cacttccgga atcgggagcg cggccgatgc aaagtgccga taaacataac gatctttgta 1020 gaaaccatcg gcgcagctat ttacccgcag gacatatcca cgccctccta catcgaagct 1080 gaaagcacga gattcttcgc cctccgagag ctgcatcagg tcggagacgc tgtcgaactt 1140 ttcgatcaga aacttctcga cagacgtcgc ggtgagttca ggcttttcca tgggtatatc 1200 tccttcttaa agttaaacaa aattatttct agagggaaac cgttgtggtc tccctatagt 1260 gagtcgtatt aatttcgcgg gatcgagatc tgatcaacct gcattaatga atcggccaac 1320 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 1380 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 1440 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 1500 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 1560 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 1620 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 1680 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct 1740 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 1800 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 1860 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 1920 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag 1980 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 2040 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 2100 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 2160 agtggaacga aaactcacgt taagggattt tggtcatgac attaacctat aaaaataggc 2220 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca 2280 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc 2340 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag 2400 agcagattgt actgagagtg caccatatgg acatattgtc gttagaacgc ggctacaatt 2460 aatacataac cttatgtatc atacacatac gatttaggtg acactataga acggcgcgcc 2520 aagcttgttg aaacatccct gaagtgtctc attttatttt atttattctt tgctgataaa 2580 aaaataaaat aaaagaagct aagcacacgg tcaaccattg ctctactgct aaaagggtta 2640 tgtgtagtgt tttactgcat aaattatgca gcaaacaaga caactcaaat taaaaaattt 2700 cctttgcttg tttttttgtt gtctctgact tgactttctt gtggaagttg gttgtataag 2760 gattgggaca ccattgtcct tcttaattta attttattct ttgctgataa aaaaaaaaat 2820 ttcatatagt gttaaataat aatttgttaa ataaccaaaa agtcaaatat gtttactctc 2880 gtttaaataa ttgagattcg tccagcaagg ctaaacgatt gtatagattt atgacaatat 2940 ttactttttt atagataaat gttatattat aataaattta tatacatata ttatatgtta 3000 tttattatta ttttaaatcc ttcaatattt tatcaaacca actcataatt ttttttttat 3060 ctgtaagaag caataaaatt aaatagaccc actttaagga tgatccaacc tttatacaga 3120 gtaagagagt tcaaatagta ccctttcata tacatatcaa ctaaaatatt agaaatatca 3180 tggatcaaac cttataaaga cattaaataa gtggataagt ataatatata aatgggtagt 3240 atataatata taaatggata caaacttctc tctttataat tgttatgtct ccttaacatc 3300 ctaatataat acataagtgg gtaatatata atatataaat ggagacaaac ttcttccatt 3360 ataattgtta tgtcttctta acacttatgt ctcgttcaca atgctaaggt tagaattgtt 3420 tagaaagtct tatagtacac atttgttttt gtactatttg aagcattcca taagccgtca 3480 cgattcagat gatttataat aataagagga aatttatcat agaacaataa ggtgcataga 3540 tagagtgtta atatatcata acatcctttg tttattcata gaagaagtga gatggagctc 3600 agttattata ctgttacatg gtcggataca atattccatg ctctccatga gctcttacac 3660 ctacatgcat tttagttcat acttgcggcc gcagtatatc ttaaattctt taatacggtg 3720 tactaggata ttgaactggt tcttgatgat gaaaacctgg gccgagattg cagctattta 3780 tagtcatagg tcttgttaac atgcatggac atttggccac ggggtggcat gcagtttgac 3840 gggtgttgaa ataaacaaaa atgaggtggc ggaagagaat acgagtttga ggttgggtta 3900 gaaacaacaa atgtgagggc tcatgatggg ttgagttggt gaatgttttg ggctgctcga 3960 ttgacacctt tgtgagtacg tgttgttgtg catggctttt ggggtccagt ttttttttct 4020 tgacgcggcg atcctgatca gctagtggat aagtgatgtc cactgtgtgt gattgcgttt 4080 ttgtttgaat tttatgaact tagacattgc tatgcaaagg atactctcat tgtgttttgt 4140 cttcttttgt tccttggctt tttcttatga tccaagagac tagtcagtgt tgtggcattc 4200 gagactacca agattaatta tgatggggga aggataagta actgattagt acggactgtt 4260 accaaattaa ttaataagcg gcaaatgaag ggcatggatc aaaagcttgg atctcctgca 4320 ggatctggcc ggccggatct cgtacggatc cgtcgacgg 4359 <210> SEQ ID NO 85 <211> LENGTH: 5147 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKR911 <400> SEQUENCE: 85 ggccgcaagt atgaactaaa atgcatgtag gtgtaagagc tcatggagag catggaatat 60 tgtatccgac catgtaacag tataataact gagctccatc tcacttcttc tatgaataaa 120 caaaggatgt tatgatatat taacactcta tctatgcacc ttattgttct atgataaatt 180 tcctcttatt attataaatc atctgaatcg tgacggctta tggaatgctt caaatagtac 240 aaaaacaaat gtgtactata agactttcta aacaattcta accttagcat tgtgaacgag 300 acataagtgt taagaagaca taacaattat aatggaagaa gtttgtctcc atttatatat 360 tatatattac ccacttatgt attatattag gatgttaagg agacataaca attataaaga 420 gagaagtttg tatccattta tatattatat actacccatt tatatattat acttatccac 480 ttatttaatg tctttataag gtttgatcca tgatatttct aatattttag ttgatatgta 540 tatgaaaggg tactatttga actctcttac tctgtataaa ggttggatca tccttaaagt 600 gggtctattt aattttattg cttcttacag ataaaaaaaa aattatgagt tggtttgata 660 aaatattgaa ggatttaaaa taataataaa taacatataa tatatgtata taaatttatt 720 ataatataac atttatctat aaaaaagtaa atattgtcat aaatctatac aatcgtttag 780 ccttgctgga cgaatctcaa ttatttaaac gagagtaaac atatttgact ttttggttat 840 ttaacaaatt attatttaac actatatgaa attttttttt ttatcagcaa agaataaaat 900 taaattaaga aggacaatgg tgtcccaatc cttatacaac caacttccac aagaaagtca 960 agtcagagac aacaaaaaaa caagcaaagg aaatttttta atttgagttg tcttgtttgc 1020 tgcataattt atgcagtaaa acactacaca taaccctttt agcagtagag caatggttga 1080 ccgtgtgctt agcttctttt attttatttt tttatcagca aagaataaat aaaataaaat 1140 gagacacttc agggatgttt caacaagctt ggcgcgccgt tctatagtgt cacctaaatc 1200 gtatgtgtat gatacataag gttatgtatt aattgtagcc gcgttctaac gacaatatgt 1260 ccatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac 1320 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca 1380 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga 1440 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgacca 1500 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag 1560 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac 1620 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa 1680 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc 1740 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag 1800 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac 1860 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc 1920 gaacgaccta caccgaactg agatacctac agcgtgagca ttgagaaagc gccacgcttc 1980 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca 2040 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc 2100 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg 2160 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct 2220 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata 2280 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc 2340 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc aggttgatca 2400 gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct 2460 agaaataatt ttgtttaact ttaagaagga gatataccca tggaaaagcc tgaactcacc 2520 gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag 2580 ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc 2640 ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt 2700 gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg 2760 acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa 2820 ctgcccgctg ttctgcagcc ggtcgcggag gctatggatg cgatcgctgc ggccgatctt 2880 agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg 2940 cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac 3000 gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac 3060 tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac 3120 aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac 3180 gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc 3240 tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc 3300 cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct 3360 tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca 3420 caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat 3480 agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagtgagg tacagcttgg 3540 atcgatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag 3600 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa 3660 ggaggaacta tatccggatg atcgggcgcg ccgtcgacgg atccgtacga gatccggccg 3720 gccagatcct gcaggagatc caagcttttg atccatgccc ttcatttgcc gcttattaat 3780 taatttggta acagtccgta ctaatcagtt acttatcctt cccccatcat aattaatctt 3840 ggtagtctcg aatgccacaa cactgactag tctcttggat cataagaaaa agccaaggaa 3900 caaaagaaga caaaacacaa tgagagtatc ctttgcatag caatgtctaa gttcataaaa 3960 ttcaaacaaa aacgcaatca cacacagtgg acatcactta tccactagct gatcaggatc 4020 gccgcgtcaa gaaaaaaaaa ctggacccca aaagccatgc acaacaacac gtactcacaa 4080 aggtgtcaat cgagcagccc aaaacattca ccaactcaac ccatcatgag ccctcacatt 4140 tgttgtttct aacccaacct caaactcgta ttctcttccg ccacctcatt tttgtttatt 4200 tcaacacccg tcaaactgca tgccaccccg tggccaaatg tccatgcatg ttaacaagac 4260 ctatgactat aaatagctgc aatctcggcc caggttttca tcatcaagaa ccagttcaat 4320 atcctagtac accgtattaa agaatttaag atatactgcg gccgcaccat ggaggtggtg 4380 aatgaaatag tctcaattgg gcaggaagtt ttacccaaag ttgattatgc ccaactctgg 4440 agtgatgcca gtcactgtga ggtgctttac ttgtccatcg catttgtcat cttgaagttc 4500 actcttggcc cccttggtcc aaaaggtcag tctcgtatga agtttgtttt caccaattac 4560 aaccttctca tgtccattta ttcgttggga tcattcctct caatggcata tgccatgtac 4620 accatcggtg ttatgtctga caactgcgag aaggcttttg acaacaacgt cttcaggatc 4680 accacgcagt tgttctattt gagcaagttc ctggagtata ttgactcctt ctatttgcca 4740 ctgatgggca agcctctgac ctggttgcaa ttcttccatc atttgggggc accgatggat 4800 atgtggctgt tctataatta ccgaaatgaa gctgtttgga tttttgtgct gttgaatggt 4860 ttcatccact ggatcatgta cggttattat tggaccagat tgatcaagct gaagttcccc 4920 atgccaaaat ccctgattac atcaatgcag atcattcaat tcaatgttgg tttctacatt 4980 gtctggaagt acaggaacat tccctgttat cgccaagatg ggatgaggat gtttggctgg 5040 ttcttcaatt acttttatgt tggcacagtc ttgtgtttgt tcttgaattt ctatgtgcaa 5100 acgtatatcg tcaggaagca caagggagcc aaaaagattc agtgagc 5147 <210> SEQ ID NO 86 <211> LENGTH: 6559 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKR680 <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (4340)..(4340) <223> OTHER INFORMATION: n is a, c, g, or t <400> SEQUENCE: 86 ggccgcgaca caagtgtgag agtactaaat aaatgctttg gttgtacgaa atcattacac 60 taaataaaat aatcaaagct tatatatgcc ttccgctaag gccgaatgca aagaaattgg 120 ttctttctcg ttatcttttg ccacttttac tagtacgtat taattactac ttaatcatct 180 ttgtttacgg ctcattatat ccggtctaga ggatccaagg ccgcgaagtt aaaagcaatg 240 ttgtcacttg tcgtactaac acatgatgtg atagtttatg ctagctagct ataacataag 300 ctgtctctga gtgtgttgta tattaataaa gatcatcact ggtgaatggt gatcgtgtac 360 gtaccctact tagtaggcaa tggaagcact tagagtgtgc tttgtgcatg gccttgcctc 420 tgttttgaga cttttgtaat gttttcgagt ttaaatcttt gcctttgcgt acgtgggcgg 480 atcccccggg ctgcaggaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc 540 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata 600 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc 660 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca 720 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac 780 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga 840 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac 900 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt 960 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct 1020 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat 1080 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg 1140 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg 1200 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc 1260 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat 1320 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact 1380 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca 1440 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact 1500 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg 1560 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg 1620 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg 1680 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg 1740 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag 1800 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc 1860 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga 1920 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat 1980 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc 2040 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 2100 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 2160 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 2220 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc 2280 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 2340 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 2400 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 2460 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 2520 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca 2580 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 2640 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 2700 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 2760 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta 2820 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag 2880 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga 2940 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg 3000 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg 3060 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc 3120 atgattacgc caagcttgca tgcctgcagg tcgactcgac gtacgtcctc gaagagaagg 3180 gttaataaca cattttttaa catttttaac acaaatttta gttatttaaa aatttattaa 3240 aaaatttaaa ataagaagag gaactcttta aataaatcta acttacaaaa tttatgattt 3300 ttaataagtt ttcaccaata aaaaatgtca taaaaatatg ttaaaaagta tattatcaat 3360 attctcttta tgataaataa aaagaaaaaa aaaataaaag ttaagtgaaa atgagattga 3420 agtgacttta ggtgtgtata aatatatcaa ccccgccaac aatttattta atccaaatat 3480 attgaagtat attattccat agcctttatt tatttatata tttattatat aaaagcttta 3540 tttgttctag gttgttcatg aaatattttt ttggttttat ctccgttgta agaaaatcat 3600 gtgctttgtg tcgccactca ctattgcagc tttttcatgc attggtcaga ttgacggttg 3660 attgtatttt tgttttttat ggttttgtgt tatgacttaa gtcttcatct ctttatctct 3720 tcatcaggtt tgatggttac ctaatatggt ccatgggtac atgcatggtt aaattaggtg 3780 gccaactttg ttgtgaacga tagaattttt tttatattaa gtaaactatt tttatattat 3840 gaaataataa taaaaaaaat attttatcat tattaacaaa atcatattag ttaatttgtt 3900 aactctataa taaaagaaat actgtaacat tcacattaca tggtaacatc tttccaccct 3960 ttcatttgtt ttttgtttga tgactttttt tcttgtttaa atttatttcc cttcttttaa 4020 atttggaata cattatcatc atatataaac taaaatacta aaaacaggat tacacaaatg 4080 ataaataata acacaaatat ttataaatct agctgcaata tatttaaact agctatatcg 4140 atattgtaaa ataaaactag ctgcattgat actgataaaa aaatatcatg tgctttctgg 4200 actgatgatg cagtatactt ttgacattgc ctttatttta tttttcagaa aagctttctt 4260 agttctgggt tcttcattat ttgtttccca tctccattgt gaattgaatc atttgcttcg 4320 tgtcacaaat acaatttagn taggtacatg cattggtcag attcacggtt tattatgtca 4380 tgacttaagt tcatggtagt acattacctg ccacgcatgc attatattgg ttagatttga 4440 taggcaaatt tggttgtcaa caatataaat ataaataatg tttttatatt acgaaataac 4500 agtgatcaaa acaaacagtt ttatctttat taacaagatt ttgtttttgt ttgatgacgt 4560 tttttaatgt ttacgctttc ccccttcttt tgaatttaga acactttatc atcataaaat 4620 caaatactaa aaaaattaca tatttcataa ataataacac aaatattttt aaaaaatctg 4680 aaataataat gaacaatatt acatattatc acgaaaattc attaataaaa atattatata 4740 aataaaatgt aatagtagtt atatgtagga aaaaagtact gcacgcataa tatatacaaa 4800 aagattaaaa tgaactatta taaataataa cactaaatta atggtgaatc atatcaaaat 4860 aatgaaaaag taaataaaat ttgtaattaa cttctatatg tattacacac acaaataata 4920 aataatagta aaaaaaatta tgataaatat ttaccatctc ataagatatt taaaataatg 4980 ataaaaatat agattatttt ttatgcaact agctagccaa aaagagaaca cgggtatata 5040 taaaaagagt acctttaaat tctactgtac ttcctttatt cctgacgttt ttatatcaag 5100 tggacatacg tgaagatttt aattatcagt ctaaatattt cattagcact taatactttt 5160 ctgttttatt cctatcctat aagtagtccc gattctccca acattgctta ttcacacaac 5220 taactaagaa agtcttccat agccccccaa gcggccgcgg gaattcgatt gaaatgaagt 5280 caaagcgcca agcgcttccc cttacaattg atggaacaac atatgatgtg tctgcctggg 5340 tcaatttcca ccctggtggt gcggaaatta tagagaatta ccaaggaagg gatgccactg 5400 atgccttcat ggttatgcac tctcaagaag ccttcgacaa gctcaagcgc atgcccaaaa 5460 tcaatcccag ttctgagttg ccaccccagg ctgcagtgaa tgaagctcaa gaggatttcc 5520 ggaagctccg agaagagttg atcgcaactg gcatgtttga tgcctccccc ctctggtact 5580 catacaaaat cagcaccaca ctgggccttg gagtgctggg ttatttcctg atggttcagt 5640 atcagatgta tttcattggg gcagtgttgc ttgggatgca ctatcaacag atgggctggc 5700 tttctcatga catttgccac caccagactt tcaagaaccg gaactggaac aacctcgtgg 5760 gactggtatt tggcaatggt ctgcaaggtt tttccgtgac atggtggaag gacagacaca 5820 atgcacatca ttcggcaacc aatgttcaag ggcacgaccc tgatattgac aacctccccc 5880 tcttagcctg gtctgaggat gacgtcacac gggcgtcacc gatttcccgc aagctcattc 5940 agttccagca gtactatttc ttggtcatct gtatcttgtt gcggttcatt tggtgtttcc 6000 agagcgtgtt gaccgtgcgc agtttgaagg acagagataa ccaattctat cgctctcagt 6060 ataagaagga ggccattggc ctcgccctgc actggacctt gaagaccctg ttccacttat 6120 tctttatgcc cagcatcctc acatcgctgt tggtgttttt cgtttcggag ctggttggcg 6180 gcttcggcat tgcgatcgtg gtgttcatga accactaccc actggagaag atcggggact 6240 cagtctggga tggccatgga ttctcggttg gccagatcca tgagaccatg aacattcggc 6300 gagggattat cacagattgg tttttcggag gcttgaatta ccagattgag caccatttgt 6360 ggccgaccct ccctcgccac aacctgacag cggttagcta ccaggtggaa cagctgtgcc 6420 agaagcacaa cctgccgtat cggaacccgc tgccccatga agggttggtc atcctgctgc 6480 gctatctggc ggtgttcgcc cggatggcgg agaagcaacc cgcggggaag gctctataag 6540 gaatcactag tgaattcgc 6559 <210> SEQ ID NO 87 <211> LENGTH: 9014 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKR913 <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (7839)..(7839) <223> OTHER INFORMATION: n is a, c, g, or t <400> SEQUENCE: 87 gtacgagatc cggccggcca gatcctgcag gagatccaag cttttgatcc atgcccttca 60 tttgccgctt attaattaat ttggtaacag tccgtactaa tcagttactt atccttcccc 120 catcataatt aatcttggta gtctcgaatg ccacaacact gactagtctc ttggatcata 180 agaaaaagcc aaggaacaaa agaagacaaa acacaatgag agtatccttt gcatagcaat 240 gtctaagttc ataaaattca aacaaaaacg caatcacaca cagtggacat cacttatcca 300 ctagctgatc aggatcgccg cgtcaagaaa aaaaaactgg accccaaaag ccatgcacaa 360 caacacgtac tcacaaaggt gtcaatcgag cagcccaaaa cattcaccaa ctcaacccat 420 catgagccct cacatttgtt gtttctaacc caacctcaaa ctcgtattct cttccgccac 480 ctcatttttg tttatttcaa cacccgtcaa actgcatgcc accccgtggc caaatgtcca 540 tgcatgttaa caagacctat gactataaat agctgcaatc tcggcccagg ttttcatcat 600 caagaaccag ttcaatatcc tagtacaccg tattaaagaa tttaagatat actgcggccg 660 caccatggag gtggtgaatg aaatagtctc aattgggcag gaagttttac ccaaagttga 720 ttatgcccaa ctctggagtg atgccagtca ctgtgaggtg ctttacttgt ccatcgcatt 780 tgtcatcttg aagttcactc ttggccccct tggtccaaaa ggtcagtctc gtatgaagtt 840 tgttttcacc aattacaacc ttctcatgtc catttattcg ttgggatcat tcctctcaat 900 ggcatatgcc atgtacacca tcggtgttat gtctgacaac tgcgagaagg cttttgacaa 960 caacgtcttc aggatcacca cgcagttgtt ctatttgagc aagttcctgg agtatattga 1020 ctccttctat ttgccactga tgggcaagcc tctgacctgg ttgcaattct tccatcattt 1080 gggggcaccg atggatatgt ggctgttcta taattaccga aatgaagctg tttggatttt 1140 tgtgctgttg aatggtttca tccactggat catgtacggt tattattgga ccagattgat 1200 caagctgaag ttccccatgc caaaatccct gattacatca atgcagatca ttcaattcaa 1260 tgttggtttc tacattgtct ggaagtacag gaacattccc tgttatcgcc aagatgggat 1320 gaggatgttt ggctggttct tcaattactt ttatgttggc acagtcttgt gtttgttctt 1380 gaatttctat gtgcaaacgt atatcgtcag gaagcacaag ggagccaaaa agattcagtg 1440 agcggccgca agtatgaact aaaatgcatg taggtgtaag agctcatgga gagcatggaa 1500 tattgtatcc gaccatgtaa cagtataata actgagctcc atctcacttc ttctatgaat 1560 aaacaaagga tgttatgata tattaacact ctatctatgc accttattgt tctatgataa 1620 atttcctctt attattataa atcatctgaa tcgtgacggc ttatggaatg cttcaaatag 1680 tacaaaaaca aatgtgtact ataagacttt ctaaacaatt ctaaccttag cattgtgaac 1740 gagacataag tgttaagaag acataacaat tataatggaa gaagtttgtc tccatttata 1800 tattatatat tacccactta tgtattatat taggatgtta aggagacata acaattataa 1860 agagagaagt ttgtatccat ttatatatta tatactaccc atttatatat tatacttatc 1920 cacttattta atgtctttat aaggtttgat ccatgatatt tctaatattt tagttgatat 1980 gtatatgaaa gggtactatt tgaactctct tactctgtat aaaggttgga tcatccttaa 2040 agtgggtcta tttaatttta ttgcttctta cagataaaaa aaaaattatg agttggtttg 2100 ataaaatatt gaaggattta aaataataat aaataacata taatatatgt atataaattt 2160 attataatat aacatttatc tataaaaaag taaatattgt cataaatcta tacaatcgtt 2220 tagccttgct ggacgaatct caattattta aacgagagta aacatatttg actttttggt 2280 tatttaacaa attattattt aacactatat gaaatttttt tttttatcag caaagaataa 2340 aattaaatta agaaggacaa tggtgtccca atccttatac aaccaacttc cacaagaaag 2400 tcaagtcaga gacaacaaaa aaacaagcaa aggaaatttt ttaatttgag ttgtcttgtt 2460 tgctgcataa tttatgcagt aaaacactac acataaccct tttagcagta gagcaatggt 2520 tgaccgtgtg cttagcttct tttattttat ttttttatca gcaaagaata aataaaataa 2580 aatgagacac ttcagggatg tttcaacaag cttggcgcgc cgttctatag tgtcacctaa 2640 atcgtatgtg tatgatacat aaggttatgt attaattgta gccgcgttct aacgacaata 2700 tgtccatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc 2760 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 2820 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 2880 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga 2940 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 3000 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 3060 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 3120 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag 3180 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 3240 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 3300 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 3360 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gcattgagaa agcgccacgc 3420 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 3480 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 3540 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 3600 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 3660 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg 3720 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag 3780 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcaggttga 3840 tcagatctcg atcccgcgaa attaatacga ctcactatag ggagaccaca acggtttccc 3900 tctagaaata attttgttta actttaagaa ggagatatac ccatggaaaa gcctgaactc 3960 accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg 4020 cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat 4080 gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac 4140 tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc 4200 ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc 4260 gaactgcccg ctgttctgca gccggtcgcg gaggctatgg atgcgatcgc tgcggccgat 4320 cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca 4380 tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg 4440 gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag 4500 gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg 4560 gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa 4620 tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg 4680 cgctacttcg agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg 4740 ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca 4800 gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt 4860 acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc 4920 gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaatagtg aggtacagct 4980 tggatcgatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct 5040 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg 5100 aaaggaggaa ctatatccgg atgatcgggc gcgccgtcga cggatccgta cgcaaaggca 5160 aagatttaaa ctcgaaaaca ttacaaaagt ctcaaaacag aggcaaggcc atgcacaaag 5220 cacactctaa gtgcttccat tgcctactaa gtagggtacg tacacgatca ccattcacca 5280 gtgatgatct ttattaatat acaacacact cagagacagc ttatgttata gctagctagc 5340 ataaactatc acatcatgtg ttagtacgac aagtgacaac attgctttta acttcgcggc 5400 cttggatcct ctagaccgga tataatgagc cgtaaacaaa gatgattaag tagtaattaa 5460 tacgtactag taaaagtggc aaaagataac gagaaagaac caatttcttt gcattcggcc 5520 ttagcggaag gcatatataa gctttgatta ttttatttag tgtaatgatt tcgtacaacc 5580 aaagcattta tttagtactc tcacacttgt gtcgcggccg cgaattcact agtgattcct 5640 tatagagcct tccccgcggg ttgcttctcc gccatccggg cgaacaccgc cagatagcgc 5700 agcaggatga ccaacccttc atggggcagc gggttccgat acggcaggtt gtgcttctgg 5760 cacagctgtt ccacctggta gctaaccgct gtcaggttgt ggcgagggag ggtcggccac 5820 aaatggtgct caatctggta attcaagcct ccgaaaaacc aatctgtgat aatccctcgc 5880 cgaatgttca tggtctcatg gatctggcca accgagaatc catggccatc ccagactgag 5940 tccccgatct tctccagtgg gtagtggttc atgaacacca cgatcgcaat gccgaagccg 6000 ccaaccagct ccgaaacgaa aaacaccaac agcgatgtga ggatgctggg cataaagaat 6060 aagtggaaca gggtcttcaa ggtccagtgc agggcgaggc caatggcctc cttcttatac 6120 tgagagcgat agaattggtt atctctgtcc ttcaaactgc gcacggtcaa cacgctctgg 6180 aaacaccaaa tgaaccgcaa caagatacag atgaccaaga aatagtactg ctggaactga 6240 atgagcttgc gggaaatcgg tgacgcccgt gtgacgtcat cctcagacca ggctaagagg 6300 gggaggttgt caatatcagg gtcgtgccct tgaacattgg ttgccgaatg atgtgcattg 6360 tgtctgtcct tccaccatgt cacggaaaaa ccttgcagac cattgccaaa taccagtccc 6420 acgaggttgt tccagttccg gttcttgaaa gtctggtggt ggcaaatgtc atgagaaagc 6480 cagcccatct gttgatagtg catcccaagc aacactgccc caatgaaata catctgatac 6540 tgaaccatca ggaaataacc cagcactcca aggcccagtg tggtgctgat tttgtatgag 6600 taccagaggg gggaggcatc aaacatgcca gttgcgatca actcttctcg gagcttccgg 6660 aaatcctctt gagcttcatt cactgcagcc tggggtggca actcagaact gggattgatt 6720 ttgggcatgc gcttgagctt gtcgaaggct tcttgagagt gcataaccat gaaggcatca 6780 gtggcatccc ttccttggta attctctata atttccgcac caccagggtg gaaattgacc 6840 caggcagaca catcatatgt tgttccatca attgtaaggg gaagcgcttg gcgctttgac 6900 ttcatttcaa tcgaattccc gcggccgctt ggggggctat ggaagacttt cttagttagt 6960 tgtgtgaata agcaatgttg ggagaatcgg gactacttat aggataggaa taaaacagaa 7020 aagtattaag tgctaatgaa atatttagac tgataattaa aatcttcacg tatgtccact 7080 tgatataaaa acgtcaggaa taaaggaagt acagtagaat ttaaaggtac tctttttata 7140 tatacccgtg ttctcttttt ggctagctag ttgcataaaa aataatctat atttttatca 7200 ttattttaaa tatcttatga gatggtaaat atttatcata atttttttta ctattattta 7260 ttatttgtgt gtgtaataca tatagaagtt aattacaaat tttatttact ttttcattat 7320 tttgatatga ttcaccatta atttagtgtt attatttata atagttcatt ttaatctttt 7380 tgtatatatt atgcgtgcag tacttttttc ctacatataa ctactattac attttattta 7440 tataatattt ttattaatga attttcgtga taatatgtaa tattgttcat tattatttca 7500 gattttttaa aaatatttgt gttattattt atgaaatatg taattttttt agtatttgat 7560 tttatgatga taaagtgttc taaattcaaa agaaggggga aagcgtaaac attaaaaaac 7620 gtcatcaaac aaaaacaaaa tcttgttaat aaagataaaa ctgtttgttt tgatcactgt 7680 tatttcgtaa tataaaaaca ttatttatat ttatattgtt gacaaccaaa tttgcctatc 7740 aaatctaacc aatataatgc atgcgtggca ggtaatgtac taccatgaac ttaagtcatg 7800 acataataaa ccgtgaatct gaccaatgca tgtacctanc taaattgtat ttgtgacacg 7860 aagcaaatga ttcaattcac aatggagatg ggaaacaaat aatgaagaac ccagaactaa 7920 gaaagctttt ctgaaaaata aaataaaggc aatgtcaaaa gtatactgca tcatcagtcc 7980 agaaagcaca tgatattttt ttatcagtat caatgcagct agttttattt tacaatatcg 8040 atatagctag tttaaatata ttgcagctag atttataaat atttgtgtta ttatttatca 8100 tttgtgtaat cctgttttta gtattttagt ttatatatga tgataatgta ttccaaattt 8160 aaaagaaggg aaataaattt aaacaagaaa aaaagtcatc aaacaaaaaa caaatgaaag 8220 ggtggaaaga tgttaccatg taatgtgaat gttacagtat ttcttttatt atagagttaa 8280 caaattaact aatatgattt tgttaataat gataaaatat tttttttatt attatttcat 8340 aatataaaaa tagtttactt aatataaaaa aaattctatc gttcacaaca aagttggcca 8400 cctaatttaa ccatgcatgt acccatggac catattaggt aaccatcaaa cctgatgaag 8460 agataaagag atgaagactt aagtcataac acaaaaccat aaaaaacaaa aatacaatca 8520 accgtcaatc tgaccaatgc atgaaaaagc tgcaatagtg agtggcgaca caaagcacat 8580 gattttctta caacggagat aaaaccaaaa aaatatttca tgaacaacct agaacaaata 8640 aagcttttat ataataaata tataaataaa taaaggctat ggaataatat acttcaatat 8700 atttggatta aataaattgt tggcggggtt gatatattta tacacaccta aagtcacttc 8760 aatctcattt tcacttaact tttatttttt ttttcttttt atttatcata aagagaatat 8820 tgataatata ctttttaaca tatttttatg acatttttta ttggtgaaaa cttattaaaa 8880 atcataaatt ttgtaagtta gatttattta aagagttcct cttcttattt taaatttttt 8940 aataaatttt taaataacta aaatttgtgt taaaaatgtt aaaaaatgtg ttattaaccc 9000 ttctcttcga ggac 9014 <210> SEQ ID NO 88 <211> LENGTH: 5561 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKR767 <400> SEQUENCE: 88 catggtcaat caatgagacg ccaacttctt aatctattga gacctgcagg tctagaaggg 60 cggatccccg ggtaccgagc tcgaattcac tggccgtcgt tttacaacgt cgtgactggg 120 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc 180 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg 240 aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat 300 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc 360 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag 420 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg 480 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg 540 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 600 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 660 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 720 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 780 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 840 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 900 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 960 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 1020 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 1080 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 1140 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 1200 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 1260 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 1320 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 1380 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 1440 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 1500 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 1560 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 1620 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 1680 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 1740 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 1800 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 1860 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 1920 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 1980 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 2040 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 2100 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 2160 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 2220 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 2280 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 2340 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc 2400 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg 2460 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt 2520 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc 2580 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc 2640 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct 2700 atgaccatga ttacgccaag cttgcatgcc tgcaggctag cctaagtacg tactcaaaat 2760 gccaacaaat aaaaaaaaag ttgctttaat aatgccaaaa caaattaata aaacacttac 2820 aacaccggat tttttttaat taaaatgtgc catttaggat aaatagttaa tatttttaat 2880 aattatttaa aaagccgtat ctactaaaat gatttttatt tggttgaaaa tattaatatg 2940 tttaaatcaa cacaatctat caaaattaaa ctaaaaaaaa aataagtgta cgtggttaac 3000 attagtacag taatataaga ggaaaatgag aaattaagaa attgaaagcg agtctaattt 3060 ttaaattatg aacctgcata tataaaagga aagaaagaat ccaggaagaa aagaaatgaa 3120 accatgcatg gtcccctcgt catcacgagt ttctgccatt tgcaatagaa acactgaaac 3180 acctttctct ttgtcactta attgagatgc cgaagccacc tcacaccatg aacttcatga 3240 ggtgtagcac ccaaggcttc catagccatg catactgaag aatgtctcaa gctcagcacc 3300 ctacttctgt gacgtgtccc tcattcacct tcctctcttc cctataaata accacgcctc 3360 aggttctccg cttcacaact caaacattct ctccattggt ccttaaacac tcatcagtca 3420 tcaccgcggc cgcatgggaa cggaccaagg aaaaaccttc acctgggaag agctggcggc 3480 ccataacacc aaggacgacc tactcttggc catccgcggc agggtgtacg atgtcacaaa 3540 gttcttgagc cgccatcctg gtggagtgga cactctcctg ctcggagctg gccgagatgt 3600 tactccggtc tttgagatgt atcacgcgtt tggggctgca gatgccatta tgaagaagta 3660 ctatgtcggt acactggtct cgaatgagct gcccatcttc ccggagccaa cggtgttcca 3720 caaaaccatc aagacgagag tcgagggcta ctttacggat cggaacattg atcccaagaa 3780 tagaccagag atctggggac gatacgctct tatctttgga tccttgatcg cttcctacta 3840 cgcgcagctc tttgtgcctt tcgttgtcga acgcacatgg cttcaggtgg tgtttgcaat 3900 catcatggga tttgcgtgcg cacaagtcgg actcaaccct cttcatgatg cgtctcactt 3960 ttcagtgacc cacaacccca ctgtctggaa gattctggga gccacgcacg actttttcaa 4020 cggagcatcg tacctggtgt ggatgtacca acatatgctc ggccatcacc cctacaccaa 4080 cattgctgga gcagatcccg acgtgtcgac gtctgagccc gatgttcgtc gtatcaagcc 4140 caaccaaaag tggtttgtca accacatcaa ccagcacatg tttgttcctt tcctgtacgg 4200 actgctggcg ttcaaggtgc gcattcagga catcaacatt ttgtactttg tcaagaccaa 4260 tgacgctatt cgtgtcaatc ccatctcgac atggcacact gtgatgttct ggggcggcaa 4320 ggctttcttt gtctggtatc gcctgattgt tcccctgcag tatctgcccc tgggcaaggt 4380 gctgctcttg ttcacggtcg cggacatggt gtcgtcttac tggctggcgc tgaccttcca 4440 ggcgaaccac gttgttgagg aagttcagtg gccgttgcct gacgagaacg ggatcatcca 4500 aaaggactgg gcagctatgc aggtcgagac tacgcaggat tacgcacacg attcgcacct 4560 ctggaccagc atcactggca gcttgaacta ccaggctgtg caccatctgt tccccaacgt 4620 gtcgcagcac cattatcccg atattctggc catcatcaag aacacctgca gcgagtacaa 4680 ggttccatac cttgtcaagg atacgttttg gcaagcattt gcttcacatt tggagcactt 4740 gcgtgttctt ggactccgtc ccaaggaaga gtaggcggcc gcatttcgca ccaaatcaat 4800 gaaagtaata atgaaaagtc tgaataagaa tacttaggct tagatgcctt tgttacttgt 4860 gtaaaataac ttgagtcatg tacctttggc ggaaacagaa taaataaaag gtgaaattcc 4920 aatgctctat gtataagtta gtaatactta atgtgttcta cggttgtttc aatatcatca 4980 aactctaatt gaaactttag aaccacaaat ctcaatcttt tcttaatgaa atgaaaaatc 5040 ttaattgtac catgtttatg ttaaacacct tacaattggt tggagaggag gaccaaccga 5100 tgggacaaca ttgggagaaa gagattcaat ggagatttgg ataggagaac aacattcttt 5160 ttcacttcaa tacaagatga gtgcaacact aaggatatgt atgagacttt cagaagctac 5220 gacaacatag atgagtgagg tggtgattcc tagcaagaaa gacattagag gaagccaaaa 5280 tcgaacaagg aagacatcaa gggcaagaga caggaccatc catctcagga aaaggagctt 5340 tgggatagtc cgagaagttg tacaagaaat tttttggagg gtgagtgatg cattgctggt 5400 gactttaact caatcaaaat tgagaaagaa agaaaaggga gggggctcac atgtgaatag 5460 aagggaaacg ggagaatttt acagttttga tctaatgggc atcccagcta gtggtaacat 5520 attcaccatg tttaaccttc acgtacgtct agaggatccc c 5561 <210> SEQ ID NO 89 <211> LENGTH: 11889 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKR916 <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (7810)..(7810) <223> OTHER INFORMATION: n is a, c, g, or t <400> SEQUENCE: 89 ggagatccaa gcttttgatc catgcccttc atttgccgct tattaattaa tttggtaaca 60 gtccgtacta atcagttact tatccttccc ccatcataat taatcttggt agtctcgaat 120 gccacaacac tgactagtct cttggatcat aagaaaaagc caaggaacaa aagaagacaa 180 aacacaatga gagtatcctt tgcatagcaa tgtctaagtt cataaaattc aaacaaaaac 240 gcaatcacac acagtggaca tcacttatcc actagctgat caggatcgcc gcgtcaagaa 300 aaaaaaactg gaccccaaaa gccatgcaca acaacacgta ctcacaaagg tgtcaatcga 360 gcagcccaaa acattcacca actcaaccca tcatgagccc tcacatttgt tgtttctaac 420 ccaacctcaa actcgtattc tcttccgcca cctcattttt gtttatttca acacccgtca 480 aactgcatgc caccccgtgg ccaaatgtcc atgcatgtta acaagaccta tgactataaa 540 tagctgcaat ctcggcccag gttttcatca tcaagaacca gttcaatatc ctagtacacc 600 gtattaaaga atttaagata tactgcggcc gcaccatgga ggtggtgaat gaaatagtct 660 caattgggca ggaagtttta cccaaagttg attatgccca actctggagt gatgccagtc 720 actgtgaggt gctttacttg tccatcgcat ttgtcatctt gaagttcact cttggccccc 780 ttggtccaaa aggtcagtct cgtatgaagt ttgttttcac caattacaac cttctcatgt 840 ccatttattc gttgggatca ttcctctcaa tggcatatgc catgtacacc atcggtgtta 900 tgtctgacaa ctgcgagaag gcttttgaca acaacgtctt caggatcacc acgcagttgt 960 tctatttgag caagttcctg gagtatattg actccttcta tttgccactg atgggcaagc 1020 ctctgacctg gttgcaattc ttccatcatt tgggggcacc gatggatatg tggctgttct 1080 ataattaccg aaatgaagct gtttggattt ttgtgctgtt gaatggtttc atccactgga 1140 tcatgtacgg ttattattgg accagattga tcaagctgaa gttccccatg ccaaaatccc 1200 tgattacatc aatgcagatc attcaattca atgttggttt ctacattgtc tggaagtaca 1260 ggaacattcc ctgttatcgc caagatggga tgaggatgtt tggctggttc ttcaattact 1320 tttatgttgg cacagtcttg tgtttgttct tgaatttcta tgtgcaaacg tatatcgtca 1380 ggaagcacaa gggagccaaa aagattcagt gagcggccgc aagtatgaac taaaatgcat 1440 gtaggtgtaa gagctcatgg agagcatgga atattgtatc cgaccatgta acagtataat 1500 aactgagctc catctcactt cttctatgaa taaacaaagg atgttatgat atattaacac 1560 tctatctatg caccttattg ttctatgata aatttcctct tattattata aatcatctga 1620 atcgtgacgg cttatggaat gcttcaaata gtacaaaaac aaatgtgtac tataagactt 1680 tctaaacaat tctaacctta gcattgtgaa cgagacataa gtgttaagaa gacataacaa 1740 ttataatgga agaagtttgt ctccatttat atattatata ttacccactt atgtattata 1800 ttaggatgtt aaggagacat aacaattata aagagagaag tttgtatcca tttatatatt 1860 atatactacc catttatata ttatacttat ccacttattt aatgtcttta taaggtttga 1920 tccatgatat ttctaatatt ttagttgata tgtatatgaa agggtactat ttgaactctc 1980 ttactctgta taaaggttgg atcatcctta aagtgggtct atttaatttt attgcttctt 2040 acagataaaa aaaaaattat gagttggttt gataaaatat tgaaggattt aaaataataa 2100 taaataacat ataatatatg tatataaatt tattataata taacatttat ctataaaaaa 2160 gtaaatattg tcataaatct atacaatcgt ttagccttgc tggacgaatc tcaattattt 2220 aaacgagagt aaacatattt gactttttgg ttatttaaca aattattatt taacactata 2280 tgaaattttt ttttttatca gcaaagaata aaattaaatt aagaaggaca atggtgtccc 2340 aatccttata caaccaactt ccacaagaaa gtcaagtcag agacaacaaa aaaacaagca 2400 aaggaaattt tttaatttga gttgtcttgt ttgctgcata atttatgcag taaaacacta 2460 cacataaccc ttttagcagt agagcaatgg ttgaccgtgt gcttagcttc ttttatttta 2520 tttttttatc agcaaagaat aaataaaata aaatgagaca cttcagggat gtttcaacaa 2580 gcttggcgcg ccgttctata gtgtcaccta aatcgtatgt gtatgataca taaggttatg 2640 tattaattgt agccgcgttc taacgacaat atgtccatat ggtgcactct cagtacaatc 2700 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc 2760 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc 2820 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg 2880 atacgcctat ttttataggt taatgtcatg accaaaatcc cttaacgtga gttttcgttc 2940 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 3000 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 3060 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 3120 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 3180 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 3240 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 3300 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 3360 ctacagcgtg agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 3420 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 3480 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 3540 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 3600 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg 3660 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag 3720 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc 3780 gcgcgttggc cgattcatta atgcaggttg atcagatctc gatcccgcga aattaatacg 3840 actcactata gggagaccac aacggtttcc ctctagaaat aattttgttt aactttaaga 3900 aggagatata cccatggaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat 3960 cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc 4020 tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg 4080 tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga 4140 agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca 4200 gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc 4260 ggaggctatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt 4320 cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga 4380 tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca 4440 ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca 4500 cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga 4560 ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag 4620 gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct 4680 tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca 4740 gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat 4800 cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt 4860 ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg 4920 tccgagggca aaggaatagt gaggtacagc ttggatcgat ccggctgcta acaaagcccg 4980 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc 5040 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gatgatcggg 5100 cgcgccgtcg acggatccgt acgcaaaggc aaagatttaa actcgaaaac attacaaaag 5160 tctcaaaaca gaggcaaggc catgcacaaa gcacactcta agtgcttcca ttgcctacta 5220 agtagggtac gtacacgatc accattcacc agtgatgatc tttattaata tacaacacac 5280 tcagagacag cttatgttat agctagctag cataaactat cacatcatgt gttagtacga 5340 caagtgacaa cattgctttt aacttcgcgg ccttggatcc tctagaccgg atataatgag 5400 ccgtaaacaa agatgattaa gtagtaatta atacgtacta gtaaaagtgg caaaagataa 5460 cgagaaagaa ccaatttctt tgcattcggc cttagcggaa ggcatatata agctttgatt 5520 attttattta gtgtaatgat ttcgtacaac caaagcattt atttagtact ctcacacttg 5580 tgtcgcggcc gcgaattcac tagtgattcc ttatagagcc ttccccgcgg gttgcttctc 5640 cgccatccgg gcgaacaccg ccagatagcg cagcaggatg accaaccctt catggggcag 5700 cgggttccga tacggcaggt tgtgcttctg gcacagctgt tccacctggt agctaaccgc 5760 tgtcaggttg tggcgaggga gggtcggcca caaatggtgc tcaatctggt aattcaagcc 5820 tccgaaaaac caatctgtga taatccctcg ccgaatgttc atggtctcat ggatctggcc 5880 aaccgagaat ccatggccat cccagactga gtccccgatc ttctccagtg ggtagtggtt 5940 catgaacacc acgatcgcaa tgccgaagcc gccaaccagc tccgaaacga aaaacaccaa 6000 cagcgatgtg aggatgctgg gcataaagaa taagtggaac agggtcttca aggtccagtg 6060 cagggcgagg ccaatggcct ccttcttata ctgagagcga tagaattggt tatctctgtc 6120 cttcaaactg cgcacggtca acacgctctg gaaacaccaa atgaaccgca acaagataca 6180 gatgaccaag aaatagtact gctggaactg aatgagcttg cgggaaatcg gtgacgcccg 6240 tgtgacgtca tcctcagacc aggctaagag ggggaggttg tcaatatcag ggtcgtgccc 6300 ttgaacattg gttgccgaat gatgtgcatt gtgtctgtcc ttccaccatg tcacggaaaa 6360 accttgcaga ccattgccaa ataccagtcc cacgaggttg ttccagttcc ggttcttgaa 6420 agtctggtgg tggcaaatgt catgagaaag ccagcccatc tgttgatagt gcatcccaag 6480 caacactgcc ccaatgaaat acatctgata ctgaaccatc aggaaataac ccagcactcc 6540 aaggcccagt gtggtgctga ttttgtatga gtaccagagg ggggaggcat caaacatgcc 6600 agttgcgatc aactcttctc ggagcttccg gaaatcctct tgagcttcat tcactgcagc 6660 ctggggtggc aactcagaac tgggattgat tttgggcatg cgcttgagct tgtcgaaggc 6720 ttcttgagag tgcataacca tgaaggcatc agtggcatcc cttccttggt aattctctat 6780 aatttccgca ccaccagggt ggaaattgac ccaggcagac acatcatatg ttgttccatc 6840 aattgtaagg ggaagcgctt ggcgctttga cttcatttca atcgaattcc cgcggccgct 6900 tggggggcta tggaagactt tcttagttag ttgtgtgaat aagcaatgtt gggagaatcg 6960 ggactactta taggatagga ataaaacaga aaagtattaa gtgctaatga aatatttaga 7020 ctgataatta aaatcttcac gtatgtccac ttgatataaa aacgtcagga ataaaggaag 7080 tacagtagaa tttaaaggta ctctttttat atatacccgt gttctctttt tggctagcta 7140 gttgcataaa aaataatcta tatttttatc attattttaa atatcttatg agatggtaaa 7200 tatttatcat aatttttttt actattattt attatttgtg tgtgtaatac atatagaagt 7260 taattacaaa ttttatttac tttttcatta ttttgatatg attcaccatt aatttagtgt 7320 tattatttat aatagttcat tttaatcttt ttgtatatat tatgcgtgca gtactttttt 7380 cctacatata actactatta cattttattt atataatatt tttattaatg aattttcgtg 7440 ataatatgta atattgttca ttattatttc agatttttta aaaatatttg tgttattatt 7500 tatgaaatat gtaatttttt tagtatttga ttttatgatg ataaagtgtt ctaaattcaa 7560 aagaaggggg aaagcgtaaa cattaaaaaa cgtcatcaaa caaaaacaaa atcttgttaa 7620 taaagataaa actgtttgtt ttgatcactg ttatttcgta atataaaaac attatttata 7680 tttatattgt tgacaaccaa atttgcctat caaatctaac caatataatg catgcgtggc 7740 aggtaatgta ctaccatgaa cttaagtcat gacataataa accgtgaatc tgaccaatgc 7800 atgtacctan ctaaattgta tttgtgacac gaagcaaatg attcaattca caatggagat 7860 gggaaacaaa taatgaagaa cccagaacta agaaagcttt tctgaaaaat aaaataaagg 7920 caatgtcaaa agtatactgc atcatcagtc cagaaagcac atgatatttt tttatcagta 7980 tcaatgcagc tagttttatt ttacaatatc gatatagcta gtttaaatat attgcagcta 8040 gatttataaa tatttgtgtt attatttatc atttgtgtaa tcctgttttt agtattttag 8100 tttatatatg atgataatgt attccaaatt taaaagaagg gaaataaatt taaacaagaa 8160 aaaaagtcat caaacaaaaa acaaatgaaa gggtggaaag atgttaccat gtaatgtgaa 8220 tgttacagta tttcttttat tatagagtta acaaattaac taatatgatt ttgttaataa 8280 tgataaaata ttttttttat tattatttca taatataaaa atagtttact taatataaaa 8340 aaaattctat cgttcacaac aaagttggcc acctaattta accatgcatg tacccatgga 8400 ccatattagg taaccatcaa acctgatgaa gagataaaga gatgaagact taagtcataa 8460 cacaaaacca taaaaaacaa aaatacaatc aaccgtcaat ctgaccaatg catgaaaaag 8520 ctgcaatagt gagtggcgac acaaagcaca tgattttctt acaacggaga taaaaccaaa 8580 aaaatatttc atgaacaacc tagaacaaat aaagctttta tataataaat atataaataa 8640 ataaaggcta tggaataata tacttcaata tatttggatt aaataaattg ttggcggggt 8700 tgatatattt atacacacct aaagtcactt caatctcatt ttcacttaac ttttattttt 8760 tttttctttt tatttatcat aaagagaata ttgataatat actttttaac atatttttat 8820 gacatttttt attggtgaaa acttattaaa aatcataaat tttgtaagtt agatttattt 8880 aaagagttcc tcttcttatt ttaaattttt taataaattt ttaaataact aaaatttgtg 8940 ttaaaaatgt taaaaaatgt gttattaacc cttctcttcg aggacgtacg agatccggcc 9000 ggccagatcc tgcaggtctc aatagattaa gaagttggcg tctcattgat tgaccatggg 9060 ggatcctcta gacgtacgtg aaggttaaac atggtgaata tgttaccact agctgggatg 9120 cccattagat caaaactgta aaattctccc gtttcccttc tattcacatg tgagccccct 9180 cccttttctt tctttctcaa ttttgattga gttaaagtca ccagcaatgc atcactcacc 9240 ctccaaaaaa tttcttgtac aacttctcgg actatcccaa agctcctttt cctgagatgg 9300 atggtcctgt ctcttgccct tgatgtcttc cttgttcgat tttggcttcc tctaatgtct 9360 ttcttgctag gaatcaccac ctcactcatc tatgttgtcg tagcttctga aagtctcata 9420 catatcctta gtgttgcact catcttgtat tgaagtgaaa aagaatgttg ttctcctatc 9480 caaatctcca ttgaatctct ttctcccaat gttgtcccat cggttggtcc tcctctccaa 9540 ccaattgtaa ggtgtttaac ataaacatgg tacaattaag atttttcatt tcattaagaa 9600 aagattgaga tttgtggttc taaagtttca attagagttt gatgatattg aaacaaccgt 9660 agaacacatt aagtattact aacttataca tagagcattg gaatttcacc ttttatttat 9720 tctgtttccg ccaaaggtac atgactcaag ttattttaca caagtaacaa aggcatctaa 9780 gcctaagtat tcttattcag acttttcatt attactttca ttgatttggt gcgaaatgcg 9840 gccgcctact cttccttggg acggagtcca agaacacgca agtgctccaa atgtgaagca 9900 aatgcttgcc aaaacgtatc cttgacaagg tatggaacct tgtactcgct gcaggtgttc 9960 ttgatgatgg ccagaatatc gggataatgg tgctgcgaca cgttggggaa cagatggtgc 10020 acagcctggt agttcaagct gccagtgatg ctggtccaga ggtgcgaatc gtgtgcgtaa 10080 tcctgcgtag tctcgacctg catagctgcc cagtcctttt ggatgatccc gttctcgtca 10140 ggcaacggcc actgaacttc ctcaacaacg tggttcgcct ggaaggtcag cgccagccag 10200 taagacgaca ccatgtccgc gaccgtgaac aagagcagca ccttgcccag gggcagatac 10260 tgcaggggaa caatcaggcg ataccagaca aagaaagcct tgccgcccca gaacatcaca 10320 gtgtgccatg tcgagatggg attgacacga atagcgtcat tggtcttgac aaagtacaaa 10380 atgttgatgt cctgaatgcg caccttgaac gccagcagtc cgtacaggaa aggaacaaac 10440 atgtgctggt tgatgtggtt gacaaaccac ttttggttgg gcttgatacg acgaacatcg 10500 ggctcagacg tcgacacgtc gggatctgct ccagcaatgt tggtgtaggg gtgatggccg 10560 agcatatgtt ggtacatcca caccaggtac gatgctccgt tgaaaaagtc gtgcgtggct 10620 cccagaatct tccagacagt ggggttgtgg gtcactgaaa agtgagacgc atcatgaaga 10680 gggttgagtc cgacttgtgc gcacgcaaat cccatgatga ttgcaaacac cacctgaagc 10740 catgtgcgtt cgacaacgaa aggcacaaag agctgcgcgt agtaggaagc gatcaaggat 10800 ccaaagataa gagcgtatcg tccccagatc tctggtctat tcttgggatc aatgttccga 10860 tccgtaaagt agccctcgac tctcgtcttg atggttttgt ggaacaccgt tggctccggg 10920 aagatgggca gctcattcga gaccagtgta ccgacatagt acttcttcat aatggcatct 10980 gcagccccaa acgcgtgata catctcaaag accggagtaa catctcggcc agctccgagc 11040 aggagagtgt ccactccacc aggatggcgg ctcaagaact ttgtgacatc gtacaccctg 11100 ccgcggatgg ccaagagtag gtcgtccttg gtgttatggg ccgccagctc ttcccaggtg 11160 aaggtttttc cttggtccgt tcccatgcgg ccgcggtgat gactgatgag tgtttaagga 11220 ccaatggaga gaatgtttga gttgtgaagc ggagaacctg aggcgtggtt atttataggg 11280 aagagaggaa ggtgaatgag ggacacgtca cagaagtagg gtgctgagct tgagacattc 11340 ttcagtatgc atggctatgg aagccttggg tgctacacct catgaagttc atggtgtgag 11400 gtggcttcgg catctcaatt aagtgacaaa gagaaaggtg tttcagtgtt tctattgcaa 11460 atggcagaaa ctcgtgatga cgaggggacc atgcatggtt tcatttcttt tcttcctgga 11520 ttctttcttt ccttttatat atgcaggttc ataatttaaa aattagactc gctttcaatt 11580 tcttaatttc tcattttcct cttatattac tgtactaatg ttaaccacgt acacttattt 11640 tttttttagt ttaattttga tagattgtgt tgatttaaac atattaatat tttcaaccaa 11700 ataaaaatca ttttagtaga tacggctttt taaataatta ttaaaaatat taactattta 11760 tcctaaatgg cacattttaa ttaaaaaaaa tccggtgttg taagtgtttt attaatttgt 11820 tttggcatta ttaaagcaac ttttttttta tttgttggca ttttgagtac gtacttaggc 11880 tagcctgca 11889 <210> SEQ ID NO 90 <211> LENGTH: 5661 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKR974 <400> SEQUENCE: 90 gtacgtctag aggatccccc atggtcaatc aatgagacgc caacttctta atctattgag 60 acctgcaggt ctagaagggc ggatccccgg gtaccgagct cgaattcact ggccgtcgtt 120 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat 180 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag 240 ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc 300 ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta 360 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg 420 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca 480 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt 540 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc 600 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa 660 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc 720 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa 780 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa 840 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg 900 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa 960 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc 1020 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc 1080 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta 1140 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag 1200 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca 1260 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata 1320 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc 1380 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca 1440 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca 1500 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg 1560 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 1620 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 1680 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 1740 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 1800 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 1860 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac 1920 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 1980 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 2040 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 2100 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 2160 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 2220 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 2280 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 2340 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc 2400 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 2460 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa 2520 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 2580 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 2640 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 2700 tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcaggctagc 2760 ctaagtacgt actcaaaatg ccaacaaata aaaaaaaagt tgctttaata atgccaaaac 2820 aaattaataa aacacttaca acaccggatt ttttttaatt aaaatgtgcc atttaggata 2880 aatagttaat atttttaata attatttaaa aagccgtatc tactaaaatg atttttattt 2940 ggttgaaaat attaatatgt ttaaatcaac acaatctatc aaaattaaac taaaaaaaaa 3000 ataagtgtac gtggttaaca ttagtacagt aatataagag gaaaatgaga aattaagaaa 3060 ttgaaagcga gtctaatttt taaattatga acctgcatat ataaaaggaa agaaagaatc 3120 caggaagaaa agaaatgaaa ccatgcatgg tcccctcgtc atcacgagtt tctgccattt 3180 gcaatagaaa cactgaaaca cctttctctt tgtcacttaa ttgagatgcc gaagccacct 3240 cacaccatga acttcatgag gtgtagcacc caaggcttcc atagccatgc atactgaaga 3300 atgtctcaag ctcagcaccc tacttctgtg acgtgtccct cattcacctt cctctcttcc 3360 ctataaataa ccacgcctca ggttctccgc ttcacaactc aaacattctc tccattggtc 3420 cttaaacact catcagtcat caccgcggcc gccaattcat ggccccgcag acggagctcc 3480 gccagcgcca cgccgccgtc gccgagacgc cggtggccgg caagaaggcc tttacatggc 3540 aggaggtcgc gcagcacaac acggcggcct cggcctggat cattatccgc ggcaaggtct 3600 acgacgtgac cgagtgggcc aacaagcacc ccggcggccg cgagatggtg ctgctgcacg 3660 ccggtcgcga ggccaccgac acgttcgact cgtaccaccc gttcagcgac aaggccgagt 3720 cgatcttgaa caagtatgag attggcacgt tcacgggccc gtccgagttt ccgaccttca 3780 agccggacac gggcttctac aaggagtgcc gcaagcgcgt tggcgagtac ttcaagaaga 3840 acaacctcca tccgcaggac ggcttcccgg gcctctggcg catgatggtc gtgtttgcgg 3900 tcgccggcct cgccttgtac ggcatgcact tttcgactat ctttgcgctg cagctcgcgg 3960 ccgcggcgct ctttggcgtc tgccaggcgc tgccgctgct ccacgtcatg cacgactcgt 4020 cgcacgcgtc gtacaccaac atgccgttct tccattacgt cgtcggccgc tttgccatgg 4080 actggtttgc cggcggctcg atggtgtcat ggctcaacca gcacgtcgtg ggccaccaca 4140 tctacacgaa cgtcgcgggc tcggacccgg atcttccggt caacatggac ggcgacatcc 4200 gccgcatcgt gaaccgccag gtgttccagc ccatgtacgc attccagcac atctaccttc 4260 cgccgctcta tggcgtgctt ggcctcaagt tccgcatcca ggacttcacc gacacgttcg 4320 gctcgcacac gaacggcccg atccgcgtca acccgcacgc gctctcgacg tggatggcca 4380 tgatcagctc caagtcgttc tgggccttct accgcgtgta ccttccgctt gccgtgctcc 4440 agatgcccat caagacgtac cttgcgatct tcttcctcgc cgagtttgtc acgggctggt 4500 acctcgcgtt caacttccaa gtaagccatg tctcgaccga gtgcggctac ccatgcggcg 4560 acgaggccaa gatggcgctc caggacgagt gggcagtctc gcaggtcaag acgtcggtcg 4620 actacgccca tggctcgtgg atgacgacgt tccttgccgg cgcgctcaac taccaggtcg 4680 tgcaccactt gttccccagc gtgtcgcagt accactaccc ggcgatcgcg cccatcatcg 4740 tcgacgtctg caaggagtac aacatcaagt acgccatctt gccggacttt acggcggcgt 4800 tcgttgccca cttgaagcac ctccgcaaca tgggccagca gggcatcgcc gccacgatcc 4860 acatgggcta actcgagctc agctagatcg cggccgcatt tcgcaccaaa tcaatgaaag 4920 taataatgaa aagtctgaat aagaatactt aggcttagat gcctttgtta cttgtgtaaa 4980 ataacttgag tcatgtacct ttggcggaaa cagaataaat aaaaggtgaa attccaatgc 5040 tctatgtata agttagtaat acttaatgtg ttctacggtt gtttcaatat catcaaactc 5100 taattgaaac tttagaacca caaatctcaa tcttttctta atgaaatgaa aaatcttaat 5160 tgtaccatgt ttatgttaaa caccttacaa ttaattggtt ggagaggagg accaaccgat 5220 gggacaacat tgggagaaag agattcaatg gagatttgga taggagaaca acattctttt 5280 tcacttcaat acaagatgag tgcaacacta aggatatgta tgagactttc agaagctacg 5340 acaacataga tgagtgaggt ggtgattcct agcaagaaag acattagagg aagccaaaat 5400 cgaacaagga agacatcaag ggcaagagac aggaccatcc atctcaggaa aaggagcttt 5460 gggatagtcc gagaagttgt acaagaaatt ttttggaggg tgagtgatgc attgctggtg 5520 actttaactc aatcaaaatt gagaaagaaa gaaaagggag ggggctcaca tgtgaataga 5580 agggaaacgg gagaatttta cagttttgat ctaatgggca tcccagctag tggtaacata 5640 ttcaccatgt ttaaccttca c 5661 <210> SEQ ID NO 91 <211> LENGTH: 1413 <212> TYPE: DNA <213> ORGANISM: Saprolegnia diclina <400> SEQUENCE: 91 atggccccgc agacggagct ccgccagcgc cacgccgccg tcgccgagac gccggtggcc 60 ggcaagaagg cctttacatg gcaggaggtc gcgcagcaca acacggcggc ctcggcctgg 120 atcattatcc gcggcaaggt ctacgacgtg accgagtggg ccaacaagca ccccggcggc 180 cgcgagatgg tgctgctgca cgccggtcgc gaggccaccg acacgttcga ctcgtaccac 240 ccgttcagcg acaaggccga gtcgatcttg aacaagtatg agattggcac gttcacgggc 300 ccgtccgagt ttccgacctt caagccggac acgggcttct acaaggagtg ccgcaagcgc 360 gttggcgagt acttcaagaa gaacaacctc catccgcagg acggcttccc gggcctctgg 420 cgcatgatgg tcgtgtttgc ggtcgccggc ctcgccttgt acggcatgca cttttcgact 480 atctttgcgc tgcagctcgc ggccgcggcg ctctttggcg tctgccaggc gctgccgctg 540 ctccacgtca tgcacgactc gtcgcacgcg tcgtacacca acatgccgtt cttccattac 600 gtcgtcggcc gctttgccat ggactggttt gccggcggct cgatggtgtc atggctcaac 660 cagcacgtcg tgggccacca catctacacg aacgtcgcgg gctcggaccc ggatcttccg 720 gtcaacatgg acggcgacat ccgccgcatc gtgaaccgcc aggtgttcca gcccatgtac 780 gcattccagc acatctacct tccgccgctc tatggcgtgc ttggcctcaa gttccgcatc 840 caggacttca ccgacacgtt cggctcgcac acgaacggcc cgatccgcgt caacccgcac 900 gcgctctcga cgtggatggc catgatcagc tccaagtcgt tctgggcctt ctaccgcgtg 960 taccttccgc ttgccgtgct ccagatgccc atcaagacgt accttgcgat cttcttcctc 1020 gccgagtttg tcacgggctg gtacctcgcg ttcaacttcc aagtaagcca tgtctcgacc 1080 gagtgcggct acccatgcgg cgacgaggcc aagatggcgc tccaggacga gtgggcagtc 1140 tcgcaggtca agacgtcggt cgactacgcc catggctcgt ggatgacgac gttccttgcc 1200 ggcgcgctca actaccaggt cgtgcaccac ttgttcccca gcgtgtcgca gtaccactac 1260 ccggcgatcg cgcccatcat cgtcgacgtc tgcaaggagt acaacatcaa gtacgccatc 1320 ttgccggact ttacggcggc gttcgttgcc cacttgaagc acctccgcaa catgggccag 1380 cagggcatcg ccgccacgat ccacatgggc taa 1413 <210> SEQ ID NO 92 <211> LENGTH: 5621 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKR1033 <400> SEQUENCE: 92 ggccgcattt cgcaccaaat caatgaaagt aataatgaaa agtctgaata agaatactta 60 ggcttagatg cctttgttac ttgtgtaaaa taacttgagt catgtacctt tggcggaaac 120 agaataaata aaaggtgaaa ttccaatgct ctatgtataa gttagtaata cttaatgtgt 180 tctacggttg tttcaatatc atcaaactct aattgaaact ttagaaccac aaatctcaat 240 cttttcttaa tgaaatgaaa aatcttaatt gtaccatgtt tatgttaaac accttacaat 300 taattggttg gagaggagga ccaaccgatg ggacaacatt gggagaaaga gattcaatgg 360 agatttggat aggagaacaa cattcttttt cacttcaata caagatgagt gcaacactaa 420 ggatatgtat gagactttca gaagctacga caacatagat gagtgaggtg gtgattccta 480 gcaagaaaga cattagagga agccaaaatc gaacaaggaa gacatcaagg gcaagagaca 540 ggaccatcca tctcaggaaa aggagctttg ggatagtccg agaagttgta caagaaattt 600 tttggagggt gagtgatgca ttgctggtga ctttaactca atcaaaattg agaaagaaag 660 aaaagggagg gggctcacat gtgaatagaa gggaaacggg agaattttac agttttgatc 720 taatgggcat cccagctagt ggtaacatat tcaccatgtt taaccttcac gtacgtctag 780 aggatccccc atggtcaatc aatgagacgc caacttctta atctattgag acctgcaggt 840 ctagaagggc ggatccccgg gtaccgagct cgaattcact ggccgtcgtt ttacaacgtc 900 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg 960 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc 1020 tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 1080 accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc 1140 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 1200 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 1260 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga 1320 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta 1380 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat 1440 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc 1500 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga 1560 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca 1620 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt 1680 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg 1740 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc 1800 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata 1860 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt 1920 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag 1980 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca 2040 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg 2100 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg 2160 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag 2220 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg 2280 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag 2340 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga 2400 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt 2460 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc 2520 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc 2580 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac 2640 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac 2700 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt 2760 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct 2820 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat 2880 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt 2940 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg 3000 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt 3060 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt 3120 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg 3180 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg 3240 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc 3300 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg 3360 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac 3420 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag 3480 gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcaggctagc ctaagtacgt 3540 actcaaaatg ccaacaaata aaaaaaaagt tgctttaata atgccaaaac aaattaataa 3600 aacacttaca acaccggatt ttttttaatt aaaatgtgcc atttaggata aatagttaat 3660 atttttaata attatttaaa aagccgtatc tactaaaatg atttttattt ggttgaaaat 3720 attaatatgt ttaaatcaac acaatctatc aaaattaaac taaaaaaaaa ataagtgtac 3780 gtggttaaca ttagtacagt aatataagag gaaaatgaga aattaagaaa ttgaaagcga 3840 gtctaatttt taaattatga acctgcatat ataaaaggaa agaaagaatc caggaagaaa 3900 agaaatgaaa ccatgcatgg tcccctcgtc atcacgagtt tctgccattt gcaatagaaa 3960 cactgaaaca cctttctctt tgtcacttaa ttgagatgcc gaagccacct cacaccatga 4020 acttcatgag gtgtagcacc caaggcttcc atagccatgc atactgaaga atgtctcaag 4080 ctcagcaccc tacttctgtg acgtgtccct cattcacctt cctctcttcc ctataaataa 4140 ccacgcctca ggttctccgc ttcacaactc aaacattctc tccattggtc cttaaacact 4200 catcagtcat caccgcggcc gcaaaccatg gctccagatg cggacaagtt gagacagcgc 4260 aaggcgcaat cgattcaaga cacggctgat tcgcaagcta ccgaactcaa gattggcacc 4320 ctgaagggct tgcaggggac agaaatcgtc attgatggag acatttacga tataaaagac 4380 tttgatcacc ccggtggtga atccatcatg acttttgggg gaaacgatgt caccgccacg 4440 tacaagatga tccaccccta ccactctaag caccatttgg agaagatgaa gaaagtggga 4500 cgagttccgg actacacctc ggaatacaag tttgatactc cctttgagcg tgaaatcaag 4560 caagaggtct tcaagattgt gcgacgaggc cgcgagtttg gaacacctgg atacttcttc 4620 cgggctttct gctacattgg acttttcttt tacttgcagt atttgtgggt cacgactccc 4680 actacctttg ccttggcgat cttctatggt gtttcgcaag ctttcattgg tttgaacgta 4740 caacatgatg ccaaccacgg agctgcctcc aagaagcctt ggatcaataa cttgctagga 4800 ttgggggctg actttatcgg aggttccaaa tggttgtgga tgaaccagca ctggacgcac 4860 cacacataca ccaaccacca tgagaaggat cccgatgcct tgggcgctga accaatgttg 4920 ttgttcaatg attatccctt gggtcaccca aagcgtactt tgattcacca cttccaggcc 4980 ttctattacc ttttcgtctt ggccggatac tgggtctctt cggtcttcaa ccctcaaatt 5040 ttggacttgc aacaccgcgg tgctcaagcg gttggaatga aaatggagaa cgattacatt 5100 gccaaaagcc gaaagtatgc catcttcttg cgtctcttgt atatttacac caacattgtc 5160 gctccgatcc aaaaccaagg cttctcgttg accgtggtcg cccacatttt gaccatgggc 5220 gtcgcttcca gtttgacttt ggcgactctt tttgccttgt cgcacaattt tgaaaacgcg 5280 gatcgcgatc ccacttacga ggcccgcaag ggaggagagc ctgtttgttg gttcaagtcg 5340 caagtcgaaa cctcgtcaac ttacggaggt ttcatctcgg gttgcttgac gggcggactc 5400 aacttccaag tggaacacca cttgttccct cgtatgagtt cggcctggta cccctacatt 5460 gcccctactg ttcgagaggt ttgcaaaaag cacggagtca agtacgcata ctatccctgg 5520 gtctggcaaa acttgatttc aactgtcaag tatctgcatc aaagcggaac tggatccaac 5580 tggaagaatg gcgccaaccc ctactcggga aaattgtaag c 5621 <210> SEQ ID NO 93 <211> LENGTH: 11949 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKR1038 <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (7810)..(7810) <223> OTHER INFORMATION: n is a, c, g, or t <400> SEQUENCE: 93 ggagatccaa gcttttgatc catgcccttc atttgccgct tattaattaa tttggtaaca 60 gtccgtacta atcagttact tatccttccc ccatcataat taatcttggt agtctcgaat 120 gccacaacac tgactagtct cttggatcat aagaaaaagc caaggaacaa aagaagacaa 180 aacacaatga gagtatcctt tgcatagcaa tgtctaagtt cataaaattc aaacaaaaac 240 gcaatcacac acagtggaca tcacttatcc actagctgat caggatcgcc gcgtcaagaa 300 aaaaaaactg gaccccaaaa gccatgcaca acaacacgta ctcacaaagg tgtcaatcga 360 gcagcccaaa acattcacca actcaaccca tcatgagccc tcacatttgt tgtttctaac 420 ccaacctcaa actcgtattc tcttccgcca cctcattttt gtttatttca acacccgtca 480 aactgcatgc caccccgtgg ccaaatgtcc atgcatgtta acaagaccta tgactataaa 540 tagctgcaat ctcggcccag gttttcatca tcaagaacca gttcaatatc ctagtacacc 600 gtattaaaga atttaagata tactgcggcc gcaccatgga ggtggtgaat gaaatagtct 660 caattgggca ggaagtttta cccaaagttg attatgccca actctggagt gatgccagtc 720 actgtgaggt gctttacttg tccatcgcat ttgtcatctt gaagttcact cttggccccc 780 ttggtccaaa aggtcagtct cgtatgaagt ttgttttcac caattacaac cttctcatgt 840 ccatttattc gttgggatca ttcctctcaa tggcatatgc catgtacacc atcggtgtta 900 tgtctgacaa ctgcgagaag gcttttgaca acaacgtctt caggatcacc acgcagttgt 960 tctatttgag caagttcctg gagtatattg actccttcta tttgccactg atgggcaagc 1020 ctctgacctg gttgcaattc ttccatcatt tgggggcacc gatggatatg tggctgttct 1080 ataattaccg aaatgaagct gtttggattt ttgtgctgtt gaatggtttc atccactgga 1140 tcatgtacgg ttattattgg accagattga tcaagctgaa gttccccatg ccaaaatccc 1200 tgattacatc aatgcagatc attcaattca atgttggttt ctacattgtc tggaagtaca 1260 ggaacattcc ctgttatcgc caagatggga tgaggatgtt tggctggttc ttcaattact 1320 tttatgttgg cacagtcttg tgtttgttct tgaatttcta tgtgcaaacg tatatcgtca 1380 ggaagcacaa gggagccaaa aagattcagt gagcggccgc aagtatgaac taaaatgcat 1440 gtaggtgtaa gagctcatgg agagcatgga atattgtatc cgaccatgta acagtataat 1500 aactgagctc catctcactt cttctatgaa taaacaaagg atgttatgat atattaacac 1560 tctatctatg caccttattg ttctatgata aatttcctct tattattata aatcatctga 1620 atcgtgacgg cttatggaat gcttcaaata gtacaaaaac aaatgtgtac tataagactt 1680 tctaaacaat tctaacctta gcattgtgaa cgagacataa gtgttaagaa gacataacaa 1740 ttataatgga agaagtttgt ctccatttat atattatata ttacccactt atgtattata 1800 ttaggatgtt aaggagacat aacaattata aagagagaag tttgtatcca tttatatatt 1860 atatactacc catttatata ttatacttat ccacttattt aatgtcttta taaggtttga 1920 tccatgatat ttctaatatt ttagttgata tgtatatgaa agggtactat ttgaactctc 1980 ttactctgta taaaggttgg atcatcctta aagtgggtct atttaatttt attgcttctt 2040 acagataaaa aaaaaattat gagttggttt gataaaatat tgaaggattt aaaataataa 2100 taaataacat ataatatatg tatataaatt tattataata taacatttat ctataaaaaa 2160 gtaaatattg tcataaatct atacaatcgt ttagccttgc tggacgaatc tcaattattt 2220 aaacgagagt aaacatattt gactttttgg ttatttaaca aattattatt taacactata 2280 tgaaattttt ttttttatca gcaaagaata aaattaaatt aagaaggaca atggtgtccc 2340 aatccttata caaccaactt ccacaagaaa gtcaagtcag agacaacaaa aaaacaagca 2400 aaggaaattt tttaatttga gttgtcttgt ttgctgcata atttatgcag taaaacacta 2460 cacataaccc ttttagcagt agagcaatgg ttgaccgtgt gcttagcttc ttttatttta 2520 tttttttatc agcaaagaat aaataaaata aaatgagaca cttcagggat gtttcaacaa 2580 gcttggcgcg ccgttctata gtgtcaccta aatcgtatgt gtatgataca taaggttatg 2640 tattaattgt agccgcgttc taacgacaat atgtccatat ggtgcactct cagtacaatc 2700 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc 2760 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc 2820 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg 2880 atacgcctat ttttataggt taatgtcatg accaaaatcc cttaacgtga gttttcgttc 2940 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 3000 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 3060 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 3120 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 3180 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 3240 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 3300 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 3360 ctacagcgtg agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 3420 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 3480 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 3540 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 3600 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg 3660 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag 3720 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc 3780 gcgcgttggc cgattcatta atgcaggttg atcagatctc gatcccgcga aattaatacg 3840 actcactata gggagaccac aacggtttcc ctctagaaat aattttgttt aactttaaga 3900 aggagatata cccatggaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat 3960 cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc 4020 tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg 4080 tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga 4140 agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca 4200 gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc 4260 ggaggctatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt 4320 cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga 4380 tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca 4440 ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca 4500 cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga 4560 ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag 4620 gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct 4680 tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca 4740 gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat 4800 cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt 4860 ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg 4920 tccgagggca aaggaatagt gaggtacagc ttggatcgat ccggctgcta acaaagcccg 4980 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc 5040 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gatgatcggg 5100 cgcgccgtcg acggatccgt acgcaaaggc aaagatttaa actcgaaaac attacaaaag 5160 tctcaaaaca gaggcaaggc catgcacaaa gcacactcta agtgcttcca ttgcctacta 5220 agtagggtac gtacacgatc accattcacc agtgatgatc tttattaata tacaacacac 5280 tcagagacag cttatgttat agctagctag cataaactat cacatcatgt gttagtacga 5340 caagtgacaa cattgctttt aacttcgcgg ccttggatcc tctagaccgg atataatgag 5400 ccgtaaacaa agatgattaa gtagtaatta atacgtacta gtaaaagtgg caaaagataa 5460 cgagaaagaa ccaatttctt tgcattcggc cttagcggaa ggcatatata agctttgatt 5520 attttattta gtgtaatgat ttcgtacaac caaagcattt atttagtact ctcacacttg 5580 tgtcgcggcc gcgaattcac tagtgattcc ttatagagcc ttccccgcgg gttgcttctc 5640 cgccatccgg gcgaacaccg ccagatagcg cagcaggatg accaaccctt catggggcag 5700 cgggttccga tacggcaggt tgtgcttctg gcacagctgt tccacctggt agctaaccgc 5760 tgtcaggttg tggcgaggga gggtcggcca caaatggtgc tcaatctggt aattcaagcc 5820 tccgaaaaac caatctgtga taatccctcg ccgaatgttc atggtctcat ggatctggcc 5880 aaccgagaat ccatggccat cccagactga gtccccgatc ttctccagtg ggtagtggtt 5940 catgaacacc acgatcgcaa tgccgaagcc gccaaccagc tccgaaacga aaaacaccaa 6000 cagcgatgtg aggatgctgg gcataaagaa taagtggaac agggtcttca aggtccagtg 6060 cagggcgagg ccaatggcct ccttcttata ctgagagcga tagaattggt tatctctgtc 6120 cttcaaactg cgcacggtca acacgctctg gaaacaccaa atgaaccgca acaagataca 6180 gatgaccaag aaatagtact gctggaactg aatgagcttg cgggaaatcg gtgacgcccg 6240 tgtgacgtca tcctcagacc aggctaagag ggggaggttg tcaatatcag ggtcgtgccc 6300 ttgaacattg gttgccgaat gatgtgcatt gtgtctgtcc ttccaccatg tcacggaaaa 6360 accttgcaga ccattgccaa ataccagtcc cacgaggttg ttccagttcc ggttcttgaa 6420 agtctggtgg tggcaaatgt catgagaaag ccagcccatc tgttgatagt gcatcccaag 6480 caacactgcc ccaatgaaat acatctgata ctgaaccatc aggaaataac ccagcactcc 6540 aaggcccagt gtggtgctga ttttgtatga gtaccagagg ggggaggcat caaacatgcc 6600 agttgcgatc aactcttctc ggagcttccg gaaatcctct tgagcttcat tcactgcagc 6660 ctggggtggc aactcagaac tgggattgat tttgggcatg cgcttgagct tgtcgaaggc 6720 ttcttgagag tgcataacca tgaaggcatc agtggcatcc cttccttggt aattctctat 6780 aatttccgca ccaccagggt ggaaattgac ccaggcagac acatcatatg ttgttccatc 6840 aattgtaagg ggaagcgctt ggcgctttga cttcatttca atcgaattcc cgcggccgct 6900 tggggggcta tggaagactt tcttagttag ttgtgtgaat aagcaatgtt gggagaatcg 6960 ggactactta taggatagga ataaaacaga aaagtattaa gtgctaatga aatatttaga 7020 ctgataatta aaatcttcac gtatgtccac ttgatataaa aacgtcagga ataaaggaag 7080 tacagtagaa tttaaaggta ctctttttat atatacccgt gttctctttt tggctagcta 7140 gttgcataaa aaataatcta tatttttatc attattttaa atatcttatg agatggtaaa 7200 tatttatcat aatttttttt actattattt attatttgtg tgtgtaatac atatagaagt 7260 taattacaaa ttttatttac tttttcatta ttttgatatg attcaccatt aatttagtgt 7320 tattatttat aatagttcat tttaatcttt ttgtatatat tatgcgtgca gtactttttt 7380 cctacatata actactatta cattttattt atataatatt tttattaatg aattttcgtg 7440 ataatatgta atattgttca ttattatttc agatttttta aaaatatttg tgttattatt 7500 tatgaaatat gtaatttttt tagtatttga ttttatgatg ataaagtgtt ctaaattcaa 7560 aagaaggggg aaagcgtaaa cattaaaaaa cgtcatcaaa caaaaacaaa atcttgttaa 7620 taaagataaa actgtttgtt ttgatcactg ttatttcgta atataaaaac attatttata 7680 tttatattgt tgacaaccaa atttgcctat caaatctaac caatataatg catgcgtggc 7740 aggtaatgta ctaccatgaa cttaagtcat gacataataa accgtgaatc tgaccaatgc 7800 atgtacctan ctaaattgta tttgtgacac gaagcaaatg attcaattca caatggagat 7860 gggaaacaaa taatgaagaa cccagaacta agaaagcttt tctgaaaaat aaaataaagg 7920 caatgtcaaa agtatactgc atcatcagtc cagaaagcac atgatatttt tttatcagta 7980 tcaatgcagc tagttttatt ttacaatatc gatatagcta gtttaaatat attgcagcta 8040 gatttataaa tatttgtgtt attatttatc atttgtgtaa tcctgttttt agtattttag 8100 tttatatatg atgataatgt attccaaatt taaaagaagg gaaataaatt taaacaagaa 8160 aaaaagtcat caaacaaaaa acaaatgaaa gggtggaaag atgttaccat gtaatgtgaa 8220 tgttacagta tttcttttat tatagagtta acaaattaac taatatgatt ttgttaataa 8280 tgataaaata ttttttttat tattatttca taatataaaa atagtttact taatataaaa 8340 aaaattctat cgttcacaac aaagttggcc acctaattta accatgcatg tacccatgga 8400 ccatattagg taaccatcaa acctgatgaa gagataaaga gatgaagact taagtcataa 8460 cacaaaacca taaaaaacaa aaatacaatc aaccgtcaat ctgaccaatg catgaaaaag 8520 ctgcaatagt gagtggcgac acaaagcaca tgattttctt acaacggaga taaaaccaaa 8580 aaaatatttc atgaacaacc tagaacaaat aaagctttta tataataaat atataaataa 8640 ataaaggcta tggaataata tacttcaata tatttggatt aaataaattg ttggcggggt 8700 tgatatattt atacacacct aaagtcactt caatctcatt ttcacttaac ttttattttt 8760 tttttctttt tatttatcat aaagagaata ttgataatat actttttaac atatttttat 8820 gacatttttt attggtgaaa acttattaaa aatcataaat tttgtaagtt agatttattt 8880 aaagagttcc tcttcttatt ttaaattttt taataaattt ttaaataact aaaatttgtg 8940 ttaaaaatgt taaaaaatgt gttattaacc cttctcttcg aggacgtacg agatccggcc 9000 ggccagatcc tgcaggtctc aatagattaa gaagttggcg tctcattgat tgaccatggg 9060 ggatcctcta gacgtacgtg aaggttaaac atggtgaata tgttaccact agctgggatg 9120 cccattagat caaaactgta aaattctccc gtttcccttc tattcacatg tgagccccct 9180 cccttttctt tctttctcaa ttttgattga gttaaagtca ccagcaatgc atcactcacc 9240 ctccaaaaaa tttcttgtac aacttctcgg actatcccaa agctcctttt cctgagatgg 9300 atggtcctgt ctcttgccct tgatgtcttc cttgttcgat tttggcttcc tctaatgtct 9360 ttcttgctag gaatcaccac ctcactcatc tatgttgtcg tagcttctga aagtctcata 9420 catatcctta gtgttgcact catcttgtat tgaagtgaaa aagaatgttg ttctcctatc 9480 caaatctcca ttgaatctct ttctcccaat gttgtcccat cggttggtcc tcctctccaa 9540 ccaattaatt gtaaggtgtt taacataaac atggtacaat taagattttt catttcatta 9600 agaaaagatt gagatttgtg gttctaaagt ttcaattaga gtttgatgat attgaaacaa 9660 ccgtagaaca cattaagtat tactaactta tacatagagc attggaattt caccttttat 9720 ttattctgtt tccgccaaag gtacatgact caagttattt tacacaagta acaaaggcat 9780 ctaagcctaa gtattcttat tcagactttt cattattact ttcattgatt tggtgcgaaa 9840 tgcggccgct tacaattttc ccgagtaggg gttggcgcca ttcttccagt tggatccagt 9900 tccgctttga tgcagatact tgacagttga aatcaagttt tgccagaccc agggatagta 9960 tgcgtacttg actccgtgct ttttgcaaac ctctcgaaca gtaggggcaa tgtaggggta 10020 ccaggccgaa ctcatacgag ggaacaagtg gtgttccact tggaagttga gtccgcccgt 10080 caagcaaccc gagatgaaac ctccgtaagt tgacgaggtt tcgacttgcg acttgaacca 10140 acaaacaggc tctcctccct tgcgggcctc gtaagtggga tcgcgatccg cgttttcaaa 10200 attgtgcgac aaggcaaaaa gagtcgccaa agtcaaactg gaagcgacgc ccatggtcaa 10260 aatgtgggcg accacggtca acgagaagcc ttggttttgg atcggagcga caatgttggt 10320 gtaaatatac aagagacgca agaagatggc atactttcgg cttttggcaa tgtaatcgtt 10380 ctccattttc attccaaccg cttgagcacc gcggtgttgc aagtccaaaa tttgagggtt 10440 gaagaccgaa gagacccagt atccggccaa gacgaaaagg taatagaagg cctggaagtg 10500 gtgaatcaaa gtacgctttg ggtgacccaa gggataatca ttgaacaaca acattggttc 10560 agcgcccaag gcatcgggat ccttctcatg gtggttggtg tatgtgtggt gcgtccagtg 10620 ctggttcatc cacaaccatt tggaacctcc gataaagtca gcccccaatc ctagcaagtt 10680 attgatccaa ggcttcttgg aggcagctcc gtggttggca tcatgttgta cgttcaaacc 10740 aatgaaagct tgcgaaacac catagaagat cgccaaggca aaggtagtgg gagtcgtgac 10800 ccacaaatac tgcaagtaaa agaaaagtcc aatgtagcag aaagcccgga agaagtatcc 10860 aggtgttcca aactcgcggc ctcgtcgcac aatcttgaag acctcttgct tgatttcacg 10920 ctcaaaggga gtatcaaact tgtattccga ggtgtagtcc ggaactcgtc ccactttctt 10980 catcttctcc aaatggtgct tagagtggta ggggtggatc atcttgtacg tggcggtgac 11040 atcgtttccc ccaaaagtca tgatggattc accaccgggg tgatcaaagt cttttatatc 11100 gtaaatgtct ccatcaatga cgatttctgt cccctgcaag cccttcaggg tgccaatctt 11160 gagttcggta gcttgcgaat cagccgtgtc ttgaatcgat tgcgccttgc gctgtctcaa 11220 cttgtccgca tctggagcca tggtttgcgg ccgcggtgat gactgatgag tgtttaagga 11280 ccaatggaga gaatgtttga gttgtgaagc ggagaacctg aggcgtggtt atttataggg 11340 aagagaggaa ggtgaatgag ggacacgtca cagaagtagg gtgctgagct tgagacattc 11400 ttcagtatgc atggctatgg aagccttggg tgctacacct catgaagttc atggtgtgag 11460 gtggcttcgg catctcaatt aagtgacaaa gagaaaggtg tttcagtgtt tctattgcaa 11520 atggcagaaa ctcgtgatga cgaggggacc atgcatggtt tcatttcttt tcttcctgga 11580 ttctttcttt ccttttatat atgcaggttc ataatttaaa aattagactc gctttcaatt 11640 tcttaatttc tcattttcct cttatattac tgtactaatg ttaaccacgt acacttattt 11700 tttttttagt ttaattttga tagattgtgt tgatttaaac atattaatat tttcaaccaa 11760 ataaaaatca ttttagtaga tacggctttt taaataatta ttaaaaatat taactattta 11820 tcctaaatgg cacattttaa ttaaaaaaaa tccggtgttg taagtgtttt attaatttgt 11880 tttggcatta ttaaagcaac ttttttttta tttgttggca ttttgagtac gtacttaggc 11940 tagcctgca 11949 <210> SEQ ID NO 94 <211> LENGTH: 8671 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKR328 <400> SEQUENCE: 94 ggatctggcc ggccggatct cgtacggatc cgtcgacggc gcgcccgatc atccggatat 60 agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa ggggttatgc 120 tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt tgttagcagc 180 cggatcgatc caagctgtac ctcactattc ctttgccctc ggacgagtgc tggggcgtcg 240 gtttccacta tcggcgagta cttctacaca gccatcggtc cagacggccg cgcttctgcg 300 ggcgatttgt gtacgcccga cagtcccggc tccggatcgg acgattgcgt cgcatcgacc 360 ctgcgcccaa gctgcatcat cgaaattgcc gtcaaccaag ctctgataga gttggtcaag 420 accaatgcgg agcatatacg cccggagccg cggcgatcct gcaagctccg gatgcctccg 480 ctcgaagtag cgcgtctgct gctccataca agccaaccac ggcctccaga agaagatgtt 540 ggcgacctcg tattgggaat ccccgaacat cgcctcgctc cagtcaatga ccgctgttat 600 gcggccattg tccgtcagga cattgttgga gccgaaatcc gcgtgcacga ggtgccggac 660 ttcggggcag tcctcggccc aaagcatcag ctcatcgaga gcctgcgcga cggacgcact 720 gacggtgtcg tccatcacag tttgccagtg atacacatgg ggatcagcaa tcgcgcatat 780 gaaatcacgc catgtagtgt attgaccgat tccttgcggt ccgaatgggc cgaacccgct 840 cgtctggcta agatcggccg cagcgatcgc atccatagcc tccgcgaccg gctgcagaac 900 agcgggcagt tcggtttcag gcaggtcttg caacgtgaca ccctgtgcac ggcgggagat 960 gcaataggtc aggctctcgc tgaattcccc aatgtcaagc acttccggaa tcgggagcgc 1020 ggccgatgca aagtgccgat aaacataacg atctttgtag aaaccatcgg cgcagctatt 1080 tacccgcagg acatatccac gccctcctac atcgaagctg aaagcacgag attcttcgcc 1140 ctccgagagc tgcatcaggt cggagacgct gtcgaacttt tcgatcagaa acttctcgac 1200 agacgtcgcg gtgagttcag gcttttccat gggtatatct ccttcttaaa gttaaacaaa 1260 attatttcta gagggaaacc gttgtggtct ccctatagtg agtcgtatta atttcgcggg 1320 atcgagatcg atccaattcc aatcccacaa aaatctgagc ttaacagcac agttgctcct 1380 ctcagagcag aatcgggtat tcaacaccct catatcaact actacgttgt gtataacggt 1440 ccacatgccg gtatatacga tgactggggt tgtacaaagg cggcaacaaa cggcgttccc 1500 ggagttgcac acaagaaatt tgccactatt acagaggcaa gagcagcagc tgacgcgtac 1560 acaacaagtc agcaaacaga caggttgaac ttcatcccca aaggagaagc tcaactcaag 1620 cccaagagct ttgctaaggc cctaacaagc ccaccaaagc aaaaagccca ctggctcacg 1680 ctaggaacca aaaggcccag cagtgatcca gccccaaaag agatctcctt tgccccggag 1740 attacaatgg acgatttcct ctatctttac gatctaggaa ggaagttcga aggtgaaggt 1800 gacgacacta tgttcaccac tgataatgag aaggttagcc tcttcaattt cagaaagaat 1860 gctgacccac agatggttag agaggcctac gcagcaggtc tcatcaagac gatctacccg 1920 agtaacaatc tccaggagat caaatacctt cccaagaagg ttaaagatgc agtcaaaaga 1980 ttcaggacta attgcatcaa gaacacagag aaagacatat ttctcaagat cagaagtact 2040 attccagtat ggacgattca aggcttgctt cataaaccaa ggcaagtaat agagattgga 2100 gtctctaaaa aggtagttcc tactgaatct aaggccatgc atggagtcta agattcaaat 2160 cgaggatcta acagaactcg ccgtgaagac tggcgaacag ttcatacaga gtcttttacg 2220 actcaatgac aagaagaaaa tcttcgtcaa catggtggag cacgacactc tggtctactc 2280 caaaaatgtc aaagatacag tctcagaaga ccaaagggct attgagactt ttcaacaaag 2340 gataatttcg ggaaacctcc tcggattcca ttgcccagct atctgtcact tcatcgaaag 2400 gacagtagaa aaggaaggtg gctcctacaa atgccatcat tgcgataaag gaaaggctat 2460 cattcaagat gcctctgccg acagtggtcc caaagatgga cccccaccca cgaggagcat 2520 cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa gtggattgat gtgacatctc 2580 cactgacgta agggatgacg cacaatccca ctatccttcg caagaccctt cctctatata 2640 aggaagttca tttcatttgg agaggacacg ctcgagctca tttctctatt acttcagcca 2700 taacaaaaga actcttttct cttcttatta aaccatgaaa aagcctgaac tcaccgcgac 2760 gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc 2820 ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg 2880 ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc 2940 ggccgcgctc ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta 3000 ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc 3060 cgctgttctg cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca 3120 gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga 3180 tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac 3240 cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc 3300 cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg 3360 ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt 3420 cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt 3480 cgagcggagg catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat 3540 tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc 3600 gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat 3660 cgcccgcaga agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg 3720 aaaccgacgc cccagcactc gtccgagggc aaaggaatag tgaggtacct aaagaaggag 3780 tgcgtcgaag cagatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgtt 3840 gccggtcttg cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt 3900 aacatgtaat gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta 3960 tacatttaat acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgc 4020 gcggtgtcat ctatgttact agatcgatgt cgaatcgatc aacctgcatt aatgaatcgg 4080 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 4140 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 4200 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 4260 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 4320 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 4380 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 4440 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc 4500 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 4560 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 4620 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 4680 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 4740 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 4800 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 4860 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 4920 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgacattaa cctataaaaa 4980 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg 5040 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca 5100 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc 5160 atcagagcag attgtactga gagtgcacca tatggacata ttgtcgttag aacgcggcta 5220 caattaatac ataaccttat gtatcataca catacgattt aggtgacact atagaacggc 5280 gcgccaagct tggatctcct gcagcccggg ggatccgccc acgtacggta ccatctgcta 5340 atattttaaa tcacatgcaa gagaggaggc atggttccat tttctacctt cacattattt 5400 gagaaaaacg aacttgttct gtgttttatt tttgcccttc acattagtac aacgtggaag 5460 actcatggtt acacagaatc atacataagt acaatgcttg tccctaagaa aacaagcact 5520 cgttgtattg aacctttacg gctcatgcgg ccgcgaattc actagtgatt gaattcgcgg 5580 ccgcttagtc cgacttggcc ttggcggccg cggccgactc tttgagcgtg aagatctgcg 5640 ccgtctcggg cacagcgccg tagttgacaa agaggtgcgc ggtcttgaag aaggccgtga 5700 tgatgggctc gtcgttcctg cgcacgaggt gcgggtacgc ggccgcaaag tgcttggtgg 5760 cttcgttgag cttgtagtgc ggaatgatcg ggaacaagtg gtggacctgg tgcgtgccaa 5820 tgtggtggct caggttgtcc acgaacgcgc cgtacgagcg gtcgacgctc gagaggttgc 5880 ccttgacgta cgtccactcc gagtcgccgt accacggcgt cgcttcgtcg ttgtggtgca 5940 agaaggtcgt aatgacgagg aacgaagcaa agacaaagag cggcgcatag tagtagaggc 6000 ccatgacggc aaagccgagc gagtatgtga ggtacgcgta cgcggcgaag aaggcggccc 6060 agacgccgag cgacacgatg acggccgacg cgcggcgaag gaggagcggg tcccacgggt 6120 caaagtggct catcgtgcgc ggggcatacc cgaccttcaa gtagacaaac cacgcaccgc 6180 cgagcgtgta gacccattgg cgcacgtcct ggaggtcctt gaccgaccgg tgcgggtaaa 6240 agatctcgtc cttatcaatg ttgcccgtgt tcttgtggtg gtggcggtgc gtcacgcgcc 6300 agctctcgaa cggcgtcaaa atcgcagagt gcatgatgca gccgatgata aagttgacgc 6360 tgtggtagcg cgagaaggcc gagtggccgc agtcgtggcc gaccgtgaag aagccccaga 6420 agatgacgcc ctgcacgtag atgtaggtgg cgcaaacgag cgcgtggagc agaacgttat 6480 cggcaatgaa cggcgtcgag cgcgccgcgt agagcagcgc cgccgaggcc gacgcgttga 6540 agatcgcgcg ggccgtgtag tagagcgaga ggccgaggtt cgactcaaag cacgcgttcg 6600 ggatcgagtg cttgagctcc gtgagcgtcg ggaactcgac cttcgtctta tcctcagtca 6660 tgcggccgct gaagtattgc ttcttagtta acctttcctt tctctctcag ctatgtgaat 6720 tcattttgct ttcgtcacaa tttatatagt gaaattggat ctttggagtt aacgccttca 6780 caggattatc gtgttagaac aatgcttttt catgttctaa ttagtagtac attacaaatg 6840 tgcactctat tcaataagca tcttttggca cgttaataaa tcatgtgaaa aaaaaatact 6900 actatttcaa agaaagtgtt gtaaaaagaa acggaaagag agctggcttc agttgttgag 6960 acttgtttgc tagtaaaaat ggtgtgaaga gtgattcatg gtgaggtggt ttttcgtccc 7020 tttctgtttg catgaaaaac aaatggcaag agatgacgta ggattccttc ccttaacgat 7080 tatctgtttt taatttcaaa tatacatata ggaatttatg aattactaag gttgtaaaat 7140 atgctggtca tttatttatg gctaaaatat ttttttttct cgtaaatata aaaatattta 7200 aaatttattt ttatcatatt ttttatcctt ataaaattat gtgtacaacc tatataaaaa 7260 aatatcatat ttaatattga ttatatgttt aatcaatata aaaaatcatt atcatatatt 7320 tagatttatt cgaatataca tctaaacaaa aaataacata ttttaatttt atgaagaaaa 7380 aaaaatattt tatcctttat ttatttaaga ttaattaata gttatgtatt gtggaaagac 7440 ttttacacat gcaatagata tactgaatca attagatgcc aatgctgagt tggaaatcac 7500 ttgaggaggg gaggagactt gccaatgctt ttcagtttca tttaaatgat ttagtggagg 7560 agatagagta gtgataaagg catgccccaa ttttggagtg tatatatgag tggaaataag 7620 agagggatag agagaaaaaa taaagagagt aaaaataatt aatgtgaaat gatatgataa 7680 aaaaataaag aaagagataa agagaaaaat gaaatgagag atagatgaaa tagagagtag 7740 atacatgttt gtttaggttt tttttaggaa ataacacatt tttttctcat cacttattac 7800 tcactgtcaa tttcctctct ttcaatcata atgatatgat ttgtttaaca aaaatgtgaa 7860 aaaacatata aagtaaaata tttttataaa ttgataaata aaaatttaca aaatttattt 7920 cttattaaat tgaatagaaa atgaaagaaa agaaaagaaa aagtatatat aaaatgatat 7980 agctttaaaa agaataaatt tttcatatca gtcttttttt aataatttag aaatatttaa 8040 gtatatagca aaaatataat gtactttaca tatgcataaa taataatttg aaaatagaac 8100 taatagaata gagaaaaaag taatataata attaactata tgaaaattta gaagggacaa 8160 tatttttaat taagaatata aacaatattt cttttcatgt aatgagggac ggatgtacgg 8220 ggccagtgtt ggagtcaaag ccaaaatagt cacggggaaa ttaatgcact gcatgactat 8280 tcgaaaaaat tcactagcct tacttagatg ttagattaat agctaggggg tgcagataat 8340 tttgaaaggc atgaaaaaca ttaatttgta cattgcaagc ttttgatgac aagctttgca 8400 attgttcaca ctaccttatg ccatttataa atagagtgat tggcatatga aggaaatcat 8460 gagagtcgaa gcgaaaaaca aagcttgaga gtgtaggaaa aatacagttt ttttggtaaa 8520 aatacagtat ttgaatagga gcgaaaaata tcctttcaaa atgatccttt tctttttttt 8580 tttttttctt gttgttcttg gtcagttatt caaaggaaaa gggattgaaa taaaaacttg 8640 catgtgggat cgtacgtcga gtcgacctgc a 8671

1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 94 <210> SEQ ID NO 1 <211> LENGTH: 1392 <212> TYPE: DNA <213> ORGANISM: Peridinium sp. CCMP626 <220> FEATURE: <221> NAME/KEY: misc_feature <223> OTHER INFORMATION: delta-5 desaturase <400> SEQUENCE: 1 atggctccag atgcggacaa gttgagacag cgcaaggcgc aatcgattca agacacggct 60 gattcgcaag ctaccgaact caagattggc accctgaagg gcttgcaggg gacagaaatc 120 gtcattgatg gagacattta cgatataaaa gactttgatc accccggtgg tgaatccatc 180 atgacttttg ggggaaacga tgtcaccgcc acgtacaaga tgatccaccc ctaccactct 240 aagcaccatt tggagaagat gaagaaagtg ggacgagttc cggactacac ctcggaatac 300 aagtttgata ctccctttga gcgtgaaatc aagcaagagg tcttcaagat tgtgcgacga 360 ggccgcgagt ttggaacacc tggatacttc ttccgggctt tctgctacat tggacttttc 420 ttttacttgc agtatttgtg ggtcacgact cccactacct ttgccttggc gatcttctat 480 ggtgtttcgc aagctttcat tggtttgaac gtacaacatg atgccaacca cggagctgcc 540 tccaagaagc cttggatcaa taacttgcta ggattggggg ctgactttat cggaggttcc 600 aaatggttgt ggatgaacca gcactggacg caccacacat acaccaacca ccatgagaag 660 gatcccgatg ccttgggcgc tgaaccaatg ttgttgttca atgattatcc cttgggtcac 720 ccaaagcgta ctttgattca ccacttccag gccttctatt accttttcgt cttggccgga 780 tactgggtct cttcggtctt caaccctcaa attttggact tgcaacaccg cggtgctcaa 840 gcggttggaa tgaaaatgga gaacgattac attgccaaaa gccgaaagta tgccatcttc 900 ttgcgtctct tgtatattta caccaacatt gtcgctccga tccaaaacca aggcttctcg 960 ttgaccgtgg tcgcccacat tttgaccatg ggcgtcgctt ccagtttgac tttggcgact 1020 ctttttgcct tgtcgcacaa ttttgaaaac gcggatcgcg atcccactta cgaggcccgc 1080 aagggaggag agcctgtttg ttggttcaag tcgcaagtcg aaacctcgtc aacttacgga 1140 ggtttcatct cgggttgctt gacgggcgga ctcaacttcc aagtggaaca ccacttgttc 1200 cctcgtatga gttcggcctg gtacccctac attgccccta ctgttcgaga ggtttgcaaa 1260 aagcacggag tcaagtacgc atactatccc tgggtctggc aaaacttgat ttcaactgtc 1320 aagtatctgc atcaaagcgg aactggatcc aactggaaga atggcgccaa cccctactcg 1380 ggaaaattgt aa 1392 <210> SEQ ID NO 2 <211> LENGTH: 463 <212> TYPE: PRT <213> ORGANISM: Peridinium sp. CCMP626 <400> SEQUENCE: 2 Met Ala Pro Asp Ala Asp Lys Leu Arg Gln Arg Lys Ala Gln Ser Ile 1 5 10 15 Gln Asp Thr Ala Asp Ser Gln Ala Thr Glu Leu Lys Ile Gly Thr Leu 20 25 30 Lys Gly Leu Gln Gly Thr Glu Ile Val Ile Asp Gly Asp Ile Tyr Asp 35 40 45 Ile Lys Asp Phe Asp His Pro Gly Gly Glu Ser Ile Met Thr Phe Gly 50 55 60 Gly Asn Asp Val Thr Ala Thr Tyr Lys Met Ile His Pro Tyr His Ser 65 70 75 80 Lys His His Leu Glu Lys Met Lys Lys Val Gly Arg Val Pro Asp Tyr 85 90 95 Thr Ser Glu Tyr Lys Phe Asp Thr Pro Phe Glu Arg Glu Ile Lys Gln 100 105 110 Glu Val Phe Lys Ile Val Arg Arg Gly Arg Glu Phe Gly Thr Pro Gly 115 120 125 Tyr Phe Phe Arg Ala Phe Cys Tyr Ile Gly Leu Phe Phe Tyr Leu Gln 130 135 140 Tyr Leu Trp Val Thr Thr Pro Thr Thr Phe Ala Leu Ala Ile Phe Tyr 145 150 155 160 Gly Val Ser Gln Ala Phe Ile Gly Leu Asn Val Gln His Asp Ala Asn 165 170 175 His Gly Ala Ala Ser Lys Lys Pro Trp Ile Asn Asn Leu Leu Gly Leu 180 185 190 Gly Ala Asp Phe Ile Gly Gly Ser Lys Trp Leu Trp Met Asn Gln His 195 200 205 Trp Thr His His Thr Tyr Thr Asn His His Glu Lys Asp Pro Asp Ala 210 215 220 Leu Gly Ala Glu Pro Met Leu Leu Phe Asn Asp Tyr Pro Leu Gly His 225 230 235 240 Pro Lys Arg Thr Leu Ile His His Phe Gln Ala Phe Tyr Tyr Leu Phe 245 250 255 Val Leu Ala Gly Tyr Trp Val Ser Ser Val Phe Asn Pro Gln Ile Leu 260 265 270 Asp Leu Gln His Arg Gly Ala Gln Ala Val Gly Met Lys Met Glu Asn 275 280 285 Asp Tyr Ile Ala Lys Ser Arg Lys Tyr Ala Ile Phe Leu Arg Leu Leu 290 295 300 Tyr Ile Tyr Thr Asn Ile Val Ala Pro Ile Gln Asn Gln Gly Phe Ser 305 310 315 320 Leu Thr Val Val Ala His Ile Leu Thr Met Gly Val Ala Ser Ser Leu 325 330 335 Thr Leu Ala Thr Leu Phe Ala Leu Ser His Asn Phe Glu Asn Ala Asp 340 345 350 Arg Asp Pro Thr Tyr Glu Ala Arg Lys Gly Gly Glu Pro Val Cys Trp 355 360 365 Phe Lys Ser Gln Val Glu Thr Ser Ser Thr Tyr Gly Gly Phe Ile Ser 370 375 380 Gly Cys Leu Thr Gly Gly Leu Asn Phe Gln Val Glu His His Leu Phe 385 390 395 400 Pro Arg Met Ser Ser Ala Trp Tyr Pro Tyr Ile Ala Pro Thr Val Arg 405 410 415 Glu Val Cys Lys Lys His Gly Val Lys Tyr Ala Tyr Tyr Pro Trp Val 420 425 430 Trp Gln Asn Leu Ile Ser Thr Val Lys Tyr Leu His Gln Ser Gly Thr 435 440 445 Gly Ser Asn Trp Lys Asn Gly Ala Asn Pro Tyr Ser Gly Lys Leu 450 455 460 <210> SEQ ID NO 3 <211> LENGTH: 1392 <212> TYPE: DNA <213> ORGANISM: Peridinium sp. CCMP626 <220> FEATURE: <221> NAME/KEY: misc_feature <223> OTHER INFORMATION: synthetic delta-5 desaturase (codon-optimized) for Yarrowia lipolytica <400> SEQUENCE: 3 atggctcccg acgccgacaa gctgcgacag cgaaaggctc agtccatcca ggacactgcc 60 gattctcagg ctaccgagct caagattggc accctgaagg gtctccaagg caccgagatc 120 gtcattgatg gcgacatcta cgacatcaaa gacttcgatc accctggagg cgaatccatc 180 atgacctttg gtggcaacga cgttactgcc acctacaaga tgattcatcc ctaccactcg 240 aagcatcacc tggagaagat gaaaaaggtc ggtcgagtgc ccgactacac ctccgagtac 300 aagttcgata ctcccttcga acgagagatc aaacaggagg tcttcaagat tgtgcgaaga 360 ggtcgagagt ttggaacacc tggctacttc tttcgagcct tctgctacat cggtctcttc 420 ttttacctgc agtatctctg ggttaccact cctaccactt tcgcccttgc tatcttctac 480 ggtgtgtctc aggccttcat tggcctgaac gtccagcacg acgccaacca cggagctgcc 540 tccaaaaagc cctggatcaa caatttgctc ggcctgggtg ccgactttat cggaggctcc 600 aagtggctct ggatgaacca gcactggacc catcacactt acaccaacca tcacgagaag 660 gatcccgacg ccctgggtgc agagcctatg ctgctcttca acgactatcc cttgggtcac 720 cccaagcgaa ccctcattca tcacttccaa gccttctact atctgtttgt ccttgctggc 780 tactgggtgt cttcggtgtt caaccctcag atcctggacc tccagcaccg aggtgcccag 840 gctgtcggca tgaagatgga gaacgactac attgccaagt ctcgaaagta cgctatcttc 900 ctgcgactcc tgtacatcta caccaacatt gtggctccca tccagaacca aggcttttcg 960 ctcaccgtcg ttgctcacat tcttactatg ggtgtcgcct ccagcctgac cctcgctact 1020 ctgttcgccc tctcccacaa cttcgagaac gcagatcggg atcccaccta cgaggctcga 1080 aagggaggcg agcctgtctg ttggttcaag tcgcaggtgg aaacctcctc tacttacggt 1140 ggcttcattt ccggttgcct tacaggcgga ctcaactttc aggtcgagca tcacctgttt 1200 cctcgaatgt cctctgcctg gtacccctac atcgctccta ccgttcgaga ggtctgcaaa 1260 aagcacggcg tcaagtacgc ctactatccc tgggtgtggc agaacctcat ctcgaccgtc 1320 aagtacctgc atcagtccgg aactggctcg aactggaaga acggtgccaa tccctactct 1380 ggcaagctgt aa 1392 <210> SEQ ID NO 4 <211> LENGTH: 563 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: 563 bp fragment of pT-12-D5 <400> SEQUENCE: 4 ggtcatcatg tttataccaa ccaccatgag aaggatcccg atgccttggg cgctgaacca 60 atgttgttgt tcaatgatta tcccttgggt cacccaaagc gtactttgat tcaccacttc 120 caggccttct attacctttt cgtcttggcc ggatactggg tctcttcggt cttcaaccct 180 caaattttgg acttgcaaca ccgcggtgct caagcggttg gaatgaaaat ggagaacgat 240 tacattgcca aaagccgaaa gtatgccatc ttcttgcgtc tcttgtatat ttacaccaac 300 attgtcgctc cgatccaaaa ccaaggcttc tcgttgaccg tggtcgccca cattttgacc 360 atgggcgtcg cttccagttt gactttggcg actctttttg cctcgtcgca caattttgaa 420 aacgcggatc gcgatcccac ttacgaggcc cgcaaggggg gagagcctgt ttgttggttc 480 aagtcgcaag tcgaaacctc gtcaacttac ggaggtttca tctcgggttg cttgacgggc 540 ggactcaact tccaagtatc aca 563

<210> SEQ ID NO 5 <211> LENGTH: 187 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: translation of 563 bp fragment of pT-12-D5 <400> SEQUENCE: 5 Gly His His Val Tyr Thr Asn His His Glu Lys Asp Pro Asp Ala Leu 1 5 10 15 Gly Ala Glu Pro Met Leu Leu Phe Asn Asp Tyr Pro Leu Gly His Pro 20 25 30 Lys Arg Thr Leu Ile His His Phe Gln Ala Phe Tyr Tyr Leu Phe Val 35 40 45 Leu Ala Gly Tyr Trp Val Ser Ser Val Phe Asn Pro Gln Ile Leu Asp 50 55 60 Leu Gln His Arg Gly Ala Gln Ala Val Gly Met Lys Met Glu Asn Asp 65 70 75 80 Tyr Ile Ala Lys Ser Arg Lys Tyr Ala Ile Phe Leu Arg Leu Leu Tyr 85 90 95 Ile Tyr Thr Asn Ile Val Ala Pro Ile Gln Asn Gln Gly Phe Ser Leu 100 105 110 Thr Val Val Ala His Ile Leu Thr Met Gly Val Ala Ser Ser Leu Thr 115 120 125 Leu Ala Thr Leu Phe Ala Ser Ser His Asn Phe Glu Asn Ala Asp Arg 130 135 140 Asp Pro Thr Tyr Glu Ala Arg Lys Gly Gly Glu Pro Val Cys Trp Phe 145 150 155 160 Lys Ser Gln Val Glu Thr Ser Ser Thr Tyr Gly Gly Phe Ile Ser Gly 165 170 175 Cys Leu Thr Gly Gly Leu Asn Phe Gln Val Ser 180 185 <210> SEQ ID NO 6 <211> LENGTH: 693 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: 693 bp fragment of pT-RD5-5'C2 <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (10)..(10) <223> OTHER INFORMATION: n is a, c, g, or t <400> SEQUENCE: 6 aatcgtcatn gatggagaca tttacgatat aaaagacttt gatcaccccg gtggtgaatc 60 catcatgact tttgggggaa acgatgtcac cgccacgtac aagatgatcc acccctacca 120 ctctaagcac catttggaga agatgaagaa agtgggacga gttccggact acacctcgga 180 atacaagttt gatactccct ttgagcgtga aatcaagyaa gaggtcttca agattgtgcg 240 acgaggccgc gagtttggaa cacctggata cttcttccgg gctttctgct acattggact 300 tttcttttac ttgcagtatt tgtgggtcac gactcccact acctttgcct tggcgatctt 360 ctatggtgtt tcgcaagctt tcattggttt gaacgtacaa catgatgcca accacggagc 420 tgcctccaag aagccttgga tcaataactt gctaggattg ggggctgact ttatcggagg 480 ttccaaatgg ttgtggatga accagcactg gacgcaccac acatacacca accaccatga 540 gaaggatccc gatgccttgg gcgctgaacc aatgttgttg ttcaatgatt atcccttggg 600 tcacccaaag cgtactttga ttcaccactt ccaggccttc tattaccttt tcgtcttggc 660 cggatactgg gtctcttcgg tcttcaaccc tca 693 <210> SEQ ID NO 7 <211> LENGTH: 511 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: 511 bp 5' extended fragment of pT-RD5-5'C2 <400> SEQUENCE: 7 aatcgtcatt gatggagaca tttacgatat aaaagacttt gatcaccccg gtggtgaatc 60 catcatgact tttgggggaa acgatgtcac cgccacgtac aagatgatcc acccctacca 120 ctctaagcac catttggaga agatgaagaa agtgggacga gttccggact acacctcgga 180 atacaagttt gatactccct ttgagcgtga aatcaagyaa gaggtcttca agattgtgcg 240 acgaggccgc gagtttggaa cacctggata cttcttccgg gctttctgct acattggact 300 tttcttttac ttgcagtatt tgtgggtcac gactcccact acctttgcct tggcgatctt 360 ctatggtgtt tcgcaagctt tcattggttt gaacgtacaa catgatgcca accacggagc 420 tgcctccaag aagccttgga tcaataactt gctaggattg ggggctgact ttatcggagg 480 ttccaaatgg ttgtggatga accagcactg g 511 <210> SEQ ID NO 8 <211> LENGTH: 358 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: 358 bp fragment of pT-RD5-5'2nd <400> SEQUENCE: 8 gatttctttc gttggcattt ttcgttggga aagactcttg caacgatggc tccagatgcg 60 gacaagttga gacagcgcaa ggcgcaatcg attcaagaca cggctgattc gcaagctacc 120 gaactcaaga ttggcaccct gaagggcttg caggggacag aaatcgtcat tgatggagac 180 atttacgata taaaagactt tgatcacccc ggtggtgaat ccatcatgac ttttggagga 240 aacgatgtca ctgccacgta caagatgatc cacccctacc actctaagca ccatttggag 300 aagatgaaga aagtgggacg agttctggac tacacctcgg aatacaagtt tgatactc 358 <210> SEQ ID NO 9 <211> LENGTH: 161 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: 161 bp 5' extended fragment of pT-RD5-5'2nd <400> SEQUENCE: 9 gatttctttc gttggcattt ttcgttggga aagactcttg caacgatggc tccagatgcg 60 gacaagttga gacagcgcaa ggcgcaatcg attcaagaca cggctgattc gcaagctacc 120 gaactcaaga ttggcaccct gaagggcttg caggggacag a 161 <210> SEQ ID NO 10 <211> LENGTH: 299 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: 299 bp fragment of pT-RD5-3' <400> SEQUENCE: 10 gaggtttcat ctcgggttgt ttgacgggcg gactcaactt tcaagtggaa caccacttgt 60 tccctcgtat gagttcggcc tggtacccct acattgcccc tgctgttcga gaggtttgca 120 aaaagcacgg agtcaagtac gcatactatc cctgggtctg gcaaaacttg atttcaactg 180 tcaagtatct gcatcaaagc ggaactggat ccaactggaa gaatggcgcc aacccctact 240 cgggaaaatt gtaaatgaat tctagtcaag atgggtcact gcattcaaaa aaaaaaaaa 299 <210> SEQ ID NO 11 <211> LENGTH: 247 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: 247 bp of 3' downstream sequence of pT-RD5-3' <400> SEQUENCE: 11 ccacttgttc cctcgtatga gttcggcctg gtacccctac attgcccctg ctgttcgaga 60 ggtttgcaaa aagcacggag tcaagtacgc atactatccc tgggtctggc aaaacttgat 120 ttcaactgtc aagtatctgc atcaaagcgg aactggatcc aactggaaga atggcgccaa 180 cccctactcg ggaaaattgt aaatgaattc tagtcaagat gggtcactgc attcaaaaaa 240 aaaaaaa 247 <210> SEQ ID NO 12 <211> LENGTH: 456 <212> TYPE: PRT <213> ORGANISM: Pythium irregulare <220> FEATURE: <221> NAME/KEY: MISC_FEATURE <223> OTHER INFORMATION: delta-5 desaturase <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: GenBank Accession No. AAL13311 <400> SEQUENCE: 12 Met Gly Thr Asp Gln Gly Lys Thr Phe Thr Trp Gln Glu Val Ala Lys 1 5 10 15 His Asn Thr Ala Lys Ser Ala Trp Val Ile Ile Arg Gly Glu Val Tyr 20 25 30 Asp Val Thr Glu Trp Ala Asp Lys His Pro Gly Gly Ser Glu Leu Ile 35 40 45 Val Leu His Ser Gly Arg Glu Cys Thr Asp Thr Phe Tyr Ser Tyr His 50 55 60 Pro Phe Ser Asn Arg Ala Asp Lys Ile Leu Ala Lys Tyr Lys Ile Gly 65 70 75 80 Lys Leu Val Gly Gly Tyr Glu Phe Pro Val Phe Lys Pro Asp Ser Gly 85 90 95 Phe Tyr Lys Glu Cys Ser Glu Arg Val Ala Glu Tyr Phe Lys Thr Asn 100 105 110 Asn Leu Asp Pro Lys Ala Ala Phe Ala Gly Leu Trp Arg Met Val Phe 115 120 125 Val Phe Ala Val Ala Ala Leu Ala Tyr Met Gly Met Asn Glu Leu Ile 130 135 140 Pro Gly Asn Val Tyr Ala Gln Tyr Ala Trp Gly Val Val Phe Gly Val 145 150 155 160 Phe Gln Ala Leu Pro Leu Leu His Val Met His Asp Ser Ser His Ala 165 170 175 Ala Cys Ser Ser Ser Pro Ala Met Trp Gln Ile Ile Gly Arg Gly Val 180 185 190 Met Asp Trp Phe Ala Gly Ala Ser Met Val Ser Trp Leu Asn Gln His 195 200 205 Val Val Gly His His Ile Tyr Thr Asn Val Ala Gly Ala Asp Pro Asp 210 215 220 Leu Pro Val Asp Phe Glu Ser Asp Val Arg Arg Ile Val His Arg Gln 225 230 235 240 Val Leu Leu Pro Ile Tyr Lys Phe Gln His Ile Tyr Leu Pro Pro Leu

245 250 255 Tyr Gly Val Leu Gly Leu Lys Phe Arg Ile Gln Asp Val Phe Glu Thr 260 265 270 Phe Val Ser Leu Thr Asn Gly Pro Val Arg Val Asn Pro His Pro Val 275 280 285 Ser Asp Trp Val Gln Met Ile Phe Ala Lys Ala Phe Trp Thr Phe Tyr 290 295 300 Arg Ile Tyr Ile Pro Leu Val Trp Leu Lys Ile Thr Pro Ser Thr Phe 305 310 315 320 Trp Gly Val Phe Phe Leu Ala Glu Phe Thr Thr Gly Trp Tyr Leu Ala 325 330 335 Phe Asn Phe Gln Val Ser His Val Ser Thr Glu Cys Glu Tyr Pro Cys 340 345 350 Gly Asp Ala Pro Ser Ala Glu Val Gly Asp Glu Trp Ala Ile Ser Gln 355 360 365 Val Lys Ser Ser Val Asp Tyr Ala His Gly Ser Pro Leu Ala Ala Phe 370 375 380 Leu Cys Gly Ala Leu Asn Tyr Gln Val Thr His His Leu Tyr Pro Gly 385 390 395 400 Ile Ser Gln Tyr His Tyr Pro Ala Ile Ala Pro Ile Ile Ile Asp Val 405 410 415 Cys Lys Lys Tyr Asn Ile Lys Tyr Thr Val Leu Pro Thr Phe Thr Glu 420 425 430 Ala Leu Leu Ala His Phe Lys His Leu Lys Asn Met Gly Glu Leu Gly 435 440 445 Lys Pro Val Glu Ile His Met Gly 450 455 <210> SEQ ID NO 13 <211> LENGTH: 477 <212> TYPE: PRT <213> ORGANISM: Phytophthora megasperma <220> FEATURE: <221> NAME/KEY: MISC_FEATURE <223> OTHER INFORMATION: delta-5 desaturase <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: GenBank Accession No. CAD53323 <400> SEQUENCE: 13 Met Ala Pro Ile Glu Thr Val Lys Asp Ala Asn Glu Gly Leu His Gln 1 5 10 15 Arg Lys Gly Ala Ala Ala Ala Ser Lys Asp Thr Thr Thr Phe Thr Trp 20 25 30 Gln Asp Val Ala Lys His Asn Thr Ala Lys Ser Ala Trp Val Thr Ile 35 40 45 Arg Gly Val Val Tyr Asp Val Thr Glu Trp Ala Asp Arg His Pro Gly 50 55 60 Gly Arg Glu Leu Val Leu Leu His Ser Gly Arg Glu Cys Thr Asp Thr 65 70 75 80 Phe Asp Ser Tyr His Pro Phe Ser Asp Arg Ala Asp Lys Ile Leu Ala 85 90 95 Lys Tyr Ala Ile Gly Lys Leu Val Gly Gly Ser Glu Phe Pro Thr Tyr 100 105 110 Lys Pro Asp Thr Gly Phe Tyr Lys Glu Cys Cys Asp Arg Val Asn Gln 115 120 125 Tyr Phe Lys Asp Asn Lys Leu Asp Pro Arg Ser Pro Tyr Ser Gly Leu 130 135 140 Trp Arg Met Ile Leu Val Ala Ile Val Gly Ala Val Ala Tyr Met Gly 145 150 155 160 Met Asn Gln Leu Leu Pro Gly Asn Ile Tyr Ala His Tyr Ala Trp Gly 165 170 175 Ala Leu Phe Gly Val Cys Gln Ala Leu Pro Leu Leu His Val Met His 180 185 190 Asp Ala Ser His Ala Ala Ile Thr Ser Ser Pro Thr Gly Trp Arg Leu 195 200 205 Ile Gly Arg Leu Ala Met Asp Trp Val Ala Gly Ala Asn Met Val Ser 210 215 220 Trp Leu Asn Gln His Val Val Gly His His Ile Tyr Thr Asn Val Ala 225 230 235 240 Gly Ala Asp Pro Asp Leu Pro Val Asp Phe Lys Ser Asp Val Arg Arg 245 250 255 Ile Val Tyr Arg Gln Val Leu Leu Pro Ile Tyr Lys Tyr Gln His Leu 260 265 270 Tyr Leu Pro Pro Leu Tyr Gly Val Leu Gly Leu Lys Phe Arg Val Gln 275 280 285 Asp Val Phe Glu Thr Phe Val Thr Leu Thr Asn Gly Pro Leu Arg Val 290 295 300 Asn Pro Leu Ser Val Gly Asp Trp Ala Glu Met Ile Leu Ser Lys Ala 305 310 315 320 Phe Trp Val Phe Tyr Arg Ile Tyr Leu Pro Leu Ala Val Leu Gln Val 325 330 335 Asp Pro Ala Arg Phe Trp Gly Val Phe Phe Leu Ala Glu Phe Ser Thr 340 345 350 Gly Trp Tyr Leu Ala Phe Asn Phe Gln Val Ser His Val Ser Thr Ala 355 360 365 Cys Glu Tyr Pro Gly Gly Asp Glu Glu Val Thr Ser Ile Asp Asp Glu 370 375 380 Trp Ala Ile Ser Gln Val Lys Ser Ser Val Asp Tyr Gly His Gly Ser 385 390 395 400 Phe Ile Thr Thr Phe Leu Thr Gly Ala Leu Asn Tyr Gln Val Thr His 405 410 415 His Leu Phe Pro Gly Val Ser Gln Tyr His Tyr Pro Ala Ile Ala Pro 420 425 430 Leu Ile Leu Asp Val Cys His Lys Tyr Lys Val Lys Tyr Asn Val Leu 435 440 445 Pro Asp Phe Thr Ala Ala Met Ala Gly His Phe Asp His Leu Val Ile 450 455 460 Met Gly Lys Met Gly Lys Arg Val Thr Ile His Met Gly 465 470 475 <210> SEQ ID NO 14 <211> LENGTH: 469 <212> TYPE: PRT <213> ORGANISM: Phaeodactylum tricornutum <220> FEATURE: <221> NAME/KEY: MISC_FEATURE <223> OTHER INFORMATION: delta-5 desaturase <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: GenBank Accession No. AAL92562 <400> SEQUENCE: 14 Met Ala Pro Asp Ala Asp Lys Leu Arg Gln Arg Gln Thr Thr Ala Val 1 5 10 15 Ala Lys His Asn Ala Ala Thr Ile Ser Thr Gln Glu Arg Leu Cys Ser 20 25 30 Leu Ser Ser Leu Lys Gly Glu Glu Val Cys Ile Asp Gly Ile Ile Tyr 35 40 45 Asp Leu Gln Ser Phe Asp His Pro Gly Gly Glu Thr Ile Lys Met Phe 50 55 60 Gly Gly Asn Asp Val Thr Val Gln Tyr Lys Met Ile His Pro Tyr His 65 70 75 80 Thr Glu Lys His Leu Glu Lys Met Lys Arg Val Gly Lys Val Thr Asp 85 90 95 Phe Val Cys Glu Tyr Lys Phe Asp Thr Glu Phe Glu Arg Glu Ile Lys 100 105 110 Arg Glu Val Phe Lys Ile Val Arg Arg Gly Lys Asp Phe Gly Thr Leu 115 120 125 Gly Trp Phe Phe Arg Ala Phe Cys Tyr Ile Ala Ile Phe Phe Tyr Leu 130 135 140 Gln Tyr His Trp Val Thr Thr Gly Thr Ser Trp Leu Leu Ala Val Ala 145 150 155 160 Tyr Gly Ile Ser Gln Ala Met Ile Gly Met Asn Val Gln His Asp Ala 165 170 175 Asn His Gly Ala Thr Ser Lys Arg Pro Trp Val Asn Asp Met Leu Gly 180 185 190 Leu Gly Ala Asp Phe Ile Gly Gly Ser Lys Trp Leu Trp Gln Glu Gln 195 200 205 His Trp Thr His His Ala Tyr Thr Asn His Ala Glu Met Asp Pro Asp 210 215 220 Ser Phe Gly Ala Glu Pro Met Leu Leu Phe Asn Asp Tyr Pro Leu Asp 225 230 235 240 His Pro Ala Arg Thr Trp Leu His Arg Phe Gln Ala Phe Phe Tyr Met 245 250 255 Pro Val Leu Ala Gly Tyr Trp Leu Ser Ala Val Phe Asn Pro Gln Ile 260 265 270 Leu Asp Leu Gln Gln Arg Gly Ala Leu Ser Val Gly Ile Arg Leu Asp 275 280 285 Asn Ala Phe Ile His Ser Arg Arg Lys Tyr Ala Val Phe Trp Arg Ala 290 295 300 Val Tyr Ile Ala Val Asn Val Ile Ala Pro Phe Tyr Thr Asn Ser Gly 305 310 315 320 Leu Glu Trp Ser Trp Arg Val Phe Gly Asn Ile Met Leu Met Gly Val 325 330 335 Ala Glu Ser Leu Ala Leu Ala Val Leu Phe Ser Leu Ser His Asn Phe 340 345 350 Glu Ser Ala Asp Arg Asp Pro Thr Ala Pro Leu Lys Lys Thr Gly Glu 355 360 365 Pro Val Asp Trp Phe Lys Thr Gln Val Glu Thr Ser Cys Thr Tyr Gly 370 375 380 Gly Phe Leu Ser Gly Cys Phe Thr Gly Gly Leu Asn Phe Gln Val Glu 385 390 395 400 His His Leu Phe Pro Arg Met Ser Ser Ala Trp Tyr Pro Tyr Ile Ala 405 410 415 Pro Lys Val Arg Glu Ile Cys Ala Lys His Gly Val His Tyr Ala Tyr 420 425 430 Tyr Pro Trp Ile His Gln Asn Phe Leu Ser Thr Val Arg Tyr Met His 435 440 445 Ala Ala Gly Thr Gly Ala Asn Trp Arg Gln Met Ala Arg Glu Asn Pro 450 455 460 Leu Thr Gly Arg Ala 465 <210> SEQ ID NO 15 <211> LENGTH: 467 <212> TYPE: PRT <213> ORGANISM: Dictyostelium discoideum

<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <223> OTHER INFORMATION: delta-5 desaturase <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: GenBank Accession No. XP_640331 <400> SEQUENCE: 15 Met Met Glu Thr Asn Asn Glu Asn Lys Glu Lys Leu Lys Leu Tyr Thr 1 5 10 15 Trp Asp Glu Val Ser Lys His Asn Gln Lys Asn Asp Leu Trp Ile Ile 20 25 30 Val Asp Gly Lys Val Tyr Asn Ile Thr Lys Trp Val Pro Leu His Pro 35 40 45 Gly Gly Glu Asp Ile Leu Leu Leu Ser Ala Gly Arg Asp Ala Thr Asn 50 55 60 Leu Phe Glu Ser Tyr His Pro Met Thr Asp Lys His Tyr Ser Leu Ile 65 70 75 80 Lys Gln Tyr Glu Ile Gly Tyr Ile Ser Ser Tyr Glu His Pro Lys Tyr 85 90 95 Val Glu Lys Ser Glu Phe Tyr Ser Thr Leu Lys Gln Arg Val Arg Lys 100 105 110 His Phe Gln Thr Ser Ser Gln Asp Pro Lys Val Ser Val Gly Val Phe 115 120 125 Thr Arg Met Val Leu Ile Tyr Leu Phe Leu Phe Val Thr Tyr Tyr Leu 130 135 140 Ser Gln Phe Ser Thr Asp Arg Phe Trp Leu Asn Cys Ile Phe Ala Val 145 150 155 160 Leu Tyr Gly Val Ala Asn Ser Leu Phe Gly Leu His Thr Met His Asp 165 170 175 Ala Cys His Thr Ala Ile Thr His Asn Pro Met Thr Trp Lys Ile Leu 180 185 190 Gly Ala Thr Phe Asp Leu Phe Ala Gly Ala Ser Phe Tyr Ala Trp Cys 195 200 205 His Gln His Val Ile Gly His His Leu Tyr Thr Asn Val Arg Asn Ala 210 215 220 Asp Pro Asp Leu Gly Gln Gly Glu Ile Asp Phe Arg Val Val Thr Pro 225 230 235 240 Tyr Gln Ala Arg Ser Trp Tyr His Lys Tyr Gln His Ile Tyr Ala Pro 245 250 255 Ile Leu Tyr Gly Val Tyr Ala Leu Lys Tyr Arg Ile Gln Asp His Glu 260 265 270 Ile Phe Thr Lys Lys Ser Asn Gly Ala Ile Arg Tyr Ser Pro Ile Ser 275 280 285 Thr Ile Asp Thr Ala Ile Phe Ile Leu Gly Lys Leu Val Phe Ile Ile 290 295 300 Ser Arg Phe Ile Leu Pro Leu Ile Tyr Asn His Ser Phe Ser His Leu 305 310 315 320 Ile Cys Phe Phe Leu Ile Ser Glu Leu Val Leu Gly Trp Tyr Leu Ala 325 330 335 Ile Ser Phe Gln Val Ser His Val Val Glu Asp Leu Gln Phe Met Ala 340 345 350 Thr Pro Glu Ile Phe Asp Gly Ala Asp His Pro Leu Pro Thr Thr Phe 355 360 365 Asn Gln Asp Trp Ala Ile Leu Gln Val Lys Thr Thr Gln Asp Tyr Ala 370 375 380 Gln Asp Ser Val Leu Ser Thr Phe Phe Ser Gly Gly Leu Asn Leu Gln 385 390 395 400 Val Ile His His Cys Phe Pro Thr Ile Ala Gln Asp Tyr Tyr Pro Gln 405 410 415 Ile Val Pro Ile Leu Lys Glu Val Cys Lys Glu Tyr Asn Val Thr Tyr 420 425 430 His Tyr Lys Pro Thr Phe Thr Glu Ala Ile Lys Ser His Ile Asn Tyr 435 440 445 Leu Tyr Lys Met Gly Asn Asp Pro Asp Tyr Val Arg Lys Pro Val Asn 450 455 460 Lys Asn Asp 465 <210> SEQ ID NO 16 <211> LENGTH: 421 <212> TYPE: PRT <213> ORGANISM: Euglena gracilis <220> FEATURE: <221> NAME/KEY: MISC_FEATURE <223> OTHER INFORMATION: delta-8 desaturase <300> PUBLICATION INFORMATION: <302> TITLE: DELTA-8 DESATURASE AND ITS USE IN MAKING POLYUNSATURATED FATTY ACIDS <310> PATENT DOCUMENT NUMBER: WO2006012325 <311> PATENT FILING DATE: 2005-06-24 <312> PUBLICATION DATE: 2006-02-02 <313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(421) <300> PUBLICATION INFORMATION: <302> TITLE: DELTA-8 DESATURASE AND ITS USE IN MAKING POLYUNSATURATED FATTY ACIDS <310> PATENT DOCUMENT NUMBER: WO2006012326 <311> PATENT FILING DATE: 2005-06-24 <312> PUBLICATION DATE: 2006-02-02 <313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(421) <400> SEQUENCE: 16 Met Lys Ser Lys Arg Gln Ala Leu Pro Leu Thr Ile Asp Gly Thr Thr 1 5 10 15 Tyr Asp Val Ser Ala Trp Val Asn Phe His Pro Gly Gly Ala Glu Ile 20 25 30 Ile Glu Asn Tyr Gln Gly Arg Asp Ala Thr Asp Ala Phe Met Val Met 35 40 45 His Ser Gln Glu Ala Phe Asp Lys Leu Lys Arg Met Pro Lys Ile Asn 50 55 60 Pro Ser Ser Glu Leu Pro Pro Gln Ala Ala Val Asn Glu Ala Gln Glu 65 70 75 80 Asp Phe Arg Lys Leu Arg Glu Glu Leu Ile Ala Thr Gly Met Phe Asp 85 90 95 Ala Ser Pro Leu Trp Tyr Ser Tyr Lys Ile Ser Thr Thr Leu Gly Leu 100 105 110 Gly Val Leu Gly Tyr Phe Leu Met Val Gln Tyr Gln Met Tyr Phe Ile 115 120 125 Gly Ala Val Leu Leu Gly Met His Tyr Gln Gln Met Gly Trp Leu Ser 130 135 140 His Asp Ile Cys His His Gln Thr Phe Lys Asn Arg Asn Trp Asn Asn 145 150 155 160 Leu Val Gly Leu Val Phe Gly Asn Gly Leu Gln Gly Phe Ser Val Thr 165 170 175 Trp Trp Lys Asp Arg His Asn Ala His His Ser Ala Thr Asn Val Gln 180 185 190 Gly His Asp Pro Asp Ile Asp Asn Leu Pro Leu Leu Ala Trp Ser Glu 195 200 205 Asp Asp Val Thr Arg Ala Ser Pro Ile Ser Arg Lys Leu Ile Gln Phe 210 215 220 Gln Gln Tyr Tyr Phe Leu Val Ile Cys Ile Leu Leu Arg Phe Ile Trp 225 230 235 240 Cys Phe Gln Ser Val Leu Thr Val Arg Ser Leu Lys Asp Arg Asp Asn 245 250 255 Gln Phe Tyr Arg Ser Gln Tyr Lys Lys Glu Ala Ile Gly Leu Ala Leu 260 265 270 His Trp Thr Leu Lys Thr Leu Phe His Leu Phe Phe Met Pro Ser Ile 275 280 285 Leu Thr Ser Leu Leu Val Phe Phe Val Ser Glu Leu Val Gly Gly Phe 290 295 300 Gly Ile Ala Ile Val Val Phe Met Asn His Tyr Pro Leu Glu Lys Ile 305 310 315 320 Gly Asp Ser Val Trp Asp Gly His Gly Phe Ser Val Gly Gln Ile His 325 330 335 Glu Thr Met Asn Ile Arg Arg Gly Ile Ile Thr Asp Trp Phe Phe Gly 340 345 350 Gly Leu Asn Tyr Gln Ile Glu His His Leu Trp Pro Thr Leu Pro Arg 355 360 365 His Asn Leu Thr Ala Val Ser Tyr Gln Val Glu Gln Leu Cys Gln Lys 370 375 380 His Asn Leu Pro Tyr Arg Asn Pro Leu Pro His Glu Gly Leu Val Ile 385 390 395 400 Leu Leu Arg Tyr Leu Ala Val Phe Ala Arg Met Ala Glu Lys Gln Pro 405 410 415 Ala Gly Lys Ala Leu 420 <210> SEQ ID NO 17 <211> LENGTH: 1269 <212> TYPE: DNA <213> ORGANISM: Pavlova lutheri CCMP459 <220> FEATURE: <221> NAME/KEY: misc_feature <223> OTHER INFORMATION: delta-8 desaturase <400> SEQUENCE: 17 atgggcaagg gtggagacgg cggcgcgcag gcggtgagcg ggaccgacgc gtctctcgct 60 gaggtgagct ccgtcgatag caagagcgtg cacgtcgtgc tctacggcaa gcgcgtggat 120 gtcacaaagt tccagaaggc acacccgggc gggagcaagg tgttccgcat cttccaggag 180 cgcgacgcga cggagcagtt cgagtcttac cactcgccca aggccatcaa gatgatggag 240 ggcatgctca agaagtcgga ggatgcgccc gcttccgtgc ccctgccctc gcggtccacc 300 atgggcacgg agttcaagga gatgattgag cgccacaaga gggctggtct ctacgaccct 360 tgcccgttgg acgagctgtt caagctcacc atcgtccttg cgcccatctt cgtgggcgcc 420 tatctcgtgc ggagcggcgt ctcgcccctc gcgggcgcgc tctccatggg ctttggcttc 480 tacctcgacg gctggcttgc tcacgactac ctgcatcacg cagtcttcaa gggctcggtc 540 aacacgctcg tcaaggcgaa caacgccatg ggatacgccc tcggcttcct ccagggctac 600 gacgtggcct ggtggcgcgc gcgccataac acgcaccacg tgtgcaccaa cgaggatggt 660 tcggacccgg acatcaagac ggcgcccctg ctcatctacg tgcgagagaa cccgtccatt 720 gccaagcggc tcaacttctt ccagcgctgg cagcagtact actatgtgcc gaccatggcc 780 atcctcgacc tctactggcg cctggagtcc atcgcgtacg tggctgtgcg cctgcctaag 840 atgtggatgc aggccgccgc tcttgccgct cactacgcgc tcctgtgctg ggtcttcgca 900 gcgcatctca acctcatccc tctcatgatg gttgcacgcg gcttcgcgac gggcatcgtt 960 gtctttgcaa cccactatgg tgaggacatc ctcgaccgcg agcacgtcga gggcatgacg 1020

ctcgtcgagc agaccgccaa gacctcccgt aacatcacgg gcggctggct agtgaacgtg 1080 ctcacgggct tcatctccct gcagaccgag catcacctct tccccatgat gcccaccggc 1140 aacctaatga ctatccagcc cgaggtacgc gacttcttca agaagcatgg cctcgagtac 1200 cgcgagggca acctcttcca gtgcgtgcac cagaacatca aggctctcgc cttcgagcac 1260 ctcctccac 1269 <210> SEQ ID NO 18 <211> LENGTH: 423 <212> TYPE: PRT <213> ORGANISM: Pavlova lutheri CCMP459 <400> SEQUENCE: 18 Met Gly Lys Gly Gly Asp Gly Gly Ala Gln Ala Val Ser Gly Thr Asp 1 5 10 15 Ala Ser Leu Ala Glu Val Ser Ser Val Asp Ser Lys Ser Val His Val 20 25 30 Val Leu Tyr Gly Lys Arg Val Asp Val Thr Lys Phe Gln Lys Ala His 35 40 45 Pro Gly Gly Ser Lys Val Phe Arg Ile Phe Gln Glu Arg Asp Ala Thr 50 55 60 Glu Gln Phe Glu Ser Tyr His Ser Pro Lys Ala Ile Lys Met Met Glu 65 70 75 80 Gly Met Leu Lys Lys Ser Glu Asp Ala Pro Ala Ser Val Pro Leu Pro 85 90 95 Ser Arg Ser Thr Met Gly Thr Glu Phe Lys Glu Met Ile Glu Arg His 100 105 110 Lys Arg Ala Gly Leu Tyr Asp Pro Cys Pro Leu Asp Glu Leu Phe Lys 115 120 125 Leu Thr Ile Val Leu Ala Pro Ile Phe Val Gly Ala Tyr Leu Val Arg 130 135 140 Ser Gly Val Ser Pro Leu Ala Gly Ala Leu Ser Met Gly Phe Gly Phe 145 150 155 160 Tyr Leu Asp Gly Trp Leu Ala His Asp Tyr Leu His His Ala Val Phe 165 170 175 Lys Gly Ser Val Asn Thr Leu Val Lys Ala Asn Asn Ala Met Gly Tyr 180 185 190 Ala Leu Gly Phe Leu Gln Gly Tyr Asp Val Ala Trp Trp Arg Ala Arg 195 200 205 His Asn Thr His His Val Cys Thr Asn Glu Asp Gly Ser Asp Pro Asp 210 215 220 Ile Lys Thr Ala Pro Leu Leu Ile Tyr Val Arg Glu Asn Pro Ser Ile 225 230 235 240 Ala Lys Arg Leu Asn Phe Phe Gln Arg Trp Gln Gln Tyr Tyr Tyr Val 245 250 255 Pro Thr Met Ala Ile Leu Asp Leu Tyr Trp Arg Leu Glu Ser Ile Ala 260 265 270 Tyr Val Ala Val Arg Leu Pro Lys Met Trp Met Gln Ala Ala Ala Leu 275 280 285 Ala Ala His Tyr Ala Leu Leu Cys Trp Val Phe Ala Ala His Leu Asn 290 295 300 Leu Ile Pro Leu Met Met Val Ala Arg Gly Phe Ala Thr Gly Ile Val 305 310 315 320 Val Phe Ala Thr His Tyr Gly Glu Asp Ile Leu Asp Arg Glu His Val 325 330 335 Glu Gly Met Thr Leu Val Glu Gln Thr Ala Lys Thr Ser Arg Asn Ile 340 345 350 Thr Gly Gly Trp Leu Val Asn Val Leu Thr Gly Phe Ile Ser Leu Gln 355 360 365 Thr Glu His His Leu Phe Pro Met Met Pro Thr Gly Asn Leu Met Thr 370 375 380 Ile Gln Pro Glu Val Arg Asp Phe Phe Lys Lys His Gly Leu Glu Tyr 385 390 395 400 Arg Glu Gly Asn Leu Phe Gln Cys Val His Gln Asn Ile Lys Ala Leu 405 410 415 Ala Phe Glu His Leu Leu His 420 <210> SEQ ID NO 19 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Conserved Region 1 <220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION: (4)..(4) <223> OTHER INFORMATION: Xaa = Ile or Val <400> SEQUENCE: 19 Gly His His Xaa Tyr Thr Asn 1 5 <210> SEQ ID NO 20 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Conserved Region 2 <220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION: (4)..(4) <223> OTHER INFORMATION: Xaa = Val or Ala <220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa = Ser or Asn <400> SEQUENCE: 20 Asn Phe Gln Xaa Xaa His Val 1 5 <210> SEQ ID NO 21 <211> LENGTH: 476 <212> TYPE: PRT <213> ORGANISM: Thalassiosira pseudonana <220> FEATURE: <221> NAME/KEY: MISC_FEATURE <223> OTHER INFORMATION: delta-8 desaturase <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: GenBank Accession No. AAX14502 <400> SEQUENCE: 21 Met Pro Pro Asn Ala Asp Ile Ser Arg Ile Arg Asn Arg Ile Pro Thr 1 5 10 15 Lys Thr Gly Thr Val Ala Ser Ala Asp Asn Asn Asp Pro Ala Thr Gln 20 25 30 Ser Val Arg Thr Leu Lys Ser Leu Lys Gly Asn Glu Val Val Ile Asn 35 40 45 Gly Thr Ile Tyr Asp Ile Ala Asp Phe Val His Pro Gly Gly Glu Val 50 55 60 Val Lys Phe Phe Gly Gly Asn Asp Val Thr Ile Gln Tyr Asn Met Ile 65 70 75 80 His Pro Tyr His Thr Gly Lys His Leu Glu Lys Met Lys Ala Val Gly 85 90 95 Lys Val Val Asp Trp Gln Ser Asp Tyr Lys Phe Asp Thr Pro Phe Glu 100 105 110 Arg Glu Ile Lys Ser Glu Val Phe Lys Ile Val Arg Arg Gly Arg Glu 115 120 125 Phe Gly Thr Thr Gly Tyr Phe Leu Arg Ala Phe Phe Tyr Ile Ala Leu 130 135 140 Phe Phe Thr Met Gln Tyr Thr Phe Ala Thr Cys Thr Thr Phe Thr Thr 145 150 155 160 Tyr Asp His Trp Tyr Gln Ser Gly Val Phe Ile Ala Ile Val Phe Gly 165 170 175 Ile Ser Gln Ala Phe Ile Gly Leu Asn Val Gln His Asp Ala Asn His 180 185 190 Gly Ala Ala Ser Lys Arg Pro Trp Val Asn Asp Leu Leu Gly Phe Gly 195 200 205 Thr Asp Leu Ile Gly Ser Asn Lys Trp Asn Trp Met Ala Gln His Trp 210 215 220 Thr His His Ala Tyr Thr Asn His Ser Glu Lys Asp Pro Asp Ser Phe 225 230 235 240 Ser Ser Glu Pro Met Phe Ala Phe Asn Asp Tyr Pro Ile Gly His Pro 245 250 255 Lys Arg Lys Trp Trp His Arg Phe Gln Gly Gly Tyr Phe Leu Phe Met 260 265 270 Leu Gly Leu Tyr Trp Leu Pro Thr Val Phe Asn Pro Gln Phe Ile Asp 275 280 285 Leu Arg Gln Arg Gly Ala Gln Tyr Val Gly Ile Gln Met Glu Asn Asp 290 295 300 Phe Ile Val Lys Arg Arg Lys Tyr Ala Val Ala Leu Arg Met Met Tyr 305 310 315 320 Ile Tyr Leu Asn Ile Val Ser Pro Phe Met Asn Asn Gly Leu Ser Trp 325 330 335 Ser Thr Phe Gly Ile Ile Met Leu Met Gly Ile Ser Glu Ser Leu Thr 340 345 350 Leu Ser Val Leu Phe Ser Leu Ser His Asn Phe Ile Asn Ser Asp Arg 355 360 365 Asp Pro Thr Ala Asp Phe Lys Lys Thr Gly Glu Gln Val Cys Trp Phe 370 375 380 Lys Ser Gln Val Glu Thr Ser Ser Thr Tyr Gly Gly Phe Ile Ser Gly 385 390 395 400 Cys Leu Thr Gly Gly Leu Asn Phe Gln Val Glu His His Leu Phe Pro 405 410 415 Arg Met Ser Ser Ala Trp Tyr Pro Tyr Ile Ala Pro Thr Val Arg Glu 420 425 430 Val Cys Lys Lys His Gly Met Ser Tyr Ala Tyr Tyr Pro Trp Ile Gly 435 440 445 Gln Asn Leu Val Ser Thr Phe Lys Tyr Met His Arg Ala Gly Ser Gly 450 455 460 Ala Asn Trp Glu Leu Lys Pro Leu Ser Gly Ser Ala 465 470 475 <210> SEQ ID NO 22 <211> LENGTH: 8165 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pZUF17 <400> SEQUENCE: 22 gtacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 60

ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 120 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 180 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 240 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 300 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 360 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 420 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 480 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 540 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 600 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 660 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 720 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 780 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 840 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 900 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 960 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 1020 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 1080 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 1140 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 1200 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 1260 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 1320 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 1380 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 1440 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 1500 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 1560 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 1620 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 1680 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 1740 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 1800 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 1860 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 1920 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 1980 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 2040 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 2100 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 2160 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 2220 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 2280 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 2340 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 2400 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 2460 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 2520 aacgcgaatt ttaacaaaat attaacgctt acaatttcca ttcgccattc aggctgcgca 2580 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 2640 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta 2700 aaacgacggc cagtgaattg taatacgact cactataggg cgaattgggt accgggcccc 2760 ccctcgaggt cgatggtgtc gataagcttg atatcgaatt catgtcacac aaaccgatct 2820 tcgcctcaag gaaacctaat tctacatccg agagactgcc gagatccagt ctacactgat 2880 taattttcgg gccaataatt taaaaaaatc gtgttatata atattatatg tattatatat 2940 atacatcatg atgatactga cagtcatgtc ccattgctaa atagacagac tccatctgcc 3000 gcctccaact gatgttctca atatttaagg ggtcatctcg cattgtttaa taataaacag 3060 actccatcta ccgcctccaa atgatgttct caaaatatat tgtatgaact tatttttatt 3120 acttagtatt attagacaac ttacttgctt tatgaaaaac acttcctatt taggaaacaa 3180 tttataatgg cagttcgttc atttaacaat ttatgtagaa taaatgttat aaatgcgtat 3240 gggaaatctt aaatatggat agcataaatg atatctgcat tgcctaattc gaaatcaaca 3300 gcaacgaaaa aaatcccttg tacaacataa atagtcatcg agaaatatca actatcaaag 3360 aacagctatt cacacgttac tattgagatt attattggac gagaatcaca cactcaactg 3420 tctttctctc ttctagaaat acaggtacaa gtatgtacta ttctcattgt tcatacttct 3480 agtcatttca tcccacatat tccttggatt tctctccaat gaatgacatt ctatcttgca 3540 aattcaacaa ttataataag atataccaaa gtagcggtat agtggcaatc aaaaagcttc 3600 tctggtgtgc ttctcgtatt tatttttatt ctaatgatcc attaaaggta tatatttatt 3660 tcttgttata taatcctttt gtttattaca tgggctggat acataaaggt attttgattt 3720 aattttttgc ttaaattcaa tcccccctcg ttcagtgtca actgtaatgg taggaaatta 3780 ccatactttt gaagaagcaa aaaaaatgaa agaaaaaaaa aatcgtattt ccaggttaga 3840 cgttccgcag aatctagaat gcggtatgcg gtacattgtt cttcgaacgt aaaagttgcg 3900 ctccctgaga tattgtacat ttttgctttt acaagtacaa gtacatcgta caactatgta 3960 ctactgttga tgcatccaca acagtttgtt ttgttttttt ttgttttttt tttttctaat 4020 gattcattac cgctatgtat acctacttgt acttgtagta agccgggtta ttggcgttca 4080 attaatcata gacttatgaa tctgcacggt gtgcgctgcg agttactttt agcttatgca 4140 tgctacttgg gtgtaatatt gggatctgtt cggaaatcaa cggatgctca atcgatttcg 4200 acagtaatta attaagtcat acacaagtca gctttcttcg agcctcatat aagtataagt 4260 agttcaacgt attagcactg tacccagcat ctccgtatcg agaaacacaa caacatgccc 4320 cattggacag atcatgcgga tacacaggtt gtgcagtatc atacatactc gatcagacag 4380 gtcgtctgac catcatacaa gctgaacaag cgctccatac ttgcacgctc tctatataca 4440 cagttaaatt acatatccat agtctaacct ctaacagtta atcttctggt aagcctccca 4500 gccagccttc tggtatcgct tggcctcctc aataggatct cggttctggc cgtacagacc 4560 tcggccgaca attatgatat ccgttccggt agacatgaca tcctcaacag ttcggtactg 4620 ctgtccgaga gcgtctccct tgtcgtcaag acccaccccg ggggtcagaa taagccagtc 4680 ctcagagtcg cccttaggtc ggttctgggc aatgaagcca accacaaact cggggtcgga 4740 tcgggcaagc tcaatggtct gcttggagta ctcgccagtg gccagagagc ccttgcaaga 4800 cagctcggcc agcatgagca gacctctggc cagcttctcg ttgggagagg ggactaggaa 4860 ctccttgtac tgggagttct cgtagtcaga gacgtcctcc ttcttctgtt cagagacagt 4920 ttcctcggca ccagctcgca ggccagcaat gattccggtt ccgggtacac cgtgggcgtt 4980 ggtgatatcg gaccactcgg cgattcggtg acaccggtac tggtgcttga cagtgttgcc 5040 aatatctgcg aactttctgt cctcgaacag gaagaaaccg tgcttaagag caagttcctt 5100 gagggggagc acagtgccgg cgtaggtgaa gtcgtcaatg atgtcgatat gggttttgat 5160 catgcacaca taaggtccga ccttatcggc aagctcaatg agctccttgg tggtggtaac 5220 atccagagaa gcacacaggt tggttttctt ggctgccacg agcttgagca ctcgagcggc 5280 aaaggcggac ttgtggacgt tagctcgagc ttcgtaggag ggcattttgg tggtgaagag 5340 gagactgaaa taaatttagt ctgcagaact ttttatcgga accttatctg gggcagtgaa 5400 gtatatgtta tggtaatagt tacgagttag ttgaacttat agatagactg gactatacgg 5460 ctatcggtcc aaattagaaa gaacgtcaat ggctctctgg gcgtcgcctt tgccgacaaa 5520 aatgtgatca tgatgaaagc cagcaatgac gttgcagctg atattgttgt cggccaaccg 5580 cgccgaaaac gcagctgtca gacccacagc ctccaacgaa gaatgtatcg tcaaagtgat 5640 ccaagcacac tcatagttgg agtcgtactc caaaggcggc aatgacgagt cagacagata 5700 ctcgtcgact caggcgacga cggaattcct gcagcccatc tgcagaattc aggagagacc 5760 gggttggcgg cgtatttgtg tcccaaaaaa cagccccaat tgccccggag aagacggcca 5820 ggccgcctag atgacaaatt caacaactca cagctgactt tctgccattg ccactagggg 5880 ggggcctttt tatatggcca agccaagctc tccacgtcgg ttgggctgca cccaacaata 5940 aatgggtagg gttgcaccaa caaagggatg ggatgggggg tagaagatac gaggataacg 6000 gggctcaatg gcacaaataa gaacgaatac tgccattaag actcgtgatc cagcgactga 6060 caccattgca tcatctaagg gcctcaaaac tacctcggaa ctgctgcgct gatctggaca 6120 ccacagaggt tccgagcact ttaggttgca ccaaatgtcc caccaggtgc aggcagaaaa 6180 cgctggaaca gcgtgtacag tttgtcttaa caaaaagtga gggcgctgag gtcgagcagg 6240 gtggtgtgac ttgttatagc ctttagagct gcgaaagcgc gtatggattt ggctcatcag 6300 gccagattga gggtctgtgg acacatgtca tgttagtgta cttcaatcgc cccctggata 6360 tagccccgac aataggccgt ggcctcattt ttttgccttc cgcacatttc cattgctcgg 6420 tacccacacc ttgcttctcc tgcacttgcc aaccttaata ctggtttaca ttgaccaaca 6480 tcttacaagc ggggggcttg tctagggtat atataaacag tggctctccc aatcggttgc 6540 cagtctcttt tttcctttct ttccccacag attcgaaatc taaactacac atcacacaat 6600 gcctgttact gacgtcctta agcgaaagtc cggtgtcatc gtcggcgacg atgtccgagc 6660 cgtgagtatc cacgacaaga tcagtgtcga gacgacgcgt tttgtgtaat gacacaatcc 6720 gaaagtcgct agcaacacac actctctaca caaactaacc cagctctcca tggctgagga 6780 taagaccaag gtcgagttcc ctaccctgac tgagctgaag cactctatcc ctaacgcttg 6840 ctttgagtcc aacctcggac tctcgctcta ctacactgcc cgagcgatct tcaacgcatc 6900 tgcctctgct gctctgctct acgctgcccg atctactccc ttcattgccg ataacgttct 6960 gctccacgct ctggtttgcg ccacctacat ctacgtgcag ggtgtcatct tctggggttt 7020 ctttaccgtc ggtcacgact gtggtcactc tgccttctcc cgataccact ccgtcaactt 7080 catcattggc tgcatcatgc actctgccat tctgactccc ttcgagtcct ggcgagtgac 7140 ccaccgacac catcacaaga acactggcaa cattgataag gacgagatct tctaccctca 7200 tcggtccgtc aaggacctcc aggacgtgcg acaatgggtc tacaccctcg gaggtgcttg 7260 gtttgtctac ctgaaggtcg gatatgctcc tcgaaccatg tcccactttg acccctggga 7320 ccctctcctg cttcgacgag cctccgctgt catcgtgtcc ctcggagtct gggctgcctt 7380 cttcgctgcc tacgcctacc tcacatactc gctcggcttt gccgtcatgg gcctctacta 7440 ctatgctcct ctctttgtct ttgcttcgtt cctcgtcatt actaccttct tgcatcacaa 7500 cgacgaagct actccctggt acggtgactc ggagtggacc tacgtcaagg gcaacctgag 7560

ctccgtcgac cgatcgtacg gagctttcgt ggacaacctg tctcaccaca ttggcaccca 7620 ccaggtccat cacttgttcc ctatcattcc ccactacaag ctcaacgaag ccaccaagca 7680 ctttgctgcc gcttaccctc acctcgtgag acgtaacgac gagcccatca ttactgcctt 7740 cttcaagacc gctcacctct ttgtcaacta cggagctgtg cccgagactg ctcagatttt 7800 caccctcaaa gagtctgccg ctgcagccaa ggccaagagc gactaagcgg ccgcaagtgt 7860 ggatggggaa gtgagtgccc ggttctgtgt gcacaattgg caatccaaga tggatggatt 7920 caacacaggg atatagcgag ctacgtggtg gtgcgaggat atagcaacgg atatttatgt 7980 ttgacacttg agaatgtacg atacaagcac tgtccaagta caatactaaa catactgtac 8040 atactcatac tcgtacccgg gcaacggttt cacttgagtg cagtggctag tgctcttact 8100 cgtacagtgt gcaatactgc gtatcatagt ctttgatgta tatcgtattc attcatgtta 8160 gttgc 8165 <210> SEQ ID NO 23 <211> LENGTH: 8480 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pDMW368 <400> SEQUENCE: 23 ggccgcaagt gtggatgggg aagtgagtgc ccggttctgt gtgcacaatt ggcaatccaa 60 gatggatgga ttcaacacag ggatatagcg agctacgtgg tggtgcgagg atatagcaac 120 ggatatttat gtttgacact tgagaatgta cgatacaagc actgtccaag tacaatacta 180 aacatactgt acatactcat actcgtaccc gggcaacggt ttcacttgag tgcagtggct 240 agtgctctta ctcgtacagt gtgcaatact gcgtatcata gtctttgatg tatatcgtat 300 tcattcatgt tagttgcgta cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 360 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 420 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 480 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 540 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 600 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 660 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 720 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 780 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 840 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 900 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 960 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 1020 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 1080 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 1140 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 1200 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 1260 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 1320 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 1380 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 1440 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 1500 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 1560 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 1620 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 1680 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 1740 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 1800 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 1860 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 1920 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 1980 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 2040 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 2100 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 2160 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 2220 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 2280 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 2340 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 2400 cccgaaaagt gccacctgac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg 2460 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct 2520 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc 2580 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg 2640 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt 2700 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg 2760 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc 2820 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttccattc 2880 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg 2940 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc 3000 ccagtcacga cgttgtaaaa cgacggccag tgaattgtaa tacgactcac tatagggcga 3060 attgggtacc gggccccccc tcgaggtcga tggtgtcgat aagcttgata tcgaattcat 3120 gtcacacaaa ccgatcttcg cctcaaggaa acctaattct acatccgaga gactgccgag 3180 atccagtcta cactgattaa ttttcgggcc aataatttaa aaaaatcgtg ttatataata 3240 ttatatgtat tatatatata catcatgatg atactgacag tcatgtccca ttgctaaata 3300 gacagactcc atctgccgcc tccaactgat gttctcaata tttaaggggt catctcgcat 3360 tgtttaataa taaacagact ccatctaccg cctccaaatg atgttctcaa aatatattgt 3420 atgaacttat ttttattact tagtattatt agacaactta cttgctttat gaaaaacact 3480 tcctatttag gaaacaattt ataatggcag ttcgttcatt taacaattta tgtagaataa 3540 atgttataaa tgcgtatggg aaatcttaaa tatggatagc ataaatgata tctgcattgc 3600 ctaattcgaa atcaacagca acgaaaaaaa tcccttgtac aacataaata gtcatcgaga 3660 aatatcaact atcaaagaac agctattcac acgttactat tgagattatt attggacgag 3720 aatcacacac tcaactgtct ttctctcttc tagaaataca ggtacaagta tgtactattc 3780 tcattgttca tacttctagt catttcatcc cacatattcc ttggatttct ctccaatgaa 3840 tgacattcta tcttgcaaat tcaacaatta taataagata taccaaagta gcggtatagt 3900 ggcaatcaaa aagcttctct ggtgtgcttc tcgtatttat ttttattcta atgatccatt 3960 aaaggtatat atttatttct tgttatataa tccttttgtt tattacatgg gctggataca 4020 taaaggtatt ttgatttaat tttttgctta aattcaatcc cccctcgttc agtgtcaact 4080 gtaatggtag gaaattacca tacttttgaa gaagcaaaaa aaatgaaaga aaaaaaaaat 4140 cgtatttcca ggttagacgt tccgcagaat ctagaatgcg gtatgcggta cattgttctt 4200 cgaacgtaaa agttgcgctc cctgagatat tgtacatttt tgcttttaca agtacaagta 4260 catcgtacaa ctatgtacta ctgttgatgc atccacaaca gtttgttttg tttttttttg 4320 tttttttttt ttctaatgat tcattaccgc tatgtatacc tacttgtact tgtagtaagc 4380 cgggttattg gcgttcaatt aatcatagac ttatgaatct gcacggtgtg cgctgcgagt 4440 tacttttagc ttatgcatgc tacttgggtg taatattggg atctgttcgg aaatcaacgg 4500 atgctcaatc gatttcgaca gtaattaatt aagtcataca caagtcagct ttcttcgagc 4560 ctcatataag tataagtagt tcaacgtatt agcactgtac ccagcatctc cgtatcgaga 4620 aacacaacaa catgccccat tggacagatc atgcggatac acaggttgtg cagtatcata 4680 catactcgat cagacaggtc gtctgaccat catacaagct gaacaagcgc tccatacttg 4740 cacgctctct atatacacag ttaaattaca tatccatagt ctaacctcta acagttaatc 4800 ttctggtaag cctcccagcc agccttctgg tatcgcttgg cctcctcaat aggatctcgg 4860 ttctggccgt acagacctcg gccgacaatt atgatatccg ttccggtaga catgacatcc 4920 tcaacagttc ggtactgctg tccgagagcg tctcccttgt cgtcaagacc caccccgggg 4980 gtcagaataa gccagtcctc agagtcgccc ttaggtcggt tctgggcaat gaagccaacc 5040 acaaactcgg ggtcggatcg ggcaagctca atggtctgct tggagtactc gccagtggcc 5100 agagagccct tgcaagacag ctcggccagc atgagcagac ctctggccag cttctcgttg 5160 ggagagggga ctaggaactc cttgtactgg gagttctcgt agtcagagac gtcctccttc 5220 ttctgttcag agacagtttc ctcggcacca gctcgcaggc cagcaatgat tccggttccg 5280 ggtacaccgt gggcgttggt gatatcggac cactcggcga ttcggtgaca ccggtactgg 5340 tgcttgacag tgttgccaat atctgcgaac tttctgtcct cgaacaggaa gaaaccgtgc 5400 ttaagagcaa gttccttgag ggggagcaca gtgccggcgt aggtgaagtc gtcaatgatg 5460 tcgatatggg ttttgatcat gcacacataa ggtccgacct tatcggcaag ctcaatgagc 5520 tccttggtgg tggtaacatc cagagaagca cacaggttgg ttttcttggc tgccacgagc 5580 ttgagcactc gagcggcaaa ggcggacttg tggacgttag ctcgagcttc gtaggagggc 5640 attttggtgg tgaagaggag actgaaataa atttagtctg cagaactttt tatcggaacc 5700 ttatctgggg cagtgaagta tatgttatgg taatagttac gagttagttg aacttataga 5760 tagactggac tatacggcta tcggtccaaa ttagaaagaa cgtcaatggc tctctgggcg 5820 tcgcctttgc cgacaaaaat gtgatcatga tgaaagccag caatgacgtt gcagctgata 5880 ttgttgtcgg ccaaccgcgc cgaaaacgca gctgtcagac ccacagcctc caacgaagaa 5940 tgtatcgtca aagtgatcca agcacactca tagttggagt cgtactccaa aggcggcaat 6000 gacgagtcag acagatactc gtcgactcag gcgacgacgg aattcctgca gcccatctgc 6060 agaattcagg agagaccggg ttggcggcgt atttgtgtcc caaaaaacag ccccaattgc 6120 cccggagaag acggccaggc cgcctagatg acaaattcaa caactcacag ctgactttct 6180 gccattgcca ctaggggggg gcctttttat atggccaagc caagctctcc acgtcggttg 6240 ggctgcaccc aacaataaat gggtagggtt gcaccaacaa agggatggga tggggggtag 6300 aagatacgag gataacgggg ctcaatggca caaataagaa cgaatactgc cattaagact 6360 cgtgatccag cgactgacac cattgcatca tctaagggcc tcaaaactac ctcggaactg 6420 ctgcgctgat ctggacacca cagaggttcc gagcacttta ggttgcacca aatgtcccac 6480 caggtgcagg cagaaaacgc tggaacagcg tgtacagttt gtcttaacaa aaagtgaggg 6540 cgctgaggtc gagcagggtg gtgtgacttg ttatagcctt tagagctgcg aaagcgcgta 6600

tggatttggc tcatcaggcc agattgaggg tctgtggaca catgtcatgt tagtgtactt 6660 caatcgcccc ctggatatag ccccgacaat aggccgtggc ctcatttttt tgccttccgc 6720 acatttccat tgctcggtac ccacaccttg cttctcctgc acttgccaac cttaatactg 6780 gtttacattg accaacatct tacaagcggg gggcttgtct agggtatata taaacagtgg 6840 ctctcccaat cggttgccag tctctttttt cctttctttc cccacagatt cgaaatctaa 6900 actacacatc acacaatgcc tgttactgac gtccttaagc gaaagtccgg tgtcatcgtc 6960 ggcgacgatg tccgagccgt gagtatccac gacaagatca gtgtcgagac gacgcgtttt 7020 gtgtaatgac acaatccgaa agtcgctagc aacacacact ctctacacaa actaacccag 7080 ctctccatgg ctccagatgc ggacaagttg agacagcgca aggcgcaatc gattcaagac 7140 acggctgatt cgcaagctac cgaactcaag attggcaccc tgaagggctt gcaggggaca 7200 gaaatcgtca ttgatggaga catttacgat ataaaagact ttgatcaccc cggtggtgaa 7260 tccatcatga cttttggggg aaacgatgtc accgccacgt acaagatgat ccacccctac 7320 cactctaagc accatttgga gaagatgaag aaagtgggac gagttccgga ctacacctcg 7380 gaatacaagt ttgatactcc ctttgagcgt gaaatcaagc aagaggtctt caagattgtg 7440 cgacgaggcc gcgagtttgg aacacctgga tacttcttcc gggctttctg ctacattgga 7500 cttttctttt acttgcagta tttgtgggtc acgactccca ctacctttgc cttggcgatc 7560 ttctatggtg tttcgcaagc tttcattggt ttgaacgtac aacatgatgc caaccacgga 7620 gctgcctcca agaagccttg gatcaataac ttgctaggat tgggggctga ctttatcgga 7680 ggttccaaat ggttgtggat gaaccagcac tggacgcacc acacatacac caaccaccat 7740 gagaaggatc ccgatgcctt gggcgctgaa ccaatgttgt tgttcaatga ttatcccttg 7800 ggtcacccaa agcgtacttt gattcaccac ttccaggcct tctattacct tttcgtcttg 7860 gccggatact gggtctcttc ggtcttcaac cctcaaattt tggacttgca acaccgcggt 7920 gctcaagcgg ttggaatgaa aatggagaac gattacattg ccaaaagccg aaagtatgcc 7980 atcttcttgc gtctcttgta tatttacacc aacattgtcg ctccgatcca aaaccaaggc 8040 ttctcgttga ccgtggtcgc ccacattttg accatgggcg tcgcttccag tttgactttg 8100 gcgactcttt ttgccttgtc gcacaatttt gaaaacgcgg atcgcgatcc cacttacgag 8160 gcccgcaagg gaggagagcc tgtttgttgg ttcaagtcgc aagtcgaaac ctcgtcaact 8220 tacggaggtt tcatctcggg ttgcttgacg ggcggactca acttccaagt ggaacaccac 8280 ttgttccctc gtatgagttc ggcctggtac ccctacattg cccctactgt tcgagaggtt 8340 tgcaaaaagc acggagtcaa gtacgcatac tatccctggg tctggcaaaa cttgatttca 8400 actgtcaagt atctgcatca aagcggaact ggatccaact ggaagaatgg cgccaacccc 8460 tactcgggaa aattgtaagc 8480 <210> SEQ ID NO 24 <211> LENGTH: 12649 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKUNF12T6E <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (2507)..(2507) <223> OTHER INFORMATION: n is a, c, g, or t <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (2512)..(2515) <223> OTHER INFORMATION: n is a, c, g, or t <400> SEQUENCE: 24 taaccctcac taaagggaac aaaagctgga gctccaccgc ggacacaata tctggtcaaa 60 tttcagtttc gttacataaa tcgttatgtc aaaggagtgt gggaggttaa gagaattatc 120 accggcaaac tatctgttaa ttgctaggta cctctagacg tccacccggg tcgcttggcg 180 gccgaagagg ccggaatctc gggccgcggt ggcggccgct tagttggtct tggacttctt 240 gggcttcttc aggtaggact ggacaaagaa gttgccgaac agagcgagca gggtgatcat 300 gtacacgccg agcagctgga ccagagcctg agggtagtcg caggggaaga ggtagtcgta 360 cagggactgc accagcatag ccatgaactg ggtcatctgc agagtggtga tgtagggctt 420 gatgggcttg acgaagccga agccctgaga ggaaaagaag tagtaggcgt acatgacggt 480 gtggacgaag gagttgagga tgacggagaa gtaggcgtcg ccaccaggag cgtacttggc 540 aatagcccac cagatggcga agatggtggc atggtggtac acgtgcagga aggagacctg 600 gttgaacttc ttgcacagga tcatgatagc ggtgtccagg aactcgtagg ccttggagac 660 gtagaacacg tagacgattc gggacatgcc ctgagcgtgg gactcgttgc ccttctccat 720 gtcgttgccg aagaccttgt agccacccag gatagcctgt cggatggtct cgacgcacat 780 gtagagggac agtccgaaga ggaacaggtt gtggagcagc ttgatggtct tcagctcgaa 840 gggcttctcc atctgcttca tgatgggaat gccgaagagc agcatggcca tgtagccgac 900 ctcgaaggcg agcatggtgg agacgtccat catgggcaga ccgtcggtca gagcgtaggg 960 cttagctccg tccatccact ggtcgacacc ggtctcgact cgtccgacca cgtcgtccca 1020 gacagaggag ttggccatgg tgaatgattc ttatactcag aaggaaatgc ttaacgattt 1080 cgggtgtgag ttgacaagga gagagagaaa agaagaggaa aggtaattcg gggacggtgg 1140 tcttttatac ccttggctaa agtcccaacc acaaagcaaa aaaattttca gtagtctatt 1200 ttgcgtccgg catgggttac ccggatggcc agacaaagaa actagtacaa agtctgaaca 1260 agcgtagatt ccagactgca gtaccctacg cccttaacgg caagtgtggg aaccggggga 1320 ggtttgatat gtggggtgaa gggggctctc gccggggttg ggcccgctac tgggtcaatt 1380 tggggtcaat tggggcaatt ggggctgttt tttgggacac aaatacgccg ccaacccggt 1440 ctctcctgaa ttctgcatcg atcgaggaag aggacaagcg gctgcttctt aagtttgtga 1500 catcagtatc caaggcacca ttgcaaggat tcaaggcttt gaacccgtca tttgccattc 1560 gtaacgctgg tagacaggtt gatcggttcc ctacggcctc cacctgtgtc aatcttctca 1620 agctgcctga ctatcaggac attgatcaac ttcggaagaa acttttgtat gccattcgat 1680 cacatgctgg tttcgatttg tcttagagga acgcatatac agtaatcata gagaataaac 1740 gatattcatt tattaaagta gatagttgag gtagaagttg taaagagtga taaatagcgg 1800 ccgcgcctac ttaagcaacg ggcttgataa cagcgggggg ggtgcccacg ttgttgcggt 1860 tgcggaagaa cagaacaccc ttaccagcac cctcggcacc agcgctgggc tcaacccact 1920 ggcacatacg cgcactgcgg tacatggcgc ggatgaagcc acgaggacca tcctggacat 1980 cagcccggta gtgcttgccc atgatgggct taatggcctc ggtggcctcg tccgcgttgt 2040 agaaggggat gctgctgacg tagtggtgga ggacatgagt ctcgatgatg ccgtggagaa 2100 ggtggcggcc gatgaagccc atctcacggt caatggtagc agcggcacca cggacgaagt 2160 tccactcgtc gttggtgtag tggggaaggg tagggtcggt gtgctggagg aaggtgatgg 2220 caacgagcca gtggttaacc cagaggtagg gaacaaagta ccagatggcc atgttgtaga 2280 aaccgaactt ctgaacgagg aagtacagag cagtggccat cagaccgata ccaatatcgc 2340 tgaggacgat gagcttagcg tcactgttct cgtacagagg gctgcgggga tcgaagtggt 2400 taacaccacc gccgaggccg ttatgcttgc ccttgccgcg accctcacgc tggcgctcgt 2460 ggtagttgtg gccggtaaca ttggtgatga ggtagttggg ccagccnacg annnnctcag 2520 taagatgagc gagctcgtgg gtcatctttc cgagacgagt agcctgctgc tcgcgggttc 2580 ggggaacgaa gaccatgtca cgctccatgt tgccagtggc cttgtggtgc tttcggtggg 2640 agatttgcca gctgaagtag gggacaagga gggaagagtg aagaacccag ccagtaatgt 2700 cgttgatgat gcgagaatcg gagaaagcac cgtgaccgca ctcatgggca ataacccaga 2760 gaccagtacc gaaaagaccc tgaagaacgg tgtacacggc ccacagacca gcgcgggcgg 2820 gggtggaggg gatatattcg ggggtcacaa agttgtacca gatgctgaaa gtggtagtca 2880 ggaggacaat gtcgcggagg atataaccgt atcccttgag agcggagcgc ttgaagcagt 2940 gcttagggat ggcattgtag atgtccttga tggtaaagtc gggaacctcg aactggttgc 3000 cgtaggtgtc gagcatgaca ccatactcgg acttgggctt ggcgatatca acctcggaca 3060 tggacgagag cgatgtggaa gaggccgagt ggcggggaga gtctgaagga gagacggcgg 3120 cagactcaga atccgtcaca gtagttgagg tgacggtgcg tctaagcgca gggttctgct 3180 tgggcagagc cgaagtggac gccatggaga gctgggttag tttgtgtaga gagtgtgtgt 3240 tgctagcgac tttcggattg tgtcattaca caaaacgcgt cgtctcgaca ctgatcttgt 3300 cgtggatact cacggctcgg acatcgtcgc cgacgatgac accggacttt cgcttaagga 3360 cgtcagtaac aggcattgtg tgatgtgtag tttagatttc gaatctgtgg ggaaagaaag 3420 gaaaaaagag actggcaacc gattgggaga gccactgttt atatataccc tagacaagcc 3480 ccccgcttgt aagatgttgg tcaatgtaaa ccagtattaa ggttggcaag tgcaggagaa 3540 gcaaggtgtg ggtaccgagc aatggaaatg tgcggaaggc aaaaaaatga ggccacggcc 3600 tattgtcggg gctatatcca gggggcgatt gaagtacact aacatgacat gtgtccacag 3660 accctcaatc tggcctgatg agccaaatcc atacgcgctt tcgcagctct aaaggctata 3720 acaagtcaca ccaccctgct cgacctcagc gccctcactt tttgttaaga caaactgtac 3780 acgctgttcc agcgttttct gcctgcacct ggtgggacat ttggtgcaac ctaaagtgct 3840 cggaacctct gtggtgtcca gatcagcgca gcagttccga ggtagttttg aggcccttag 3900 atgatgcaat ggtgtcagtc gctggatcac gagtcttaat ggcagtattc gttcttattt 3960 gtgccattga gccccgttat cctcgtatct tctacccccc atcccatccc tttgttggtg 4020 caaccctacc catttattgt tgggtgcagc ccaaccgacg tggagagctt ggcttggcca 4080 tataaaaagg ccccccccta gtggcaatgg cagaaagtca gctgtgagtt gttgaatttg 4140 tcatctaggc ggcctggccg tcttctccgg ggcaattgtt cctctatagt actgcgtaca 4200 ctgtttaaac agtgtacgca gatctgcgac gacggaattc ctgcagccca tctgcagaat 4260 tcaggagaga ccgggttggc ggcgtatttg tgtcccaaaa aacagcccca attgccccaa 4320 ttgaccccaa attgacccag tagcgggccc aaccccggcg agagccccct tcaccccaca 4380 tatcaaacct cccccggttc ccacacttgc cgttaagggc gtagggtact gcagtctgga 4440 atctacgctt gttcagactt tgtactagtt tctttgtctg gccatccggg taacccatgc 4500 cggacgcaaa atagactact gaaaattttt ttgctttgtg gttgggactt tagccaaggg 4560 tataaaagac caccgtcccc gaattacctt tcctcttctt ttctctctct ccttgtcaac 4620 tcacacccga aatcgttaag catttccttc tgagtataag aatcattcac catggctgcc 4680 gctccctctg tgcgaacctt tacccgagcc gaggttctga acgctgaggc tctgaacgag 4740 ggcaagaagg acgctgaggc tcccttcctg atgatcatcg acaacaaggt gtacgacgtc 4800 cgagagttcg tccctgacca tcctggaggc tccgtgattc tcacccacgt tggcaaggac 4860 ggcaccgacg tctttgacac ctttcatccc gaggctgctt gggagactct cgccaacttc 4920 tacgttggag acattgacga gtccgaccga gacatcaaga acgatgactt tgccgctgag 4980 gtccgaaagc tgcgaaccct gttccagtct ctcggctact acgactcctc taaggcctac 5040

tacgccttca aggtctcctt caacctctgc atctggggac tgtccaccgt cattgtggcc 5100 aagtggggtc agacctccac cctcgccaac gtgctctctg ctgccctgct cggcctgttc 5160 tggcagcagt gcggatggct ggctcacgac tttctgcacc accaggtctt ccaggaccga 5220 ttctggggtg atctcttcgg agccttcctg ggaggtgtct gccagggctt ctcctcttcc 5280 tggtggaagg acaagcacaa cactcaccat gccgctccca acgtgcatgg cgaggatcct 5340 gacattgaca cccaccctct cctgacctgg tccgagcacg ctctggagat gttctccgac 5400 gtccccgatg aggagctgac ccgaatgtgg tctcgattca tggtcctgaa ccagacctgg 5460 ttctacttcc ccattctctc cttcgctcga ctgtcttggt gcctccagtc cattctcttt 5520 gtgctgccca acggtcaggc tcacaagccc tccggagctc gagtgcccat ctccctggtc 5580 gagcagctgt ccctcgccat gcactggacc tggtacctcg ctaccatgtt cctgttcatc 5640 aaggatcctg tcaacatgct cgtgtacttc ctggtgtctc aggctgtgtg cggaaacctg 5700 ctcgccatcg tgttctccct caaccacaac ggtatgcctg tgatctccaa ggaggaggct 5760 gtcgacatgg atttctttac caagcagatc atcactggtc gagatgtcca tcctggactg 5820 ttcgccaact ggttcaccgg tggcctgaac taccagatcg agcatcacct gttcccttcc 5880 atgcctcgac acaacttctc caagatccag cctgccgtcg agaccctgtg caagaagtac 5940 aacgtccgat accacaccac tggtatgatc gagggaactg ccgaggtctt ctcccgactg 6000 aacgaggtct ccaaggccac ctccaagatg ggcaaggctc agtaagcggc cgcatgagaa 6060 gataaatata taaatacatt gagatattaa atgcgctaga ttagagagcc tcatactgct 6120 cggagagaag ccaagacgag tactcaaagg ggattacacc atccatatcc acagacacaa 6180 gctggggaaa ggttctatat acactttccg gaataccgta gtttccgatg ttatcaatgg 6240 gggcagccag gatttcaggc acttcggtgt ctcggggtga aatggcgttc ttggcctcca 6300 tcaagtcgta ccatgtcttc atttgcctgt caaagtaaaa cagaagcaga tgaagaatga 6360 acttgaagtg aaggaattta aattgccccg gagaagacgg ccaggccgcc tagatgacaa 6420 attcaacaac tcacagctga ctttctgcca ttgccactag gggggggcct ttttatatgg 6480 ccaagccaag ctctccacgt cggttgggct gcacccaaca ataaatgggt agggttgcac 6540 caacaaaggg atgggatggg gggtagaaga tacgaggata acggggctca atggcacaaa 6600 taagaacgaa tactgccatt aagactcgtg atccagcgac tgacaccatt gcatcatcta 6660 agggcctcaa aactacctcg gaactgctgc gctgatctgg acaccacaga ggttccgagc 6720 actttaggtt gcaccaaatg tcccaccagg tgcaggcaga aaacgctgga acagcgtgta 6780 cagtttgtct taacaaaaag tgagggcgct gaggtcgagc agggtggtgt gacttgttat 6840 agcctttaga gctgcgaaag cgcgtatgga tttggctcat caggccagat tgagggtctg 6900 tggacacatg tcatgttagt gtacttcaat cgccccctgg atatagcccc gacaataggc 6960 cgtggcctca tttttttgcc ttccgcacat ttccattgct cggtacccac accttgcttc 7020 tcctgcactt gccaacctta atactggttt acattgacca acatcttaca agcggggggc 7080 ttgtctaggg tatatataaa cagtggctct cccaatcggt tgccagtctc ttttttcctt 7140 tctttcccca cagattcgaa atctaaacta cacatcacac aatgcctgtt actgacgtcc 7200 ttaagcgaaa gtccggtgtc atcgtcggcg acgatgtccg agccgtgagt atccacgaca 7260 agatcagtgt cgagacgacg cgttttgtgt aatgacacaa tccgaaagtc gctagcaaca 7320 cacactctct acacaaacta acccagctct ccatggagtc cattgctccc ttcctgccct 7380 ccaagatgcc tcaggacctg ttcatggacc tcgccagcgc tatcggtgtc cgagctgctc 7440 cctacgtcga tcccctggag gctgccctgg ttgcccaggc cgagaagtac attcccacca 7500 ttgtccatca cactcgaggc ttcctggttg ccgtggagtc tcccctggct cgagagctgc 7560 ctctgatgaa ccccttccac gtgctcctga tcgtgctcgc ctacctggtc accgtgtttg 7620 tgggtatgca gatcatgaag aactttgaac gattcgaggt caagaccttc tccctcctgc 7680 acaacttctg tctggtctcc atctccgcct acatgtgcgg tggcatcctg tacgaggctt 7740 atcaggccaa ctatggactg tttgagaacg ctgccgatca caccttcaag ggtctcccta 7800 tggctaagat gatctggctc ttctacttct ccaagatcat ggagtttgtc gacaccatga 7860 tcatggtcct caagaagaac aaccgacaga tttcctttct gcacgtgtac caccactctt 7920 ccatcttcac catctggtgg ctggtcacct tcgttgctcc caacggtgaa gcctacttct 7980 ctgctgccct gaactccttc atccacgtca tcatgtacgg ctactacttt ctgtctgccc 8040 tgggcttcaa gcaggtgtcg ttcatcaagt tctacatcac tcgatcccag atgacccagt 8100 tctgcatgat gtctgtccag tcttcctggg acatgtacgc catgaaggtc cttggccgac 8160 ctggataccc cttcttcatc accgctctgc tctggttcta catgtggacc atgctcggtc 8220 tcttctacaa cttttaccga aagaacgcca agctcgccaa gcaggccaag gctgacgctg 8280 ccaaggagaa ggccagaaag ctccagtaag cggccgcaag tgtggatggg gaagtgagtg 8340 cccggttctg tgtgcacaat tggcaatcca agatggatgg attcaacaca gggatatagc 8400 gagctacgtg gtggtgcgag gatatagcaa cggatattta tgtttgacac ttgagaatgt 8460 acgatacaag cactgtccaa gtacaatact aaacatactg tacatactca tactcgtacc 8520 cgggcaacgg tttcacttga gtgcagtggc tagtgctctt actcgtacag tgtgcaatac 8580 tgcgtatcat agtctttgat gtatatcgta ttcattcatg ttagttgcgt acgaagtcgt 8640 caatgatgtc gatatgggtt ttgatcatgc acacataagg tccgacctta tcggcaagct 8700 caatgagctc cttggtggtg gtaacatcca gagaagcaca caggttggtt ttcttggctg 8760 ccacgagctt gagcactcga gcggcaaagg cggacttgtg gacgttagct cgagcttcgt 8820 aggagggcat tttggtggtg aagaggagac tgaaataaat ttagtctgca gaacttttta 8880 tcggaacctt atctggggca gtgaagtata tgttatggta atagttacga gttagttgaa 8940 cttatagata gactggacta tacggctatc ggtccaaatt agaaagaacg tcaatggctc 9000 tctgggcgtc gcctttgccg acaaaaatgt gatcatgatg aaagccagca atgacgttgc 9060 agctgatatt gttgtcggcc aaccgcgccg aaaacgcagc tgtcagaccc acagcctcca 9120 acgaagaatg tatcgtcaaa gtgatccaag cacactcata gttggagtcg tactccaaag 9180 gcggcaatga cgagtcagac agatactcgt cgaccttttc cttgggaacc accaccgtca 9240 gcccttctga ctcacgtatt gtagccaccg acacaggcaa cagtccgtgg atagcagaat 9300 atgtcttgtc ggtccatttc tcaccaactt taggcgtcaa gtgaatgttg cagaagaagt 9360 atgtgccttc attgagaatc ggtgttgctg atttcaataa agtcttgaga tcagtttggc 9420 gcgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg 9480 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 9540 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 9600 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 9660 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 9720 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 9780 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 9840 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 9900 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 9960 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 10020 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 10080 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt 10140 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 10200 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 10260 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 10320 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 10380 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 10440 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 10500 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 10560 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 10620 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 10680 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac 10740 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 10800 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 10860 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 10920 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 10980 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat 11040 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 11100 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 11160 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 11220 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 11280 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 11340 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 11400 aaaagtgcca cctgatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca 11460 tcaggaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag 11520 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac 11580 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga 11640 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc 11700 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg 11760 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa 11820 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac 11880 caccacaccc gccgcgctta atgcgccgct acagggcgcg tccattcgcc attcaggctg 11940 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa 12000 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt 12060 tgtaaaacga cggccagtga attgtaatac gactcactat agggcgaatt gggcccgacg 12120 tcgcatgcag tggtggtatt gtgactgggg atgtagttga gaataagtca tacacaagtc 12180 agctttcttc gagcctcata taagtataag tagttcaacg tattagcact gtacccagca 12240 tctccgtatc gagaaacaca acaacatgcc ccattggaca gatcatgcgg atacacaggt 12300 tgtgcagtat catacatact cgatcagaca ggtcgtctga ccatcataca agctgaacaa 12360 gcgctccata cttgcacgct ctctatatac acagttaaat tacatatcca tagtctaacc 12420 tctaacagtt aatcttctgg taagcctccc agccagcctt ctggtatcgc ttggcctcct 12480 caataggatc tcggttctgg ccgtacagac ctcggccgac aattatgata tccgttccgg 12540 tagacatgac atcctcaaca gttcggtact gctgtccgag agcgtctccc ttgtcgtcaa 12600

gacccacccc gggggtcaga ataagccagt cctcagagtc gcccttaat 12649 <210> SEQ ID NO 25 <211> LENGTH: 819 <212> TYPE: DNA <213> ORGANISM: Thraustochytrium aureum <220> FEATURE: <221> NAME/KEY: misc_feature <223> OTHER INFORMATION: synthetic elongase (codon-optimized) for Yarrowia lipolytica <400> SEQUENCE: 25 atggccaact cctctgtctg ggacgacgtg gtcggacgag tcgagaccgg tgtcgaccag 60 tggatggacg gagctaagcc ctacgctctg accgacggtc tgcccatgat ggacgtctcc 120 accatgctcg ccttcgaggt cggctacatg gccatgctgc tcttcggcat tcccatcatg 180 aagcagatgg agaagccctt cgagctgaag accatcaagc tgctccacaa cctgttcctc 240 ttcggactgt ccctctacat gtgcgtcgag accatccgac aggctatcct gggtggctac 300 aaggtcttcg gcaacgacat ggagaagggc aacgagtccc acgctcaggg catgtcccga 360 atcgtctacg tgttctacgt ctccaaggcc tacgagttcc tggacaccgc tatcatgatc 420 ctgtgcaaga agttcaacca ggtctccttc ctgcacgtgt accaccatgc caccatcttc 480 gccatctggt gggctattgc caagtacgct cctggtggcg acgcctactt ctccgtcatc 540 ctcaactcct tcgtccacac cgtcatgtac gcctactact tcttttcctc tcagggcttc 600 ggcttcgtca agcccatcaa gccctacatc accactctgc agatgaccca gttcatggct 660 atgctggtgc agtccctgta cgactacctc ttcccctgcg actaccctca ggctctggtc 720 cagctgctcg gcgtgtacat gatcaccctg ctcgctctgt tcggcaactt ctttgtccag 780 tcctacctga agaagcccaa gaagtccaag accaactaa 819 <210> SEQ ID NO 26 <211> LENGTH: 272 <212> TYPE: PRT <213> ORGANISM: Thraustochytrium aureum <400> SEQUENCE: 26 Met Ala Asn Ser Ser Val Trp Asp Asp Val Val Gly Arg Val Glu Thr 1 5 10 15 Gly Val Asp Gln Trp Met Asp Gly Ala Lys Pro Tyr Ala Leu Thr Asp 20 25 30 Gly Leu Pro Met Met Asp Val Ser Thr Met Leu Ala Phe Glu Val Gly 35 40 45 Tyr Met Ala Met Leu Leu Phe Gly Ile Pro Ile Met Lys Gln Met Glu 50 55 60 Lys Pro Phe Glu Leu Lys Thr Ile Lys Leu Leu His Asn Leu Phe Leu 65 70 75 80 Phe Gly Leu Ser Leu Tyr Met Cys Val Glu Thr Ile Arg Gln Ala Ile 85 90 95 Leu Gly Gly Tyr Lys Val Phe Gly Asn Asp Met Glu Lys Gly Asn Glu 100 105 110 Ser His Ala Gln Gly Met Ser Arg Ile Val Tyr Val Phe Tyr Val Ser 115 120 125 Lys Ala Tyr Glu Phe Leu Asp Thr Ala Ile Met Ile Leu Cys Lys Lys 130 135 140 Phe Asn Gln Val Ser Phe Leu His Val Tyr His His Ala Thr Ile Phe 145 150 155 160 Ala Ile Trp Trp Ala Ile Ala Lys Tyr Ala Pro Gly Gly Asp Ala Tyr 165 170 175 Phe Ser Val Ile Leu Asn Ser Phe Val His Thr Val Met Tyr Ala Tyr 180 185 190 Tyr Phe Phe Ser Ser Gln Gly Phe Gly Phe Val Lys Pro Ile Lys Pro 195 200 205 Tyr Ile Thr Thr Leu Gln Met Thr Gln Phe Met Ala Met Leu Val Gln 210 215 220 Ser Leu Tyr Asp Tyr Leu Phe Pro Cys Asp Tyr Pro Gln Ala Leu Val 225 230 235 240 Gln Leu Leu Gly Val Tyr Met Ile Thr Leu Leu Ala Leu Phe Gly Asn 245 250 255 Phe Phe Val Gln Ser Tyr Leu Lys Lys Pro Lys Lys Ser Lys Thr Asn 260 265 270 <210> SEQ ID NO 27 <211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer AP <400> SEQUENCE: 27 ggccacgcgt cgactagtac tttttttttt ttttttt 37 <210> SEQ ID NO 28 <211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Smart IV oligonucleotide primer <400> SEQUENCE: 28 aagcagtggt atcaacgcag agtggccatt acggccggg 39 <210> SEQ ID NO 29 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer CDSIII 5' <400> SEQUENCE: 29 aagcagtggt atcaacgcag agt 23 <210> SEQ ID NO 30 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Degenerate primer 5-1A <400> SEQUENCE: 30 gghcaycayr tbtayacaaa 20 <210> SEQ ID NO 31 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Degenerate primer 5-1B <400> SEQUENCE: 31 gghcaycayr tbtayaccaa 20 <210> SEQ ID NO 32 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Degenerate primer 5-1C <400> SEQUENCE: 32 gghcaycayr tbtayacgaa 20 <210> SEQ ID NO 33 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Degenerate primer 5-1D <400> SEQUENCE: 33 gghcaycayr tbtayactaa 20 <210> SEQ ID NO 34 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Degenerate primer 5-4AR <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (9)..(9) <223> OTHER INFORMATION: n is a, c, g, or t <400> SEQUENCE: 34 acrtgrytna cytgraagtt 20 <210> SEQ ID NO 35 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Degenerate primer 5-4BR <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (9)..(9) <223> OTHER INFORMATION: n is a, c, g, or t <400> SEQUENCE: 35 acrtgrytna cytgraaatt 20 <210> SEQ ID NO 36 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Degenerate primer 5-4CR <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (6)..(6) <223> OTHER INFORMATION: n is a, c, g, or t <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (9)..(9) <223> OTHER INFORMATION: n is a, c, g, or t <400> SEQUENCE: 36 acrtgngana cytgraagtt 20 <210> SEQ ID NO 37 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Degenerate primer 5-4DR <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (6)..(6) <223> OTHER INFORMATION: n is a, c, g, or t <220> FEATURE: <221> NAME/KEY: misc_feature

<222> LOCATION: (9)..(9) <223> OTHER INFORMATION: n is a, c, g, or t <400> SEQUENCE: 37 acrtgngana cytgraaatt 20 <210> SEQ ID NO 38 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer ODMW520 <400> SEQUENCE: 38 cgttctccat tttcattcca accgc 25 <210> SEQ ID NO 39 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer ODMW521 <400> SEQUENCE: 39 ggttgaagac cgaagagacc cagtatcc 28 <210> SEQ ID NO 40 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer DNR CDS 5' <400> SEQUENCE: 40 caacgcagag tggccattac gg 22 <210> SEQ ID NO 41 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer ODMW541 <400> SEQUENCE: 41 ctcgcggcct cgtcgcacaa tcttgaag 28 <210> SEQ ID NO 42 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer ODMW542 <400> SEQUENCE: 42 gagtatcaaa cttgtattcc gaggtgtag 29 <210> SEQ ID NO 43 <211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer ODMW523 <400> SEQUENCE: 43 ggttcaagtc gcaagtcgaa acctcg 26 <210> SEQ ID NO 44 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer AUAP <400> SEQUENCE: 44 ggccacgcgt cgactagtac 20 <210> SEQ ID NO 45 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer ODMW524 <400> SEQUENCE: 45 caacttacgg aggtttcatc tcggg 25 <210> SEQ ID NO 46 <211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer YL807 <400> SEQUENCE: 46 tttccatggc tccagatgcg gacaagttga g 31 <210> SEQ ID NO 47 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer YL810 <400> SEQUENCE: 47 caaggcatcg ggatccttct catggtggt 29 <210> SEQ ID NO 48 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer YL808 <400> SEQUENCE: 48 tttgcggccg cttacaattt tcccgagtag gggttggcgc ca 42 <210> SEQ ID NO 49 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer YL809 <400> SEQUENCE: 49 accaccatga gaaggatccc gatgccttg 29 <210> SEQ ID NO 50 <211> LENGTH: 4112 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pRD5S <400> SEQUENCE: 50 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa 420 tgcatctaga tccatggctc ccgacgccga caagctgcga cagcgaaagg ctcagtccat 480 ccaggacact gccgattctc aggctaccga gctcaagatt ggcaccctga agggtctcca 540 aggcaccgag atcgtcattg atggcgacat ctacgacatc aaagacttcg atcaccctgg 600 aggcgaatcc atcatgacct ttggtggcaa cgacgttact gccacctaca agatgattca 660 tccctaccac tcgaagcatc acctggagaa gatgaaaaag gtcggtcgag tgcccgacta 720 cacctccgag tacaagttcg atactccctt cgaacgagag atcaaacagg aggtcttcaa 780 gattgtgcga agaggtcgag agtttggaac acctggctac ttctttcgag ccttctgcta 840 catcggtctc ttcttttacc tgcagtatct ctgggttacc actcctacca ctttcgccct 900 tgctatcttc tacggtgtgt ctcaggcctt cattggcctg aacgtccagc acgacgccaa 960 ccacggagct gcctccaaaa agccctggat caacaatttg ctcggcctgg gtgccgactt 1020 tatcggaggc tccaagtggc tctggatgaa ccagcactgg acccatcaca cttacaccaa 1080 ccatcacgag aaggatcccg acgccctggg tgcagagcct atgctgctct tcaacgacta 1140 tcccttgggt caccccaagc gaaccctcat tcatcacttc caagccttct actatctgtt 1200 tgtccttgct ggctactggg tgtcttcggt gttcaaccct cagatcctgg acctccagca 1260 ccgaggtgcc caggctgtcg gcatgaagat ggagaacgac tacattgcca agtctcgaaa 1320 gtacgctatc ttcctgcgac tcctgtacat ctacaccaac attgtggctc ccatccagaa 1380 ccaaggcttt tcgctcaccg tcgttgctca cattcttact atgggtgtcg cctccagcct 1440 gaccctcgct actctgttcg ccctctccca caacttcgag aacgcagatc gggatcccac 1500 ctacgaggct cgaaagggag gcgagcctgt ctgttggttc aagtcgcagg tggaaacctc 1560 ctctacttac ggtggcttca tttccggttg ccttacaggc ggactcaact ttcaggtcga 1620 gcatcacctg tttcctcgaa tgtcctctgc ctggtacccc tacatcgctc ctaccgttcg 1680 agaggtctgc aaaaagcacg gcgtcaagta cgcctactat ccctgggtgt ggcagaacct 1740 catctcgacc gtcaagtacc tgcatcagtc cggaactggc tcgaactgga agaacggtgc 1800 caatccctac tctggcaagc tgtaagcggc cgcatcggat cccgggcccg tcgactgcag 1860 aggcctgcat gcaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta 1920 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc 1980 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 2040 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 2100 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 2160 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 2220 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 2280 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 2340 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 2400 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 2460 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 2520 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 2580 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 2640 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 2700

gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct 2760 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 2820 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 2880 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 2940 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 3000 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 3060 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 3120 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 3180 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 3240 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 3300 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 3360 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 3420 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 3480 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 3540 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 3600 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 3660 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 3720 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 3780 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 3840 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 3900 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 3960 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 4020 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta 4080 taaaaatagg cgtatcacga ggccctttcg tc 4112 <210> SEQ ID NO 51 <211> LENGTH: 8480 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pZURD5S <400> SEQUENCE: 51 catggctccc gacgccgaca agctgcgaca gcgaaaggct cagtccatcc aggacactgc 60 cgattctcag gctaccgagc tcaagattgg caccctgaag ggtctccaag gcaccgagat 120 cgtcattgat ggcgacatct acgacatcaa agacttcgat caccctggag gcgaatccat 180 catgaccttt ggtggcaacg acgttactgc cacctacaag atgattcatc cctaccactc 240 gaagcatcac ctggagaaga tgaaaaaggt cggtcgagtg cccgactaca cctccgagta 300 caagttcgat actcccttcg aacgagagat caaacaggag gtcttcaaga ttgtgcgaag 360 aggtcgagag tttggaacac ctggctactt ctttcgagcc ttctgctaca tcggtctctt 420 cttttacctg cagtatctct gggttaccac tcctaccact ttcgcccttg ctatcttcta 480 cggtgtgtct caggccttca ttggcctgaa cgtccagcac gacgccaacc acggagctgc 540 ctccaaaaag ccctggatca acaatttgct cggcctgggt gccgacttta tcggaggctc 600 caagtggctc tggatgaacc agcactggac ccatcacact tacaccaacc atcacgagaa 660 ggatcccgac gccctgggtg cagagcctat gctgctcttc aacgactatc ccttgggtca 720 ccccaagcga accctcattc atcacttcca agccttctac tatctgtttg tccttgctgg 780 ctactgggtg tcttcggtgt tcaaccctca gatcctggac ctccagcacc gaggtgccca 840 ggctgtcggc atgaagatgg agaacgacta cattgccaag tctcgaaagt acgctatctt 900 cctgcgactc ctgtacatct acaccaacat tgtggctccc atccagaacc aaggcttttc 960 gctcaccgtc gttgctcaca ttcttactat gggtgtcgcc tccagcctga ccctcgctac 1020 tctgttcgcc ctctcccaca acttcgagaa cgcagatcgg gatcccacct acgaggctcg 1080 aaagggaggc gagcctgtct gttggttcaa gtcgcaggtg gaaacctcct ctacttacgg 1140 tggcttcatt tccggttgcc ttacaggcgg actcaacttt caggtcgagc atcacctgtt 1200 tcctcgaatg tcctctgcct ggtaccccta catcgctcct accgttcgag aggtctgcaa 1260 aaagcacggc gtcaagtacg cctactatcc ctgggtgtgg cagaacctca tctcgaccgt 1320 caagtacctg catcagtccg gaactggctc gaactggaag aacggtgcca atccctactc 1380 tggcaagctg taagcggccg caagtgtgga tggggaagtg agtgcccggt tctgtgtgca 1440 caattggcaa tccaagatgg atggattcaa cacagggata tagcgagcta cgtggtggtg 1500 cgaggatata gcaacggata tttatgtttg acacttgaga atgtacgata caagcactgt 1560 ccaagtacaa tactaaacat actgtacata ctcatactcg tacccgggca acggtttcac 1620 ttgagtgcag tggctagtgc tcttactcgt acagtgtgca atactgcgta tcatagtctt 1680 tgatgtatat cgtattcatt catgttagtt gcgtacgagc cggaagcata aagtgtaaag 1740 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt 1800 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 1860 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 1920 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 1980 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 2040 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 2100 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 2160 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 2220 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 2280 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 2340 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 2400 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 2460 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct 2520 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 2580 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 2640 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 2700 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 2760 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 2820 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 2880 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 2940 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 3000 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 3060 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 3120 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 3180 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 3240 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 3300 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 3360 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 3420 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 3480 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 3540 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 3600 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 3660 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg 3720 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 3780 ttccgcgcac atttccccga aaagtgccac ctgacgcgcc ctgtagcggc gcattaagcg 3840 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg 3900 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 3960 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 4020 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 4080 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 4140 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt 4200 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc 4260 ttacaatttc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc 4320 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt 4380 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgaat tgtaatacga 4440 ctcactatag ggcgaattgg gtaccgggcc ccccctcgag gtcgatggtg tcgataagct 4500 tgatatcgaa ttcatgtcac acaaaccgat cttcgcctca aggaaaccta attctacatc 4560 cgagagactg ccgagatcca gtctacactg attaattttc gggccaataa tttaaaaaaa 4620 tcgtgttata taatattata tgtattatat atatacatca tgatgatact gacagtcatg 4680 tcccattgct aaatagacag actccatctg ccgcctccaa ctgatgttct caatatttaa 4740 ggggtcatct cgcattgttt aataataaac agactccatc taccgcctcc aaatgatgtt 4800 ctcaaaatat attgtatgaa cttattttta ttacttagta ttattagaca acttacttgc 4860 tttatgaaaa acacttccta tttaggaaac aatttataat ggcagttcgt tcatttaaca 4920 atttatgtag aataaatgtt ataaatgcgt atgggaaatc ttaaatatgg atagcataaa 4980 tgatatctgc attgcctaat tcgaaatcaa cagcaacgaa aaaaatccct tgtacaacat 5040 aaatagtcat cgagaaatat caactatcaa agaacagcta ttcacacgtt actattgaga 5100 ttattattgg acgagaatca cacactcaac tgtctttctc tcttctagaa atacaggtac 5160 aagtatgtac tattctcatt gttcatactt ctagtcattt catcccacat attccttgga 5220 tttctctcca atgaatgaca ttctatcttg caaattcaac aattataata agatatacca 5280 aagtagcggt atagtggcaa tcaaaaagct tctctggtgt gcttctcgta tttattttta 5340 ttctaatgat ccattaaagg tatatattta tttcttgtta tataatcctt ttgtttatta 5400 catgggctgg atacataaag gtattttgat ttaatttttt gcttaaattc aatcccccct 5460 cgttcagtgt caactgtaat ggtaggaaat taccatactt ttgaagaagc aaaaaaaatg 5520 aaagaaaaaa aaaatcgtat ttccaggtta gacgttccgc agaatctaga atgcggtatg 5580 cggtacattg ttcttcgaac gtaaaagttg cgctccctga gatattgtac atttttgctt 5640 ttacaagtac aagtacatcg tacaactatg tactactgtt gatgcatcca caacagtttg 5700 ttttgttttt ttttgttttt tttttttcta atgattcatt accgctatgt atacctactt 5760 gtacttgtag taagccgggt tattggcgtt caattaatca tagacttatg aatctgcacg 5820

gtgtgcgctg cgagttactt ttagcttatg catgctactt gggtgtaata ttgggatctg 5880 ttcggaaatc aacggatgct caatcgattt cgacagtaat taattaagtc atacacaagt 5940 cagctttctt cgagcctcat ataagtataa gtagttcaac gtattagcac tgtacccagc 6000 atctccgtat cgagaaacac aacaacatgc cccattggac agatcatgcg gatacacagg 6060 ttgtgcagta tcatacatac tcgatcagac aggtcgtctg accatcatac aagctgaaca 6120 agcgctccat acttgcacgc tctctatata cacagttaaa ttacatatcc atagtctaac 6180 ctctaacagt taatcttctg gtaagcctcc cagccagcct tctggtatcg cttggcctcc 6240 tcaataggat ctcggttctg gccgtacaga cctcggccga caattatgat atccgttccg 6300 gtagacatga catcctcaac agttcggtac tgctgtccga gagcgtctcc cttgtcgtca 6360 agacccaccc cgggggtcag aataagccag tcctcagagt cgcccttagg tcggttctgg 6420 gcaatgaagc caaccacaaa ctcggggtcg gatcgggcaa gctcaatggt ctgcttggag 6480 tactcgccag tggccagaga gcccttgcaa gacagctcgg ccagcatgag cagacctctg 6540 gccagcttct cgttgggaga ggggactagg aactccttgt actgggagtt ctcgtagtca 6600 gagacgtcct ccttcttctg ttcagagaca gtttcctcgg caccagctcg caggccagca 6660 atgattccgg ttccgggtac accgtgggcg ttggtgatat cggaccactc ggcgattcgg 6720 tgacaccggt actggtgctt gacagtgttg ccaatatctg cgaactttct gtcctcgaac 6780 aggaagaaac cgtgcttaag agcaagttcc ttgaggggga gcacagtgcc ggcgtaggtg 6840 aagtcgtcaa tgatgtcgat atgggttttg atcatgcaca cataaggtcc gaccttatcg 6900 gcaagctcaa tgagctcctt ggtggtggta acatccagag aagcacacag gttggttttc 6960 ttggctgcca cgagcttgag cactcgagcg gcaaaggcgg acttgtggac gttagctcga 7020 gcttcgtagg agggcatttt ggtggtgaag aggagactga aataaattta gtctgcagaa 7080 ctttttatcg gaaccttatc tggggcagtg aagtatatgt tatggtaata gttacgagtt 7140 agttgaactt atagatagac tggactatac ggctatcggt ccaaattaga aagaacgtca 7200 atggctctct gggcgtcgcc tttgccgaca aaaatgtgat catgatgaaa gccagcaatg 7260 acgttgcagc tgatattgtt gtcggccaac cgcgccgaaa acgcagctgt cagacccaca 7320 gcctccaacg aagaatgtat cgtcaaagtg atccaagcac actcatagtt ggagtcgtac 7380 tccaaaggcg gcaatgacga gtcagacaga tactcgtcga ctcaggcgac gacggaattc 7440 ctgcagccca tctgcagaat tcaggagaga ccgggttggc ggcgtatttg tgtcccaaaa 7500 aacagcccca attgccccgg agaagacggc caggccgcct agatgacaaa ttcaacaact 7560 cacagctgac tttctgccat tgccactagg ggggggcctt tttatatggc caagccaagc 7620 tctccacgtc ggttgggctg cacccaacaa taaatgggta gggttgcacc aacaaaggga 7680 tgggatgggg ggtagaagat acgaggataa cggggctcaa tggcacaaat aagaacgaat 7740 actgccatta agactcgtga tccagcgact gacaccattg catcatctaa gggcctcaaa 7800 actacctcgg aactgctgcg ctgatctgga caccacagag gttccgagca ctttaggttg 7860 caccaaatgt cccaccaggt gcaggcagaa aacgctggaa cagcgtgtac agtttgtctt 7920 aacaaaaagt gagggcgctg aggtcgagca gggtggtgtg acttgttata gcctttagag 7980 ctgcgaaagc gcgtatggat ttggctcatc aggccagatt gagggtctgt ggacacatgt 8040 catgttagtg tacttcaatc gccccctgga tatagccccg acaataggcc gtggcctcat 8100 ttttttgcct tccgcacatt tccattgctc ggtacccaca ccttgcttct cctgcacttg 8160 ccaaccttaa tactggttta cattgaccaa catcttacaa gcggggggct tgtctagggt 8220 atatataaac agtggctctc ccaatcggtt gccagtctct tttttccttt ctttccccac 8280 agattcgaaa tctaaactac acatcacaca atgcctgtta ctgacgtcct taagcgaaag 8340 tccggtgtca tcgtcggcga cgatgtccga gccgtgagta tccacgacaa gatcagtgtc 8400 gagacgacgc gttttgtgta atgacacaat ccgaaagtcg ctagcaacac acactctcta 8460 cacaaactaa cccagctctc 8480 <210> SEQ ID NO 52 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer T7 <400> SEQUENCE: 52 ggaaacagct atgaccatg 19 <210> SEQ ID NO 53 <211> LENGTH: 459 <212> TYPE: PRT <213> ORGANISM: Rhizopus stolonifer <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: GenBank Accession No. AAX22052 <400> SEQUENCE: 53 Met Ser Thr Leu Asp Arg Gln Ser Ile Phe Thr Ile Lys Glu Leu Glu 1 5 10 15 Ser Ile Ser Gln Arg Ile His Asp Gly Asp Glu Glu Ala Met Lys Phe 20 25 30 Ile Ile Ile Asp Lys Lys Val Tyr Asp Val Thr Glu Phe Ile Glu Asp 35 40 45 His Pro Gly Gly Ala Gln Val Leu Leu Thr His Val Gly Lys Asp Ala 50 55 60 Ser Asp Val Phe His Ala Met His Pro Glu Ser Ala Tyr Glu Val Leu 65 70 75 80 Asn Asn Tyr Phe Val Gly Asp Val Gln Glu Thr Val Val Thr Glu Lys 85 90 95 Ser Ser Ser Ala Gln Phe Ala Val Glu Met Arg Gln Leu Arg Asp Gln 100 105 110 Leu Lys Lys Glu Gly Tyr Phe His Ser Ser Lys Leu Phe Tyr Ala Tyr 115 120 125 Lys Val Leu Ser Thr Leu Ala Ile Cys Ile Ala Gly Leu Ser Leu Leu 130 135 140 Tyr Ala Tyr Gly Arg Thr Ser Thr Leu Ala Val Val Ala Ser Ala Ile 145 150 155 160 Thr Val Gly Ile Phe Trp Gln Gln Cys Gly Trp Leu Ala His Asp Phe 165 170 175 Gly His His Gln Cys Phe Glu Asp Arg Thr Trp Asn Asp Val Leu Val 180 185 190 Val Phe Leu Gly Asn Phe Cys Gln Gly Phe Ser Leu Ser Trp Trp Lys 195 200 205 Asn Lys His Asn Thr His His Ala Ser Thr Asn Val His Gly Gln Asp 210 215 220 Pro Asp Ile Asp Thr Ala Pro Val Leu Leu Trp Asp Glu Tyr Ala Ser 225 230 235 240 Ala Ala Tyr Tyr Ala Ser Leu Asp Gln Glu Pro Thr Met Val Ser Arg 245 250 255 Phe Leu Ala Glu Gln Val Leu Pro His Gln Thr Arg Tyr Phe Phe Phe 260 265 270 Ile Leu Ala Phe Ala Arg Leu Ser Trp Ala Leu Gln Ser Leu Ser Tyr 275 280 285 Ser Phe Lys Lys Glu Ser Ile Asn Lys Ser Arg Gln Leu Asn Leu Phe 290 295 300 Glu Arg Val Cys Ile Val Gly His Trp Ala Leu Phe Ala Phe Cys Ile 305 310 315 320 Tyr Ser Trp Cys Ser Asn Val Tyr His Met Val Leu Phe Phe Leu Val 325 330 335 Ser Gln Ala Thr Thr Gly Tyr Thr Leu Ala Leu Val Phe Ala Leu Asn 340 345 350 His Asn Gly Met Pro Val Ile Thr Glu Glu Lys Ala Glu Ser Met Glu 355 360 365 Phe Phe Glu Ile Gln Val Ile Thr Gly Arg Asp Val Thr Leu Ser Pro 370 375 380 Leu Gly Asp Trp Phe Met Gly Gly Leu Asn Tyr Gln Ile Glu His His 385 390 395 400 Val Phe Pro Asn Met Pro Arg His Asn Leu Pro Thr Val Lys Pro Met 405 410 415 Val Lys Ser Leu Cys Gln Lys Tyr Asp Ile Asn Tyr His Asp Thr Gly 420 425 430 Phe Leu Lys Gly Thr Leu Glu Val Leu Gln Thr Leu Asp Ile Thr Ser 435 440 445 Lys Leu Ser Leu Gln Leu Ser Lys Lys Ser Phe 450 455 <210> SEQ ID NO 54 <211> LENGTH: 695 <212> TYPE: DNA <213> ORGANISM: Pavlova lutheri <400> SEQUENCE: 54 agggccaagg gtgccaacca ccttccacgt gagactacac accgtaggcc gatgggcaag 60 ggtggagacg gcggcgcgca ggcggtgagc gggaccgacg cgtctctcgc tgaggtgagc 120 tccgtcgata gcaagagcgt gcacgtcgtg ctctacggca agcgcgtgga tgtcacaaag 180 ttccagggct acgacgtggc ctggtggcgc gcgcgccata acacgcacca cgtgtgcacc 240 aacgaggatg gttcggaccc ggacatcaag acggcgcccc tgctcatcta cgtgcgagag 300 aacccgtcca ttgccaagcg gctcaacttc ttccagcgct ggcagcagta ctactatgtg 360 ccgaccatgg ccatcctcga cctctactgg cgcctggagt ccatcgcgta cgtggctgtg 420 cgcctgccta agatgtggat gcaggccgcc gctcttgccg ctcactacgc gctcctgtgc 480 tgggtcttcg cagcgcatct caacctcatc cctctcatga tggttgcacg cggcttcgcg 540 acgggcatcg ttgtctttgc aacccactat ggtgaggaca tcctcgaccg cgagcacgtc 600 gagggcatga cgctcgtcga gcagaccgcc aagacctccc gtaacatcac gggcggctgg 660 ctagtgaacg tgctcacggg cttcatctcc ctgca 695 <210> SEQ ID NO 55 <211> LENGTH: 1106 <212> TYPE: DNA <213> ORGANISM: Pavlova lutheri <400> SEQUENCE: 55 agggccaagg gtgccaacca ccttccacgt gagactacac accgtaggcc gatgggcaag 60 ggtggagacg gcggcgcgca ggcggtgagc gggaccgacg cgtctctcgc tgaggtgagc 120 tccgtcgata gcaagagcgt gcacgtcgtg ctctacggca agcgcgtgga tgtcacaaag 180 ttccagggct acgacgtggc ctggtggcgc gcgcgccata acacgcacca cgtgtgcacc 240 aacgaggatg gttcggaccc ggacatcaag acggcgcccc tgctcatcta cgtgcgagag 300 aacccgtcca ttgccaagcg gctcaacttc ttccagcgct ggcagcagta ctactatgtg 360

ccgaccatgg ccatcctcga cctctactgg cgcctggagt ccatcgcgta cgtggctgtg 420 cgcctgccta agatgtggat gcaggccgcc gctcttgccg ctcactacgc gctcctgtgc 480 tgggtcttcg cagcgcatct caacctcatc cctctcatga tggttgcacg cggcttcgcg 540 acgggcatcg ttgtctttgc aacccactat ggtgaggaca tcctcgaccg cgagcacgtc 600 gagggcatga cgctcgtcga gcagaccgcc aagacctccc gtaacatcac gggcggctgg 660 ctagtgaacg tgctcacggg cttcatctcc ctgcagaccg agcatcacct cttccccatg 720 atgcccaccg gcaacctaat gactatccag cccgaggtac gcgacttctt caagaagcat 780 ggcctcgagt accgcgaggg caacctcttc cagtgcgtgc accagaacat caaggctctc 840 gccttcgagc acctcctcca ctgagcgtca ccactcaagc gtcctaagtg cacaggtact 900 gtcttctgac cgatggccgc gcggctccct cggctggcag tggggccaac gagtggcctc 960 gcgggatcgg gcacgatcgg gcctccatga aacttcagtg ttcagagaca agccgacaac 1020 ctccgcatcg tgagaaatct tttaaagcag tatgttccat cacgccgctt ttgcagtcaa 1080 taacattacc caaaaaaaaa aaaaaa 1106 <210> SEQ ID NO 56 <211> LENGTH: 287 <212> TYPE: PRT <213> ORGANISM: Pavlova lutheri <400> SEQUENCE: 56 Arg Ala Lys Gly Ala Asn His Leu Pro Arg Glu Thr Thr His Arg Arg 1 5 10 15 Pro Met Gly Lys Gly Gly Asp Gly Gly Ala Gln Ala Val Ser Gly Thr 20 25 30 Asp Ala Ser Leu Ala Glu Val Ser Ser Val Asp Ser Lys Ser Val His 35 40 45 Val Val Leu Tyr Gly Lys Arg Val Asp Val Thr Lys Phe Gln Gly Tyr 50 55 60 Asp Val Ala Trp Trp Arg Ala Arg His Asn Thr His His Val Cys Thr 65 70 75 80 Asn Glu Asp Gly Ser Asp Pro Asp Ile Lys Thr Ala Pro Leu Leu Ile 85 90 95 Tyr Val Arg Glu Asn Pro Ser Ile Ala Lys Arg Leu Asn Phe Phe Gln 100 105 110 Arg Trp Gln Gln Tyr Tyr Tyr Val Pro Thr Met Ala Ile Leu Asp Leu 115 120 125 Tyr Trp Arg Leu Glu Ser Ile Ala Tyr Val Ala Val Arg Leu Pro Lys 130 135 140 Met Trp Met Gln Ala Ala Ala Leu Ala Ala His Tyr Ala Leu Leu Cys 145 150 155 160 Trp Val Phe Ala Ala His Leu Asn Leu Ile Pro Leu Met Met Val Ala 165 170 175 Arg Gly Phe Ala Thr Gly Ile Val Val Phe Ala Thr His Tyr Gly Glu 180 185 190 Asp Ile Leu Asp Arg Glu His Val Glu Gly Met Thr Leu Val Glu Gln 195 200 205 Thr Ala Lys Thr Ser Arg Asn Ile Thr Gly Gly Trp Leu Val Asn Val 210 215 220 Leu Thr Gly Phe Ile Ser Leu Gln Thr Glu His His Leu Phe Pro Met 225 230 235 240 Met Pro Thr Gly Asn Leu Met Thr Ile Gln Pro Glu Val Arg Asp Phe 245 250 255 Phe Lys Lys His Gly Leu Glu Tyr Arg Glu Gly Asn Leu Phe Gln Cys 260 265 270 Val His Gln Asn Ile Lys Ala Leu Ala Phe Glu His Leu Leu His 275 280 285 <210> SEQ ID NO 57 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer SeqE <400> SEQUENCE: 57 cgacacactc caatctttcc 20 <210> SEQ ID NO 58 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer SeqW <400> SEQUENCE: 58 ggtggctgga gttagacatc 20 <210> SEQ ID NO 59 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer AP1 <400> SEQUENCE: 59 gtaatacgac tcactatagg gc 22 <210> SEQ ID NO 60 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer GSP PvDES <400> SEQUENCE: 60 ctgcgaagac ccagcacagg 20 <210> SEQ ID NO 61 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer M13-28Rev <400> SEQUENCE: 61 gtaatacgac tcactatagg gc 22 <210> SEQ ID NO 62 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer PavDes seq <400> SEQUENCE: 62 ttgtggcgct caatcatctc c 21 <210> SEQ ID NO 63 <211> LENGTH: 1294 <212> TYPE: DNA <213> ORGANISM: Pavlova lutheri <400> SEQUENCE: 63 ctttgcgagc gcggcgcaga cgattgcggc ccgtagtgat cgcggtgcgc attgctgtgt 60 ttctagtttt gctgacgccc ggcccgataa tgacaccttc tcccgtttga aatactaata 120 agtaactata ttataatatt caaaggtggc gactatggat ctccttttct aaagttcagc 180 ggaattggga atcggagaaa tttcgagata tgtcataatc acgtgctcta tctcgaatga 240 accgcggccg gtgagcgatt actcgggaag ccaattccta ttaacgagtc aggggggatc 300 tttgaggtga gtcggccacg cagagagagc aaggaatcat cctcatccgc cgttctcgag 360 aaagagccaa gggtgccaac caccttccac gtgagactac acaccgtagg ccgatgggca 420 agggtggaga cggcggcgcg caggcggcga gcgggaccga cgcatctctc gctgaggtga 480 gctccgtcga tagcaagagc gtgcgcgtcg tgctctacgg caagcgcgtg gatgtcacaa 540 agttccagag ggcacacccg ggcgggagca aggtgttccg catcttccag gagcgcgacg 600 cgacggagca gttcgagtct taccactcgc ccaaggccat caagatgatg gagggcatgc 660 tcaagaagtc ggaggatgcg cccgcttccg tgcccctgcc ctcgcggtcc accatgggca 720 cggagttcaa ggagatgatt gagcgccaca agagggctgg tctctacgac ccttgcccgt 780 tggacgagct gttcaagctc accatcgtcc ttgcgcccat cttcgtgggc gcctatctcg 840 tgcggagcgg cgtctcgccc ctcgcgggcg cgctctccat gggctttggc ttctacctcg 900 acggctggct tgctcacrac tacctgcatc acgcagtctt caagggctcg gtcaacacgc 960 tcgtcaaggc gaacaacgcc atgggatacg ccctcggctt cctccagggc tacgacgtgg 1020 cctggtggcg cgcgcgccat aacacgcacc acgtgtgcac caacgaggat ggttcggacc 1080 cggacatcaa gacggcgccc ctgctcatct acgtgcgaga gaacccgtcc attgccaagc 1140 ggctcaactt cttccagcgc tggcagcagt actactatgt gccgaccatg gccatcctcg 1200 acctctactg gcgcctggag tccatcgcgt acgtggctgt gcgcctgcct aagatgtgga 1260 tgcaggccgc cgctcttgcc gctcactacg cgct 1294 <210> SEQ ID NO 64 <211> LENGTH: 1927 <212> TYPE: DNA <213> ORGANISM: Pavlova lutheri <400> SEQUENCE: 64 ctttgcgagc gcggcgcaga cgattgcggc ccgtagtgat cgcggtgcgc attgctgtgt 60 ttctagtttt gctgacgccc ggcccgataa tgacaccttc tcccgtttga aatactaata 120 agtaactata ttataatatt caaaggtggc gactatggat ctccttttct aaagttcagc 180 ggaattggga atcggagaaa tttcgagata tgtcataatc acgtgctcta tctcgaatga 240 accgcggccg gtgagcgatt actcgggaag ccaattccta ttaacgagtc aggggggatc 300 tttgaggtga gtcggccacg cagagagagc aaggaatcat cctcatccgc cgttctcgag 360 aaagagccaa gggtgccaac caccttccac gtgagactac acaccgtagg ccgatgggca 420 agggtggaga cggcggcgcg caggcggtga gcgggaccga cgcgtctctc gctgaggtga 480 gctccgtcga tagcaagagc gtgcacgtcg tgctctacgg caagcgcgtg gatgtcacaa 540 agttccagaa ggcacacccg ggcgggagca aggtgttccg catcttccag gagcgcgacg 600 cgacggagca gttcgagtct taccactcgc ccaaggccat caagatgatg gagggcatgc 660 tcaagaagtc ggaggatgcg cccgcttccg tgcccctgcc ctcgcggtcc accatgggca 720 cggagttcaa ggagatgatt gagcgccaca agagggctgg tctctacgac ccttgcccgt 780 tggacgagct gttcaagctc accatcgtcc ttgcgcccat cttcgtgggc gcctatctcg 840 tgcggagcgg cgtctcgccc ctcgcgggcg cgctctccat gggctttggc ttctacctcg 900

acggctggct tgctcacgac tacctgcatc acgcagtctt caagggctcg gtcaacacgc 960 tcgtcaaggc gaacaacgcc atgggatacg ccctcggctt cctccagggc tacgacgtgg 1020 cctggtggcg cgcgcgccat aacacgcacc acgtgtgcac caacgaggat ggttcggacc 1080 cggacatcaa gacggcgccc ctgctcatct acgtgcgaga gaacccgtcc attgccaagc 1140 ggctcaactt cttccagcgc tggcagcagt actactatgt gccgaccatg gccatcctcg 1200 acctctactg gcgcctggag tccatcgcgt acgtggctgt gcgcctgcct aagatgtgga 1260 tgcaggccgc cgctcttgcc gctcactacg cgctcctgtg ctgggtcttc gcagcgcatc 1320 tcaacctcat ccctctcatg atggttgcac gcggcttcgc gacgggcatc gttgtctttg 1380 caacccacta tggtgaggac atcctcgacc gcgagcacgt cgagggcatg acgctcgtcg 1440 agcagaccgc caagacctcc cgtaacatca cgggcggctg gctagtgaac gtgctcacgg 1500 gcttcatctc cctgcagacc gagcatcacc tcttccccat gatgcccacc ggcaacctaa 1560 tgactatcca gcccgaggta cgcgacttct tcaagaagca tggcctcgag taccgcgagg 1620 gcaacctctt ccagtgcgtg caccagaaca tcaaggctct cgccttcgag cacctcctcc 1680 actgagcgtc accactcaag cgtcctaagt gcacaggtac tgtcttctga ccgatggccg 1740 cgcggctccc tcggctggca gtggggccaa cgagtggcct cgcgggatcg ggcacgatcg 1800 ggcctccatg aaacttcagt gttcagagac aagccgacaa cctccgcatc gtgagaaatc 1860 ttttaaagca gtatgttcca tcacgccgct tttgcagtca ataacattac ccaaaaaaaa 1920 aaaaaaa 1927 <210> SEQ ID NO 65 <211> LENGTH: 459 <212> TYPE: PRT <213> ORGANISM: Rhizopus stolonifer <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: GenBank Accession No. ABB96724 <400> SEQUENCE: 65 Met Ser Thr Leu Asp Arg Gln Ser Ile Phe Thr Ile Lys Glu Leu Glu 1 5 10 15 Ser Ile Ser Gln Arg Ile His Asp Gly Asp Glu Glu Ala Met Lys Phe 20 25 30 Ile Ile Ile Asp Lys Lys Val Tyr Asp Val Thr Glu Phe Ile Glu Asp 35 40 45 His Pro Gly Gly Ala Gln Val Leu Leu Thr His Val Gly Lys Asp Ala 50 55 60 Ser Asp Val Phe His Ala Met His Pro Glu Ser Ala Tyr Glu Val Leu 65 70 75 80 Asn Asn Tyr Phe Val Gly Asp Val Gln Glu Thr Val Val Thr Glu Lys 85 90 95 Ser Ser Ser Ala Gln Phe Ala Val Glu Met Arg Gln Leu Arg Asp Gln 100 105 110 Leu Lys Lys Glu Gly Tyr Phe His Ser Ser Lys Leu Phe Tyr Ala Tyr 115 120 125 Lys Val Leu Ser Thr Leu Ala Ile Cys Ile Ala Gly Leu Ser Pro Leu 130 135 140 Tyr Ala Tyr Gly Arg Thr Ser Thr Leu Ala Val Val Ala Ser Ala Ile 145 150 155 160 Thr Val Gly Ile Phe Trp Gln Gln Cys Gly Trp Leu Ala His Asp Phe 165 170 175 Gly His His Gln Cys Phe Glu Asp Arg Thr Trp Asn Asp Val Leu Val 180 185 190 Val Phe Leu Gly Asn Phe Cys Gln Gly Phe Ser Leu Ser Trp Trp Lys 195 200 205 Asn Lys His Asn Thr His His Ala Ser Thr Asn Val His Gly Gln Asp 210 215 220 Pro Asp Ile Asp Thr Ala Pro Val Leu Leu Trp Asp Glu Tyr Ala Ser 225 230 235 240 Ala Ala Tyr Tyr Ala Ser Leu Asp Gln Glu Pro Thr Met Val Ser Arg 245 250 255 Phe Leu Ala Glu Gln Val Leu Pro His Gln Thr Arg Tyr Phe Phe Phe 260 265 270 Ile Leu Ala Phe Ala Arg Leu Ser Trp Ala Leu Gln Ser Leu Ser Tyr 275 280 285 Ser Phe Lys Lys Glu Ser Ile Asn Lys Ser Arg Gln Leu Asn Leu Phe 290 295 300 Glu Arg Val Cys Ile Val Gly His Trp Ala Leu Ser Ala Phe Cys Ile 305 310 315 320 Tyr Ser Trp Cys Ser Asn Val Tyr His Met Val Leu Phe Phe Leu Val 325 330 335 Ser Gln Ala Thr Thr Gly Tyr Thr Leu Ala Leu Val Phe Ala Leu Asn 340 345 350 His Asn Gly Met Pro Val Ile Thr Glu Glu Lys Ala Glu Ser Met Glu 355 360 365 Phe Phe Glu Ile Gln Val Ile Thr Gly Arg Asp Val Thr Leu Ser Pro 370 375 380 Leu Gly Asp Trp Phe Met Gly Gly Leu Asn Tyr Gln Ile Glu His His 385 390 395 400 Val Phe Pro Asn Met Pro Arg His Asn Leu Pro Thr Val Lys Pro Met 405 410 415 Val Lys Ser Leu Cys Gln Lys Tyr Asp Ile Asn Tyr His Asp Thr Gly 420 425 430 Phe Leu Lys Gly Thr Leu Glu Val Leu Gln Thr Leu Asp Ile Thr Ser 435 440 445 Lys Leu Ser Leu Gln Leu Ser Lys Lys Ser Phe 450 455 <210> SEQ ID NO 66 <211> LENGTH: 427 <212> TYPE: PRT <213> ORGANISM: Pavlova salina <300> PUBLICATION INFORMATION: <302> TITLE: SYNTHESIS OF LONG-CHAIN POLYUNSATURATED FATTY ACIDS BY RECOMBINANT CELLS <310> PATENT DOCUMENT NUMBER: WO 2005/103253 <311> PATENT FILING DATE: 2005-04-22 <312> PUBLICATION DATE: 2005-11-03 <313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(427) <400> SEQUENCE: 66 Met Gly Arg Gly Gly Asp Ser Ser Gly Gln Ala His Pro Ala Ala Glu 1 5 10 15 Leu Ala Val Pro Ser Asp Arg Ala Glu Val Ser Asn Ala Asp Ser Lys 20 25 30 Ala Leu His Ile Val Leu Tyr Gly Lys Arg Val Asp Val Thr Lys Phe 35 40 45 Gln Arg Thr His Pro Gly Gly Ser Lys Val Phe Arg Ile Phe Gln Asp 50 55 60 Arg Asp Ala Thr Glu Gln Phe Glu Ser Tyr His Ser Lys Arg Ala Ile 65 70 75 80 Lys Met Met Glu Gly Met Leu Lys Lys Ser Glu Asp Ala Pro Ala Asp 85 90 95 Thr Pro Leu Pro Ser Gln Ser Pro Met Gly Lys Asp Phe Lys Ala Met 100 105 110 Ile Glu Arg His Val Ala Ala Gly Tyr Tyr Asp Pro Cys Pro Leu Asp 115 120 125 Glu Leu Phe Lys Leu Ser Leu Val Leu Leu Pro Thr Phe Ala Gly Met 130 135 140 Tyr Met Leu Lys Ala Gly Val Gly Ser Pro Leu Cys Gly Ala Leu Met 145 150 155 160 Val Ser Phe Gly Trp Tyr Leu Asp Gly Trp Leu Ala His Asp Tyr Leu 165 170 175 His His Ser Val Phe Lys Gly Ser Val Ala Arg Thr Val Gly Trp Asn 180 185 190 Asn Ala Ala Gly Tyr Phe Leu Gly Phe Val Gln Gly Tyr Ala Val Glu 195 200 205 Trp Trp Arg Ala Arg His Asn Thr His His Val Cys Thr Asn Glu Asp 210 215 220 Gly Ser Asp Pro Asp Ile Lys Thr Ala Pro Leu Leu Ile Tyr Val Arg 225 230 235 240 Asn Lys Pro Ser Ile Ala Lys Arg Leu Asn Ala Phe Gln Arg Tyr Gln 245 250 255 Gln Tyr Tyr Tyr Val Pro Val Met Ala Ile Leu Asp Leu Tyr Trp Arg 260 265 270 Leu Glu Ser Ile Ala Tyr Val Ala Met Arg Leu Pro Lys Met Leu Pro 275 280 285 Gln Ala Leu Ala Leu Val Ala His Tyr Ala Ile Val Ala Trp Val Phe 290 295 300 Ala Gly Asn Tyr His Leu Leu Pro Leu Val Thr Val Leu Arg Gly Phe 305 310 315 320 Gly Thr Gly Ile Thr Val Phe Ala Thr His Tyr Gly Glu Asp Ile Leu 325 330 335 Asp Ala Asp Gln Val Arg His Met Thr Leu Val Glu Gln Thr Ala Leu 340 345 350 Thr Ser Arg Asn Ile Ser Gly Gly Trp Leu Val Asn Val Leu Thr Gly 355 360 365 Phe Ile Ser Leu Gln Thr Glu His His Leu Phe Pro Met Met Pro Thr 370 375 380 Gly Asn Leu Met Thr Ile Gln Pro Glu Val Arg Ala Phe Phe Lys Lys 385 390 395 400 His Gly Leu Glu Tyr Arg Glu Gly Asn Leu Ile Glu Cys Val Arg Gln 405 410 415 Asn Ile Arg Ala Leu Ala Phe Glu His Leu Leu 420 425 <210> SEQ ID NO 67 <211> LENGTH: 1338 <212> TYPE: DNA <213> ORGANISM: Mortierella alpina <400> SEQUENCE: 67 atgggaacgg accaaggaaa aaccttcacc tgggaagagc tggcggccca taacaccaag 60 gacgacctac tcttggccat ccgcggcagg gtgtacgatg tcacaaagtt cttgagccgc 120 catcctggtg gagtggacac tctcctgctc ggagctggcc gagatgttac tccggtcttt 180 gagatgtatc acgcgtttgg ggctgcagat gccattatga agaagtacta tgtcggtaca 240 ctggtctcga atgagctgcc catcttcccg gagccaacgg tgttccacaa aaccatcaag 300 acgagagtcg agggctactt tacggatcgg aacattgatc ccaagaatag accagagatc 360 tggggacgat acgctcttat ctttggatcc ttgatcgctt cctactacgc gcagctcttt 420

gtgcctttcg ttgtcgaacg cacatggctt caggtggtgt ttgcaatcat catgggattt 480 gcgtgcgcac aagtcggact caaccctctt catgatgcgt ctcacttttc agtgacccac 540 aaccccactg tctggaagat tctgggagcc acgcacgact ttttcaacgg agcatcgtac 600 ctggtgtgga tgtaccaaca tatgctcggc catcacccct acaccaacat tgctggagca 660 gatcccgacg tgtcgacgtc tgagcccgat gttcgtcgta tcaagcccaa ccaaaagtgg 720 tttgtcaacc acatcaacca gcacatgttt gttcctttcc tgtacggact gctggcgttc 780 aaggtgcgca ttcaggacat caacattttg tactttgtca agaccaatga cgctattcgt 840 gtcaatccca tctcgacatg gcacactgtg atgttctggg gcggcaaggc tttctttgtc 900 tggtatcgcc tgattgttcc cctgcagtat ctgcccctgg gcaaggtgct gctcttgttc 960 acggtcgcgg acatggtgtc gtcttactgg ctggcgctga ccttccaggc gaaccacgtt 1020 gttgaggaag ttcagtggcc gttgcctgac gagaacggga tcatccaaaa ggactgggca 1080 gctatgcagg tcgagactac gcaggattac gcacacgatt cgcacctctg gaccagcatc 1140 actggcagct tgaactacca ggctgtgcac catctgttcc ccaacgtgtc gcagcaccat 1200 tatcccgata ttctggccat catcaagaac acctgcagcg agtacaaggt tccatacctt 1260 gtcaaggata cgttttggca agcatttgct tcacatttgg agcacttgcg tgttcttgga 1320 ctccgtccca aggaagag 1338 <210> SEQ ID NO 68 <211> LENGTH: 446 <212> TYPE: PRT <213> ORGANISM: Mortierella alpina <400> SEQUENCE: 68 Met Gly Thr Asp Gln Gly Lys Thr Phe Thr Trp Glu Glu Leu Ala Ala 1 5 10 15 His Asn Thr Lys Asp Asp Leu Leu Leu Ala Ile Arg Gly Arg Val Tyr 20 25 30 Asp Val Thr Lys Phe Leu Ser Arg His Pro Gly Gly Val Asp Thr Leu 35 40 45 Leu Leu Gly Ala Gly Arg Asp Val Thr Pro Val Phe Glu Met Tyr His 50 55 60 Ala Phe Gly Ala Ala Asp Ala Ile Met Lys Lys Tyr Tyr Val Gly Thr 65 70 75 80 Leu Val Ser Asn Glu Leu Pro Ile Phe Pro Glu Pro Thr Val Phe His 85 90 95 Lys Thr Ile Lys Thr Arg Val Glu Gly Tyr Phe Thr Asp Arg Asn Ile 100 105 110 Asp Pro Lys Asn Arg Pro Glu Ile Trp Gly Arg Tyr Ala Leu Ile Phe 115 120 125 Gly Ser Leu Ile Ala Ser Tyr Tyr Ala Gln Leu Phe Val Pro Phe Val 130 135 140 Val Glu Arg Thr Trp Leu Gln Val Val Phe Ala Ile Ile Met Gly Phe 145 150 155 160 Ala Cys Ala Gln Val Gly Leu Asn Pro Leu His Asp Ala Ser His Phe 165 170 175 Ser Val Thr His Asn Pro Thr Val Trp Lys Ile Leu Gly Ala Thr His 180 185 190 Asp Phe Phe Asn Gly Ala Ser Tyr Leu Val Trp Met Tyr Gln His Met 195 200 205 Leu Gly His His Pro Tyr Thr Asn Ile Ala Gly Ala Asp Pro Asp Val 210 215 220 Ser Thr Ser Glu Pro Asp Val Arg Arg Ile Lys Pro Asn Gln Lys Trp 225 230 235 240 Phe Val Asn His Ile Asn Gln His Met Phe Val Pro Phe Leu Tyr Gly 245 250 255 Leu Leu Ala Phe Lys Val Arg Ile Gln Asp Ile Asn Ile Leu Tyr Phe 260 265 270 Val Lys Thr Asn Asp Ala Ile Arg Val Asn Pro Ile Ser Thr Trp His 275 280 285 Thr Val Met Phe Trp Gly Gly Lys Ala Phe Phe Val Trp Tyr Arg Leu 290 295 300 Ile Val Pro Leu Gln Tyr Leu Pro Leu Gly Lys Val Leu Leu Leu Phe 305 310 315 320 Thr Val Ala Asp Met Val Ser Ser Tyr Trp Leu Ala Leu Thr Phe Gln 325 330 335 Ala Asn His Val Val Glu Glu Val Gln Trp Pro Leu Pro Asp Glu Asn 340 345 350 Gly Ile Ile Gln Lys Asp Trp Ala Ala Met Gln Val Glu Thr Thr Gln 355 360 365 Asp Tyr Ala His Asp Ser His Leu Trp Thr Ser Ile Thr Gly Ser Leu 370 375 380 Asn Tyr Gln Ala Val His His Leu Phe Pro Asn Val Ser Gln His His 385 390 395 400 Tyr Pro Asp Ile Leu Ala Ile Ile Lys Asn Thr Cys Ser Glu Tyr Lys 405 410 415 Val Pro Tyr Leu Val Lys Asp Thr Phe Trp Gln Ala Phe Ala Ser His 420 425 430 Leu Glu His Leu Arg Val Leu Gly Leu Arg Pro Lys Glu Glu 435 440 445 <210> SEQ ID NO 69 <211> LENGTH: 6473 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pY5-22 <400> SEQUENCE: 69 ggtggagctc cagcttttgt tccctttagt gagggttaat ttcgagcttg gcgtaatcat 60 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacgtacgag 120 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg 180 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 240 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca 300 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 360 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 420 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 480 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 540 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 600 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 660 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 720 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 780 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 840 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 900 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 960 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 1020 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 1080 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 1140 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 1200 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 1260 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 1320 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 1380 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 1440 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 1500 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 1560 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 1620 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 1680 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 1740 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 1800 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 1860 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 1920 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 1980 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 2040 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat 2100 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 2160 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgcgc 2220 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 2280 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 2340 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 2400 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc 2460 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 2520 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga 2580 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 2640 attttaacaa aatattaacg cttacaattt ccattcgcca ttcaggctgc gcaactgttg 2700 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc 2760 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac 2820 ggccagtgaa ttgtaatacg actcactata gggcgaattg ggtaccgggc cccccctcga 2880 ggtcgatggt gtcgataagc ttgatatcga attcatgtca cacaaaccga tcttcgcctc 2940 aaggaaacct aattctacat ccgagagact gccgagatcc agtctacact gattaatttt 3000 cgggccaata atttaaaaaa atcgtgttat ataatattat atgtattata tatatacatc 3060 atgatgatac tgacagtcat gtcccattgc taaatagaca gactccatct gccgcctcca 3120 actgatgttc tcaatattta aggggtcatc tcgcattgtt taataataaa cagactccat 3180 ctaccgcctc caaatgatgt tctcaaaata tattgtatga acttattttt attacttagt 3240 attattagac aacttacttg ctttatgaaa aacacttcct atttaggaaa caatttataa 3300 tggcagttcg ttcatttaac aatttatgta gaataaatgt tataaatgcg tatgggaaat 3360 cttaaatatg gatagcataa atgatatctg cattgcctaa ttcgaaatca acagcaacga 3420 aaaaaatccc ttgtacaaca taaatagtca tcgagaaata tcaactatca aagaacagct 3480 attcacacgt tactattgag attattattg gacgagaatc acacactcaa ctgtctttct 3540

ctcttctaga aatacaggta caagtatgta ctattctcat tgttcatact tctagtcatt 3600 tcatcccaca tattccttgg atttctctcc aatgaatgac attctatctt gcaaattcaa 3660 caattataat aagatatacc aaagtagcgg tatagtggca atcaaaaagc ttctctggtg 3720 tgcttctcgt atttattttt attctaatga tccattaaag gtatatattt atttcttgtt 3780 atataatcct tttgtttatt acatgggctg gatacataaa ggtattttga tttaattttt 3840 tgcttaaatt caatcccccc tcgttcagtg tcaactgtaa tggtaggaaa ttaccatact 3900 tttgaagaag caaaaaaaat gaaagaaaaa aaaaatcgta tttccaggtt agacgttccg 3960 cagaatctag aatgcggtat gcggtacatt gttcttcgaa cgtaaaagtt gcgctccctg 4020 agatattgta catttttgct tttacaagta caagtacatc gtacaactat gtactactgt 4080 tgatgcatcc acaacagttt gttttgtttt tttttgtttt ttttttttct aatgattcat 4140 taccgctatg tatacctact tgtacttgta gtaagccggg ttattggcgt tcaattaatc 4200 atagacttat gaatctgcac ggtgtgcgct gcgagttact tttagcttat gcatgctact 4260 tgggtgtaat attgggatct gttcggaaat caacggatgc tcaaccgatt tcgacagtaa 4320 ttaattaagt catacacaag tcagctttct tcgagcctca tataagtata agtagttcaa 4380 cgtattagca ctgtacccag catctccgta tcgagaaaca caacaacatg ccccattgga 4440 cagatcatgc ggatacacag gttgtgcagt atcatacata ctcgatcaga caggtcgtct 4500 gaccatcata caagctgaac aagcgctcca tacttgcacg ctctctatat acacagttaa 4560 attacatatc catagtctaa cctctaacag ttaatcttct ggtaagcctc ccagccagcc 4620 ttctggtatc gcttggcctc ctcaatagga tctcggttct ggccgtacag acctcggccg 4680 acaattatga tatccgttcc ggtagacatg acatcctcaa cagttcggta ctgctgtccg 4740 agagcgtctc ccttgtcgtc aagacccacc ccgggggtca gaataagcca gtcctcagag 4800 tcgcccttag gtcggttctg ggcaatgaag ccaaccacaa actcggggtc ggatcgggca 4860 agctcaatgg tctgcttgga gtactcgcca gtggccagag agcccttgca agacagctcg 4920 gccagcatga gcagacctct ggccagcttc tcgttgggag aggggactag gaactccttg 4980 tactgggagt tctcgtagtc agagacgtcc tccttcttct gttcagagac agtttcctcg 5040 gcaccagctc gcaggccagc aatgattccg gttccgggta caccgtgggc gttggtgata 5100 tcggaccact cggcgattcg gtgacaccgg tactggtgct tgacagtgtt gccaatatct 5160 gcgaactttc tgtcctcgaa caggaagaaa ccgtgcttaa gagcaagttc cttgaggggg 5220 agcacagtgc cggcgtaggt gaagtcgtca atgatgtcga tatgggtttt gatcatgcac 5280 acataaggtc cgaccttatc ggcaagctca atgagctcct tggtggtggt aacatccaga 5340 gaagcacaca ggttggtttt cttggctgcc acgagcttga gcactcgagc ggcaaaggcg 5400 gacttgtgga cgttagctcg agcttcgtag gagggcattt tggtggtgaa gaggagactg 5460 aaataaattt agtctgcaga actttttatc ggaaccttat ctggggcagt gaagtatatg 5520 ttatggtaat agttacgagt tagttgaact tatagataga ctggactata cggctatcgg 5580 tccaaattag aaagaacgtc aatggctctc tgggcgtcgc ctttgccgac aaaaatgtga 5640 tcatgatgaa agccagcaat gacgttgcag ctgatattgt tgtcggccaa ccgcgccgaa 5700 aacgcagctg tcagacccac agcctccaac gaagaatgta tcgtcaaagt gatccaagca 5760 cactcatagt tggagtcgta ctccaaaggc ggcaatgacg agtcagacag atactcgtcg 5820 actcaggcga cgacggaatt cctgcagccc atctgcagaa ttcaggagag accgggttgg 5880 cggcgtattt gtgtcccaaa aaacagcccc aattgcccca attgacccca aattgaccca 5940 gtagcgggcc caaccccggc gagagccccc ttcaccccac atatcaaacc tcccccggtt 6000 cccacacttg ccgttaaggg cgtagggtac tgcagtctgg aatctacgct tgttcagact 6060 ttgtactagt ttctttgtct ggccatccgg gtaacccatg ccggacgcaa aatagactac 6120 tgaaaatttt tttgctttgt ggttgggact ttagccaagg gtataaaaga ccaccgtccc 6180 cgaattacct ttcctcttct tttctctctc tccttgtcaa ctcacacccg aaatcgttaa 6240 gcatttcctt ctgagtataa gaatcattca ccatggatcc actagttcta gagcggccgc 6300 caccgcggcc cgagattccg gcctcttcgg ccgccaagcg acccgggtgg acgtctagag 6360 gtacctagca attaacagat agtttgccgg tgataattct cttaacctcc cacactcctt 6420 tgacataacg atttatgtaa cgaaactgaa atttgaccag atattgtgtc cgc 6473 <210> SEQ ID NO 70 <211> LENGTH: 6970 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pY5-22GPD <400> SEQUENCE: 70 tcgacgcagt aggatgtcct gcacgggtct ttttgtgggg tgtggagaaa ggggtgcttg 60 gagatggaag ccggtagaac cgggctgctt gtgcttggag atggaagccg gtagaaccgg 120 gctgcttggg gggatttggg gccgctgggc tccaaagagg ggtaggcatt tcgttggggt 180 tacgtaattg cggcatttgg gtcctgcgcg catgtcccat tggtcagaat tagtccggat 240 aggagactta tcagccaatc acagcgccgg atccacctgt aggttgggtt gggtgggagc 300 acccctccac agagtagagt caaacagcag cagcaacatg atagttgggg gtgtgcgtgt 360 taaaggaaaa aaaagaagct tgggttatat tcccgctcta tttagaggtt gcgggataga 420 cgccgacgga gggcaatggc gccatggaac cttgcggata tcgatacgcc gcggcggact 480 gcgtccgaac cagctccagc agcgtttttt ccgggccatt gagccgactg cgaccccgcc 540 aacgtgtctt ggcccacgca ctcatgtcat gttggtgttg ggaggccact ttttaagtag 600 cacaaggcac ctagctcgca gcaaggtgtc cgaaccaaag aagcggctgc agtggtgcaa 660 acggggcgga aacggcggga aaaagccacg ggggcacgaa ttgaggcacg ccctcgaatt 720 tgagacgagt cacggcccca ttcgcccgcg caatggctcg ccaacgcccg gtcttttgca 780 ccacatcagg ttaccccaag ccaaaccttt gtgttaaaaa gcttaacata ttataccgaa 840 cgtaggtttg ggcgggcttg ctccgtctgt ccaaggcaac atttatataa gggtctgcat 900 cgccggctca attgaatctt ttttcttctt ctcttctcta tattcattct tgaattaaac 960 acacatcaat ccgcggccgc caccgcggcc cgagattccg gcctcttcgg ccgccaagcg 1020 acccgggtgg acgtctagag gtacctagca attaacagat agtttgccgg tgataattct 1080 cttaacctcc cacactcctt tgacataacg atttatgtaa cgaaactgaa atttgaccag 1140 atattgtgtc cgcggtggag ctccagcttt tgttcccttt agtgagggtt aatttcgagc 1200 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 1260 cacaacgtac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 1320 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 1380 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 1440 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 1500 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 1560 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 1620 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 1680 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 1740 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 1800 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 1860 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 1920 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 1980 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 2040 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc 2100 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 2160 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 2220 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 2280 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 2340 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 2400 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 2460 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 2520 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 2580 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 2640 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 2700 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 2760 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 2820 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 2880 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 2940 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 3000 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 3060 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 3120 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 3180 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 3240 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 3300 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 3360 ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc 3420 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt 3480 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc 3540 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt 3600 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt 3660 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt 3720 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa 3780 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttccattcg ccattcaggc 3840 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga 3900 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac 3960 gttgtaaaac gacggccagt gaattgtaat acgactcact atagggcgaa ttgggtaccg 4020 ggccccccct cgaggtcgat ggtgtcgata agcttgatat cgaattcatg tcacacaaac 4080 cgatcttcgc ctcaaggaaa cctaattcta catccgagag actgccgaga tccagtctac 4140 actgattaat tttcgggcca ataatttaaa aaaatcgtgt tatataatat tatatgtatt 4200 atatatatac atcatgatga tactgacagt catgtcccat tgctaaatag acagactcca 4260

tctgccgcct ccaactgatg ttctcaatat ttaaggggtc atctcgcatt gtttaataat 4320 aaacagactc catctaccgc ctccaaatga tgttctcaaa atatattgta tgaacttatt 4380 tttattactt agtattatta gacaacttac ttgctttatg aaaaacactt cctatttagg 4440 aaacaattta taatggcagt tcgttcattt aacaatttat gtagaataaa tgttataaat 4500 gcgtatggga aatcttaaat atggatagca taaatgatat ctgcattgcc taattcgaaa 4560 tcaacagcaa cgaaaaaaat cccttgtaca acataaatag tcatcgagaa atatcaacta 4620 tcaaagaaca gctattcaca cgttactatt gagattatta ttggacgaga atcacacact 4680 caactgtctt tctctcttct agaaatacag gtacaagtat gtactattct cattgttcat 4740 acttctagtc atttcatccc acatattcct tggatttctc tccaatgaat gacattctat 4800 cttgcaaatt caacaattat aataagatat accaaagtag cggtatagtg gcaatcaaaa 4860 agcttctctg gtgtgcttct cgtatttatt tttattctaa tgatccatta aaggtatata 4920 tttatttctt gttatataat ccttttgttt attacatggg ctggatacat aaaggtattt 4980 tgatttaatt ttttgcttaa attcaatccc ccctcgttca gtgtcaactg taatggtagg 5040 aaattaccat acttttgaag aagcaaaaaa aatgaaagaa aaaaaaaatc gtatttccag 5100 gttagacgtt ccgcagaatc tagaatgcgg tatgcggtac attgttcttc gaacgtaaaa 5160 gttgcgctcc ctgagatatt gtacattttt gcttttacaa gtacaagtac atcgtacaac 5220 tatgtactac tgttgatgca tccacaacag tttgttttgt ttttttttgt tttttttttt 5280 tctaatgatt cattaccgct atgtatacct acttgtactt gtagtaagcc gggttattgg 5340 cgttcaatta atcatagact tatgaatctg cacggtgtgc gctgcgagtt acttttagct 5400 tatgcatgct acttgggtgt aatattggga tctgttcgga aatcaacgga tgctcaaccg 5460 atttcgacag taattaatta agtcatacac aagtcagctt tcttcgagcc tcatataagt 5520 ataagtagtt caacgtatta gcactgtacc cagcatctcc gtatcgagaa acacaacaac 5580 atgccccatt ggacagatca tgcggataca caggttgtgc agtatcatac atactcgatc 5640 agacaggtcg tctgaccatc atacaagctg aacaagcgct ccatacttgc acgctctcta 5700 tatacacagt taaattacat atccatagtc taacctctaa cagttaatct tctggtaagc 5760 ctcccagcca gccttctggt atcgcttggc ctcctcaata ggatctcggt tctggccgta 5820 cagacctcgg ccgacaatta tgatatccgt tccggtagac atgacatcct caacagttcg 5880 gtactgctgt ccgagagcgt ctcccttgtc gtcaagaccc accccggggg tcagaataag 5940 ccagtcctca gagtcgccct taggtcggtt ctgggcaatg aagccaacca caaactcggg 6000 gtcggatcgg gcaagctcaa tggtctgctt ggagtactcg ccagtggcca gagagccctt 6060 gcaagacagc tcggccagca tgagcagacc tctggccagc ttctcgttgg gagaggggac 6120 taggaactcc ttgtactggg agttctcgta gtcagagacg tcctccttct tctgttcaga 6180 gacagtttcc tcggcaccag ctcgcaggcc agcaatgatt ccggttccgg gtacaccgtg 6240 ggcgttggtg atatcggacc actcggcgat tcggtgacac cggtactggt gcttgacagt 6300 gttgccaata tctgcgaact ttctgtcctc gaacaggaag aaaccgtgct taagagcaag 6360 ttccttgagg gggagcacag tgccggcgta ggtgaagtcg tcaatgatgt cgatatgggt 6420 tttgatcatg cacacataag gtccgacctt atcggcaagc tcaatgagct ccttggtggt 6480 ggtaacatcc agagaagcac acaggttggt tttcttggct gccacgagct tgagcactcg 6540 agcggcaaag gcggacttgt ggacgttagc tcgagcttcg taggagggca ttttggtggt 6600 gaagaggaga ctgaaataaa tttagtctgc agaacttttt atcggaacct tatctggggc 6660 agtgaagtat atgttatggt aatagttacg agttagttga acttatagat agactggact 6720 atacggctat cggtccaaat tagaaagaac gtcaatggct ctctgggcgt cgcctttgcc 6780 gacaaaaatg tgatcatgat gaaagccagc aatgacgttg cagctgatat tgttgtcggc 6840 caaccgcgcc gaaaacgcag ctgtcagacc cacagcctcc aacgaagaat gtatcgtcaa 6900 agtgatccaa gcacactcat agttggagtc gtactccaaa ggcggcaatg acgagtcaga 6960 cagatactcg 6970 <210> SEQ ID NO 71 <211> LENGTH: 968 <212> TYPE: DNA <213> ORGANISM: Yarrowia lipolytica <300> PUBLICATION INFORMATION: <302> TITLE: GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE AND PHOSPHOGLYCERATE MUTASE PROMOTERS FOR GENE EXPRESSION IN OLEAGINOUS YEAST <310> PATENT DOCUMENT NUMBER: US-2005-0014270-A1 <311> PATENT FILING DATE: 2004-06-16 <312> PUBLICATION DATE: 2005-01-20 <313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(968) <400> SEQUENCE: 71 tcgacgcagt aggatgtcct gcacgggtct ttttgtgggg tgtggagaaa ggggtgcttg 60 gagatggaag ccggtagaac cgggctgctt gtgcttggag atggaagccg gtagaaccgg 120 gctgcttggg gggatttggg gccgctgggc tccaaagagg ggtaggcatt tcgttggggt 180 tacgtaattg cggcatttgg gtcctgcgcg catgtcccat tggtcagaat tagtccggat 240 aggagactta tcagccaatc acagcgccgg atccacctgt aggttgggtt gggtgggagc 300 acccctccac agagtagagt caaacagcag cagcaacatg atagttgggg gtgtgcgtgt 360 taaaggaaaa aaaagaagct tgggttatat tcccgctcta tttagaggtt gcgggataga 420 cgccgacgga gggcaatggc gccatggaac cttgcggata tcgatacgcc gcggcggact 480 gcgtccgaac cagctccagc agcgtttttt ccgggccatt gagccgactg cgaccccgcc 540 aacgtgtctt ggcccacgca ctcatgtcat gttggtgttg ggaggccact ttttaagtag 600 cacaaggcac ctagctcgca gcaaggtgtc cgaaccaaag aagcggctgc agtggtgcaa 660 acggggcgga aacggcggga aaaagccacg ggggcacgaa ttgaggcacg ccctcgaatt 720 tgagacgagt cacggcccca ttcgcccgcg caatggctcg ccaacgcccg gtcttttgca 780 ccacatcagg ttaccccaag ccaaaccttt gtgttaaaaa gcttaacata ttataccgaa 840 cgtaggtttg ggcgggcttg ctccgtctgt ccaaggcaac atttatataa gggtctgcat 900 cgccggctca attgaatctt ttttcttctt ctcttctcta tattcattct tgaattaaac 960 acacatca 968 <210> SEQ ID NO 72 <211> LENGTH: 8630 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pYZDE2-S <400> SEQUENCE: 72 ggtggagctc cagcttttgt tccctttagt gagggttaat ttcgagcttg gcgtaatcat 60 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacgtacgag 120 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg 180 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 240 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca 300 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 360 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 420 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 480 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 540 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 600 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 660 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 720 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 780 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 840 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 900 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 960 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 1020 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 1080 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 1140 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 1200 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 1260 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 1320 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 1380 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 1440 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 1500 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 1560 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 1620 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 1680 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 1740 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 1800 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 1860 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 1920 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 1980 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 2040 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat 2100 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 2160 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgcgc 2220 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 2280 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 2340 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 2400 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc 2460 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 2520 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga 2580 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 2640 attttaacaa aatattaacg cttacaattt ccattcgcca ttcaggctgc gcaactgttg 2700 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc 2760 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac 2820 ggccagtgaa ttgtaatacg actcactata gggcgaattg ggtaccgggc cccccctcga 2880 ggtcgatggt gtcgataagc ttgatatcga attcatgtca cacaaaccga tcttcgcctc 2940 aaggaaacct aattctacat ccgagagact gccgagatcc agtctacact gattaatttt 3000

cgggccaata atttaaaaaa atcgtgttat ataatattat atgtattata tatatacatc 3060 atgatgatac tgacagtcat gtcccattgc taaatagaca gactccatct gccgcctcca 3120 actgatgttc tcaatattta aggggtcatc tcgcattgtt taataataaa cagactccat 3180 ctaccgcctc caaatgatgt tctcaaaata tattgtatga acttattttt attacttagt 3240 attattagac aacttacttg ctttatgaaa aacacttcct atttaggaaa caatttataa 3300 tggcagttcg ttcatttaac aatttatgta gaataaatgt tataaatgcg tatgggaaat 3360 cttaaatatg gatagcataa atgatatctg cattgcctaa ttcgaaatca acagcaacga 3420 aaaaaatccc ttgtacaaca taaatagtca tcgagaaata tcaactatca aagaacagct 3480 attcacacgt tactattgag attattattg gacgagaatc acacactcaa ctgtctttct 3540 ctcttctaga aatacaggta caagtatgta ctattctcat tgttcatact tctagtcatt 3600 tcatcccaca tattccttgg atttctctcc aatgaatgac attctatctt gcaaattcaa 3660 caattataat aagatatacc aaagtagcgg tatagtggca atcaaaaagc ttctctggtg 3720 tgcttctcgt atttattttt attctaatga tccattaaag gtatatattt atttcttgtt 3780 atataatcct tttgtttatt acatgggctg gatacataaa ggtattttga tttaattttt 3840 tgcttaaatt caatcccccc tcgttcagtg tcaactgtaa tggtaggaaa ttaccatact 3900 tttgaagaag caaaaaaaat gaaagaaaaa aaaaatcgta tttccaggtt agacgttccg 3960 cagaatctag aatgcggtat gcggtacatt gttcttcgaa cgtaaaagtt gcgctccctg 4020 agatattgta catttttgct tttacaagta caagtacatc gtacaactat gtactactgt 4080 tgatgcatcc acaacagttt gttttgtttt tttttgtttt ttttttttct aatgattcat 4140 taccgctatg tatacctact tgtacttgta gtaagccggg ttattggcgt tcaattaatc 4200 atagacttat gaatctgcac ggtgtgcgct gcgagttact tttagcttat gcatgctact 4260 tgggtgtaat attgggatct gttcggaaat caacggatgc tcaaccgatt tcgacagtaa 4320 taatttgaat cgaatcggag cctaaaatga acccgagtat atctcataaa attctcggtg 4380 agaggtctgt gactgtcagt acaaggtgcc ttcattatgc cctcaacctt accatacctc 4440 actgaatgta gtgtacctct aaaaatgaaa tacagtgcca aaagccaagg cactgagctc 4500 gtctaacgga cttgatatac aaccaattaa aacaaatgaa aagaaataca gttctttgta 4560 tcatttgtaa caattaccct gtacaaacta aggtattgaa atcccacaat attcccaaag 4620 tccacccctt tccaaattgt catgcctaca actcatatac caagcactaa cctaccaaac 4680 accactaaaa ccccacaaaa tatatcttac cgaatataca gtaacaagct accaccacac 4740 tcgttgggtg cagtcgccag cttaaagata tctatccaca tcagccacaa ctcccttcct 4800 ttaataaacc gactacaccc ttggctattg aggttatgag tgaatatact gtagacaaga 4860 cactttcaag aagactgttt ccaaaacgta ccactgtcct ccactacaaa cacacccaat 4920 ctgcttcttc tagtcaaggt tgctacaccg gtaaattata aatcatcatt tcattagcag 4980 ggcagggccc tttttataga gtcttataca ctagcggacc ctgccggtag accaacccgc 5040 aggcgcgtca gtttgctcct tccatcaatg cgtcgtagaa acgacttact ccttcttgag 5100 cagctccttg accttgttgg caacaagtct ccgacctcgg aggtggagga agagcctccg 5160 atatcggcgg tagtgatacc agcctcgacg gactccttga cggcagcctc aacagcgtca 5220 ccggcgggct tcatgttaag agagaacttg agcatcatgg cggcagacag aatggtggca 5280 atggggttga ccttctgctt gccgagatcg ggggcagatc cgtgacaggg ctcgtacaga 5340 ccgaacgcct cgttggtgtc gggcagagaa gccagagagg cggagggcag cagacccaga 5400 gaaccgggga tgacggaggc ctcgtcggag atgatatcgc caaacatgtt ggtggtgatg 5460 atgataccat tcatcttgga gggctgcttg atgaggatca tggcggccga gtcgatcagc 5520 tggtggttga gctcgagctg ggggaattcg tccttgagga ctcgagtgac agtctttcgc 5580 caaagtcgag aggaggccag cacgttggcc ttgtcaagag accacacggg aagagggggg 5640 ttgtgctgaa gggccaggaa ggcggccatt cgggcaattc gctcaacctc aggaacggag 5700 taggtctcgg tgtcggaagc gacgccagat ccgtcatcct cctttcgctc tccaaagtag 5760 atacctccga cgagctctcg gacaatgatg aagtcggtgc cctcaacgtt tcggatgggg 5820 gagagatcgg cgagcttggg cgacagcagc tggcagggtc gcaggttggc gtacaggttc 5880 aggtcctttc gcagcttgag gagaccctgc tcgggtcgca cgtcggttcg tccgtcggga 5940 gtggtccata cggtgttggc agcgcctccg acagcaccga gcataataga gtcagccttt 6000 cggcagatgt cgagagtagc gtcggtgatg ggctcgccct ccttctcaat ggcagctcct 6060 ccaatgagtc ggtcctcaaa cacaaactcg gtgccggagg cctcagcaac agacttgagc 6120 accttgacgg cctcggcaat cacctcgggg ccacagaagt cgccgccgag aagaacaatc 6180 ttcttggagt cagtcttggt cttcttagtt tcgggttcca ttgtggatgt gtgtggttgt 6240 atgtgtgatg tggtgtgtgg agtgaaaatc tgtggctggc aaacgctctt gtatatatac 6300 gcacttttgc ccgtgctatg tggaagacta aacctccgaa gattgtgact caggtagtgc 6360 ggtatcggct agggacccaa accttgtcga tgccgatagc gctatcgaac gtaccccagc 6420 cggccgggag tatgtcggag gggacatacg agatcgtcaa gggtttgtgg ccaactggta 6480 aatttaaatg atgtcgacgc agtaggatgt cctgcacggg tctttttgtg gggtgtggag 6540 aaaggggtgc ttggagatgg aagccggtag aaccgggctg cttgtgcttg gagatggaag 6600 ccggtagaac cgggctgctt ggggggattt ggggccgctg ggctccaaag aggggtaggc 6660 atttcgttgg ggttacgtaa ttgcggcatt tgggtcctgc gcgcatgtcc cattggtcag 6720 aattagtccg gataggagac ttatcagcca atcacagcgc cggatccacc tgtaggttgg 6780 gttgggtggg agcacccctc cacagagtag agtcaaacag cagcagcaac atgatagttg 6840 ggggtgtgcg tgttaaagga aaaaaaagaa gcttgggtta tattcccgct ctatttagag 6900 gttgcgggat agacgccgac ggagggcaat ggcgccatgg aaccttgcgg atatcgatac 6960 gccgcggcgg actgcgtccg aaccagctcc agcagcgttt tttccgggcc attgagccga 7020 ctgcgacccc gccaacgtgt cttggcccac gcactcatgt catgttggtg ttgggaggcc 7080 actttttaag tagcacaagg cacctagctc gcagcaaggt gtccgaacca aagaagcggc 7140 tgcagtggtg caaacggggc ggaaacggcg ggaaaaagcc acgggggcac gaattgaggc 7200 acgccctcga atttgagacg agtcacggcc ccattcgccc gcgcaatggc tcgccaacgc 7260 ccggtctttt gcaccacatc aggttacccc aagccaaacc tttgtgttaa aaagcttaac 7320 atattatacc gaacgtaggt ttgggcgggc ttgctccgtc tgtccaaggc aacatttata 7380 taagggtctg catcgccggc tcaattgaat cttttttctt cttctcttct ctatattcat 7440 tcttgaatta aacacacatc aatccatggc aaacagcagc gtgtgggatg atgtggtggg 7500 ccgcgtggag accggcgtgg accagtggat ggatggcgcc aagccgtacg cactcaccga 7560 tgggctcccg atgatggacg tgtccaccat gctggcattc gaggtgggat acatggccat 7620 gctgctcttc ggcatcccga tcatgaagca gatggagaag ccttttgagc tcaagaccat 7680 caagctcttg cacaacttgt ttctcttcgg actttccttg tacatgtgcg tggagaccat 7740 ccgccaggct atcctcggag gctacaaagt gtttggaaac gacatggaga agggcaacga 7800 gtctcatgct cagggcatgt ctcgcatcgt gtacgtgttc tacgtgtcca aggcatacga 7860 gttcttggat accgccatca tgatcctttg caagaagttc aaccaggttt ccttcttgca 7920 tgtgtaccac catgccactc atttttgcca tctggtgggc tatccgccaa gtacgctcca 7980 ggaggtgatg cgtacttttt cagtgatcct caactctttc gtgcacaccg tcatgtacgg 8040 catactactt cttctcctcc caagggttcg ggttcgtgaa gccaatcaag ccgtacatca 8100 ccacccttca gatgacccag ttcatggcaa tgcttgtgca gtccttgtac gactacctct 8160 tcccatgcga ctacccacag gctcttgtgc agctccttgg agtgtacatg atcaccttgc 8220 ttgccctctt cggcaacttt tttgtgcaga gctatcttaa aaagccaaaa aagagcaaga 8280 ccaactaaaa ctgcctgcat gatatgccgc tcgccggcgt tcgaattgac tcagaaagcg 8340 agttaaggcg acacgcaaac tctatatttt ttcaaacgtg ttgccgtcac tcattcgcca 8400 tctgtttact acgtgtctgt tcaatgagca tgttcttgaa tctaaagaat ctcgaatgtt 8460 ttttaaaaaa agaattcgat atcaagctta cgcgtcgacc cgggtggacg tctagaggta 8520 cctagcaatt aacagatagt ttgccggtga taattctctt aacctcccac actcctttga 8580 cataacgatt tatgtaacga aactgaaatt tgaccagata ttgtgtccgc 8630 <210> SEQ ID NO 73 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer GPDsense <400> SEQUENCE: 73 atacgagatc gtcaaggg 18 <210> SEQ ID NO 74 <211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer GPDantisense <400> SEQUENCE: 74 gcggccgcgg attgatgtgt gtttaa 26 <210> SEQ ID NO 75 <211> LENGTH: 6339 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKR136 <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (4078)..(4078) <223> OTHER INFORMATION: n is a, c, g, or t <400> SEQUENCE: 75 ggccgcgaca caagtgtgag agtactaaat aaatgctttg gttgtacgaa atcattacac 60 taaataaaat aatcaaagct tatatatgcc ttccgctaag gccgaatgca aagaaattgg 120 ttctttctcg ttatcttttg ccacttttac tagtacgtat taattactac ttaatcatct 180 ttgtttacgg ctcattatat ccgtacgtcg agtcgacctg caggcatgca agcttggcgt 240 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca 300 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat 360 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt 420 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct 480 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 540 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 600 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 660 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 720

caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 780 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 840 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 900 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 960 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 1020 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 1080 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 1140 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 1200 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 1260 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 1320 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 1380 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 1440 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 1500 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 1560 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 1620 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 1680 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 1740 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 1800 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 1860 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 1920 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 1980 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 2040 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 2100 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 2160 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 2220 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 2280 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 2340 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 2400 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc 2460 cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg 2520 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt 2580 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac 2640 tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca 2700 tcaggcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct 2760 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa 2820 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattc gagctcggta 2880 cccggggatc ctctagacgt acgtcctcga agagaagggt taataacaca ttttttaaca 2940 tttttaacac aaattttagt tatttaaaaa tttattaaaa aatttaaaat aagaagagga 3000 actctttaaa taaatctaac ttacaaaatt tatgattttt aataagtttt caccaataaa 3060 aaatgtcata aaaatatgtt aaaaagtata ttatcaatat tctctttatg ataaataaaa 3120 agaaaaaaaa aataaaagtt aagtgaaaat gagattgaag tgactttagg tgtgtataaa 3180 tatatcaacc ccgccaacaa tttatttaat ccaaatatat tgaagtatat tattccatag 3240 cctttattta tttatatatt tattatataa aagctttatt tgttctaggt tgttcatgaa 3300 atattttttt ggttttatct ccgttgtaag aaaatcatgt gctttgtgtc gccactcact 3360 attgcagctt tttcatgcat tggtcagatt gacggttgat tgtatttttg ttttttatgg 3420 ttttgtgtta tgacttaagt cttcatctct ttatctcttc atcaggtttg atggttacct 3480 aatatggtcc atgggtacat gcatggttaa attaggtggc caactttgtt gtgaacgata 3540 gaattttttt tatattaagt aaactatttt tatattatga aataataata aaaaaaatat 3600 tttatcatta ttaacaaaat catattagtt aatttgttaa ctctataata aaagaaatac 3660 tgtaacattc acattacatg gtaacatctt tccacccttt catttgtttt ttgtttgatg 3720 actttttttc ttgtttaaat ttatttccct tcttttaaat ttggaataca ttatcatcat 3780 atataaacta aaatactaaa aacaggatta cacaaatgat aaataataac acaaatattt 3840 ataaatctag ctgcaatata tttaaactag ctatatcgat attgtaaaat aaaactagct 3900 gcattgatac tgataaaaaa atatcatgtg ctttctggac tgatgatgca gtatactttt 3960 gacattgcct ttattttatt tttcagaaaa gctttcttag ttctgggttc ttcattattt 4020 gtttcccatc tccattgtga attgaatcat ttgcttcgtg tcacaaatac aatttagnta 4080 ggtacatgca ttggtcagat tcacggttta ttatgtcatg acttaagttc atggtagtac 4140 attacctgcc acgcatgcat tatattggtt agatttgata ggcaaatttg gttgtcaaca 4200 atataaatat aaataatgtt tttatattac gaaataacag tgatcaaaac aaacagtttt 4260 atctttatta acaagatttt gtttttgttt gatgacgttt tttaatgttt acgctttccc 4320 ccttcttttg aatttagaac actttatcat cataaaatca aatactaaaa aaattacata 4380 tttcataaat aataacacaa atatttttaa aaaatctgaa ataataatga acaatattac 4440 atattatcac gaaaattcat taataaaaat attatataaa taaaatgtaa tagtagttat 4500 atgtaggaaa aaagtactgc acgcataata tatacaaaaa gattaaaatg aactattata 4560 aataataaca ctaaattaat ggtgaatcat atcaaaataa tgaaaaagta aataaaattt 4620 gtaattaact tctatatgta ttacacacac aaataataaa taatagtaaa aaaaattatg 4680 ataaatattt accatctcat aagatattta aaataatgat aaaaatatag attatttttt 4740 atgcaactag ctagccaaaa agagaacacg ggtatatata aaaagagtac ctttaaattc 4800 tactgtactt cctttattcc tgacgttttt atatcaagtg gacatacgtg aagattttaa 4860 ttatcagtct aaatatttca ttagcactta atacttttct gttttattcc tatcctataa 4920 gtagtcccga ttctcccaac attgcttatt cacacaacta actaagaaag tcttccatag 4980 ccccccaagc ggccgcatgg gaacggacca aggaaaaacc ttcacctggg aagagctggc 5040 ggcccataac accaaggacg acctactctt ggccatccgc ggcagggtgt acgatgtcac 5100 aaagttcttg agccgccatc ctggtggagt ggacactctc ctgctcggag ctggccgaga 5160 tgttactccg gtctttgaga tgtatcacgc gtttggggct gcagatgcca ttatgaagaa 5220 gtactatgtc ggtacactgg tctcgaatga gctgcccatc ttcccggagc caacggtgtt 5280 ccacaaaacc atcaagacga gagtcgaggg ctactttacg gatcggaaca ttgatcccaa 5340 gaatagacca gagatctggg gacgatacgc tcttatcttt ggatccttga tcgcttccta 5400 ctacgcgcag ctctttgtgc ctttcgttgt cgaacgcaca tggcttcagg tggtgtttgc 5460 aatcatcatg ggatttgcgt gcgcacaagt cggactcaac cctcttcatg atgcgtctca 5520 cttttcagtg acccacaacc ccactgtctg gaagattctg ggagccacgc acgacttttt 5580 caacggagca tcgtacctgg tgtggatgta ccaacatatg ctcggccatc acccctacac 5640 caacattgct ggagcagatc ccgacgtgtc gacgtctgag cccgatgttc gtcgtatcaa 5700 gcccaaccaa aagtggtttg tcaaccacat caaccagcac atgtttgttc ctttcctgta 5760 cggactgctg gcgttcaagg tgcgcattca ggacatcaac attttgtact ttgtcaagac 5820 caatgacgct attcgtgtca atcccatctc gacatggcac actgtgatgt tctggggcgg 5880 caaggctttc tttgtctggt atcgcctgat tgttcccctg cagtatctgc ccctgggcaa 5940 ggtgctgctc ttgttcacgg tcgcggacat ggtgtcgtct tactggctgg cgctgacctt 6000 ccaggcgaac cacgttgttg aggaagttca gtggccgttg cctgacgaga acgggatcat 6060 ccaaaaggac tgggcagcta tgcaggtcga gactacgcag gattacgcac acgattcgca 6120 cctctggacc agcatcactg gcagcttgaa ctaccaggct gtgcaccatc tgttccccaa 6180 cgtgtcgcag caccattatc ccgatattct ggccatcatc aagaacacct gcagcgagta 6240 caaggttcca taccttgtca aggatacgtt ttggcaagca tttgcttcac atttggagca 6300 cttgcgtgtt cttggactcc gtcccaagga agagtaggc 6339 <210> SEQ ID NO 76 <211> LENGTH: 8319 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pY98 <400> SEQUENCE: 76 ggccgccacc gcggcccgag attccggcct cttcggccgc caagcgaccc gggtggacgt 60 ctagaggtac ctagcaatta acagatagtt tgccggtgat aattctctta acctcccaca 120 ctcctttgac ataacgattt atgtaacgaa actgaaattt gaccagatat tgtgtccgcg 180 gtggagctcc agcttttgtt ccctttagtg agggttaatt tcgagcttgg cgtaatcatg 240 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acgtacgagc 300 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc 360 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat 420 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac 480 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 540 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca 600 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 660 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 720 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 780 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag 840 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 900 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 960 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 1020 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 1080 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 1140 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 1200 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc 1260 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag 1320 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata 1380 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat 1440 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg 1500 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc 1560 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc 1620

aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc 1680 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc 1740 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc 1800 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa 1860 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat 1920 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata 1980 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca 2040 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag 2100 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc 2160 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc 2220 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata 2280 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta 2340 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgcgcc 2400 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact 2460 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc 2520 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt 2580 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc 2640 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt 2700 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat 2760 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa 2820 ttttaacaaa atattaacgc ttacaatttc cattcgccat tcaggctgcg caactgttgg 2880 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct 2940 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg 3000 gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtaccgggcc ccccctcgag 3060 gtcgatggtg tcgataagct tgatatcgaa ttcatgtcac acaaaccgat cttcgcctca 3120 aggaaaccta attctacatc cgagagactg ccgagatcca gtctacactg attaattttc 3180 gggccaataa tttaaaaaaa tcgtgttata taatattata tgtattatat atatacatca 3240 tgatgatact gacagtcatg tcccattgct aaatagacag actccatctg ccgcctccaa 3300 ctgatgttct caatatttaa ggggtcatct cgcattgttt aataataaac agactccatc 3360 taccgcctcc aaatgatgtt ctcaaaatat attgtatgaa cttattttta ttacttagta 3420 ttattagaca acttacttgc tttatgaaaa acacttccta tttaggaaac aatttataat 3480 ggcagttcgt tcatttaaca atttatgtag aataaatgtt ataaatgcgt atgggaaatc 3540 ttaaatatgg atagcataaa tgatatctgc attgcctaat tcgaaatcaa cagcaacgaa 3600 aaaaatccct tgtacaacat aaatagtcat cgagaaatat caactatcaa agaacagcta 3660 ttcacacgtt actattgaga ttattattgg acgagaatca cacactcaac tgtctttctc 3720 tcttctagaa atacaggtac aagtatgtac tattctcatt gttcatactt ctagtcattt 3780 catcccacat attccttgga tttctctcca atgaatgaca ttctatcttg caaattcaac 3840 aattataata agatatacca aagtagcggt atagtggcaa tcaaaaagct tctctggtgt 3900 gcttctcgta tttattttta ttctaatgat ccattaaagg tatatattta tttcttgtta 3960 tataatcctt ttgtttatta catgggctgg atacataaag gtattttgat ttaatttttt 4020 gcttaaattc aatcccccct cgttcagtgt caactgtaat ggtaggaaat taccatactt 4080 ttgaagaagc aaaaaaaatg aaagaaaaaa aaaatcgtat ttccaggtta gacgttccgc 4140 agaatctaga atgcggtatg cggtacattg ttcttcgaac gtaaaagttg cgctccctga 4200 gatattgtac atttttgctt ttacaagtac aagtacatcg tacaactatg tactactgtt 4260 gatgcatcca caacagtttg ttttgttttt ttttgttttt tttttttcta atgattcatt 4320 accgctatgt atacctactt gtacttgtag taagccgggt tattggcgtt caattaatca 4380 tagacttatg aatctgcacg gtgtgcgctg cgagttactt ttagcttatg catgctactt 4440 gggtgtaata ttgggatctg ttcggaaatc aacggatgct caaccgattt cgacagtaat 4500 taattaagtc atacacaagt cagctttctt cgagcctcat ataagtataa gtagttcaac 4560 gtattagcac tgtacccagc atctccgtat cgagaaacac aacaacatgc cccattggac 4620 agatcatgcg gatacacagg ttgtgcagta tcatacatac tcgatcagac aggtcgtctg 4680 accatcatac aagctgaaca agcgctccat acttgcacgc tctctatata cacagttaaa 4740 ttacatatcc atagtctaac ctctaacagt taatcttctg gtaagcctcc cagccagcct 4800 tctggtatcg cttggcctcc tcaataggat ctcggttctg gccgtacaga cctcggccga 4860 caattatgat atccgttccg gtagacatga catcctcaac agttcggtac tgctgtccga 4920 gagcgtctcc cttgtcgtca agacccaccc cgggggtcag aataagccag tcctcagagt 4980 cgcccttagg tcggttctgg gcaatgaagc caaccacaaa ctcggggtcg gatcgggcaa 5040 gctcaatggt ctgcttggag tactcgccag tggccagaga gcccttgcaa gacagctcgg 5100 ccagcatgag cagacctctg gccagcttct cgttgggaga ggggactagg aactccttgt 5160 actgggagtt ctcgtagtca gagacgtcct ccttcttctg ttcagagaca gtttcctcgg 5220 caccagctcg caggccagca atgattccgg ttccgggtac accgtgggcg ttggtgatat 5280 cggaccactc ggcgattcgg tgacaccggt actggtgctt gacagtgttg ccaatatctg 5340 cgaactttct gtcctcgaac aggaagaaac cgtgcttaag agcaagttcc ttgaggggga 5400 gcacagtgcc ggcgtaggtg aagtcgtcaa tgatgtcgat atgggttttg atcatgcaca 5460 cataaggtcc gaccttatcg gcaagctcaa tgagctcctt ggtggtggta acatccagag 5520 aagcacacag gttggttttc ttggctgcca cgagcttgag cactcgagcg gcaaaggcgg 5580 acttgtggac gttagctcga gcttcgtagg agggcatttt ggtggtgaag aggagactga 5640 aataaattta gtctgcagaa ctttttatcg gaaccttatc tggggcagtg aagtatatgt 5700 tatggtaata gttacgagtt agttgaactt atagatagac tggactatac ggctatcggt 5760 ccaaattaga aagaacgtca atggctctct gggcgtcgcc tttgccgaca aaaatgtgat 5820 catgatgaaa gccagcaatg acgttgcagc tgatattgtt gtcggccaac cgcgccgaaa 5880 acgcagctgt cagacccaca gcctccaacg aagaatgtat cgtcaaagtg atccaagcac 5940 actcatagtt ggagtcgtac tccaaaggcg gcaatgacga gtcagacaga tactcgtcga 6000 cgcagtagga tgtcctgcac gggtcttttt gtggggtgtg gagaaagggg tgcttggaga 6060 tggaagccgg tagaaccggg ctgcttgtgc ttggagatgg aagccggtag aaccgggctg 6120 cttgggggga tttggggccg ctgggctcca aagaggggta ggcatttcgt tggggttacg 6180 taattgcggc atttgggtcc tgcgcgcatg tcccattggt cagaattagt ccggatagga 6240 gacttatcag ccaatcacag cgccggatcc acctgtaggt tgggttgggt gggagcaccc 6300 ctccacagag tagagtcaaa cagcagcagc aacatgatag ttgggggtgt gcgtgttaaa 6360 ggaaaaaaaa gaagcttggg ttatattccc gctctattta gaggttgcgg gatagacgcc 6420 gacggagggc aatggcgcca tggaaccttg cggatatcga tacgccgcgg cggactgcgt 6480 ccgaaccagc tccagcagcg ttttttccgg gccattgagc cgactgcgac cccgccaacg 6540 tgtcttggcc cacgcactca tgtcatgttg gtgttgggag gccacttttt aagtagcaca 6600 aggcacctag ctcgcagcaa ggtgtccgaa ccaaagaagc ggctgcagtg gtgcaaacgg 6660 ggcggaaacg gcgggaaaaa gccacggggg cacgaattga ggcacgccct cgaatttgag 6720 acgagtcacg gccccattcg cccgcgcaat ggctcgccaa cgcccggtct tttgcaccac 6780 atcaggttac cccaagccaa acctttgtgt taaaaagctt aacatattat accgaacgta 6840 ggtttgggcg ggcttgctcc gtctgtccaa ggcaacattt atataagggt ctgcatcgcc 6900 ggctcaattg aatctttttt cttcttctct tctctatatt cattcttgaa ttaaacacac 6960 atcaatccgc ggccgcatgg gaacggacca aggaaaaacc ttcacctggg aagagctggc 7020 ggcccataac accaaggacg acctactctt ggccatccgc ggcagggtgt acgatgtcac 7080 aaagttcttg agccgccatc ctggtggagt ggacactctc ctgctcggag ctggccgaga 7140 tgttactccg gtctttgaga tgtatcacgc gtttggggct gcagatgcca ttatgaagaa 7200 gtactatgtc ggtacactgg tctcgaatga gctgcccatc ttcccggagc caacggtgtt 7260 ccacaaaacc atcaagacga gagtcgaggg ctactttacg gatcggaaca ttgatcccaa 7320 gaatagacca gagatctggg gacgatacgc tcttatcttt ggatccttga tcgcttccta 7380 ctacgcgcag ctctttgtgc ctttcgttgt cgaacgcaca tggcttcagg tggtgtttgc 7440 aatcatcatg ggatttgcgt gcgcacaagt cggactcaac cctcttcatg atgcgtctca 7500 cttttcagtg acccacaacc ccactgtctg gaagattctg ggagccacgc acgacttttt 7560 caacggagca tcgtacctgg tgtggatgta ccaacatatg ctcggccatc acccctacac 7620 caacattgct ggagcagatc ccgacgtgtc gacgtctgag cccgatgttc gtcgtatcaa 7680 gcccaaccaa aagtggtttg tcaaccacat caaccagcac atgtttgttc ctttcctgta 7740 cggactgctg gcgttcaagg tgcgcattca ggacatcaac attttgtact ttgtcaagac 7800 caatgacgct attcgtgtca atcccatctc gacatggcac actgtgatgt tctggggcgg 7860 caaggctttc tttgtctggt atcgcctgat tgttcccctg cagtatctgc ccctgggcaa 7920 ggtgctgctc ttgttcacgg tcgcggacat ggtgtcgtct tactggctgg cgctgacctt 7980 ccaggcgaac cacgttgttg aggaagttca gtggccgttg cctgacgaga acgggatcat 8040 ccaaaaggac tgggcagcta tgcaggtcga gactacgcag gattacgcac acgattcgca 8100 cctctggacc agcatcactg gcagcttgaa ctaccaggct gtgcaccatc tgttccccaa 8160 cgtgtcgcag caccattatc ccgatattct ggccatcatc aagaacacct gcagcgagta 8220 caaggttcca taccttgtca aggatacgtt ttggcaagca tttgcttcac atttggagca 8280 cttgcgtgtt cttggactcc gtcccaagga agagtaggc 8319 <210> SEQ ID NO 77 <211> LENGTH: 774 <212> TYPE: DNA <213> ORGANISM: Euglena gracilis <400> SEQUENCE: 77 atggaggtgg tgaatgaaat agtctcaatt gggcaggaag ttttacccaa agttgattat 60 gcccaactct ggagtgatgc cagtcactgt gaggtgcttt acttgtccat cgcatttgtc 120 atcttgaagt tcactcttgg cccccttggt ccaaaaggtc agtctcgtat gaagtttgtt 180 ttcaccaatt acaaccttct catgtccatt tattcgttgg gatcattcct ctcaatggca 240 tatgccatgt acaccatcgg tgttatgtct gacaactgcg agaaggcttt tgacaacaac 300 gtcttcagga tcaccacgca gttgttctat ttgagcaagt tcctggagta tattgactcc 360 ttctatttgc cactgatggg caagcctctg acctggttgc aattcttcca tcatttgggg 420 gcaccgatgg atatgtggct gttctataat taccgaaatg aagctgtttg gatttttgtg 480 ctgttgaatg gtttcatcca ctggatcatg tacggttatt attggaccag attgatcaag 540

ctgaagttcc ccatgccaaa atccctgatt acatcaatgc agatcattca attcaatgtt 600 ggtttctaca ttgtctggaa gtacaggaac attccctgtt atcgccaaga tgggatgagg 660 atgtttggct ggttcttcaa ttacttttat gttggcacag tcttgtgttt gttcttgaat 720 ttctatgtgc aaacgtatat cgtcaggaag cacaagggag ccaaaaagat tcag 774 <210> SEQ ID NO 78 <211> LENGTH: 1263 <212> TYPE: DNA <213> ORGANISM: Euglena gracilis <400> SEQUENCE: 78 atgaagtcaa agcgccaagc gcttcccctt acaattgatg gaacaacata tgatgtgtct 60 gcctgggtca atttccaccc tggtggtgcg gaaattatag agaattacca aggaagggat 120 gccactgatg ccttcatggt tatgcactct caagaagcct tcgacaagct caagcgcatg 180 cccaaaatca atcccagttc tgagttgcca ccccaggctg cagtgaatga agctcaagag 240 gatttccgga agctccgaga agagttgatc gcaactggca tgtttgatgc ctcccccctc 300 tggtactcat acaaaatcag caccacactg ggccttggag tgctgggtta tttcctgatg 360 gttcagtatc agatgtattt cattggggca gtgttgcttg ggatgcacta tcaacagatg 420 ggctggcttt ctcatgacat ttgccaccac cagactttca agaaccggaa ctggaacaac 480 ctcgtgggac tggtatttgg caatggtctg caaggttttt ccgtgacatg gtggaaggac 540 agacacaatg cacatcattc ggcaaccaat gttcaagggc acgaccctga tattgacaac 600 ctccccctct tagcctggtc tgaggatgac gtcacacggg cgtcaccgat ttcccgcaag 660 ctcattcagt tccagcagta ctatttcttg gtcatctgta tcttgttgcg gttcatttgg 720 tgtttccaga gcgtgttgac cgtgcgcagt ttgaaggaca gagataacca attctatcgc 780 tctcagtata agaaggaggc cattggcctc gccctgcact ggaccttgaa gaccctgttc 840 cacttattct ttatgcccag catcctcaca tcgctgttgg tgtttttcgt ttcggagctg 900 gttggcggct tcggcattgc gatcgtggtg ttcatgaacc actacccact ggagaagatc 960 ggggactcag tctgggatgg ccatggattc tcggttggcc agatccatga gaccatgaac 1020 attcggcgag ggattatcac agattggttt ttcggaggct tgaattacca gattgagcac 1080 catttgtggc cgaccctccc tcgccacaac ctgacagcgg ttagctacca ggtggaacag 1140 ctgtgccaga agcacaacct gccgtatcgg aacccgctgc cccatgaagg gttggtcatc 1200 ctgctgcgct atctggcggt gttcgcccgg atggcggaga agcaacccgc ggggaaggct 1260 cta 1263 <210> SEQ ID NO 79 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer oEugEL1-1 <400> SEQUENCE: 79 agcggccgca ccatggaggt ggtgaatgaa 30 <210> SEQ ID NO 80 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer oEugEL1-2 <400> SEQUENCE: 80 tgcggccgct cactgaatct ttttggctcc 30 <210> SEQ ID NO 81 <211> LENGTH: 4311 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKR906 <400> SEQUENCE: 81 agcggccgca ccatggaggt ggtgaatgaa atagtctcaa ttgggcagga agttttaccc 60 aaagttgatt atgcccaact ctggagtgat gccagtcact gtgaggtgct ttacttgtcc 120 atcgcatttg tcatcttgaa gttcactctt ggcccccttg gtccaaaagg tcagtctcgt 180 atgaagtttg ttttcaccaa ttacaacctt ctcatgtcca tttattcgtt gggatcattc 240 ctctcaatgg catatgccat gtacaccatc ggtgttatgt ctgacaactg cgagaaggct 300 tttgacaaca acgtcttcag gatcaccacg cagttgttct atttgagcaa gttcctggag 360 tatattgact ccttctattt gccactgatg ggcaagcctc tgacctggtt gcaattcttc 420 catcatttgg gggcaccgat ggatatgtgg ctgttctata attaccgaaa tgaagctgtt 480 tggatttttg tgctgttgaa tggtttcatc cactggatca tgtacggtta ttattggacc 540 agattgatca agctgaagtt ccccatgcca aaatccctga ttacatcaat gcagatcatt 600 caattcaatg ttggtttcta cattgtctgg aagtacagga acattccctg ttatcgccaa 660 gatgggatga ggatgtttgg ctggttcttc aattactttt atgttggcac agtcttgtgt 720 ttgttcttga atttctatgt gcaaacgtat atcgtcagga agcacaaggg agccaaaaag 780 attcagtgag cggccgcacc tgaattccag cacactggcg gccgttacta gtggatccga 840 gctcggtacc aagcttgatg catagcttga gtattctaac gcgtcaccta aatagcttgg 900 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca 960 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca 1020 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 1080 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt 1140 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 1200 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 1260 caaaaggcca gcaaaagccc aggaaccgta aaaaggccgc gttgctggcg tttttccata 1320 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 1380 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 1440 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 1500 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 1560 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 1620 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 1680 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 1740 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 1800 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 1860 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 1920 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 1980 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt agcacgtgtc 2040 agtcctgctc ctcggccacg aagtgcacgc agttgccggc cgggtcgcgc agggcgaact 2100 cccgccccca cggctgctcg ccgatctcgg tcatggccgg cccggaggcg tcccggaagt 2160 tcgtggacac gacctccgac cactcggcgt acagctcgtc caggccgcgc acccacaccc 2220 aggccagggt gttgtccggc accacctggt cctggaccgc gctgatgaac agggtcacgt 2280 cgtcccggac cacaccggcg aagtcgtcct ccacgaagtc ccgggagaac ccgagccggt 2340 cggtccagaa ctcgaccgct ccggcgacgt cgcgcgcggt gagcaccgga acggcactgg 2400 tcaacttggc catggtggcc ctcctcacgt gctattattg aagcatttat cagggttatt 2460 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 2520 gcacatttcc ccgaaaagtg ccacctgtat gcggtgtgaa ataccgcaca gatgcgtaag 2580 gagaaaatac cgcatcagga aattgtaagc gttaataatt cagaagaact cgtcaagaag 2640 gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg 2700 gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg ctatgtcctg 2760 atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc ggccattttc 2820 caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct cgccgtcggg 2880 catgctcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat gctcttcgtc 2940 cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct cgatgcgatg 3000 tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc 3060 atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga gatcctgccc 3120 cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt cgagcacagc 3180 tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt cttgcagttc 3240 attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct gcgctgacag 3300 ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat agccgaatag 3360 cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa 3420 cgatcctcat cctgtctctt gatcagagct tgatcccctg cgccatcaga tccttggcgg 3480 cgagaaagcc atccagttta ctttgcaggg cttcccaacc ttaccagagg gcgccccagc 3540 tggcaattcc ggttcgcttg ctgtccataa aaccgcccag tctagctatc gccatgtaag 3600 cccactgcaa gctacctgct ttctctttgc gcttgcgttt tcccttgtcc agatagccca 3660 gtagctgaca ttcatccggg gtcagcaccg tttctgcgga ctggctttct acgtgaaaag 3720 gatctaggtg aagatccttt ttgataatct catgcctgac atttatattc cccagaacat 3780 caggttaatg gcgtttttga tgtcattttc gcggtggctg agatcagcca cttcttcccc 3840 gataacggag accggcacac tggccatatc ggtggtcatc atgcgccagc tttcatcccc 3900 gatatgcacc accgggtaaa gttcacggga gactttatct gacagcagac gtgcactggc 3960 cagggggatc accatccgtc gccccggcgt gtcaataata tcactctgta catccacaaa 4020 cagacgataa cggctctctc ttttataggt gtaaacctta aactgccgta cgtataggct 4080 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 4140 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg 4200 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tgggccctct 4260 agatgcatgc tcgagcggcc gccagtgtga tggatatctg cagaattcag g 4311 <210> SEQ ID NO 82 <211> LENGTH: 7085 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKR72 <400> SEQUENCE: 82 gtacggatcc gtcgacggcg cgcccgatca tccggatata gttcctcctt tcagcaaaaa 60 acccctcaag acccgtttag aggccccaag gggttatgct agttattgct cagcggtggc 120

agcagccaac tcagcttcct ttcgggcttt gttagcagcc ggatcgatcc aagctgtacc 180 tcactattcc tttgccctcg gacgagtgct ggggcgtcgg tttccactat cggcgagtac 240 ttctacacag ccatcggtcc agacggccgc gcttctgcgg gcgatttgtg tacgcccgac 300 agtcccggct ccggatcgga cgattgcgtc gcatcgaccc tgcgcccaag ctgcatcatc 360 gaaattgccg tcaaccaagc tctgatagag ttggtcaaga ccaatgcgga gcatatacgc 420 ccggagccgc ggcgatcctg caagctccgg atgcctccgc tcgaagtagc gcgtctgctg 480 ctccatacaa gccaaccacg gcctccagaa gaagatgttg gcgacctcgt attgggaatc 540 cccgaacatc gcctcgctcc agtcaatgac cgctgttatg cggccattgt ccgtcaggac 600 attgttggag ccgaaatccg cgtgcacgag gtgccggact tcggggcagt cctcggccca 660 aagcatcagc tcatcgagag cctgcgcgac ggacgcactg acggtgtcgt ccatcacagt 720 ttgccagtga tacacatggg gatcagcaat cgcgcatatg aaatcacgcc atgtagtgta 780 ttgaccgatt ccttgcggtc cgaatgggcc gaacccgctc gtctggctaa gatcggccgc 840 agcgatcgca tccatagcct ccgcgaccgg ctgcagaaca gcgggcagtt cggtttcagg 900 caggtcttgc aacgtgacac cctgtgcacg gcgggagatg caataggtca ggctctcgct 960 gaattcccca atgtcaagca cttccggaat cgggagcgcg gccgatgcaa agtgccgata 1020 aacataacga tctttgtaga aaccatcggc gcagctattt acccgcagga catatccacg 1080 ccctcctaca tcgaagctga aagcacgaga ttcttcgccc tccgagagct gcatcaggtc 1140 ggagacgctg tcgaactttt cgatcagaaa cttctcgaca gacgtcgcgg tgagttcagg 1200 cttttccatg ggtatatctc cttcttaaag ttaaacaaaa ttatttctag agggaaaccg 1260 ttgtggtctc cctatagtga gtcgtattaa tttcgcggga tcgagatcga tccaattcca 1320 atcccacaaa aatctgagct taacagcaca gttgctcctc tcagagcaga atcgggtatt 1380 caacaccctc atatcaacta ctacgttgtg tataacggtc cacatgccgg tatatacgat 1440 gactggggtt gtacaaaggc ggcaacaaac ggcgttcccg gagttgcaca caagaaattt 1500 gccactatta cagaggcaag agcagcagct gacgcgtaca caacaagtca gcaaacagac 1560 aggttgaact tcatccccaa aggagaagct caactcaagc ccaagagctt tgctaaggcc 1620 ctaacaagcc caccaaagca aaaagcccac tggctcacgc taggaaccaa aaggcccagc 1680 agtgatccag ccccaaaaga gatctccttt gccccggaga ttacaatgga cgatttcctc 1740 tatctttacg atctaggaag gaagttcgaa ggtgaaggtg acgacactat gttcaccact 1800 gataatgaga aggttagcct cttcaatttc agaaagaatg ctgacccaca gatggttaga 1860 gaggcctacg cagcaggtct catcaagacg atctacccga gtaacaatct ccaggagatc 1920 aaataccttc ccaagaaggt taaagatgca gtcaaaagat tcaggactaa ttgcatcaag 1980 aacacagaga aagacatatt tctcaagatc agaagtacta ttccagtatg gacgattcaa 2040 ggcttgcttc ataaaccaag gcaagtaata gagattggag tctctaaaaa ggtagttcct 2100 actgaatcta aggccatgca tggagtctaa gattcaaatc gaggatctaa cagaactcgc 2160 cgtgaagact ggcgaacagt tcatacagag tcttttacga ctcaatgaca agaagaaaat 2220 cttcgtcaac atggtggagc acgacactct ggtctactcc aaaaatgtca aagatacagt 2280 ctcagaagac caaagggcta ttgagacttt tcaacaaagg ataatttcgg gaaacctcct 2340 cggattccat tgcccagcta tctgtcactt catcgaaagg acagtagaaa aggaaggtgg 2400 ctcctacaaa tgccatcatt gcgataaagg aaaggctatc attcaagatg cctctgccga 2460 cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag aagacgttcc 2520 aaccacgtct tcaaagcaag tggattgatg tgacatctcc actgacgtaa gggatgacgc 2580 acaatcccac tatccttcgc aagacccttc ctctatataa ggaagttcat ttcatttgga 2640 gaggacacgc tcgagctcat ttctctatta cttcagccat aacaaaagaa ctcttttctc 2700 ttcttattaa accatgaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat 2760 cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc 2820 tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg 2880 tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga 2940 agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca 3000 gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc 3060 ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt 3120 cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga 3180 tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca 3240 ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca 3300 cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga 3360 ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag 3420 gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct 3480 tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca 3540 gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat 3600 cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt 3660 ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg 3720 tccgagggca aaggaatagt gaggtaccta aagaaggagt gcgtcgaagc agatcgttca 3780 aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc 3840 atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta 3900 tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa 3960 aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta 4020 gatcgatgtc gaatcgatca acctgcatta atgaatcggc caacgcgcgg ggagaggcgg 4080 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 4140 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 4200 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4260 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 4320 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 4380 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4440 ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc 4500 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4560 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4620 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 4680 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 4740 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4800 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 4860 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 4920 acgttaaggg attttggtca tgacattaac ctataaaaat aggcgtatca cgaggccctt 4980 tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac 5040 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc 5100 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag 5160 agtgcaccat atggacatat tgtcgttaga acgcggctac aattaataca taaccttatg 5220 tatcatacac atacgattta ggtgacacta tagaacggcg cgccaagctt gttgaaacat 5280 ccctgaagtg tctcatttta ttttatttat tctttgctga taaaaaaata aaataaaaga 5340 agctaagcac acggtcaacc attgctctac tgctaaaagg gttatgtgta gtgttttact 5400 gcataaatta tgcagcaaac aagacaactc aaattaaaaa atttcctttg cttgtttttt 5460 tgttgtctct gacttgactt tcttgtggaa gttggttgta taaggattgg gacaccattg 5520 tccttcttaa tttaatttta ttctttgctg ataaaaaaaa aaatttcata tagtgttaaa 5580 taataatttg ttaaataacc aaaaagtcaa atatgtttac tctcgtttaa ataattgaga 5640 ttcgtccagc aaggctaaac gattgtatag atttatgaca atatttactt ttttatagat 5700 aaatgttata ttataataaa tttatataca tatattatat gttatttatt attattttaa 5760 atccttcaat attttatcaa accaactcat aatttttttt ttatctgtaa gaagcaataa 5820 aattaaatag acccacttta aggatgatcc aacctttata cagagtaaga gagttcaaat 5880 agtacccttt catatacata tcaactaaaa tattagaaat atcatggatc aaaccttata 5940 aagacattaa ataagtggat aagtataata tataaatggg tagtatataa tatataaatg 6000 gatacaaact tctctcttta taattgttat gtctccttaa catcctaata taatacataa 6060 gtgggtaata tataatatat aaatggagac aaacttcttc cattataatt gttatgtctt 6120 cttaacactt atgtctcgtt cacaatgcta aggttagaat tgtttagaaa gtcttatagt 6180 acacatttgt ttttgtacta tttgaagcat tccataagcc gtcacgattc agatgattta 6240 taataataag aggaaattta tcatagaaca ataaggtgca tagatagagt gttaatatat 6300 cataacatcc tttgtttatt catagaagaa gtgagatgga gctcagttat tatactgtta 6360 catggtcgga tacaatattc catgctctcc atgagctctt acacctacat gcattttagt 6420 tcatacttgc ggccgcagta tatcttaaat tctttaatac ggtgtactag gatattgaac 6480 tggttcttga tgatgaaaac ctgggccgag attgcagcta tttatagtca taggtcttgt 6540 taacatgcat ggacatttgg ccacggggtg gcatgcagtt tgacgggtgt tgaaataaac 6600 aaaaatgagg tggcggaaga gaatacgagt ttgaggttgg gttagaaaca acaaatgtga 6660 gggctcatga tgggttgagt tggtgaatgt tttgggctgc tcgattgaca cctttgtgag 6720 tacgtgttgt tgtgcatggc ttttggggtc cagttttttt ttcttgacgc ggcgatcctg 6780 atcagctagt ggataagtga tgtccactgt gtgtgattgc gtttttgttt gaattttatg 6840 aacttagaca ttgctatgca aaggatactc tcattgtgtt ttgtcttctt ttgttccttg 6900 gctttttctt atgatccaag agactagtca gtgttgtggc attcgagact accaagatta 6960 attatgatgg gggaaggata agtaactgat tagtacggac tgttaccaaa ttaattaata 7020 agcggcaaat gaagggcatg gatcaaaagc ttggatctcc tgcaggatct ggccggccgg 7080 atctc 7085 <210> SEQ ID NO 83 <211> LENGTH: 2540 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKS102 <400> SEQUENCE: 83 cgatcatccg gatatagttc ctcctttcag caaaaaaccc ctcaagaccc gtttagaggc 60 cccaaggggt tatgctagtt attgctcagc ggtggcagca gccaactcag cttcctttcg 120 ggctttgtta gcagccggat cgatccaagc tgtacctcac tattcctttg ccctcggacg 180

agtgctgggg cgtcggtttc cactatcggc gagtacttct acacagccat cggtccagac 240 ggccgcgctt ctgcgggcga tttgtgtacg cccgacagtc ccggctccgg atcggacgat 300 tgcgtcgcat cgaccctgcg cccaagctgc atcatcgaaa ttgccgtcaa ccaagctctg 360 atagagttgg tcaagaccaa tgcggagcat atacgcccgg agccgcggcg atcctgcaag 420 ctccggatgc ctccgctcga agtagcgcgt ctgctgctcc atacaagcca accacggcct 480 ccagaagaag atgttggcga cctcgtattg ggaatccccg aacatcgcct cgctccagtc 540 aatgaccgct gttatgcggc cattgtccgt caggacattg ttggagccga aatccgcgtg 600 cacgaggtgc cggacttcgg ggcagtcctc ggcccaaagc atcagctcat cgagagcctg 660 cgcgacggac gcactgacgg tgtcgtccat cacagtttgc cagtgataca catggggatc 720 agcaatcgcg catatgaaat cacgccatgt agtgtattga ccgattcctt gcggtccgaa 780 tgggccgaac ccgctcgtct ggctaagatc ggccgcagcg atcgcatcca tagcctccgc 840 gaccggctgc agaacagcgg gcagttcggt ttcaggcagg tcttgcaacg tgacaccctg 900 tgcacggcgg gagatgcaat aggtcaggct ctcgctgaat tccccaatgt caagcacttc 960 cggaatcggg agcgcggccg atgcaaagtg ccgataaaca taacgatctt tgtagaaacc 1020 atcggcgcag ctatttaccc gcaggacata tccacgccct cctacatcga agctgaaagc 1080 acgagattct tcgccctccg agagctgcat caggtcggag acgctgtcga acttttcgat 1140 cagaaacttc tcgacagacg tcgcggtgag ttcaggcttt tccatgggta tatctccttc 1200 ttaaagttaa acaaaattat ttctagaggg aaaccgttgt ggtctcccta tagtgagtcg 1260 tattaatttc gcgggatcga gatctgatca acctgcatta atgaatcggc caacgcgcgg 1320 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 1380 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 1440 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 1500 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 1560 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 1620 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 1680 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt 1740 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 1800 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 1860 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 1920 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg 1980 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 2040 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 2100 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 2160 acgaaaactc acgttaaggg attttggtca tgacattaac ctataaaaat aggcgtatca 2220 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc 2280 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg 2340 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga 2400 ttgtactgag agtgcaccat atggacatat tgtcgttaga acgcggctac aattaataca 2460 taaccttatg tatcatacac atacgattta ggtgacacta tagaacggcg cgccaagctt 2520 ggatccgtcg acggcgcgcc 2540 <210> SEQ ID NO 84 <211> LENGTH: 4359 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKR197 <400> SEQUENCE: 84 cgcgcccgat catccggata tagttcctcc tttcagcaaa aaacccctca agacccgttt 60 agaggcccca aggggttatg ctagttattg ctcagcggtg gcagcagcca actcagcttc 120 ctttcgggct ttgttagcag ccggatcgat ccaagctgta cctcactatt cctttgccct 180 cggacgagtg ctggggcgtc ggtttccact atcggcgagt acttctacac agccatcggt 240 ccagacggcc gcgcttctgc gggcgatttg tgtacgcccg acagtcccgg ctccggatcg 300 gacgattgcg tcgcatcgac cctgcgccca agctgcatca tcgaaattgc cgtcaaccaa 360 gctctgatag agttggtcaa gaccaatgcg gagcatatac gcccggagcc gcggcgatcc 420 tgcaagctcc ggatgcctcc gctcgaagta gcgcgtctgc tgctccatac aagccaacca 480 cggcctccag aagaagatgt tggcgacctc gtattgggaa tccccgaaca tcgcctcgct 540 ccagtcaatg accgctgtta tgcggccatt gtccgtcagg acattgttgg agccgaaatc 600 cgcgtgcacg aggtgccgga cttcggggca gtcctcggcc caaagcatca gctcatcgag 660 agcctgcgcg acggacgcac tgacggtgtc gtccatcaca gtttgccagt gatacacatg 720 gggatcagca atcgcgcata tgaaatcacg ccatgtagtg tattgaccga ttccttgcgg 780 tccgaatggg ccgaacccgc tcgtctggct aagatcggcc gcagcgatcg catccatagc 840 ctccgcgacc ggctgcagaa cagcgggcag ttcggtttca ggcaggtctt gcaacgtgac 900 accctgtgca cggcgggaga tgcaataggt caggctctcg ctgaattccc caatgtcaag 960 cacttccgga atcgggagcg cggccgatgc aaagtgccga taaacataac gatctttgta 1020 gaaaccatcg gcgcagctat ttacccgcag gacatatcca cgccctccta catcgaagct 1080 gaaagcacga gattcttcgc cctccgagag ctgcatcagg tcggagacgc tgtcgaactt 1140 ttcgatcaga aacttctcga cagacgtcgc ggtgagttca ggcttttcca tgggtatatc 1200 tccttcttaa agttaaacaa aattatttct agagggaaac cgttgtggtc tccctatagt 1260 gagtcgtatt aatttcgcgg gatcgagatc tgatcaacct gcattaatga atcggccaac 1320 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 1380 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 1440 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 1500 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 1560 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 1620 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 1680 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct 1740 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 1800 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 1860 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 1920 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag 1980 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 2040 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 2100 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 2160 agtggaacga aaactcacgt taagggattt tggtcatgac attaacctat aaaaataggc 2220 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca 2280 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc 2340 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag 2400 agcagattgt actgagagtg caccatatgg acatattgtc gttagaacgc ggctacaatt 2460 aatacataac cttatgtatc atacacatac gatttaggtg acactataga acggcgcgcc 2520 aagcttgttg aaacatccct gaagtgtctc attttatttt atttattctt tgctgataaa 2580 aaaataaaat aaaagaagct aagcacacgg tcaaccattg ctctactgct aaaagggtta 2640 tgtgtagtgt tttactgcat aaattatgca gcaaacaaga caactcaaat taaaaaattt 2700 cctttgcttg tttttttgtt gtctctgact tgactttctt gtggaagttg gttgtataag 2760 gattgggaca ccattgtcct tcttaattta attttattct ttgctgataa aaaaaaaaat 2820 ttcatatagt gttaaataat aatttgttaa ataaccaaaa agtcaaatat gtttactctc 2880 gtttaaataa ttgagattcg tccagcaagg ctaaacgatt gtatagattt atgacaatat 2940 ttactttttt atagataaat gttatattat aataaattta tatacatata ttatatgtta 3000 tttattatta ttttaaatcc ttcaatattt tatcaaacca actcataatt ttttttttat 3060 ctgtaagaag caataaaatt aaatagaccc actttaagga tgatccaacc tttatacaga 3120 gtaagagagt tcaaatagta ccctttcata tacatatcaa ctaaaatatt agaaatatca 3180 tggatcaaac cttataaaga cattaaataa gtggataagt ataatatata aatgggtagt 3240 atataatata taaatggata caaacttctc tctttataat tgttatgtct ccttaacatc 3300 ctaatataat acataagtgg gtaatatata atatataaat ggagacaaac ttcttccatt 3360 ataattgtta tgtcttctta acacttatgt ctcgttcaca atgctaaggt tagaattgtt 3420 tagaaagtct tatagtacac atttgttttt gtactatttg aagcattcca taagccgtca 3480 cgattcagat gatttataat aataagagga aatttatcat agaacaataa ggtgcataga 3540 tagagtgtta atatatcata acatcctttg tttattcata gaagaagtga gatggagctc 3600 agttattata ctgttacatg gtcggataca atattccatg ctctccatga gctcttacac 3660 ctacatgcat tttagttcat acttgcggcc gcagtatatc ttaaattctt taatacggtg 3720 tactaggata ttgaactggt tcttgatgat gaaaacctgg gccgagattg cagctattta 3780 tagtcatagg tcttgttaac atgcatggac atttggccac ggggtggcat gcagtttgac 3840 gggtgttgaa ataaacaaaa atgaggtggc ggaagagaat acgagtttga ggttgggtta 3900 gaaacaacaa atgtgagggc tcatgatggg ttgagttggt gaatgttttg ggctgctcga 3960 ttgacacctt tgtgagtacg tgttgttgtg catggctttt ggggtccagt ttttttttct 4020 tgacgcggcg atcctgatca gctagtggat aagtgatgtc cactgtgtgt gattgcgttt 4080 ttgtttgaat tttatgaact tagacattgc tatgcaaagg atactctcat tgtgttttgt 4140 cttcttttgt tccttggctt tttcttatga tccaagagac tagtcagtgt tgtggcattc 4200 gagactacca agattaatta tgatggggga aggataagta actgattagt acggactgtt 4260 accaaattaa ttaataagcg gcaaatgaag ggcatggatc aaaagcttgg atctcctgca 4320 ggatctggcc ggccggatct cgtacggatc cgtcgacgg 4359 <210> SEQ ID NO 85 <211> LENGTH: 5147 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKR911 <400> SEQUENCE: 85 ggccgcaagt atgaactaaa atgcatgtag gtgtaagagc tcatggagag catggaatat 60 tgtatccgac catgtaacag tataataact gagctccatc tcacttcttc tatgaataaa 120 caaaggatgt tatgatatat taacactcta tctatgcacc ttattgttct atgataaatt 180

tcctcttatt attataaatc atctgaatcg tgacggctta tggaatgctt caaatagtac 240 aaaaacaaat gtgtactata agactttcta aacaattcta accttagcat tgtgaacgag 300 acataagtgt taagaagaca taacaattat aatggaagaa gtttgtctcc atttatatat 360 tatatattac ccacttatgt attatattag gatgttaagg agacataaca attataaaga 420 gagaagtttg tatccattta tatattatat actacccatt tatatattat acttatccac 480 ttatttaatg tctttataag gtttgatcca tgatatttct aatattttag ttgatatgta 540 tatgaaaggg tactatttga actctcttac tctgtataaa ggttggatca tccttaaagt 600 gggtctattt aattttattg cttcttacag ataaaaaaaa aattatgagt tggtttgata 660 aaatattgaa ggatttaaaa taataataaa taacatataa tatatgtata taaatttatt 720 ataatataac atttatctat aaaaaagtaa atattgtcat aaatctatac aatcgtttag 780 ccttgctgga cgaatctcaa ttatttaaac gagagtaaac atatttgact ttttggttat 840 ttaacaaatt attatttaac actatatgaa attttttttt ttatcagcaa agaataaaat 900 taaattaaga aggacaatgg tgtcccaatc cttatacaac caacttccac aagaaagtca 960 agtcagagac aacaaaaaaa caagcaaagg aaatttttta atttgagttg tcttgtttgc 1020 tgcataattt atgcagtaaa acactacaca taaccctttt agcagtagag caatggttga 1080 ccgtgtgctt agcttctttt attttatttt tttatcagca aagaataaat aaaataaaat 1140 gagacacttc agggatgttt caacaagctt ggcgcgccgt tctatagtgt cacctaaatc 1200 gtatgtgtat gatacataag gttatgtatt aattgtagcc gcgttctaac gacaatatgt 1260 ccatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac 1320 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca 1380 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga 1440 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgacca 1500 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag 1560 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac 1620 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa 1680 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc 1740 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag 1800 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac 1860 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc 1920 gaacgaccta caccgaactg agatacctac agcgtgagca ttgagaaagc gccacgcttc 1980 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca 2040 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc 2100 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg 2160 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct 2220 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata 2280 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc 2340 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc aggttgatca 2400 gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct 2460 agaaataatt ttgtttaact ttaagaagga gatataccca tggaaaagcc tgaactcacc 2520 gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag 2580 ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc 2640 ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt 2700 gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg 2760 acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa 2820 ctgcccgctg ttctgcagcc ggtcgcggag gctatggatg cgatcgctgc ggccgatctt 2880 agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg 2940 cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac 3000 gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac 3060 tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac 3120 aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac 3180 gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc 3240 tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc 3300 cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct 3360 tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca 3420 caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat 3480 agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagtgagg tacagcttgg 3540 atcgatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag 3600 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa 3660 ggaggaacta tatccggatg atcgggcgcg ccgtcgacgg atccgtacga gatccggccg 3720 gccagatcct gcaggagatc caagcttttg atccatgccc ttcatttgcc gcttattaat 3780 taatttggta acagtccgta ctaatcagtt acttatcctt cccccatcat aattaatctt 3840 ggtagtctcg aatgccacaa cactgactag tctcttggat cataagaaaa agccaaggaa 3900 caaaagaaga caaaacacaa tgagagtatc ctttgcatag caatgtctaa gttcataaaa 3960 ttcaaacaaa aacgcaatca cacacagtgg acatcactta tccactagct gatcaggatc 4020 gccgcgtcaa gaaaaaaaaa ctggacccca aaagccatgc acaacaacac gtactcacaa 4080 aggtgtcaat cgagcagccc aaaacattca ccaactcaac ccatcatgag ccctcacatt 4140 tgttgtttct aacccaacct caaactcgta ttctcttccg ccacctcatt tttgtttatt 4200 tcaacacccg tcaaactgca tgccaccccg tggccaaatg tccatgcatg ttaacaagac 4260 ctatgactat aaatagctgc aatctcggcc caggttttca tcatcaagaa ccagttcaat 4320 atcctagtac accgtattaa agaatttaag atatactgcg gccgcaccat ggaggtggtg 4380 aatgaaatag tctcaattgg gcaggaagtt ttacccaaag ttgattatgc ccaactctgg 4440 agtgatgcca gtcactgtga ggtgctttac ttgtccatcg catttgtcat cttgaagttc 4500 actcttggcc cccttggtcc aaaaggtcag tctcgtatga agtttgtttt caccaattac 4560 aaccttctca tgtccattta ttcgttggga tcattcctct caatggcata tgccatgtac 4620 accatcggtg ttatgtctga caactgcgag aaggcttttg acaacaacgt cttcaggatc 4680 accacgcagt tgttctattt gagcaagttc ctggagtata ttgactcctt ctatttgcca 4740 ctgatgggca agcctctgac ctggttgcaa ttcttccatc atttgggggc accgatggat 4800 atgtggctgt tctataatta ccgaaatgaa gctgtttgga tttttgtgct gttgaatggt 4860 ttcatccact ggatcatgta cggttattat tggaccagat tgatcaagct gaagttcccc 4920 atgccaaaat ccctgattac atcaatgcag atcattcaat tcaatgttgg tttctacatt 4980 gtctggaagt acaggaacat tccctgttat cgccaagatg ggatgaggat gtttggctgg 5040 ttcttcaatt acttttatgt tggcacagtc ttgtgtttgt tcttgaattt ctatgtgcaa 5100 acgtatatcg tcaggaagca caagggagcc aaaaagattc agtgagc 5147 <210> SEQ ID NO 86 <211> LENGTH: 6559 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKR680 <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (4340)..(4340) <223> OTHER INFORMATION: n is a, c, g, or t <400> SEQUENCE: 86 ggccgcgaca caagtgtgag agtactaaat aaatgctttg gttgtacgaa atcattacac 60 taaataaaat aatcaaagct tatatatgcc ttccgctaag gccgaatgca aagaaattgg 120 ttctttctcg ttatcttttg ccacttttac tagtacgtat taattactac ttaatcatct 180 ttgtttacgg ctcattatat ccggtctaga ggatccaagg ccgcgaagtt aaaagcaatg 240 ttgtcacttg tcgtactaac acatgatgtg atagtttatg ctagctagct ataacataag 300 ctgtctctga gtgtgttgta tattaataaa gatcatcact ggtgaatggt gatcgtgtac 360 gtaccctact tagtaggcaa tggaagcact tagagtgtgc tttgtgcatg gccttgcctc 420 tgttttgaga cttttgtaat gttttcgagt ttaaatcttt gcctttgcgt acgtgggcgg 480 atcccccggg ctgcaggaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc 540 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata 600 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc 660 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca 720 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac 780 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga 840 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac 900 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt 960 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct 1020 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat 1080 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg 1140 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg 1200 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc 1260 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat 1320 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact 1380 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca 1440 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact 1500 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg 1560 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg 1620 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg 1680 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg 1740 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag 1800 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc 1860 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga 1920 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat 1980 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc 2040 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 2100

accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 2160 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 2220 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc 2280 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 2340 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 2400 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 2460 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 2520 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca 2580 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 2640 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 2700 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 2760 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta 2820 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag 2880 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga 2940 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg 3000 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg 3060 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc 3120 atgattacgc caagcttgca tgcctgcagg tcgactcgac gtacgtcctc gaagagaagg 3180 gttaataaca cattttttaa catttttaac acaaatttta gttatttaaa aatttattaa 3240 aaaatttaaa ataagaagag gaactcttta aataaatcta acttacaaaa tttatgattt 3300 ttaataagtt ttcaccaata aaaaatgtca taaaaatatg ttaaaaagta tattatcaat 3360 attctcttta tgataaataa aaagaaaaaa aaaataaaag ttaagtgaaa atgagattga 3420 agtgacttta ggtgtgtata aatatatcaa ccccgccaac aatttattta atccaaatat 3480 attgaagtat attattccat agcctttatt tatttatata tttattatat aaaagcttta 3540 tttgttctag gttgttcatg aaatattttt ttggttttat ctccgttgta agaaaatcat 3600 gtgctttgtg tcgccactca ctattgcagc tttttcatgc attggtcaga ttgacggttg 3660 attgtatttt tgttttttat ggttttgtgt tatgacttaa gtcttcatct ctttatctct 3720 tcatcaggtt tgatggttac ctaatatggt ccatgggtac atgcatggtt aaattaggtg 3780 gccaactttg ttgtgaacga tagaattttt tttatattaa gtaaactatt tttatattat 3840 gaaataataa taaaaaaaat attttatcat tattaacaaa atcatattag ttaatttgtt 3900 aactctataa taaaagaaat actgtaacat tcacattaca tggtaacatc tttccaccct 3960 ttcatttgtt ttttgtttga tgactttttt tcttgtttaa atttatttcc cttcttttaa 4020 atttggaata cattatcatc atatataaac taaaatacta aaaacaggat tacacaaatg 4080 ataaataata acacaaatat ttataaatct agctgcaata tatttaaact agctatatcg 4140 atattgtaaa ataaaactag ctgcattgat actgataaaa aaatatcatg tgctttctgg 4200 actgatgatg cagtatactt ttgacattgc ctttatttta tttttcagaa aagctttctt 4260 agttctgggt tcttcattat ttgtttccca tctccattgt gaattgaatc atttgcttcg 4320 tgtcacaaat acaatttagn taggtacatg cattggtcag attcacggtt tattatgtca 4380 tgacttaagt tcatggtagt acattacctg ccacgcatgc attatattgg ttagatttga 4440 taggcaaatt tggttgtcaa caatataaat ataaataatg tttttatatt acgaaataac 4500 agtgatcaaa acaaacagtt ttatctttat taacaagatt ttgtttttgt ttgatgacgt 4560 tttttaatgt ttacgctttc ccccttcttt tgaatttaga acactttatc atcataaaat 4620 caaatactaa aaaaattaca tatttcataa ataataacac aaatattttt aaaaaatctg 4680 aaataataat gaacaatatt acatattatc acgaaaattc attaataaaa atattatata 4740 aataaaatgt aatagtagtt atatgtagga aaaaagtact gcacgcataa tatatacaaa 4800 aagattaaaa tgaactatta taaataataa cactaaatta atggtgaatc atatcaaaat 4860 aatgaaaaag taaataaaat ttgtaattaa cttctatatg tattacacac acaaataata 4920 aataatagta aaaaaaatta tgataaatat ttaccatctc ataagatatt taaaataatg 4980 ataaaaatat agattatttt ttatgcaact agctagccaa aaagagaaca cgggtatata 5040 taaaaagagt acctttaaat tctactgtac ttcctttatt cctgacgttt ttatatcaag 5100 tggacatacg tgaagatttt aattatcagt ctaaatattt cattagcact taatactttt 5160 ctgttttatt cctatcctat aagtagtccc gattctccca acattgctta ttcacacaac 5220 taactaagaa agtcttccat agccccccaa gcggccgcgg gaattcgatt gaaatgaagt 5280 caaagcgcca agcgcttccc cttacaattg atggaacaac atatgatgtg tctgcctggg 5340 tcaatttcca ccctggtggt gcggaaatta tagagaatta ccaaggaagg gatgccactg 5400 atgccttcat ggttatgcac tctcaagaag ccttcgacaa gctcaagcgc atgcccaaaa 5460 tcaatcccag ttctgagttg ccaccccagg ctgcagtgaa tgaagctcaa gaggatttcc 5520 ggaagctccg agaagagttg atcgcaactg gcatgtttga tgcctccccc ctctggtact 5580 catacaaaat cagcaccaca ctgggccttg gagtgctggg ttatttcctg atggttcagt 5640 atcagatgta tttcattggg gcagtgttgc ttgggatgca ctatcaacag atgggctggc 5700 tttctcatga catttgccac caccagactt tcaagaaccg gaactggaac aacctcgtgg 5760 gactggtatt tggcaatggt ctgcaaggtt tttccgtgac atggtggaag gacagacaca 5820 atgcacatca ttcggcaacc aatgttcaag ggcacgaccc tgatattgac aacctccccc 5880 tcttagcctg gtctgaggat gacgtcacac gggcgtcacc gatttcccgc aagctcattc 5940 agttccagca gtactatttc ttggtcatct gtatcttgtt gcggttcatt tggtgtttcc 6000 agagcgtgtt gaccgtgcgc agtttgaagg acagagataa ccaattctat cgctctcagt 6060 ataagaagga ggccattggc ctcgccctgc actggacctt gaagaccctg ttccacttat 6120 tctttatgcc cagcatcctc acatcgctgt tggtgttttt cgtttcggag ctggttggcg 6180 gcttcggcat tgcgatcgtg gtgttcatga accactaccc actggagaag atcggggact 6240 cagtctggga tggccatgga ttctcggttg gccagatcca tgagaccatg aacattcggc 6300 gagggattat cacagattgg tttttcggag gcttgaatta ccagattgag caccatttgt 6360 ggccgaccct ccctcgccac aacctgacag cggttagcta ccaggtggaa cagctgtgcc 6420 agaagcacaa cctgccgtat cggaacccgc tgccccatga agggttggtc atcctgctgc 6480 gctatctggc ggtgttcgcc cggatggcgg agaagcaacc cgcggggaag gctctataag 6540 gaatcactag tgaattcgc 6559 <210> SEQ ID NO 87 <211> LENGTH: 9014 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKR913 <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (7839)..(7839) <223> OTHER INFORMATION: n is a, c, g, or t <400> SEQUENCE: 87 gtacgagatc cggccggcca gatcctgcag gagatccaag cttttgatcc atgcccttca 60 tttgccgctt attaattaat ttggtaacag tccgtactaa tcagttactt atccttcccc 120 catcataatt aatcttggta gtctcgaatg ccacaacact gactagtctc ttggatcata 180 agaaaaagcc aaggaacaaa agaagacaaa acacaatgag agtatccttt gcatagcaat 240 gtctaagttc ataaaattca aacaaaaacg caatcacaca cagtggacat cacttatcca 300 ctagctgatc aggatcgccg cgtcaagaaa aaaaaactgg accccaaaag ccatgcacaa 360 caacacgtac tcacaaaggt gtcaatcgag cagcccaaaa cattcaccaa ctcaacccat 420 catgagccct cacatttgtt gtttctaacc caacctcaaa ctcgtattct cttccgccac 480 ctcatttttg tttatttcaa cacccgtcaa actgcatgcc accccgtggc caaatgtcca 540 tgcatgttaa caagacctat gactataaat agctgcaatc tcggcccagg ttttcatcat 600 caagaaccag ttcaatatcc tagtacaccg tattaaagaa tttaagatat actgcggccg 660 caccatggag gtggtgaatg aaatagtctc aattgggcag gaagttttac ccaaagttga 720 ttatgcccaa ctctggagtg atgccagtca ctgtgaggtg ctttacttgt ccatcgcatt 780 tgtcatcttg aagttcactc ttggccccct tggtccaaaa ggtcagtctc gtatgaagtt 840 tgttttcacc aattacaacc ttctcatgtc catttattcg ttgggatcat tcctctcaat 900 ggcatatgcc atgtacacca tcggtgttat gtctgacaac tgcgagaagg cttttgacaa 960 caacgtcttc aggatcacca cgcagttgtt ctatttgagc aagttcctgg agtatattga 1020 ctccttctat ttgccactga tgggcaagcc tctgacctgg ttgcaattct tccatcattt 1080 gggggcaccg atggatatgt ggctgttcta taattaccga aatgaagctg tttggatttt 1140 tgtgctgttg aatggtttca tccactggat catgtacggt tattattgga ccagattgat 1200 caagctgaag ttccccatgc caaaatccct gattacatca atgcagatca ttcaattcaa 1260 tgttggtttc tacattgtct ggaagtacag gaacattccc tgttatcgcc aagatgggat 1320 gaggatgttt ggctggttct tcaattactt ttatgttggc acagtcttgt gtttgttctt 1380 gaatttctat gtgcaaacgt atatcgtcag gaagcacaag ggagccaaaa agattcagtg 1440 agcggccgca agtatgaact aaaatgcatg taggtgtaag agctcatgga gagcatggaa 1500 tattgtatcc gaccatgtaa cagtataata actgagctcc atctcacttc ttctatgaat 1560 aaacaaagga tgttatgata tattaacact ctatctatgc accttattgt tctatgataa 1620 atttcctctt attattataa atcatctgaa tcgtgacggc ttatggaatg cttcaaatag 1680 tacaaaaaca aatgtgtact ataagacttt ctaaacaatt ctaaccttag cattgtgaac 1740 gagacataag tgttaagaag acataacaat tataatggaa gaagtttgtc tccatttata 1800 tattatatat tacccactta tgtattatat taggatgtta aggagacata acaattataa 1860 agagagaagt ttgtatccat ttatatatta tatactaccc atttatatat tatacttatc 1920 cacttattta atgtctttat aaggtttgat ccatgatatt tctaatattt tagttgatat 1980 gtatatgaaa gggtactatt tgaactctct tactctgtat aaaggttgga tcatccttaa 2040 agtgggtcta tttaatttta ttgcttctta cagataaaaa aaaaattatg agttggtttg 2100 ataaaatatt gaaggattta aaataataat aaataacata taatatatgt atataaattt 2160 attataatat aacatttatc tataaaaaag taaatattgt cataaatcta tacaatcgtt 2220 tagccttgct ggacgaatct caattattta aacgagagta aacatatttg actttttggt 2280 tatttaacaa attattattt aacactatat gaaatttttt tttttatcag caaagaataa 2340 aattaaatta agaaggacaa tggtgtccca atccttatac aaccaacttc cacaagaaag 2400 tcaagtcaga gacaacaaaa aaacaagcaa aggaaatttt ttaatttgag ttgtcttgtt 2460 tgctgcataa tttatgcagt aaaacactac acataaccct tttagcagta gagcaatggt 2520 tgaccgtgtg cttagcttct tttattttat ttttttatca gcaaagaata aataaaataa 2580 aatgagacac ttcagggatg tttcaacaag cttggcgcgc cgttctatag tgtcacctaa 2640

atcgtatgtg tatgatacat aaggttatgt attaattgta gccgcgttct aacgacaata 2700 tgtccatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc 2760 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 2820 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 2880 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga 2940 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 3000 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 3060 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 3120 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag 3180 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 3240 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 3300 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 3360 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gcattgagaa agcgccacgc 3420 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 3480 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 3540 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 3600 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 3660 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg 3720 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag 3780 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcaggttga 3840 tcagatctcg atcccgcgaa attaatacga ctcactatag ggagaccaca acggtttccc 3900 tctagaaata attttgttta actttaagaa ggagatatac ccatggaaaa gcctgaactc 3960 accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg 4020 cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat 4080 gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac 4140 tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc 4200 ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc 4260 gaactgcccg ctgttctgca gccggtcgcg gaggctatgg atgcgatcgc tgcggccgat 4320 cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca 4380 tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg 4440 gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag 4500 gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg 4560 gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa 4620 tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg 4680 cgctacttcg agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg 4740 ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca 4800 gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt 4860 acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc 4920 gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaatagtg aggtacagct 4980 tggatcgatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct 5040 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg 5100 aaaggaggaa ctatatccgg atgatcgggc gcgccgtcga cggatccgta cgcaaaggca 5160 aagatttaaa ctcgaaaaca ttacaaaagt ctcaaaacag aggcaaggcc atgcacaaag 5220 cacactctaa gtgcttccat tgcctactaa gtagggtacg tacacgatca ccattcacca 5280 gtgatgatct ttattaatat acaacacact cagagacagc ttatgttata gctagctagc 5340 ataaactatc acatcatgtg ttagtacgac aagtgacaac attgctttta acttcgcggc 5400 cttggatcct ctagaccgga tataatgagc cgtaaacaaa gatgattaag tagtaattaa 5460 tacgtactag taaaagtggc aaaagataac gagaaagaac caatttcttt gcattcggcc 5520 ttagcggaag gcatatataa gctttgatta ttttatttag tgtaatgatt tcgtacaacc 5580 aaagcattta tttagtactc tcacacttgt gtcgcggccg cgaattcact agtgattcct 5640 tatagagcct tccccgcggg ttgcttctcc gccatccggg cgaacaccgc cagatagcgc 5700 agcaggatga ccaacccttc atggggcagc gggttccgat acggcaggtt gtgcttctgg 5760 cacagctgtt ccacctggta gctaaccgct gtcaggttgt ggcgagggag ggtcggccac 5820 aaatggtgct caatctggta attcaagcct ccgaaaaacc aatctgtgat aatccctcgc 5880 cgaatgttca tggtctcatg gatctggcca accgagaatc catggccatc ccagactgag 5940 tccccgatct tctccagtgg gtagtggttc atgaacacca cgatcgcaat gccgaagccg 6000 ccaaccagct ccgaaacgaa aaacaccaac agcgatgtga ggatgctggg cataaagaat 6060 aagtggaaca gggtcttcaa ggtccagtgc agggcgaggc caatggcctc cttcttatac 6120 tgagagcgat agaattggtt atctctgtcc ttcaaactgc gcacggtcaa cacgctctgg 6180 aaacaccaaa tgaaccgcaa caagatacag atgaccaaga aatagtactg ctggaactga 6240 atgagcttgc gggaaatcgg tgacgcccgt gtgacgtcat cctcagacca ggctaagagg 6300 gggaggttgt caatatcagg gtcgtgccct tgaacattgg ttgccgaatg atgtgcattg 6360 tgtctgtcct tccaccatgt cacggaaaaa ccttgcagac cattgccaaa taccagtccc 6420 acgaggttgt tccagttccg gttcttgaaa gtctggtggt ggcaaatgtc atgagaaagc 6480 cagcccatct gttgatagtg catcccaagc aacactgccc caatgaaata catctgatac 6540 tgaaccatca ggaaataacc cagcactcca aggcccagtg tggtgctgat tttgtatgag 6600 taccagaggg gggaggcatc aaacatgcca gttgcgatca actcttctcg gagcttccgg 6660 aaatcctctt gagcttcatt cactgcagcc tggggtggca actcagaact gggattgatt 6720 ttgggcatgc gcttgagctt gtcgaaggct tcttgagagt gcataaccat gaaggcatca 6780 gtggcatccc ttccttggta attctctata atttccgcac caccagggtg gaaattgacc 6840 caggcagaca catcatatgt tgttccatca attgtaaggg gaagcgcttg gcgctttgac 6900 ttcatttcaa tcgaattccc gcggccgctt ggggggctat ggaagacttt cttagttagt 6960 tgtgtgaata agcaatgttg ggagaatcgg gactacttat aggataggaa taaaacagaa 7020 aagtattaag tgctaatgaa atatttagac tgataattaa aatcttcacg tatgtccact 7080 tgatataaaa acgtcaggaa taaaggaagt acagtagaat ttaaaggtac tctttttata 7140 tatacccgtg ttctcttttt ggctagctag ttgcataaaa aataatctat atttttatca 7200 ttattttaaa tatcttatga gatggtaaat atttatcata atttttttta ctattattta 7260 ttatttgtgt gtgtaataca tatagaagtt aattacaaat tttatttact ttttcattat 7320 tttgatatga ttcaccatta atttagtgtt attatttata atagttcatt ttaatctttt 7380 tgtatatatt atgcgtgcag tacttttttc ctacatataa ctactattac attttattta 7440 tataatattt ttattaatga attttcgtga taatatgtaa tattgttcat tattatttca 7500 gattttttaa aaatatttgt gttattattt atgaaatatg taattttttt agtatttgat 7560 tttatgatga taaagtgttc taaattcaaa agaaggggga aagcgtaaac attaaaaaac 7620 gtcatcaaac aaaaacaaaa tcttgttaat aaagataaaa ctgtttgttt tgatcactgt 7680 tatttcgtaa tataaaaaca ttatttatat ttatattgtt gacaaccaaa tttgcctatc 7740 aaatctaacc aatataatgc atgcgtggca ggtaatgtac taccatgaac ttaagtcatg 7800 acataataaa ccgtgaatct gaccaatgca tgtacctanc taaattgtat ttgtgacacg 7860 aagcaaatga ttcaattcac aatggagatg ggaaacaaat aatgaagaac ccagaactaa 7920 gaaagctttt ctgaaaaata aaataaaggc aatgtcaaaa gtatactgca tcatcagtcc 7980 agaaagcaca tgatattttt ttatcagtat caatgcagct agttttattt tacaatatcg 8040 atatagctag tttaaatata ttgcagctag atttataaat atttgtgtta ttatttatca 8100 tttgtgtaat cctgttttta gtattttagt ttatatatga tgataatgta ttccaaattt 8160 aaaagaaggg aaataaattt aaacaagaaa aaaagtcatc aaacaaaaaa caaatgaaag 8220 ggtggaaaga tgttaccatg taatgtgaat gttacagtat ttcttttatt atagagttaa 8280 caaattaact aatatgattt tgttaataat gataaaatat tttttttatt attatttcat 8340 aatataaaaa tagtttactt aatataaaaa aaattctatc gttcacaaca aagttggcca 8400 cctaatttaa ccatgcatgt acccatggac catattaggt aaccatcaaa cctgatgaag 8460 agataaagag atgaagactt aagtcataac acaaaaccat aaaaaacaaa aatacaatca 8520 accgtcaatc tgaccaatgc atgaaaaagc tgcaatagtg agtggcgaca caaagcacat 8580 gattttctta caacggagat aaaaccaaaa aaatatttca tgaacaacct agaacaaata 8640 aagcttttat ataataaata tataaataaa taaaggctat ggaataatat acttcaatat 8700 atttggatta aataaattgt tggcggggtt gatatattta tacacaccta aagtcacttc 8760 aatctcattt tcacttaact tttatttttt ttttcttttt atttatcata aagagaatat 8820 tgataatata ctttttaaca tatttttatg acatttttta ttggtgaaaa cttattaaaa 8880 atcataaatt ttgtaagtta gatttattta aagagttcct cttcttattt taaatttttt 8940 aataaatttt taaataacta aaatttgtgt taaaaatgtt aaaaaatgtg ttattaaccc 9000 ttctcttcga ggac 9014 <210> SEQ ID NO 88 <211> LENGTH: 5561 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKR767 <400> SEQUENCE: 88 catggtcaat caatgagacg ccaacttctt aatctattga gacctgcagg tctagaaggg 60 cggatccccg ggtaccgagc tcgaattcac tggccgtcgt tttacaacgt cgtgactggg 120 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc 180 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg 240 aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat 300 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc 360 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag 420 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg 480 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg 540 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 600 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 660 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 720 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 780

atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 840 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 900 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 960 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 1020 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 1080 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 1140 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 1200 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 1260 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 1320 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 1380 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 1440 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 1500 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 1560 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 1620 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 1680 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 1740 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 1800 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 1860 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 1920 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 1980 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 2040 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 2100 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 2160 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 2220 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 2280 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 2340 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc 2400 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg 2460 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt 2520 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc 2580 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc 2640 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct 2700 atgaccatga ttacgccaag cttgcatgcc tgcaggctag cctaagtacg tactcaaaat 2760 gccaacaaat aaaaaaaaag ttgctttaat aatgccaaaa caaattaata aaacacttac 2820 aacaccggat tttttttaat taaaatgtgc catttaggat aaatagttaa tatttttaat 2880 aattatttaa aaagccgtat ctactaaaat gatttttatt tggttgaaaa tattaatatg 2940 tttaaatcaa cacaatctat caaaattaaa ctaaaaaaaa aataagtgta cgtggttaac 3000 attagtacag taatataaga ggaaaatgag aaattaagaa attgaaagcg agtctaattt 3060 ttaaattatg aacctgcata tataaaagga aagaaagaat ccaggaagaa aagaaatgaa 3120 accatgcatg gtcccctcgt catcacgagt ttctgccatt tgcaatagaa acactgaaac 3180 acctttctct ttgtcactta attgagatgc cgaagccacc tcacaccatg aacttcatga 3240 ggtgtagcac ccaaggcttc catagccatg catactgaag aatgtctcaa gctcagcacc 3300 ctacttctgt gacgtgtccc tcattcacct tcctctcttc cctataaata accacgcctc 3360 aggttctccg cttcacaact caaacattct ctccattggt ccttaaacac tcatcagtca 3420 tcaccgcggc cgcatgggaa cggaccaagg aaaaaccttc acctgggaag agctggcggc 3480 ccataacacc aaggacgacc tactcttggc catccgcggc agggtgtacg atgtcacaaa 3540 gttcttgagc cgccatcctg gtggagtgga cactctcctg ctcggagctg gccgagatgt 3600 tactccggtc tttgagatgt atcacgcgtt tggggctgca gatgccatta tgaagaagta 3660 ctatgtcggt acactggtct cgaatgagct gcccatcttc ccggagccaa cggtgttcca 3720 caaaaccatc aagacgagag tcgagggcta ctttacggat cggaacattg atcccaagaa 3780 tagaccagag atctggggac gatacgctct tatctttgga tccttgatcg cttcctacta 3840 cgcgcagctc tttgtgcctt tcgttgtcga acgcacatgg cttcaggtgg tgtttgcaat 3900 catcatggga tttgcgtgcg cacaagtcgg actcaaccct cttcatgatg cgtctcactt 3960 ttcagtgacc cacaacccca ctgtctggaa gattctggga gccacgcacg actttttcaa 4020 cggagcatcg tacctggtgt ggatgtacca acatatgctc ggccatcacc cctacaccaa 4080 cattgctgga gcagatcccg acgtgtcgac gtctgagccc gatgttcgtc gtatcaagcc 4140 caaccaaaag tggtttgtca accacatcaa ccagcacatg tttgttcctt tcctgtacgg 4200 actgctggcg ttcaaggtgc gcattcagga catcaacatt ttgtactttg tcaagaccaa 4260 tgacgctatt cgtgtcaatc ccatctcgac atggcacact gtgatgttct ggggcggcaa 4320 ggctttcttt gtctggtatc gcctgattgt tcccctgcag tatctgcccc tgggcaaggt 4380 gctgctcttg ttcacggtcg cggacatggt gtcgtcttac tggctggcgc tgaccttcca 4440 ggcgaaccac gttgttgagg aagttcagtg gccgttgcct gacgagaacg ggatcatcca 4500 aaaggactgg gcagctatgc aggtcgagac tacgcaggat tacgcacacg attcgcacct 4560 ctggaccagc atcactggca gcttgaacta ccaggctgtg caccatctgt tccccaacgt 4620 gtcgcagcac cattatcccg atattctggc catcatcaag aacacctgca gcgagtacaa 4680 ggttccatac cttgtcaagg atacgttttg gcaagcattt gcttcacatt tggagcactt 4740 gcgtgttctt ggactccgtc ccaaggaaga gtaggcggcc gcatttcgca ccaaatcaat 4800 gaaagtaata atgaaaagtc tgaataagaa tacttaggct tagatgcctt tgttacttgt 4860 gtaaaataac ttgagtcatg tacctttggc ggaaacagaa taaataaaag gtgaaattcc 4920 aatgctctat gtataagtta gtaatactta atgtgttcta cggttgtttc aatatcatca 4980 aactctaatt gaaactttag aaccacaaat ctcaatcttt tcttaatgaa atgaaaaatc 5040 ttaattgtac catgtttatg ttaaacacct tacaattggt tggagaggag gaccaaccga 5100 tgggacaaca ttgggagaaa gagattcaat ggagatttgg ataggagaac aacattcttt 5160 ttcacttcaa tacaagatga gtgcaacact aaggatatgt atgagacttt cagaagctac 5220 gacaacatag atgagtgagg tggtgattcc tagcaagaaa gacattagag gaagccaaaa 5280 tcgaacaagg aagacatcaa gggcaagaga caggaccatc catctcagga aaaggagctt 5340 tgggatagtc cgagaagttg tacaagaaat tttttggagg gtgagtgatg cattgctggt 5400 gactttaact caatcaaaat tgagaaagaa agaaaaggga gggggctcac atgtgaatag 5460 aagggaaacg ggagaatttt acagttttga tctaatgggc atcccagcta gtggtaacat 5520 attcaccatg tttaaccttc acgtacgtct agaggatccc c 5561 <210> SEQ ID NO 89 <211> LENGTH: 11889 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKR916 <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (7810)..(7810) <223> OTHER INFORMATION: n is a, c, g, or t <400> SEQUENCE: 89 ggagatccaa gcttttgatc catgcccttc atttgccgct tattaattaa tttggtaaca 60 gtccgtacta atcagttact tatccttccc ccatcataat taatcttggt agtctcgaat 120 gccacaacac tgactagtct cttggatcat aagaaaaagc caaggaacaa aagaagacaa 180 aacacaatga gagtatcctt tgcatagcaa tgtctaagtt cataaaattc aaacaaaaac 240 gcaatcacac acagtggaca tcacttatcc actagctgat caggatcgcc gcgtcaagaa 300 aaaaaaactg gaccccaaaa gccatgcaca acaacacgta ctcacaaagg tgtcaatcga 360 gcagcccaaa acattcacca actcaaccca tcatgagccc tcacatttgt tgtttctaac 420 ccaacctcaa actcgtattc tcttccgcca cctcattttt gtttatttca acacccgtca 480 aactgcatgc caccccgtgg ccaaatgtcc atgcatgtta acaagaccta tgactataaa 540 tagctgcaat ctcggcccag gttttcatca tcaagaacca gttcaatatc ctagtacacc 600 gtattaaaga atttaagata tactgcggcc gcaccatgga ggtggtgaat gaaatagtct 660 caattgggca ggaagtttta cccaaagttg attatgccca actctggagt gatgccagtc 720 actgtgaggt gctttacttg tccatcgcat ttgtcatctt gaagttcact cttggccccc 780 ttggtccaaa aggtcagtct cgtatgaagt ttgttttcac caattacaac cttctcatgt 840 ccatttattc gttgggatca ttcctctcaa tggcatatgc catgtacacc atcggtgtta 900 tgtctgacaa ctgcgagaag gcttttgaca acaacgtctt caggatcacc acgcagttgt 960 tctatttgag caagttcctg gagtatattg actccttcta tttgccactg atgggcaagc 1020 ctctgacctg gttgcaattc ttccatcatt tgggggcacc gatggatatg tggctgttct 1080 ataattaccg aaatgaagct gtttggattt ttgtgctgtt gaatggtttc atccactgga 1140 tcatgtacgg ttattattgg accagattga tcaagctgaa gttccccatg ccaaaatccc 1200 tgattacatc aatgcagatc attcaattca atgttggttt ctacattgtc tggaagtaca 1260 ggaacattcc ctgttatcgc caagatggga tgaggatgtt tggctggttc ttcaattact 1320 tttatgttgg cacagtcttg tgtttgttct tgaatttcta tgtgcaaacg tatatcgtca 1380 ggaagcacaa gggagccaaa aagattcagt gagcggccgc aagtatgaac taaaatgcat 1440 gtaggtgtaa gagctcatgg agagcatgga atattgtatc cgaccatgta acagtataat 1500 aactgagctc catctcactt cttctatgaa taaacaaagg atgttatgat atattaacac 1560 tctatctatg caccttattg ttctatgata aatttcctct tattattata aatcatctga 1620 atcgtgacgg cttatggaat gcttcaaata gtacaaaaac aaatgtgtac tataagactt 1680 tctaaacaat tctaacctta gcattgtgaa cgagacataa gtgttaagaa gacataacaa 1740 ttataatgga agaagtttgt ctccatttat atattatata ttacccactt atgtattata 1800 ttaggatgtt aaggagacat aacaattata aagagagaag tttgtatcca tttatatatt 1860 atatactacc catttatata ttatacttat ccacttattt aatgtcttta taaggtttga 1920 tccatgatat ttctaatatt ttagttgata tgtatatgaa agggtactat ttgaactctc 1980 ttactctgta taaaggttgg atcatcctta aagtgggtct atttaatttt attgcttctt 2040 acagataaaa aaaaaattat gagttggttt gataaaatat tgaaggattt aaaataataa 2100 taaataacat ataatatatg tatataaatt tattataata taacatttat ctataaaaaa 2160 gtaaatattg tcataaatct atacaatcgt ttagccttgc tggacgaatc tcaattattt 2220 aaacgagagt aaacatattt gactttttgg ttatttaaca aattattatt taacactata 2280 tgaaattttt ttttttatca gcaaagaata aaattaaatt aagaaggaca atggtgtccc 2340

aatccttata caaccaactt ccacaagaaa gtcaagtcag agacaacaaa aaaacaagca 2400 aaggaaattt tttaatttga gttgtcttgt ttgctgcata atttatgcag taaaacacta 2460 cacataaccc ttttagcagt agagcaatgg ttgaccgtgt gcttagcttc ttttatttta 2520 tttttttatc agcaaagaat aaataaaata aaatgagaca cttcagggat gtttcaacaa 2580 gcttggcgcg ccgttctata gtgtcaccta aatcgtatgt gtatgataca taaggttatg 2640 tattaattgt agccgcgttc taacgacaat atgtccatat ggtgcactct cagtacaatc 2700 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc 2760 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc 2820 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg 2880 atacgcctat ttttataggt taatgtcatg accaaaatcc cttaacgtga gttttcgttc 2940 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 3000 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 3060 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 3120 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 3180 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 3240 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 3300 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 3360 ctacagcgtg agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 3420 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 3480 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 3540 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 3600 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg 3660 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag 3720 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc 3780 gcgcgttggc cgattcatta atgcaggttg atcagatctc gatcccgcga aattaatacg 3840 actcactata gggagaccac aacggtttcc ctctagaaat aattttgttt aactttaaga 3900 aggagatata cccatggaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat 3960 cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc 4020 tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg 4080 tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga 4140 agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca 4200 gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc 4260 ggaggctatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt 4320 cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga 4380 tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca 4440 ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca 4500 cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga 4560 ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag 4620 gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct 4680 tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca 4740 gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat 4800 cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt 4860 ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg 4920 tccgagggca aaggaatagt gaggtacagc ttggatcgat ccggctgcta acaaagcccg 4980 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc 5040 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gatgatcggg 5100 cgcgccgtcg acggatccgt acgcaaaggc aaagatttaa actcgaaaac attacaaaag 5160 tctcaaaaca gaggcaaggc catgcacaaa gcacactcta agtgcttcca ttgcctacta 5220 agtagggtac gtacacgatc accattcacc agtgatgatc tttattaata tacaacacac 5280 tcagagacag cttatgttat agctagctag cataaactat cacatcatgt gttagtacga 5340 caagtgacaa cattgctttt aacttcgcgg ccttggatcc tctagaccgg atataatgag 5400 ccgtaaacaa agatgattaa gtagtaatta atacgtacta gtaaaagtgg caaaagataa 5460 cgagaaagaa ccaatttctt tgcattcggc cttagcggaa ggcatatata agctttgatt 5520 attttattta gtgtaatgat ttcgtacaac caaagcattt atttagtact ctcacacttg 5580 tgtcgcggcc gcgaattcac tagtgattcc ttatagagcc ttccccgcgg gttgcttctc 5640 cgccatccgg gcgaacaccg ccagatagcg cagcaggatg accaaccctt catggggcag 5700 cgggttccga tacggcaggt tgtgcttctg gcacagctgt tccacctggt agctaaccgc 5760 tgtcaggttg tggcgaggga gggtcggcca caaatggtgc tcaatctggt aattcaagcc 5820 tccgaaaaac caatctgtga taatccctcg ccgaatgttc atggtctcat ggatctggcc 5880 aaccgagaat ccatggccat cccagactga gtccccgatc ttctccagtg ggtagtggtt 5940 catgaacacc acgatcgcaa tgccgaagcc gccaaccagc tccgaaacga aaaacaccaa 6000 cagcgatgtg aggatgctgg gcataaagaa taagtggaac agggtcttca aggtccagtg 6060 cagggcgagg ccaatggcct ccttcttata ctgagagcga tagaattggt tatctctgtc 6120 cttcaaactg cgcacggtca acacgctctg gaaacaccaa atgaaccgca acaagataca 6180 gatgaccaag aaatagtact gctggaactg aatgagcttg cgggaaatcg gtgacgcccg 6240 tgtgacgtca tcctcagacc aggctaagag ggggaggttg tcaatatcag ggtcgtgccc 6300 ttgaacattg gttgccgaat gatgtgcatt gtgtctgtcc ttccaccatg tcacggaaaa 6360 accttgcaga ccattgccaa ataccagtcc cacgaggttg ttccagttcc ggttcttgaa 6420 agtctggtgg tggcaaatgt catgagaaag ccagcccatc tgttgatagt gcatcccaag 6480 caacactgcc ccaatgaaat acatctgata ctgaaccatc aggaaataac ccagcactcc 6540 aaggcccagt gtggtgctga ttttgtatga gtaccagagg ggggaggcat caaacatgcc 6600 agttgcgatc aactcttctc ggagcttccg gaaatcctct tgagcttcat tcactgcagc 6660 ctggggtggc aactcagaac tgggattgat tttgggcatg cgcttgagct tgtcgaaggc 6720 ttcttgagag tgcataacca tgaaggcatc agtggcatcc cttccttggt aattctctat 6780 aatttccgca ccaccagggt ggaaattgac ccaggcagac acatcatatg ttgttccatc 6840 aattgtaagg ggaagcgctt ggcgctttga cttcatttca atcgaattcc cgcggccgct 6900 tggggggcta tggaagactt tcttagttag ttgtgtgaat aagcaatgtt gggagaatcg 6960 ggactactta taggatagga ataaaacaga aaagtattaa gtgctaatga aatatttaga 7020 ctgataatta aaatcttcac gtatgtccac ttgatataaa aacgtcagga ataaaggaag 7080 tacagtagaa tttaaaggta ctctttttat atatacccgt gttctctttt tggctagcta 7140 gttgcataaa aaataatcta tatttttatc attattttaa atatcttatg agatggtaaa 7200 tatttatcat aatttttttt actattattt attatttgtg tgtgtaatac atatagaagt 7260 taattacaaa ttttatttac tttttcatta ttttgatatg attcaccatt aatttagtgt 7320 tattatttat aatagttcat tttaatcttt ttgtatatat tatgcgtgca gtactttttt 7380 cctacatata actactatta cattttattt atataatatt tttattaatg aattttcgtg 7440 ataatatgta atattgttca ttattatttc agatttttta aaaatatttg tgttattatt 7500 tatgaaatat gtaatttttt tagtatttga ttttatgatg ataaagtgtt ctaaattcaa 7560 aagaaggggg aaagcgtaaa cattaaaaaa cgtcatcaaa caaaaacaaa atcttgttaa 7620 taaagataaa actgtttgtt ttgatcactg ttatttcgta atataaaaac attatttata 7680 tttatattgt tgacaaccaa atttgcctat caaatctaac caatataatg catgcgtggc 7740 aggtaatgta ctaccatgaa cttaagtcat gacataataa accgtgaatc tgaccaatgc 7800 atgtacctan ctaaattgta tttgtgacac gaagcaaatg attcaattca caatggagat 7860 gggaaacaaa taatgaagaa cccagaacta agaaagcttt tctgaaaaat aaaataaagg 7920 caatgtcaaa agtatactgc atcatcagtc cagaaagcac atgatatttt tttatcagta 7980 tcaatgcagc tagttttatt ttacaatatc gatatagcta gtttaaatat attgcagcta 8040 gatttataaa tatttgtgtt attatttatc atttgtgtaa tcctgttttt agtattttag 8100 tttatatatg atgataatgt attccaaatt taaaagaagg gaaataaatt taaacaagaa 8160 aaaaagtcat caaacaaaaa acaaatgaaa gggtggaaag atgttaccat gtaatgtgaa 8220 tgttacagta tttcttttat tatagagtta acaaattaac taatatgatt ttgttaataa 8280 tgataaaata ttttttttat tattatttca taatataaaa atagtttact taatataaaa 8340 aaaattctat cgttcacaac aaagttggcc acctaattta accatgcatg tacccatgga 8400 ccatattagg taaccatcaa acctgatgaa gagataaaga gatgaagact taagtcataa 8460 cacaaaacca taaaaaacaa aaatacaatc aaccgtcaat ctgaccaatg catgaaaaag 8520 ctgcaatagt gagtggcgac acaaagcaca tgattttctt acaacggaga taaaaccaaa 8580 aaaatatttc atgaacaacc tagaacaaat aaagctttta tataataaat atataaataa 8640 ataaaggcta tggaataata tacttcaata tatttggatt aaataaattg ttggcggggt 8700 tgatatattt atacacacct aaagtcactt caatctcatt ttcacttaac ttttattttt 8760 tttttctttt tatttatcat aaagagaata ttgataatat actttttaac atatttttat 8820 gacatttttt attggtgaaa acttattaaa aatcataaat tttgtaagtt agatttattt 8880 aaagagttcc tcttcttatt ttaaattttt taataaattt ttaaataact aaaatttgtg 8940 ttaaaaatgt taaaaaatgt gttattaacc cttctcttcg aggacgtacg agatccggcc 9000 ggccagatcc tgcaggtctc aatagattaa gaagttggcg tctcattgat tgaccatggg 9060 ggatcctcta gacgtacgtg aaggttaaac atggtgaata tgttaccact agctgggatg 9120 cccattagat caaaactgta aaattctccc gtttcccttc tattcacatg tgagccccct 9180 cccttttctt tctttctcaa ttttgattga gttaaagtca ccagcaatgc atcactcacc 9240 ctccaaaaaa tttcttgtac aacttctcgg actatcccaa agctcctttt cctgagatgg 9300 atggtcctgt ctcttgccct tgatgtcttc cttgttcgat tttggcttcc tctaatgtct 9360 ttcttgctag gaatcaccac ctcactcatc tatgttgtcg tagcttctga aagtctcata 9420 catatcctta gtgttgcact catcttgtat tgaagtgaaa aagaatgttg ttctcctatc 9480 caaatctcca ttgaatctct ttctcccaat gttgtcccat cggttggtcc tcctctccaa 9540 ccaattgtaa ggtgtttaac ataaacatgg tacaattaag atttttcatt tcattaagaa 9600 aagattgaga tttgtggttc taaagtttca attagagttt gatgatattg aaacaaccgt 9660 agaacacatt aagtattact aacttataca tagagcattg gaatttcacc ttttatttat 9720 tctgtttccg ccaaaggtac atgactcaag ttattttaca caagtaacaa aggcatctaa 9780 gcctaagtat tcttattcag acttttcatt attactttca ttgatttggt gcgaaatgcg 9840

gccgcctact cttccttggg acggagtcca agaacacgca agtgctccaa atgtgaagca 9900 aatgcttgcc aaaacgtatc cttgacaagg tatggaacct tgtactcgct gcaggtgttc 9960 ttgatgatgg ccagaatatc gggataatgg tgctgcgaca cgttggggaa cagatggtgc 10020 acagcctggt agttcaagct gccagtgatg ctggtccaga ggtgcgaatc gtgtgcgtaa 10080 tcctgcgtag tctcgacctg catagctgcc cagtcctttt ggatgatccc gttctcgtca 10140 ggcaacggcc actgaacttc ctcaacaacg tggttcgcct ggaaggtcag cgccagccag 10200 taagacgaca ccatgtccgc gaccgtgaac aagagcagca ccttgcccag gggcagatac 10260 tgcaggggaa caatcaggcg ataccagaca aagaaagcct tgccgcccca gaacatcaca 10320 gtgtgccatg tcgagatggg attgacacga atagcgtcat tggtcttgac aaagtacaaa 10380 atgttgatgt cctgaatgcg caccttgaac gccagcagtc cgtacaggaa aggaacaaac 10440 atgtgctggt tgatgtggtt gacaaaccac ttttggttgg gcttgatacg acgaacatcg 10500 ggctcagacg tcgacacgtc gggatctgct ccagcaatgt tggtgtaggg gtgatggccg 10560 agcatatgtt ggtacatcca caccaggtac gatgctccgt tgaaaaagtc gtgcgtggct 10620 cccagaatct tccagacagt ggggttgtgg gtcactgaaa agtgagacgc atcatgaaga 10680 gggttgagtc cgacttgtgc gcacgcaaat cccatgatga ttgcaaacac cacctgaagc 10740 catgtgcgtt cgacaacgaa aggcacaaag agctgcgcgt agtaggaagc gatcaaggat 10800 ccaaagataa gagcgtatcg tccccagatc tctggtctat tcttgggatc aatgttccga 10860 tccgtaaagt agccctcgac tctcgtcttg atggttttgt ggaacaccgt tggctccggg 10920 aagatgggca gctcattcga gaccagtgta ccgacatagt acttcttcat aatggcatct 10980 gcagccccaa acgcgtgata catctcaaag accggagtaa catctcggcc agctccgagc 11040 aggagagtgt ccactccacc aggatggcgg ctcaagaact ttgtgacatc gtacaccctg 11100 ccgcggatgg ccaagagtag gtcgtccttg gtgttatggg ccgccagctc ttcccaggtg 11160 aaggtttttc cttggtccgt tcccatgcgg ccgcggtgat gactgatgag tgtttaagga 11220 ccaatggaga gaatgtttga gttgtgaagc ggagaacctg aggcgtggtt atttataggg 11280 aagagaggaa ggtgaatgag ggacacgtca cagaagtagg gtgctgagct tgagacattc 11340 ttcagtatgc atggctatgg aagccttggg tgctacacct catgaagttc atggtgtgag 11400 gtggcttcgg catctcaatt aagtgacaaa gagaaaggtg tttcagtgtt tctattgcaa 11460 atggcagaaa ctcgtgatga cgaggggacc atgcatggtt tcatttcttt tcttcctgga 11520 ttctttcttt ccttttatat atgcaggttc ataatttaaa aattagactc gctttcaatt 11580 tcttaatttc tcattttcct cttatattac tgtactaatg ttaaccacgt acacttattt 11640 tttttttagt ttaattttga tagattgtgt tgatttaaac atattaatat tttcaaccaa 11700 ataaaaatca ttttagtaga tacggctttt taaataatta ttaaaaatat taactattta 11760 tcctaaatgg cacattttaa ttaaaaaaaa tccggtgttg taagtgtttt attaatttgt 11820 tttggcatta ttaaagcaac ttttttttta tttgttggca ttttgagtac gtacttaggc 11880 tagcctgca 11889 <210> SEQ ID NO 90 <211> LENGTH: 5661 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKR974 <400> SEQUENCE: 90 gtacgtctag aggatccccc atggtcaatc aatgagacgc caacttctta atctattgag 60 acctgcaggt ctagaagggc ggatccccgg gtaccgagct cgaattcact ggccgtcgtt 120 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat 180 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag 240 ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc 300 ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta 360 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg 420 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca 480 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt 540 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc 600 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa 660 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc 720 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa 780 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa 840 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg 900 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa 960 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc 1020 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc 1080 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta 1140 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag 1200 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca 1260 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata 1320 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc 1380 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca 1440 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca 1500 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg 1560 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 1620 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 1680 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 1740 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 1800 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 1860 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac 1920 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 1980 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 2040 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 2100 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 2160 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 2220 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 2280 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 2340 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc 2400 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 2460 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa 2520 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 2580 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 2640 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 2700 tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcaggctagc 2760 ctaagtacgt actcaaaatg ccaacaaata aaaaaaaagt tgctttaata atgccaaaac 2820 aaattaataa aacacttaca acaccggatt ttttttaatt aaaatgtgcc atttaggata 2880 aatagttaat atttttaata attatttaaa aagccgtatc tactaaaatg atttttattt 2940 ggttgaaaat attaatatgt ttaaatcaac acaatctatc aaaattaaac taaaaaaaaa 3000 ataagtgtac gtggttaaca ttagtacagt aatataagag gaaaatgaga aattaagaaa 3060 ttgaaagcga gtctaatttt taaattatga acctgcatat ataaaaggaa agaaagaatc 3120 caggaagaaa agaaatgaaa ccatgcatgg tcccctcgtc atcacgagtt tctgccattt 3180 gcaatagaaa cactgaaaca cctttctctt tgtcacttaa ttgagatgcc gaagccacct 3240 cacaccatga acttcatgag gtgtagcacc caaggcttcc atagccatgc atactgaaga 3300 atgtctcaag ctcagcaccc tacttctgtg acgtgtccct cattcacctt cctctcttcc 3360 ctataaataa ccacgcctca ggttctccgc ttcacaactc aaacattctc tccattggtc 3420 cttaaacact catcagtcat caccgcggcc gccaattcat ggccccgcag acggagctcc 3480 gccagcgcca cgccgccgtc gccgagacgc cggtggccgg caagaaggcc tttacatggc 3540 aggaggtcgc gcagcacaac acggcggcct cggcctggat cattatccgc ggcaaggtct 3600 acgacgtgac cgagtgggcc aacaagcacc ccggcggccg cgagatggtg ctgctgcacg 3660 ccggtcgcga ggccaccgac acgttcgact cgtaccaccc gttcagcgac aaggccgagt 3720 cgatcttgaa caagtatgag attggcacgt tcacgggccc gtccgagttt ccgaccttca 3780 agccggacac gggcttctac aaggagtgcc gcaagcgcgt tggcgagtac ttcaagaaga 3840 acaacctcca tccgcaggac ggcttcccgg gcctctggcg catgatggtc gtgtttgcgg 3900 tcgccggcct cgccttgtac ggcatgcact tttcgactat ctttgcgctg cagctcgcgg 3960 ccgcggcgct ctttggcgtc tgccaggcgc tgccgctgct ccacgtcatg cacgactcgt 4020 cgcacgcgtc gtacaccaac atgccgttct tccattacgt cgtcggccgc tttgccatgg 4080 actggtttgc cggcggctcg atggtgtcat ggctcaacca gcacgtcgtg ggccaccaca 4140 tctacacgaa cgtcgcgggc tcggacccgg atcttccggt caacatggac ggcgacatcc 4200 gccgcatcgt gaaccgccag gtgttccagc ccatgtacgc attccagcac atctaccttc 4260 cgccgctcta tggcgtgctt ggcctcaagt tccgcatcca ggacttcacc gacacgttcg 4320 gctcgcacac gaacggcccg atccgcgtca acccgcacgc gctctcgacg tggatggcca 4380 tgatcagctc caagtcgttc tgggccttct accgcgtgta ccttccgctt gccgtgctcc 4440 agatgcccat caagacgtac cttgcgatct tcttcctcgc cgagtttgtc acgggctggt 4500 acctcgcgtt caacttccaa gtaagccatg tctcgaccga gtgcggctac ccatgcggcg 4560 acgaggccaa gatggcgctc caggacgagt gggcagtctc gcaggtcaag acgtcggtcg 4620 actacgccca tggctcgtgg atgacgacgt tccttgccgg cgcgctcaac taccaggtcg 4680 tgcaccactt gttccccagc gtgtcgcagt accactaccc ggcgatcgcg cccatcatcg 4740 tcgacgtctg caaggagtac aacatcaagt acgccatctt gccggacttt acggcggcgt 4800 tcgttgccca cttgaagcac ctccgcaaca tgggccagca gggcatcgcc gccacgatcc 4860 acatgggcta actcgagctc agctagatcg cggccgcatt tcgcaccaaa tcaatgaaag 4920 taataatgaa aagtctgaat aagaatactt aggcttagat gcctttgtta cttgtgtaaa 4980 ataacttgag tcatgtacct ttggcggaaa cagaataaat aaaaggtgaa attccaatgc 5040 tctatgtata agttagtaat acttaatgtg ttctacggtt gtttcaatat catcaaactc 5100 taattgaaac tttagaacca caaatctcaa tcttttctta atgaaatgaa aaatcttaat 5160

tgtaccatgt ttatgttaaa caccttacaa ttaattggtt ggagaggagg accaaccgat 5220 gggacaacat tgggagaaag agattcaatg gagatttgga taggagaaca acattctttt 5280 tcacttcaat acaagatgag tgcaacacta aggatatgta tgagactttc agaagctacg 5340 acaacataga tgagtgaggt ggtgattcct agcaagaaag acattagagg aagccaaaat 5400 cgaacaagga agacatcaag ggcaagagac aggaccatcc atctcaggaa aaggagcttt 5460 gggatagtcc gagaagttgt acaagaaatt ttttggaggg tgagtgatgc attgctggtg 5520 actttaactc aatcaaaatt gagaaagaaa gaaaagggag ggggctcaca tgtgaataga 5580 agggaaacgg gagaatttta cagttttgat ctaatgggca tcccagctag tggtaacata 5640 ttcaccatgt ttaaccttca c 5661 <210> SEQ ID NO 91 <211> LENGTH: 1413 <212> TYPE: DNA <213> ORGANISM: Saprolegnia diclina <400> SEQUENCE: 91 atggccccgc agacggagct ccgccagcgc cacgccgccg tcgccgagac gccggtggcc 60 ggcaagaagg cctttacatg gcaggaggtc gcgcagcaca acacggcggc ctcggcctgg 120 atcattatcc gcggcaaggt ctacgacgtg accgagtggg ccaacaagca ccccggcggc 180 cgcgagatgg tgctgctgca cgccggtcgc gaggccaccg acacgttcga ctcgtaccac 240 ccgttcagcg acaaggccga gtcgatcttg aacaagtatg agattggcac gttcacgggc 300 ccgtccgagt ttccgacctt caagccggac acgggcttct acaaggagtg ccgcaagcgc 360 gttggcgagt acttcaagaa gaacaacctc catccgcagg acggcttccc gggcctctgg 420 cgcatgatgg tcgtgtttgc ggtcgccggc ctcgccttgt acggcatgca cttttcgact 480 atctttgcgc tgcagctcgc ggccgcggcg ctctttggcg tctgccaggc gctgccgctg 540 ctccacgtca tgcacgactc gtcgcacgcg tcgtacacca acatgccgtt cttccattac 600 gtcgtcggcc gctttgccat ggactggttt gccggcggct cgatggtgtc atggctcaac 660 cagcacgtcg tgggccacca catctacacg aacgtcgcgg gctcggaccc ggatcttccg 720 gtcaacatgg acggcgacat ccgccgcatc gtgaaccgcc aggtgttcca gcccatgtac 780 gcattccagc acatctacct tccgccgctc tatggcgtgc ttggcctcaa gttccgcatc 840 caggacttca ccgacacgtt cggctcgcac acgaacggcc cgatccgcgt caacccgcac 900 gcgctctcga cgtggatggc catgatcagc tccaagtcgt tctgggcctt ctaccgcgtg 960 taccttccgc ttgccgtgct ccagatgccc atcaagacgt accttgcgat cttcttcctc 1020 gccgagtttg tcacgggctg gtacctcgcg ttcaacttcc aagtaagcca tgtctcgacc 1080 gagtgcggct acccatgcgg cgacgaggcc aagatggcgc tccaggacga gtgggcagtc 1140 tcgcaggtca agacgtcggt cgactacgcc catggctcgt ggatgacgac gttccttgcc 1200 ggcgcgctca actaccaggt cgtgcaccac ttgttcccca gcgtgtcgca gtaccactac 1260 ccggcgatcg cgcccatcat cgtcgacgtc tgcaaggagt acaacatcaa gtacgccatc 1320 ttgccggact ttacggcggc gttcgttgcc cacttgaagc acctccgcaa catgggccag 1380 cagggcatcg ccgccacgat ccacatgggc taa 1413 <210> SEQ ID NO 92 <211> LENGTH: 5621 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKR1033 <400> SEQUENCE: 92 ggccgcattt cgcaccaaat caatgaaagt aataatgaaa agtctgaata agaatactta 60 ggcttagatg cctttgttac ttgtgtaaaa taacttgagt catgtacctt tggcggaaac 120 agaataaata aaaggtgaaa ttccaatgct ctatgtataa gttagtaata cttaatgtgt 180 tctacggttg tttcaatatc atcaaactct aattgaaact ttagaaccac aaatctcaat 240 cttttcttaa tgaaatgaaa aatcttaatt gtaccatgtt tatgttaaac accttacaat 300 taattggttg gagaggagga ccaaccgatg ggacaacatt gggagaaaga gattcaatgg 360 agatttggat aggagaacaa cattcttttt cacttcaata caagatgagt gcaacactaa 420 ggatatgtat gagactttca gaagctacga caacatagat gagtgaggtg gtgattccta 480 gcaagaaaga cattagagga agccaaaatc gaacaaggaa gacatcaagg gcaagagaca 540 ggaccatcca tctcaggaaa aggagctttg ggatagtccg agaagttgta caagaaattt 600 tttggagggt gagtgatgca ttgctggtga ctttaactca atcaaaattg agaaagaaag 660 aaaagggagg gggctcacat gtgaatagaa gggaaacggg agaattttac agttttgatc 720 taatgggcat cccagctagt ggtaacatat tcaccatgtt taaccttcac gtacgtctag 780 aggatccccc atggtcaatc aatgagacgc caacttctta atctattgag acctgcaggt 840 ctagaagggc ggatccccgg gtaccgagct cgaattcact ggccgtcgtt ttacaacgtc 900 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg 960 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc 1020 tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 1080 accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc 1140 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 1200 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 1260 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga 1320 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta 1380 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat 1440 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc 1500 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga 1560 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca 1620 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt 1680 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg 1740 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc 1800 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata 1860 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt 1920 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag 1980 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca 2040 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg 2100 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg 2160 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag 2220 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg 2280 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag 2340 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga 2400 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt 2460 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc 2520 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc 2580 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac 2640 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac 2700 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt 2760 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct 2820 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat 2880 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt 2940 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg 3000 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt 3060 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt 3120 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg 3180 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg 3240 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc 3300 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg 3360 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac 3420 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag 3480 gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcaggctagc ctaagtacgt 3540 actcaaaatg ccaacaaata aaaaaaaagt tgctttaata atgccaaaac aaattaataa 3600 aacacttaca acaccggatt ttttttaatt aaaatgtgcc atttaggata aatagttaat 3660 atttttaata attatttaaa aagccgtatc tactaaaatg atttttattt ggttgaaaat 3720 attaatatgt ttaaatcaac acaatctatc aaaattaaac taaaaaaaaa ataagtgtac 3780 gtggttaaca ttagtacagt aatataagag gaaaatgaga aattaagaaa ttgaaagcga 3840 gtctaatttt taaattatga acctgcatat ataaaaggaa agaaagaatc caggaagaaa 3900 agaaatgaaa ccatgcatgg tcccctcgtc atcacgagtt tctgccattt gcaatagaaa 3960 cactgaaaca cctttctctt tgtcacttaa ttgagatgcc gaagccacct cacaccatga 4020 acttcatgag gtgtagcacc caaggcttcc atagccatgc atactgaaga atgtctcaag 4080 ctcagcaccc tacttctgtg acgtgtccct cattcacctt cctctcttcc ctataaataa 4140 ccacgcctca ggttctccgc ttcacaactc aaacattctc tccattggtc cttaaacact 4200 catcagtcat caccgcggcc gcaaaccatg gctccagatg cggacaagtt gagacagcgc 4260 aaggcgcaat cgattcaaga cacggctgat tcgcaagcta ccgaactcaa gattggcacc 4320 ctgaagggct tgcaggggac agaaatcgtc attgatggag acatttacga tataaaagac 4380 tttgatcacc ccggtggtga atccatcatg acttttgggg gaaacgatgt caccgccacg 4440 tacaagatga tccaccccta ccactctaag caccatttgg agaagatgaa gaaagtggga 4500 cgagttccgg actacacctc ggaatacaag tttgatactc cctttgagcg tgaaatcaag 4560 caagaggtct tcaagattgt gcgacgaggc cgcgagtttg gaacacctgg atacttcttc 4620 cgggctttct gctacattgg acttttcttt tacttgcagt atttgtgggt cacgactccc 4680 actacctttg ccttggcgat cttctatggt gtttcgcaag ctttcattgg tttgaacgta 4740 caacatgatg ccaaccacgg agctgcctcc aagaagcctt ggatcaataa cttgctagga 4800 ttgggggctg actttatcgg aggttccaaa tggttgtgga tgaaccagca ctggacgcac 4860 cacacataca ccaaccacca tgagaaggat cccgatgcct tgggcgctga accaatgttg 4920 ttgttcaatg attatccctt gggtcaccca aagcgtactt tgattcacca cttccaggcc 4980

ttctattacc ttttcgtctt ggccggatac tgggtctctt cggtcttcaa ccctcaaatt 5040 ttggacttgc aacaccgcgg tgctcaagcg gttggaatga aaatggagaa cgattacatt 5100 gccaaaagcc gaaagtatgc catcttcttg cgtctcttgt atatttacac caacattgtc 5160 gctccgatcc aaaaccaagg cttctcgttg accgtggtcg cccacatttt gaccatgggc 5220 gtcgcttcca gtttgacttt ggcgactctt tttgccttgt cgcacaattt tgaaaacgcg 5280 gatcgcgatc ccacttacga ggcccgcaag ggaggagagc ctgtttgttg gttcaagtcg 5340 caagtcgaaa cctcgtcaac ttacggaggt ttcatctcgg gttgcttgac gggcggactc 5400 aacttccaag tggaacacca cttgttccct cgtatgagtt cggcctggta cccctacatt 5460 gcccctactg ttcgagaggt ttgcaaaaag cacggagtca agtacgcata ctatccctgg 5520 gtctggcaaa acttgatttc aactgtcaag tatctgcatc aaagcggaac tggatccaac 5580 tggaagaatg gcgccaaccc ctactcggga aaattgtaag c 5621 <210> SEQ ID NO 93 <211> LENGTH: 11949 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKR1038 <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (7810)..(7810) <223> OTHER INFORMATION: n is a, c, g, or t <400> SEQUENCE: 93 ggagatccaa gcttttgatc catgcccttc atttgccgct tattaattaa tttggtaaca 60 gtccgtacta atcagttact tatccttccc ccatcataat taatcttggt agtctcgaat 120 gccacaacac tgactagtct cttggatcat aagaaaaagc caaggaacaa aagaagacaa 180 aacacaatga gagtatcctt tgcatagcaa tgtctaagtt cataaaattc aaacaaaaac 240 gcaatcacac acagtggaca tcacttatcc actagctgat caggatcgcc gcgtcaagaa 300 aaaaaaactg gaccccaaaa gccatgcaca acaacacgta ctcacaaagg tgtcaatcga 360 gcagcccaaa acattcacca actcaaccca tcatgagccc tcacatttgt tgtttctaac 420 ccaacctcaa actcgtattc tcttccgcca cctcattttt gtttatttca acacccgtca 480 aactgcatgc caccccgtgg ccaaatgtcc atgcatgtta acaagaccta tgactataaa 540 tagctgcaat ctcggcccag gttttcatca tcaagaacca gttcaatatc ctagtacacc 600 gtattaaaga atttaagata tactgcggcc gcaccatgga ggtggtgaat gaaatagtct 660 caattgggca ggaagtttta cccaaagttg attatgccca actctggagt gatgccagtc 720 actgtgaggt gctttacttg tccatcgcat ttgtcatctt gaagttcact cttggccccc 780 ttggtccaaa aggtcagtct cgtatgaagt ttgttttcac caattacaac cttctcatgt 840 ccatttattc gttgggatca ttcctctcaa tggcatatgc catgtacacc atcggtgtta 900 tgtctgacaa ctgcgagaag gcttttgaca acaacgtctt caggatcacc acgcagttgt 960 tctatttgag caagttcctg gagtatattg actccttcta tttgccactg atgggcaagc 1020 ctctgacctg gttgcaattc ttccatcatt tgggggcacc gatggatatg tggctgttct 1080 ataattaccg aaatgaagct gtttggattt ttgtgctgtt gaatggtttc atccactgga 1140 tcatgtacgg ttattattgg accagattga tcaagctgaa gttccccatg ccaaaatccc 1200 tgattacatc aatgcagatc attcaattca atgttggttt ctacattgtc tggaagtaca 1260 ggaacattcc ctgttatcgc caagatggga tgaggatgtt tggctggttc ttcaattact 1320 tttatgttgg cacagtcttg tgtttgttct tgaatttcta tgtgcaaacg tatatcgtca 1380 ggaagcacaa gggagccaaa aagattcagt gagcggccgc aagtatgaac taaaatgcat 1440 gtaggtgtaa gagctcatgg agagcatgga atattgtatc cgaccatgta acagtataat 1500 aactgagctc catctcactt cttctatgaa taaacaaagg atgttatgat atattaacac 1560 tctatctatg caccttattg ttctatgata aatttcctct tattattata aatcatctga 1620 atcgtgacgg cttatggaat gcttcaaata gtacaaaaac aaatgtgtac tataagactt 1680 tctaaacaat tctaacctta gcattgtgaa cgagacataa gtgttaagaa gacataacaa 1740 ttataatgga agaagtttgt ctccatttat atattatata ttacccactt atgtattata 1800 ttaggatgtt aaggagacat aacaattata aagagagaag tttgtatcca tttatatatt 1860 atatactacc catttatata ttatacttat ccacttattt aatgtcttta taaggtttga 1920 tccatgatat ttctaatatt ttagttgata tgtatatgaa agggtactat ttgaactctc 1980 ttactctgta taaaggttgg atcatcctta aagtgggtct atttaatttt attgcttctt 2040 acagataaaa aaaaaattat gagttggttt gataaaatat tgaaggattt aaaataataa 2100 taaataacat ataatatatg tatataaatt tattataata taacatttat ctataaaaaa 2160 gtaaatattg tcataaatct atacaatcgt ttagccttgc tggacgaatc tcaattattt 2220 aaacgagagt aaacatattt gactttttgg ttatttaaca aattattatt taacactata 2280 tgaaattttt ttttttatca gcaaagaata aaattaaatt aagaaggaca atggtgtccc 2340 aatccttata caaccaactt ccacaagaaa gtcaagtcag agacaacaaa aaaacaagca 2400 aaggaaattt tttaatttga gttgtcttgt ttgctgcata atttatgcag taaaacacta 2460 cacataaccc ttttagcagt agagcaatgg ttgaccgtgt gcttagcttc ttttatttta 2520 tttttttatc agcaaagaat aaataaaata aaatgagaca cttcagggat gtttcaacaa 2580 gcttggcgcg ccgttctata gtgtcaccta aatcgtatgt gtatgataca taaggttatg 2640 tattaattgt agccgcgttc taacgacaat atgtccatat ggtgcactct cagtacaatc 2700 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc 2760 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc 2820 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg 2880 atacgcctat ttttataggt taatgtcatg accaaaatcc cttaacgtga gttttcgttc 2940 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 3000 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 3060 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 3120 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 3180 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 3240 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 3300 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 3360 ctacagcgtg agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 3420 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 3480 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 3540 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 3600 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg 3660 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag 3720 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc 3780 gcgcgttggc cgattcatta atgcaggttg atcagatctc gatcccgcga aattaatacg 3840 actcactata gggagaccac aacggtttcc ctctagaaat aattttgttt aactttaaga 3900 aggagatata cccatggaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat 3960 cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc 4020 tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg 4080 tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga 4140 agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca 4200 gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc 4260 ggaggctatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt 4320 cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga 4380 tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca 4440 ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca 4500 cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga 4560 ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag 4620 gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct 4680 tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca 4740 gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat 4800 cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt 4860 ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg 4920 tccgagggca aaggaatagt gaggtacagc ttggatcgat ccggctgcta acaaagcccg 4980 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc 5040 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gatgatcggg 5100 cgcgccgtcg acggatccgt acgcaaaggc aaagatttaa actcgaaaac attacaaaag 5160 tctcaaaaca gaggcaaggc catgcacaaa gcacactcta agtgcttcca ttgcctacta 5220 agtagggtac gtacacgatc accattcacc agtgatgatc tttattaata tacaacacac 5280 tcagagacag cttatgttat agctagctag cataaactat cacatcatgt gttagtacga 5340 caagtgacaa cattgctttt aacttcgcgg ccttggatcc tctagaccgg atataatgag 5400 ccgtaaacaa agatgattaa gtagtaatta atacgtacta gtaaaagtgg caaaagataa 5460 cgagaaagaa ccaatttctt tgcattcggc cttagcggaa ggcatatata agctttgatt 5520 attttattta gtgtaatgat ttcgtacaac caaagcattt atttagtact ctcacacttg 5580 tgtcgcggcc gcgaattcac tagtgattcc ttatagagcc ttccccgcgg gttgcttctc 5640 cgccatccgg gcgaacaccg ccagatagcg cagcaggatg accaaccctt catggggcag 5700 cgggttccga tacggcaggt tgtgcttctg gcacagctgt tccacctggt agctaaccgc 5760 tgtcaggttg tggcgaggga gggtcggcca caaatggtgc tcaatctggt aattcaagcc 5820 tccgaaaaac caatctgtga taatccctcg ccgaatgttc atggtctcat ggatctggcc 5880 aaccgagaat ccatggccat cccagactga gtccccgatc ttctccagtg ggtagtggtt 5940 catgaacacc acgatcgcaa tgccgaagcc gccaaccagc tccgaaacga aaaacaccaa 6000 cagcgatgtg aggatgctgg gcataaagaa taagtggaac agggtcttca aggtccagtg 6060 cagggcgagg ccaatggcct ccttcttata ctgagagcga tagaattggt tatctctgtc 6120 cttcaaactg cgcacggtca acacgctctg gaaacaccaa atgaaccgca acaagataca 6180 gatgaccaag aaatagtact gctggaactg aatgagcttg cgggaaatcg gtgacgcccg 6240 tgtgacgtca tcctcagacc aggctaagag ggggaggttg tcaatatcag ggtcgtgccc 6300 ttgaacattg gttgccgaat gatgtgcatt gtgtctgtcc ttccaccatg tcacggaaaa 6360 accttgcaga ccattgccaa ataccagtcc cacgaggttg ttccagttcc ggttcttgaa 6420 agtctggtgg tggcaaatgt catgagaaag ccagcccatc tgttgatagt gcatcccaag 6480

caacactgcc ccaatgaaat acatctgata ctgaaccatc aggaaataac ccagcactcc 6540 aaggcccagt gtggtgctga ttttgtatga gtaccagagg ggggaggcat caaacatgcc 6600 agttgcgatc aactcttctc ggagcttccg gaaatcctct tgagcttcat tcactgcagc 6660 ctggggtggc aactcagaac tgggattgat tttgggcatg cgcttgagct tgtcgaaggc 6720 ttcttgagag tgcataacca tgaaggcatc agtggcatcc cttccttggt aattctctat 6780 aatttccgca ccaccagggt ggaaattgac ccaggcagac acatcatatg ttgttccatc 6840 aattgtaagg ggaagcgctt ggcgctttga cttcatttca atcgaattcc cgcggccgct 6900 tggggggcta tggaagactt tcttagttag ttgtgtgaat aagcaatgtt gggagaatcg 6960 ggactactta taggatagga ataaaacaga aaagtattaa gtgctaatga aatatttaga 7020 ctgataatta aaatcttcac gtatgtccac ttgatataaa aacgtcagga ataaaggaag 7080 tacagtagaa tttaaaggta ctctttttat atatacccgt gttctctttt tggctagcta 7140 gttgcataaa aaataatcta tatttttatc attattttaa atatcttatg agatggtaaa 7200 tatttatcat aatttttttt actattattt attatttgtg tgtgtaatac atatagaagt 7260 taattacaaa ttttatttac tttttcatta ttttgatatg attcaccatt aatttagtgt 7320 tattatttat aatagttcat tttaatcttt ttgtatatat tatgcgtgca gtactttttt 7380 cctacatata actactatta cattttattt atataatatt tttattaatg aattttcgtg 7440 ataatatgta atattgttca ttattatttc agatttttta aaaatatttg tgttattatt 7500 tatgaaatat gtaatttttt tagtatttga ttttatgatg ataaagtgtt ctaaattcaa 7560 aagaaggggg aaagcgtaaa cattaaaaaa cgtcatcaaa caaaaacaaa atcttgttaa 7620 taaagataaa actgtttgtt ttgatcactg ttatttcgta atataaaaac attatttata 7680 tttatattgt tgacaaccaa atttgcctat caaatctaac caatataatg catgcgtggc 7740 aggtaatgta ctaccatgaa cttaagtcat gacataataa accgtgaatc tgaccaatgc 7800 atgtacctan ctaaattgta tttgtgacac gaagcaaatg attcaattca caatggagat 7860 gggaaacaaa taatgaagaa cccagaacta agaaagcttt tctgaaaaat aaaataaagg 7920 caatgtcaaa agtatactgc atcatcagtc cagaaagcac atgatatttt tttatcagta 7980 tcaatgcagc tagttttatt ttacaatatc gatatagcta gtttaaatat attgcagcta 8040 gatttataaa tatttgtgtt attatttatc atttgtgtaa tcctgttttt agtattttag 8100 tttatatatg atgataatgt attccaaatt taaaagaagg gaaataaatt taaacaagaa 8160 aaaaagtcat caaacaaaaa acaaatgaaa gggtggaaag atgttaccat gtaatgtgaa 8220 tgttacagta tttcttttat tatagagtta acaaattaac taatatgatt ttgttaataa 8280 tgataaaata ttttttttat tattatttca taatataaaa atagtttact taatataaaa 8340 aaaattctat cgttcacaac aaagttggcc acctaattta accatgcatg tacccatgga 8400 ccatattagg taaccatcaa acctgatgaa gagataaaga gatgaagact taagtcataa 8460 cacaaaacca taaaaaacaa aaatacaatc aaccgtcaat ctgaccaatg catgaaaaag 8520 ctgcaatagt gagtggcgac acaaagcaca tgattttctt acaacggaga taaaaccaaa 8580 aaaatatttc atgaacaacc tagaacaaat aaagctttta tataataaat atataaataa 8640 ataaaggcta tggaataata tacttcaata tatttggatt aaataaattg ttggcggggt 8700 tgatatattt atacacacct aaagtcactt caatctcatt ttcacttaac ttttattttt 8760 tttttctttt tatttatcat aaagagaata ttgataatat actttttaac atatttttat 8820 gacatttttt attggtgaaa acttattaaa aatcataaat tttgtaagtt agatttattt 8880 aaagagttcc tcttcttatt ttaaattttt taataaattt ttaaataact aaaatttgtg 8940 ttaaaaatgt taaaaaatgt gttattaacc cttctcttcg aggacgtacg agatccggcc 9000 ggccagatcc tgcaggtctc aatagattaa gaagttggcg tctcattgat tgaccatggg 9060 ggatcctcta gacgtacgtg aaggttaaac atggtgaata tgttaccact agctgggatg 9120 cccattagat caaaactgta aaattctccc gtttcccttc tattcacatg tgagccccct 9180 cccttttctt tctttctcaa ttttgattga gttaaagtca ccagcaatgc atcactcacc 9240 ctccaaaaaa tttcttgtac aacttctcgg actatcccaa agctcctttt cctgagatgg 9300 atggtcctgt ctcttgccct tgatgtcttc cttgttcgat tttggcttcc tctaatgtct 9360 ttcttgctag gaatcaccac ctcactcatc tatgttgtcg tagcttctga aagtctcata 9420 catatcctta gtgttgcact catcttgtat tgaagtgaaa aagaatgttg ttctcctatc 9480 caaatctcca ttgaatctct ttctcccaat gttgtcccat cggttggtcc tcctctccaa 9540 ccaattaatt gtaaggtgtt taacataaac atggtacaat taagattttt catttcatta 9600 agaaaagatt gagatttgtg gttctaaagt ttcaattaga gtttgatgat attgaaacaa 9660 ccgtagaaca cattaagtat tactaactta tacatagagc attggaattt caccttttat 9720 ttattctgtt tccgccaaag gtacatgact caagttattt tacacaagta acaaaggcat 9780 ctaagcctaa gtattcttat tcagactttt cattattact ttcattgatt tggtgcgaaa 9840 tgcggccgct tacaattttc ccgagtaggg gttggcgcca ttcttccagt tggatccagt 9900 tccgctttga tgcagatact tgacagttga aatcaagttt tgccagaccc agggatagta 9960 tgcgtacttg actccgtgct ttttgcaaac ctctcgaaca gtaggggcaa tgtaggggta 10020 ccaggccgaa ctcatacgag ggaacaagtg gtgttccact tggaagttga gtccgcccgt 10080 caagcaaccc gagatgaaac ctccgtaagt tgacgaggtt tcgacttgcg acttgaacca 10140 acaaacaggc tctcctccct tgcgggcctc gtaagtggga tcgcgatccg cgttttcaaa 10200 attgtgcgac aaggcaaaaa gagtcgccaa agtcaaactg gaagcgacgc ccatggtcaa 10260 aatgtgggcg accacggtca acgagaagcc ttggttttgg atcggagcga caatgttggt 10320 gtaaatatac aagagacgca agaagatggc atactttcgg cttttggcaa tgtaatcgtt 10380 ctccattttc attccaaccg cttgagcacc gcggtgttgc aagtccaaaa tttgagggtt 10440 gaagaccgaa gagacccagt atccggccaa gacgaaaagg taatagaagg cctggaagtg 10500 gtgaatcaaa gtacgctttg ggtgacccaa gggataatca ttgaacaaca acattggttc 10560 agcgcccaag gcatcgggat ccttctcatg gtggttggtg tatgtgtggt gcgtccagtg 10620 ctggttcatc cacaaccatt tggaacctcc gataaagtca gcccccaatc ctagcaagtt 10680 attgatccaa ggcttcttgg aggcagctcc gtggttggca tcatgttgta cgttcaaacc 10740 aatgaaagct tgcgaaacac catagaagat cgccaaggca aaggtagtgg gagtcgtgac 10800 ccacaaatac tgcaagtaaa agaaaagtcc aatgtagcag aaagcccgga agaagtatcc 10860 aggtgttcca aactcgcggc ctcgtcgcac aatcttgaag acctcttgct tgatttcacg 10920 ctcaaaggga gtatcaaact tgtattccga ggtgtagtcc ggaactcgtc ccactttctt 10980 catcttctcc aaatggtgct tagagtggta ggggtggatc atcttgtacg tggcggtgac 11040 atcgtttccc ccaaaagtca tgatggattc accaccgggg tgatcaaagt cttttatatc 11100 gtaaatgtct ccatcaatga cgatttctgt cccctgcaag cccttcaggg tgccaatctt 11160 gagttcggta gcttgcgaat cagccgtgtc ttgaatcgat tgcgccttgc gctgtctcaa 11220 cttgtccgca tctggagcca tggtttgcgg ccgcggtgat gactgatgag tgtttaagga 11280 ccaatggaga gaatgtttga gttgtgaagc ggagaacctg aggcgtggtt atttataggg 11340 aagagaggaa ggtgaatgag ggacacgtca cagaagtagg gtgctgagct tgagacattc 11400 ttcagtatgc atggctatgg aagccttggg tgctacacct catgaagttc atggtgtgag 11460 gtggcttcgg catctcaatt aagtgacaaa gagaaaggtg tttcagtgtt tctattgcaa 11520 atggcagaaa ctcgtgatga cgaggggacc atgcatggtt tcatttcttt tcttcctgga 11580 ttctttcttt ccttttatat atgcaggttc ataatttaaa aattagactc gctttcaatt 11640 tcttaatttc tcattttcct cttatattac tgtactaatg ttaaccacgt acacttattt 11700 tttttttagt ttaattttga tagattgtgt tgatttaaac atattaatat tttcaaccaa 11760 ataaaaatca ttttagtaga tacggctttt taaataatta ttaaaaatat taactattta 11820 tcctaaatgg cacattttaa ttaaaaaaaa tccggtgttg taagtgtttt attaatttgt 11880 tttggcatta ttaaagcaac ttttttttta tttgttggca ttttgagtac gtacttaggc 11940 tagcctgca 11949 <210> SEQ ID NO 94 <211> LENGTH: 8671 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Plasmid pKR328 <400> SEQUENCE: 94 ggatctggcc ggccggatct cgtacggatc cgtcgacggc gcgcccgatc atccggatat 60 agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa ggggttatgc 120 tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt tgttagcagc 180 cggatcgatc caagctgtac ctcactattc ctttgccctc ggacgagtgc tggggcgtcg 240 gtttccacta tcggcgagta cttctacaca gccatcggtc cagacggccg cgcttctgcg 300 ggcgatttgt gtacgcccga cagtcccggc tccggatcgg acgattgcgt cgcatcgacc 360 ctgcgcccaa gctgcatcat cgaaattgcc gtcaaccaag ctctgataga gttggtcaag 420 accaatgcgg agcatatacg cccggagccg cggcgatcct gcaagctccg gatgcctccg 480 ctcgaagtag cgcgtctgct gctccataca agccaaccac ggcctccaga agaagatgtt 540 ggcgacctcg tattgggaat ccccgaacat cgcctcgctc cagtcaatga ccgctgttat 600 gcggccattg tccgtcagga cattgttgga gccgaaatcc gcgtgcacga ggtgccggac 660 ttcggggcag tcctcggccc aaagcatcag ctcatcgaga gcctgcgcga cggacgcact 720 gacggtgtcg tccatcacag tttgccagtg atacacatgg ggatcagcaa tcgcgcatat 780 gaaatcacgc catgtagtgt attgaccgat tccttgcggt ccgaatgggc cgaacccgct 840 cgtctggcta agatcggccg cagcgatcgc atccatagcc tccgcgaccg gctgcagaac 900 agcgggcagt tcggtttcag gcaggtcttg caacgtgaca ccctgtgcac ggcgggagat 960 gcaataggtc aggctctcgc tgaattcccc aatgtcaagc acttccggaa tcgggagcgc 1020 ggccgatgca aagtgccgat aaacataacg atctttgtag aaaccatcgg cgcagctatt 1080 tacccgcagg acatatccac gccctcctac atcgaagctg aaagcacgag attcttcgcc 1140 ctccgagagc tgcatcaggt cggagacgct gtcgaacttt tcgatcagaa acttctcgac 1200 agacgtcgcg gtgagttcag gcttttccat gggtatatct ccttcttaaa gttaaacaaa 1260 attatttcta gagggaaacc gttgtggtct ccctatagtg agtcgtatta atttcgcggg 1320 atcgagatcg atccaattcc aatcccacaa aaatctgagc ttaacagcac agttgctcct 1380 ctcagagcag aatcgggtat tcaacaccct catatcaact actacgttgt gtataacggt 1440 ccacatgccg gtatatacga tgactggggt tgtacaaagg cggcaacaaa cggcgttccc 1500 ggagttgcac acaagaaatt tgccactatt acagaggcaa gagcagcagc tgacgcgtac 1560 acaacaagtc agcaaacaga caggttgaac ttcatcccca aaggagaagc tcaactcaag 1620 cccaagagct ttgctaaggc cctaacaagc ccaccaaagc aaaaagccca ctggctcacg 1680

ctaggaacca aaaggcccag cagtgatcca gccccaaaag agatctcctt tgccccggag 1740 attacaatgg acgatttcct ctatctttac gatctaggaa ggaagttcga aggtgaaggt 1800 gacgacacta tgttcaccac tgataatgag aaggttagcc tcttcaattt cagaaagaat 1860 gctgacccac agatggttag agaggcctac gcagcaggtc tcatcaagac gatctacccg 1920 agtaacaatc tccaggagat caaatacctt cccaagaagg ttaaagatgc agtcaaaaga 1980 ttcaggacta attgcatcaa gaacacagag aaagacatat ttctcaagat cagaagtact 2040 attccagtat ggacgattca aggcttgctt cataaaccaa ggcaagtaat agagattgga 2100 gtctctaaaa aggtagttcc tactgaatct aaggccatgc atggagtcta agattcaaat 2160 cgaggatcta acagaactcg ccgtgaagac tggcgaacag ttcatacaga gtcttttacg 2220 actcaatgac aagaagaaaa tcttcgtcaa catggtggag cacgacactc tggtctactc 2280 caaaaatgtc aaagatacag tctcagaaga ccaaagggct attgagactt ttcaacaaag 2340 gataatttcg ggaaacctcc tcggattcca ttgcccagct atctgtcact tcatcgaaag 2400 gacagtagaa aaggaaggtg gctcctacaa atgccatcat tgcgataaag gaaaggctat 2460 cattcaagat gcctctgccg acagtggtcc caaagatgga cccccaccca cgaggagcat 2520 cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa gtggattgat gtgacatctc 2580 cactgacgta agggatgacg cacaatccca ctatccttcg caagaccctt cctctatata 2640 aggaagttca tttcatttgg agaggacacg ctcgagctca tttctctatt acttcagcca 2700 taacaaaaga actcttttct cttcttatta aaccatgaaa aagcctgaac tcaccgcgac 2760 gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc 2820 ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg 2880 ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc 2940 ggccgcgctc ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta 3000 ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc 3060 cgctgttctg cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca 3120 gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga 3180 tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac 3240 cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc 3300 cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg 3360 ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt 3420 cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt 3480 cgagcggagg catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat 3540 tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc 3600 gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat 3660 cgcccgcaga agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg 3720 aaaccgacgc cccagcactc gtccgagggc aaaggaatag tgaggtacct aaagaaggag 3780 tgcgtcgaag cagatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgtt 3840 gccggtcttg cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt 3900 aacatgtaat gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta 3960 tacatttaat acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgc 4020 gcggtgtcat ctatgttact agatcgatgt cgaatcgatc aacctgcatt aatgaatcgg 4080 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 4140 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 4200 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 4260 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 4320 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 4380 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 4440 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc 4500 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 4560 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 4620 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 4680 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 4740 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 4800 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 4860 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 4920 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgacattaa cctataaaaa 4980 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg 5040 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca 5100 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc 5160 atcagagcag attgtactga gagtgcacca tatggacata ttgtcgttag aacgcggcta 5220 caattaatac ataaccttat gtatcataca catacgattt aggtgacact atagaacggc 5280 gcgccaagct tggatctcct gcagcccggg ggatccgccc acgtacggta ccatctgcta 5340 atattttaaa tcacatgcaa gagaggaggc atggttccat tttctacctt cacattattt 5400 gagaaaaacg aacttgttct gtgttttatt tttgcccttc acattagtac aacgtggaag 5460 actcatggtt acacagaatc atacataagt acaatgcttg tccctaagaa aacaagcact 5520 cgttgtattg aacctttacg gctcatgcgg ccgcgaattc actagtgatt gaattcgcgg 5580 ccgcttagtc cgacttggcc ttggcggccg cggccgactc tttgagcgtg aagatctgcg 5640 ccgtctcggg cacagcgccg tagttgacaa agaggtgcgc ggtcttgaag aaggccgtga 5700 tgatgggctc gtcgttcctg cgcacgaggt gcgggtacgc ggccgcaaag tgcttggtgg 5760 cttcgttgag cttgtagtgc ggaatgatcg ggaacaagtg gtggacctgg tgcgtgccaa 5820 tgtggtggct caggttgtcc acgaacgcgc cgtacgagcg gtcgacgctc gagaggttgc 5880 ccttgacgta cgtccactcc gagtcgccgt accacggcgt cgcttcgtcg ttgtggtgca 5940 agaaggtcgt aatgacgagg aacgaagcaa agacaaagag cggcgcatag tagtagaggc 6000 ccatgacggc aaagccgagc gagtatgtga ggtacgcgta cgcggcgaag aaggcggccc 6060 agacgccgag cgacacgatg acggccgacg cgcggcgaag gaggagcggg tcccacgggt 6120 caaagtggct catcgtgcgc ggggcatacc cgaccttcaa gtagacaaac cacgcaccgc 6180 cgagcgtgta gacccattgg cgcacgtcct ggaggtcctt gaccgaccgg tgcgggtaaa 6240 agatctcgtc cttatcaatg ttgcccgtgt tcttgtggtg gtggcggtgc gtcacgcgcc 6300 agctctcgaa cggcgtcaaa atcgcagagt gcatgatgca gccgatgata aagttgacgc 6360 tgtggtagcg cgagaaggcc gagtggccgc agtcgtggcc gaccgtgaag aagccccaga 6420 agatgacgcc ctgcacgtag atgtaggtgg cgcaaacgag cgcgtggagc agaacgttat 6480 cggcaatgaa cggcgtcgag cgcgccgcgt agagcagcgc cgccgaggcc gacgcgttga 6540 agatcgcgcg ggccgtgtag tagagcgaga ggccgaggtt cgactcaaag cacgcgttcg 6600 ggatcgagtg cttgagctcc gtgagcgtcg ggaactcgac cttcgtctta tcctcagtca 6660 tgcggccgct gaagtattgc ttcttagtta acctttcctt tctctctcag ctatgtgaat 6720 tcattttgct ttcgtcacaa tttatatagt gaaattggat ctttggagtt aacgccttca 6780 caggattatc gtgttagaac aatgcttttt catgttctaa ttagtagtac attacaaatg 6840 tgcactctat tcaataagca tcttttggca cgttaataaa tcatgtgaaa aaaaaatact 6900 actatttcaa agaaagtgtt gtaaaaagaa acggaaagag agctggcttc agttgttgag 6960 acttgtttgc tagtaaaaat ggtgtgaaga gtgattcatg gtgaggtggt ttttcgtccc 7020 tttctgtttg catgaaaaac aaatggcaag agatgacgta ggattccttc ccttaacgat 7080 tatctgtttt taatttcaaa tatacatata ggaatttatg aattactaag gttgtaaaat 7140 atgctggtca tttatttatg gctaaaatat ttttttttct cgtaaatata aaaatattta 7200 aaatttattt ttatcatatt ttttatcctt ataaaattat gtgtacaacc tatataaaaa 7260 aatatcatat ttaatattga ttatatgttt aatcaatata aaaaatcatt atcatatatt 7320 tagatttatt cgaatataca tctaaacaaa aaataacata ttttaatttt atgaagaaaa 7380 aaaaatattt tatcctttat ttatttaaga ttaattaata gttatgtatt gtggaaagac 7440 ttttacacat gcaatagata tactgaatca attagatgcc aatgctgagt tggaaatcac 7500 ttgaggaggg gaggagactt gccaatgctt ttcagtttca tttaaatgat ttagtggagg 7560 agatagagta gtgataaagg catgccccaa ttttggagtg tatatatgag tggaaataag 7620 agagggatag agagaaaaaa taaagagagt aaaaataatt aatgtgaaat gatatgataa 7680 aaaaataaag aaagagataa agagaaaaat gaaatgagag atagatgaaa tagagagtag 7740 atacatgttt gtttaggttt tttttaggaa ataacacatt tttttctcat cacttattac 7800 tcactgtcaa tttcctctct ttcaatcata atgatatgat ttgtttaaca aaaatgtgaa 7860 aaaacatata aagtaaaata tttttataaa ttgataaata aaaatttaca aaatttattt 7920 cttattaaat tgaatagaaa atgaaagaaa agaaaagaaa aagtatatat aaaatgatat 7980 agctttaaaa agaataaatt tttcatatca gtcttttttt aataatttag aaatatttaa 8040 gtatatagca aaaatataat gtactttaca tatgcataaa taataatttg aaaatagaac 8100 taatagaata gagaaaaaag taatataata attaactata tgaaaattta gaagggacaa 8160 tatttttaat taagaatata aacaatattt cttttcatgt aatgagggac ggatgtacgg 8220 ggccagtgtt ggagtcaaag ccaaaatagt cacggggaaa ttaatgcact gcatgactat 8280 tcgaaaaaat tcactagcct tacttagatg ttagattaat agctaggggg tgcagataat 8340 tttgaaaggc atgaaaaaca ttaatttgta cattgcaagc ttttgatgac aagctttgca 8400 attgttcaca ctaccttatg ccatttataa atagagtgat tggcatatga aggaaatcat 8460 gagagtcgaa gcgaaaaaca aagcttgaga gtgtaggaaa aatacagttt ttttggtaaa 8520 aatacagtat ttgaatagga gcgaaaaata tcctttcaaa atgatccttt tctttttttt 8580 tttttttctt gttgttcttg gtcagttatt caaaggaaaa gggattgaaa taaaaacttg 8640 catgtgggat cgtacgtcga gtcgacctgc a 8671

Claims

1-21. (canceled)

22. Oil or by-products obtained from a transgenic seed obtained from an oilseed plant produced by a method comprising: (a) transforming an oilseed plant cell with a recombinant DNA construct comprising an isolated polynucleotide comprising: (i) a nucleotide sequence encoding a polypeptide having delta-5 desaturase activity, wherein the polypeptide has at least 80% amino acid identity, based on the Clustal W method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:2; (ii) a nucleotide sequence encoding a polypeptide having delta-5 desaturase activity, wherein the nucleotide sequence has at least 80% sequence identity, based on the BLASTN method of alignment, when compared to a nucleotide sequence as set forth in SEQ ID NO:1 or SEQ ID NO:3; (iii) a nucleotide sequence encoding a polypeptide having delta-5 desaturase activity, wherein the nucleotide sequence selectively hybridizes under stringent conditions to a nucleotide sequence as set forth in SEQ ID NO:1 or SEQ ID NO:3; or (iv) a complement of the nucleotide sequence of (a), (b) or (c), wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary, operably linked to at least one regulatory sequence and at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8 desaturase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase a delta-9 desaturase a delta-9 elongase a C.sub.14/16 elongase a C.sub.16/18 elongase, a C.sub.18/20 elongase and a C.sub.20/22 elongase; (b) regenerating an oilseed plant from the transformed cell of step (a); and (c) selecting those seeds obtained from the plants of step (b) having an altered level of polyunsaturated fatty acids when compared to the level in seeds obtained from a nontransformed oilseed plant.

23. The oil or by-products of claim 22, wherein the oilseed plant is selected from the group consisting of soybean, Brassica species, sunflower, maize, cotton, flax, and safflower.

24. (canceled)

25. Food or feed which incorporates the oil of claim 22.

26. Food or feed which incorporates the oil of claim 23.

27-29. (canceled)

30. Food or feed comprising an ingredient derived from the processing of a transgenic seed obtained from an oilseed plant produced by a method comprising: (a) transforming an oilseed plant cell with a recombinant DNA construct comprising an isolated polynucleotide comprising: (i) a nucleotide sequence encoding a polypeptide having delta-5 desaturase activity, wherein the polypeptide has at least 80% amino acid identity, based on the Clustal W method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:2; (ii) a nucleotide sequence encoding a polypeptide having delta-5 desaturase activity, wherein the nucleotide sequence has at least 80% sequence identity, based on the BLASTN method of alignment, when compared to a nucleotide sequence as set forth in SEQ ID NO:1 or SEQ ID NO:3; (iii) a nucleotide sequence encoding a polypeptide having delta-5 desaturase activity, wherein the nucleotide sequence selectively hybridizes under stringent conditions to a nucleotide sequence as set forth in SEQ ID NO:1 or SEQ ID NO:3; or (iv) a complement of the nucleotide sequence of (a), (b) or (c), wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary, operably linked to at least one regulatory sequence and at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8 desaturase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a delta-9 desaturase, a delta-9 elongase, a C.sub.14/16 elongase, a C.sub.16/18 elongase, a C.sub.18/20 elongase and a C.sub.20/22 elongase; (b) regenerating an oilseed plant from the transformed cell of step (a); and (c) selecting those seeds obtained from the plants of step (b) having an altered level of polyunsaturated fatty acids when compared to the level in seeds obtained from a nontransformed oilseed plant.

31. The food or feed of claim 30, wherein the oilseed plant is selected from the group consisting of soybean, Brassica species, sunflower, maize, cotton, flax, and safflower.

32. The oil or by-products of claim 22 wherein the by-product is lecithin.

33. The oil or by-products of claim 23 wherein the by-product is lecithin.

34-36. (canceled)

37. The oil or by-products of claim 22, wherein the nucleotide sequence comprises SEQ ID NO:1 or SEQ ID NO:3.

38. The oil or by-products of claim 22, wherein the amino acid sequence of the polypeptide comprises (a) SEQ ID NO:2; or (b) an amino acid sequence that differs from the amino acid sequences in (a) by at least one conservative amino acid substitution.

39. The oil or by-products of claim 30, wherein the nucleotide sequence comprises SEQ ID NO:1 or SEQ ID NO:3.

40. The oil or by-products of claim 30, wherein the amino acid sequence of the polypeptide comprises (a) SEQ ID NO:2; or (b) an amino acid sequence that differs from the amino acid sequences in (a) by at least one conservative amino acid substitution.