Delta-5 Desaturases And Their Use In Making Polyunsaturated Fatty Acids

Abstract

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

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 60/915,733, filed May 3, 2007, the entire content of which is herein incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention is in the field of biotechnology, in particular, this pertains to polynucleotide sequences encoding delta-5 desaturases and the use of these desaturases 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 elongation and desaturation 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 et al., Amer. J. Clin. Nutr. 28:958-966 (1975); Dyerberg et al., Lancet. 2(8081):117-119 (1978); Shimokawa, H., World Rev. Nutr. Diet 88:100-108 (2001); von Schacky et al., World Rev. Nutr. Diet 88:90-99 (2001)). 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] Today, a variety of different hosts including plants, algae, fungi and yeast are being investigated as means for commercial PUFA production via numerous divergent efforts. Although the natural PUFA-producing abilities of the host organisms are sometimes essential to a given methodology, genetic engineering has also proven 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 effect 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) all require expression of either 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) and/or eicosatrienoic acid (ETrA; 20:3 .omega.-3)) or 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.-linoleic acid (GLA; 18:3 .omega.-6) and/or stearidonic acid (STA; 18:4 .omega.-3) (FIG. 1). A delta-6 elongase is also known as a C.sub.18/20 elongase.

[0005] The delta-8 desaturase enzymes identified thus far have the ability to convert both EDA to dihomo-.gamma.-linolenic acid (DGLA; 20:3) and ETrA to eicosatetraenoic acid (ETA; 20:4) (wherein ARA are EPA are subsequently synthesized from DGLA and ETA, respectively, following reaction with a delta-5 desaturase, while DHA synthesis requires subsequent expression of an additional C.sub.20/22 elongase and a delta-4 desaturase).

[0006] Based on the role delta-8 desaturase enzymes play in the synthesis of e.g., ARA, EPA and DHA, there has been effort to identify and characterize these enzymes. Initial efforts on the isolation and characterization of delta-8 desaturases from Euglena gracilis; and, several sequence variations within the Euglena gracilis delta-8 desaturase have been reported (see, e.g., Wallis et al., Arch. Biochem. and Biophys. 365(2):307-316 (1999); PCT Publication No. WO 2000/34439; U.S. Pat. No. 6,825,017; PCT Publication No. WO 2004/057001). Also, Applicants' Assignee's co-pending applications having U.S. application Ser. Nos. 11/166,003 and 11/166,993 filed Jun. 24, 2005 (Attorney Docket Nos. BB-1547 and CL-3150, respectively (PCT Publication Nos. WO 2006/012325 and WO 2006/012326; both published Feb. 2, 2006)) discloses amino acid and nucleic acid sequences for a Euglena gracilis delta-8 desaturase.

[0007] More recently, PCT Publication No. WO 2005/103253 (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 U.S. patent application Ser. No. 11/737,772 (filed Apr. 20, 2007; Attorney Docket No. BB-1566) discloses amino acid and nucleic acid sequences for a delta-8 desaturase enzyme from Pavlova lutheri (CCMP459). U.S. patent application Ser. No. 11/876,115 (filed Oct. 22, 2007; Attorney Docket No. BB-1574) discloses amino acid and nucleic acid sequences for a delta-8 desaturase enzyme from Tetruetreptia pomquetensis CCMP1491, Eutreptiella sp. CCMP389 and Eutreptiella cf.sub.--gymnastica CCMP1594.

[0008] Based on the utility of expressing delta-8 desaturases in conjunction with delta-9 elongases, there has also been effort to identify and characterize delta-9 elongases from various sources. Most delta-9 elongase enzymes identified so far have the ability to convert both LA to EDA and ALA to ETrA (wherein DGLA and ETA are subsequently synthesized from EDA and ETrA, respectively, following reaction with a .DELTA.8 desaturase; ARA and EPA are subsequently synthesized from DGLA and ETA, respectively, following reaction with a .DELTA.5 desaturase; and, DHA synthesis requires subsequent expression of an additional C.sub.20/22 elongase and a .DELTA.4 desaturase). A delta-9 elongase from Isochrysis galbana has been publicly available (described in GenBank Accession No. AAL37626, as well as PCT Publication No. WO 02/077213). Applicants' Assignee's co-pending application having U.S. application Ser. No. 11/601,563 (filed Nov. 16, 2006, which published May 24, 2007; Attorney Docket No. BB-1562), discloses a delta-9 elongase from Euglena gracilis. Applicants' Assignee's co-pending application having U.S. application Ser. No. 11/601,564 filed Nov. 16, 2006 (Attorney Docket No. CL-3600), discloses a delta-9 elongase from Eutreptiella sp. CCMP389.

[0009] Most delta-5 desaturase enzymes identified so far have the primary ability to convert dihomo-gamma-linolenic acid [20:3, DGLA] to ARA, with secondary activity in converting eicosatetraenoic acid [20:4, ETA] to EPA (where DHA 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-linoleic acid ["GLA"; 18:3 .omega.-6] and/or stearidonic acid ["STA"; 18:4 .omega.-3]) 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] and/or eicosatrienoic acid ["ETrA"; 20:3 .omega.-3]) (FIG. 1).

[0010] Furthermore, based on the role delta-5 desaturase enzymes play in the synthesis of e.g., ARA, EPA and DHA, there has also been an effort to identify and characterize these enzymes from various sources. As such, delta-5 desaturases have been disclosed in both the open literature (e.g., GenBank Accession Nos. AF199596, AF226273, AF320509, AB072976, AF489588, AJ510244, AF419297, AF07879, AF067654 and AB022097) and the patent literature (e.g., U.S. Pat. No. 5,972,664 and U.S. Pat. No. 6,075,183).

[0011] Applicants' Assignee has a number of patent applications concerning the production of PUFAs in oleaginous yeasts (i.e., Yarrowia lipolytica), including: PCT Publication Nos. WO 2004/101757 and WO 2004/101753 (both published Nov. 25, 2004); U.S. application Ser. No. 11/265,761 (filed Nov. 2, 2005); U.S. application Ser. No. 11/264,784 (filed Nov. 1, 2005); and U.S. application Ser. No. 11/264,737 (filed Nov. 1, 2005).

[0012] Relatedly, PCT Publication No. WO 2004/071467 (published Aug. 26, 2004; Attorney Docket No. BB-1538) concerns the production of PUFAs in plants, while PCT Publication No. WO 2004/071178 (published Aug. 26, 2004) concerns annexin promoters and their use in expression of transgenes in plants; both are Applicants' Assignee's copending applications.

SUMMARY OF THE INVENTION

[0013] The present invention concerns an isolated polynucleotide comprising: [0014] (a) 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 V method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:13; [0015] (b) 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:12; [0016] (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:12; or [0017] (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.

[0018] 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.

[0019] In a third embodiment, the invention concerns a plant cell comprising in its genome the recombinant DNA construct of the invention.

[0020] In a fourth embodiment, the invention concerns a method for transforming a plant cell, comprising transforming a plant cell with a recombinant construct of the invention or an isolated polynucleotide of the invention and selecting those plant cells transformed with the recombinant construct or the isolated polynucleotide.

[0021] 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.

[0022] In a sixth embodiment, the invention concerns a method for making long-chain polyunsaturated fatty acids in a plant cell comprising: [0023] (a) transforming a plant cell with the recombinant construct of the invention; and [0024] (b) selecting those transformed plant cells that make long-chain polyunsaturated fatty acids.

[0025] In a seventh embodiment, the invention concerns a method for producing at least one polyunsaturated fatty acid in an oilseed plant cell comprising: [0026] (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; [0027] (b) regenerating an oilseed plant from the transformed cell of step (a); and [0028] (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.

[0029] 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.

[0030] In a ninth embodiment, the invention concerns an oilseed plant comprising: [0031] (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 [0032] (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.18/20 elongase and a C.sub.20/22 elongase.

[0033] 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.

[0034] 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.

[0035] 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.

Biological Deposits

[0036] The following plasmid has been deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, and bears the following designation, Accession Number and date of deposit (Table 1).

TABLE-US-00001 TABLE 1 ATCC Deposit Plasmid Accession Number Date of Deposit pKR72 PTA-6019 May 28, 2004

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS

[0037] The invention can be more fully understood from the following detailed description and the accompanying drawings and Sequence Listing, which form a part of this application.

[0038] FIG. 1 is a representative omega-3 and omega-6 fatty acid pathway providing for the conversion of myristic acid through various intermediates to DHA.

[0039] FIG. 2A is a schematic of EgD5.

[0040] FIG. 2B is a map of plasmid pZUF17 (SEQ ID NO:7).

[0041] FIG. 2C is a map of plasmid pDMW267 (SEQ ID NO:8).

[0042] FIG. 3 is a map of plasmid pY115 (SEQ ID NO:19).

[0043] FIG. 4 is a map of plasmid pY159 (SEQ ID NO:23).

[0044] FIG. 5 is a map of plasmid pY169 (SEQ ID NO:24).

[0045] FIG. 6 are the fatty acid profiles for Yarrowia lipolytica expressing pY169.

[0046] FIG. 7 is a map of pKR1153 (SEQ ID NO:44).

[0047] FIGS. 8A, 8B and 8C shows a comparison of the nucleotide sequences of EaD5 (same as EaD5Des1) (SEQ ID NO:12) and EaD5S (SEQ ID NO:45).

[0048] FIG. 9 is a map of plasmid pEaD5S (SEQ ID NO:46).

[0049] FIG. 10 shows a chromatogram of the lipid profile of an Euglena anabaena cell extract as described in the Examples.

[0050] FIG. 11 shows ten events having the highest average correct delta-5 desaturase activities (average of the 5 somatic soybean embryos analyzed) from approximately 30 events transformed with pKR1153 (Experiment MSE2140). 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 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. The activity of the delta-5 desaturase is expressed as percent delta-5 desaturation ("% delta-5 desat"), calculated according to the following formula: ([product]/[substrate+product])*100. More specifically, the percent delta-5 desaturation was determined as: ([ARA+EPA]/[DGLA+ETA+ARA+EPA])*100.

[0051] The sequence descriptions summarize the Sequences Listing attached hereto. The Sequence Listing contains one letter codes for nucleotide sequence characters and the single and three letter codes for amino acids as defined in the IUPAC-IUB standards described in Nucleic Acids Research 13:3021-3030 (1985) and in the Biochemical Journal 219(2):345-373 (1984).

[0052] SEQ ID NO:1 is the cDNA sequence of the Euglena anabaena delta-5 desaturase 1 (EaD5Des1).

[0053] SEQ ID NO:2 is the nucleotide sequence of the Euglena gracilis delta-5 desaturase coding sequence (EgD5).

[0054] SEQ ID NO:3 is the nucleotide sequence of the Euglena gracilis delta-5 desaturase oligonucleotide YL794.

[0055] SEQ ID NO:4 is the nucleotide sequence of the Euglena gracilis delta-5 desaturase oligonucleotide YL797.

[0056] SEQ ID NO:5 is the nucleotide sequence of the Euglena gracilis delta-5 desaturase oligonucleotide YL796.

[0057] SEQ ID NO:6 is the nucleotide sequence of the Euglena gracilis delta-5 desaturase oligonucleotide YL795.

[0058] SEQ ID NO:7 is the nucleotide sequence of plasmid pZUF17.

[0059] SEQ ID NO:8 is the nucleotide sequence of plasmid pDMW367.

[0060] SEQ ID NO:9 is the nucleotide sequence of the M13F universal primer.

[0061] SEQ ID NO:10 is the nucleotide sequence of M13-28Rev.

[0062] SEQ ID NO:11 is the nucleotide sequence of plasmid pLF119.

[0063] SEQ ID NO:12 is the nucleotide sequence of the Euglena anabaena delta-5 desaturase 1 coding sequence (EaD5Des1).

[0064] SEQ ID NO:13 is the amino acid sequence of the Euglena anabaena delta-5 desaturase 1 (EaD5Des1).

[0065] SEQ ID NO:14 is the amino acid sequence of the Thalassiosira pseudonana delta-8 fatty acid desaturase.

[0066] SEQ ID NO:15 is the amino acid sequence of the Phaeodactylum tricornutum delta-5 fatty acid desaturase.

[0067] SEQ ID NO:16 is the amino acid sequence of the Euglena gracilis delta-5 desaturase (EgD5).

[0068] SEQ ID NO:17 is the nucleotide sequence of plasmid pDMW263.

[0069] SEQ ID NO:18 is the nucleotide sequence of plasmid pDMW237.

[0070] SEQ ID NO:19 is the nucleotide sequence of plasmid pY115.

[0071] SEQ ID NO:20 is the nucleotide sequence of oligonucleotide oYFBA1.

[0072] SEQ ID NO:21 is the nucleotide sequence of oligonucleotide oYFBA1-6.

[0073] SEQ ID NO:22 is the nucleotide sequence of plasmid pY158.

[0074] SEQ ID NO:23 is the nucleotide sequence of plasmid pY159.

[0075] SEQ ID NO:24 is the nucleotide sequence of plasmid pY169.

[0076] SEQ ID NO:25 is the nucleotide sequence of the Euglena gracilis delta-9 elongase (EgD9e).

[0077] SEQ ID NO:26 is the nucleotide sequence of the Euglena gracilis delta-8 desaturase (EgD8).

[0078] SEQ ID NO:27 is the nucleotide sequence of the Euglena gracilis elongase sense oligonucleotide oEugEL1-1.

[0079] SEQ ID NO:28 is the nucleotide sequence of the Euglena gracilis elongase anti-sense oligonucleotide oEugEL1-2.

[0080] SEQ ID NO:29 is the nucleotide sequence of plasmid pKR906.

[0081] SEQ ID NO:30 is the nucleotide sequence of plasmid pKR72.

[0082] SEQ ID NO:31 is the nucleotide sequence of plasmid KS102.

[0083] SEQ ID NO:32 is the nucleotide sequence of plasmid pKR197.

[0084] SEQ ID NO:33 is the nucleotide sequence of plasmid pKR911.

[0085] SEQ ID NO:34 is the nucleotide sequence of plasmid pKR680.

[0086] SEQ ID NO:35 is the nucleotide sequence of plasmid pKR913.

[0087] SEQ ID NO:36 is the nucleotide sequence of oligonucleotide oEAd5-1-1.

[0088] SEQ ID NO:37 is the nucleotide sequence of oligonucleotide oEAd5-1-2.

[0089] SEQ ID NO:38 is the nucleotide sequence of plasmid pKR1136.

[0090] SEQ ID NO:39 is the nucleotide sequence of plasmid pKR767.

[0091] SEQ ID NO:40 is the nucleotide sequence of the Mortierella alpine delta-5 desaturase coding sequence (MaD5).

[0092] SEQ ID NO:41 is the nucleotide sequence of plasmid pKR974.

[0093] SEQ ID NO:42 is the nucleotide sequence of the Saprolegnia diclina delta-5 desaturase coding sequence (SdD5).

[0094] SEQ ID NO:43 is the nucleotide sequence of plasmid pKR1139.

[0095] SEQ ID NO:44 is the nucleotide sequence of plasmid pKR1153.

[0096] SEQ ID NO:45 is the nucleotide sequence of the codon-optimized Euglena anabaena delta-5 desaturase gene (EaD5S).

[0097] SEQ ID NO:46 is the nucleotide sequence of plasmid pEaD5S.

DETAILED DESCRIPTION OF THE INVENTION

[0098] The disclosure of each reference set forth herein is hereby incorporated by reference in its entirety.

[0099] 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.

[0100] The present invention relates to delta-5 desaturase enzymes and nucleic acid for encoding the same isolated from Euglena anabaena. These are useful for, inter alia, for the manipulation of biochemical pathways for the production of PUFAs. Thus, the subject invention finds many applications.

[0101] 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). In this case, the PUFAs are generally administered orally but can be administered by any route by which they may be successfully absorbed, e.g., parenterally (e.g., subcutaneously, intramuscularly or intravenously), rectally, vaginally or topically (e.g., as a skin ointment or lotion).

[0102] 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.

[0103] In the context of this disclosure, a number of terms and abbreviations are used. The following definitions are provided.

[0104] "Open reading frame" is abbreviated ORF.

[0105] "Polymerase chain reaction" is abbreviated PCR.

[0106] "American Type Culture Collection" is abbreviated ATCC.

[0107] "Polyunsaturated fatty acid(s)" is abbreviated PUFA(s).

[0108] "Triacylglycerols" are abbreviated TAGs.

[0109] 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. 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 PCT Publication No. WO 2004/101757.

[0110] 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.

[0111] 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 remainder of the specification, and each compounds' chemical name.

TABLE-US-00002 TABLE 2 Nomenclature of Polyunsaturated Fatty Acids and Precursors Common Shorthand Name Abbreviation Chemical Name Notation myristic -- tetradecanoic 14:0 palmitic PA 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- GLA cis-6,9,12- 18:3 .omega.-6 linolenic octadecatrienoic eicosadienoic EDA cis-11,14-eicosadienoic 20:2 .omega.-6 dihomo- DGLA cis-8,11,14-eicosatrienoic 20:3 .omega.-6 gamma- linolenic sciadonic SCI cis-5,11,14-eicosatrienoic 20:3b .omega.-6 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 ERA cis-11,14,17- 20:3 .omega.-3 eicosatrienoic eicosa- ETA cis-8,11,14,17- 20:4 .omega.-3 tetraenoic eicosatetraenoic juniperonic JUP cis-5,11,14,17- 20:4b .omega.-3 eicosatrienoic 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

[0112] 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, and involve a step by step modification of the initial substance to shape it into a product having the exact chemical structure desired.

[0113] 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). Simplistically, 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 reticulim membrane. More specifically, "PUFA biosynthetic pathway enzyme" refers 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.

[0114] 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, omega-6 fatty acids.

[0115] 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 PUFA biosynthetic pathway enzyme genes are required, as a number of fatty acid products will only require the expression of a subset of the genes of this pathway.

[0116] The term "delta-9 elongase/delta-8 desaturase pathway" refers to a biosynthetic pathway for production of long-chain PUFAs. This pathway, at a minimum, comprises a delta-9 elongase and a delta-8 desaturase, thereby enabling biosynthesis of DGLA and/or ETA from LA and ALA, respectively. With expression of other desaturases and elongases, ARA, EPA, DPA and DHA may also be synthesized. This pathway may be advantageous in some embodiments, as the biosynthesis of GLA and/or STA is excluded.

[0117] 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.

[0118] 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 (SCI) 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.

[0119] 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.

[0120] "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.

[0121] 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.

[0122] "Desaturase" is 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 will desaturate a fatty acid between the fifth and sixth carbon atom numbered from the carboxyl-terminal end of the molecule and that can, for example, catalyze the conversion of DGLA to ARA and/or ETA to EPA. Other useful fatty acid desaturases include, for example: (1) delta-8 desaturases that catalyze the conversion of EDA to DGLA and/or ERA to ETA; (2) delta-6 desaturases that catalyze the conversion of LA to GLA and/or ALA to STA; (3) delta-4 desaturases that catalyze the conversion of DPA to DHA; (4) delta-12 desaturases that catalyze the conversion of oleic acid to LA; (5) delta-15 desaturases that catalyze the conversion of LA to ALA and/or GLA to STA; (6) delta-17 desaturases that catalyze the conversion of ARA to EPA and/or DGLA to ETA; and (7) delta-9 desaturases that catalyze the conversion of palmitic acid to palmitoleic acid (16:1) and/or stearic acid to oleic acid (18:1). 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.

[0123] 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].

[0124] For the purposes herein, the terms "EaD5Des1" or "EaD5" refers to a delta-5 desaturase enzyme (SEQ ID NO:13) isolated from Euglena anabaena, encoded by SEQ ID NO:12 herein. Likewise, the term "EaD5S" (SEQ ID NO:45) refers to a delta-5 desaturase codon-optimized for expression in Yarrowia lipolytica.

[0125] For the purposes herein, the term "IgD9e" refers to a delta-9 elongase (SEQ ID NO:15) (NCBI Accession No. AAL37626 [GI 17226123], locus AAL37626, CDS AF390174; GenBank Accession No. AF390174) isolated from Isochrysis galbana. In contrast, the term "IgD9eS" refers to a synthetic (codon-optimized) delta-9 elongase derived from the DNA sequence of the Isochrysis galbana delta-9 elongase which can be used for expression in Yarrowia lipolytica.

[0126] Similarly for the purposes herein, the term "EgD9e" refers to a delta-9 elongase isolated from Euglena gracilis. EgD9e is described in U.S. application Ser. No. 11/601,563 (filed Nov. 16, 2006, which published May 24, 2007; Attorney Docket No. BB-1562).

[0127] Similarly, the term "EgD8" refers to a delta-8 desaturase enzyme isolated from Euglena gracilis. EgD8 is 100% identical and functionally equivalent to "Eg5", as described in PCT Publication Nos. WO 2006/012325 and WO 2006/012326 (SEQ ID NO:2 of U.S. Publication No. 20050287652-A1).

[0128] The term "elongase system" refers to a suite of four enzymes that are responsible for elongation of a fatty acid carbon chain to produce a fatty acid that is two carbons longer than the fatty acid substrate that the elongase system acts upon. More specifically, the process of elongation occurs in association with fatty acid synthase, whereby CoA is the acyl carrier (Lassner et al., Plant Cell 8:281-292 (1996)). In the first step, which has been found to be both substrate-specific and also rate-limiting, malonyl-CoA is condensed with a long-chain acyl-CoA to yield carbon dioxide (CO.sub.2) and a .beta.-ketoacyl-CoA (where the acyl moiety has been elongated by two carbon atoms). Subsequent reactions include reduction to .beta.-hydroxyacyl-CoA, dehydration to an enoyl-CoA and a second reduction to yield the elongated acyl-CoA. Examples of reactions catalyzed by elongase systems are the conversion of GLA to DGLA, STA to ETA, LA to EDA, ALA to ETrA and EPA to DPA.

[0129] For the purposes herein, an enzyme catalyzing the first condensation reaction (i.e., conversion of malonyl-CoA and long-chain acyl-CoA to .beta.-ketoacyl-CoA) will be referred to generically as an "elongase". In general, the substrate selectivity of elongases is somewhat broad but segregated by both chain length and the degree of unsaturation. Accordingly, elongases can have different specificities. 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 will utilize a C.sub.18 substrate (e.g., GLA, STA) and a C.sub.20/22 elongase will utilize a C.sub.20 substrate (e.g., EPA). Similarly, a "delta-9 elongase" may be able to catalyze the conversion of LA to EDA and/or ALA to ETrA. It is important to note that some elongases have broad specificity and thus a single enzyme may be capable of catalyzing several elongase reactions. Thus, for example, a delta-9 elongase may also act as a C.sub.16/18 elongase, C.sub.18/20 elongase and/or C.sub.20/22 elongase and may have alternate, but not preferred, specificities for delta-5 and delta-6 fatty acids such as EPA and/or GLA, respectively.

[0130] 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: [0131] 1. small aliphatic, nonpolar or slightly polar residues: Ala [A], Ser [S], Thr [T] (Pro [P], Gly [G]); [0132] 2. polar, negatively charged residues and their amides: Asp [D], Asn [N], Glu [E], Gln [Q]; [0133] 3. polar, positively charged residues: His [H], Arg [R], Lys [K]; [0134] 4. large aliphatic, nonpolar residues: Met [M], Leu [L], Ile [I], Val [V] (Cys [C]); and, [0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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% SDS (sodium dodecyl sulphate) at 37.degree. C., and a wash in 1.times. 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% SDS at 37.degree. C., and a wash in 0.1.times.SSC at 60 to 65.degree. C.

[0144] 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, 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.

[0145] "Sequence identity" or "identity" in the context of nucleic acid or polypeptide sequences refers to the nucleic acid bases or amino acid residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window.

[0146] Thus, "percentage of sequence identity" refers to the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the results by 100 to yield the percentage of sequence identity. Useful examples of percent sequence identities include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 50% to 100%. These identities can be determined using any of the programs described herein.

[0147] Sequence alignments and percent identity or similarity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the MegAlign.TM. program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). 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.

[0148] The "Clustal V method of alignment" corresponds to the alignment method labeled Clustal V (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al. (1992) Comput. Appl. Biosci. 8:189-191) and found in the MegAlign.TM. program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). 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 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.

[0149] "BLASTN method of alignment" is an algorithm provided by the National Center for Biotechnology Information (NCBI) to compare nucleotide sequences using default parameters.

[0150] 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. Useful examples of percent identities include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 50% to 100%. Indeed, any integer amino acid identity from 50% to 100% may be useful in describing the present invention, such as 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 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%. Also, of interest is any full-length or partial complement of this isolated nucleotide fragment.

[0151] "Gene" refers to a nucleic acid fragment that expresses a specific protein, including 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. A "foreign" gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A "transgene" is a gene that has been introduced into the genome by a transformation procedure.

[0152] The term "genome" as it applies to a plant cells encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular components (e.g., mitochondrial, plastid) of the cell.

[0153] 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.

[0154] An "allele" is 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 that plant is homozygous at that locus. If the alleles present at a given locus on a chromosome differ that plant is heterozygous at that locus.

[0155] "Coding sequence" refers to a DNA sequence that codes for a specific amino acid sequence. "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, but are not limited to: promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures.

[0156] "Promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. The promoter sequence consists 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. 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 conditions. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of some variation may have identical promoter activity. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". 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).

[0157] "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)).

[0158] "3' non-coding sequences", "transcription terminator" or "termination sequences" refer to DNA sequences located downstream of a coding sequence and include 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 use of different 3' non-coding sequences is exemplified by Ingelbrecht, I. L., et al. Plant Cell 1:671-680 (1989).

[0159] "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. A RNA transcript is referred to as the mature RNA when it is a RNA sequence derived from post-transcriptional processing of the primary transcript. "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 DNA that is complementary to, and synthesized from, a mRNA template using the enzyme reverse transcriptase. The cDNA can be single-stranded or converted into double-stranded form using the Klenow fragment of DNA polymerase I. "Sense" RNA refers to RNA transcript that includes the mRNA and can be translated into protein within a cell or in vitro. "Antisense RNA" refers to an 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). 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, introns, or the coding sequence. "Functional RNA" refers to antisense RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect on cellular processes. The terms "complement" and "reverse complement" are used interchangeably herein with respect to mRNA transcripts, and are meant to define the antisense RNA of the message.

[0160] 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 regulated by the other. For example, a promoter is operably linked with a coding sequence when it is capable of regulating 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 a sense or antisense orientation. In another example, the complementary RNA regions of the invention can be operably linked, either directly or indirectly, 5' to the target mRNA, or 3' to the target mRNA, or within the target mRNA, or a first complementary region is 5' and its complement is 3' to the target mRNA.

[0161] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989). Transformation methods are well known to those skilled in the art and are described infra.

[0162] "PCR" or "polymerase chain reaction" is a technique for the synthesis of large quantities of specific DNA segments and consists of a series of repetitive cycles (Perkin Elmer Cetus Instruments, Norwalk, Conn.). Typically, the double-stranded DNA is heat denatured, the two primers complementary to the 3' boundaries of the target segment are annealed at low temperature and then extended at an intermediate temperature. One set of these three consecutive steps is referred to as a "cycle".

[0163] The term "recombinant" refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.

[0164] 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. "Transformation cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitates transformation of a particular host 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 (i.e., to a discrete nucleic acid fragment into which a nucleic acid sequence or fragment can be moved.)

[0165] 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.

[0166] 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]).

[0167] 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).

[0168] "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.

[0169] "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.

[0170] 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.

[0171] "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 02/00904, 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)).

[0172] 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). A class of plants identified as oleaginous are commonly referred to as "oilseed" plants. Examples of oilseed plants include, but are not limited to: soybean (Glycine and Soja sp.), flax (Linum sp.), rapeseed (Brassica sp.), maize, cotton, safflower (Carthamus sp.) and sunflower (Helianthus sp.).

[0173] Within oleaginous microorganisms the cellular oil or TAG content generally 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. Microbiol. 57:419-25 (1991)). The term "oleaginous yeast" refers to those microorganisms classified as yeasts that make oil. 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.

[0174] The term "Euglenophyceae" refers to a group of unicellular colorless or photosynthetic flagellates ("euglenoids") found living in freshwater, marine, soil, and parasitic environments. The class is characterized by solitary unicells, wherein most are free-swimming and have two flagella (one of which may be nonemergent) arising from an anterior invagination known as a reservoir. Photosynthetic euglenoids contain one to many grass-green chloroplasts, which vary from minute disks to expanded plates or ribbons. Colorless euglenoids depend on osmotrophy or phagotrophy for nutrient assimilation. About 1000 species have been described and classified into about 40 genera and 6 orders. Examples of Euglenophyceae include, but are no means limited to, the following genera: Euglena, Eutreptiella and Tetruetreptia.

[0175] The term "plant" refers to whole plants, plant organs, plant tissues, seeds, plant cells, seeds and progeny of the same. Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores. "Progeny" comprises any subsequent generation of a plant.

An Overview: Microbial Biosynthesis of Fatty Acids and Triacylglycerols

[0176] 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).

[0177] 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

[0178] The metabolic process wherein oleic acid is converted to long chain 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 long chain omega-3/omega-6 fatty acid.

[0179] 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-9 elongase/delta-8 desaturase pathway", long chain omega-6 fatty acids are formed as follows: (1) LA is converted to EDA by a delta-9 elongase; (2) EDA is converted to DGLA by a delta-8 desaturase; and (3) DGLA is converted to ARA by a delta-5 desaturase. Alternatively, the "delta-9 elongase/delta-8 desaturase pathway" can be utilized for formation of long chain 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 ETrA by a delta-9 elongase; (3) ETrA is converted to ETA by a delta-8 desaturase; (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.

[0180] Alternate pathways for the biosynthesis of omega-3/omega-6 fatty acids utilize a delta-6 desaturase and C.sub.18/20 elongase (also known as delta-6 elongase, the terms can be used interchangeably) (i.e., the "delta-6 desaturase/delta-6 elongase pathway"). More specifically, LA and ALA may be converted to GLA and STA, respectively, by a delta-6 desaturase; then, a C.sub.18/20 elongase converts GLA to DGLA and/or STA to ETA.

[0181] It is contemplated that the particular functionalities required to be introduced into 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). For example, expression of the delta-9 elongase/delta-8 desaturase pathway may be preferred in some embodiments, as opposed to expression of the delta-6 desaturase/delta-6 elongase pathway, since PUFAs produced via the former pathway are devoid of GLA.

[0182] 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) cofactors 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).

[0183] 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 unpurified 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, consideration of each enzyme's conversion efficiency is also a variable when optimizing biosynthesis of a desired fatty acid that must be considered in light of the final desired lipid profile of the product.

[0184] 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 Novel Delta-5 Desaturases

[0185] In the present invention, nucleotide sequences encoding delta-5 desaturases have been isolated from Euglena anabaena (designated herein as "EaD5Des1").

[0186] Thus, the present invention concerns an isolated polynucleotide comprising: [0187] (a) 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 V method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:13 [EaD5Des1]; [0188] (b) 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:12 [EaD5Des1]; or, [0189] (c) a complement of the nucleotide sequence of (a) or (b), wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.

[0190] In still another aspect, this invention concerns an isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having delta-5 desaturase activity, wherein the nucleotide sequence has at least 90% sequence identity, based on the BLASTN method of alignment, when compared to a nucleotide sequence as set forth in SEQ ID NO:12.

[0191] The instant EaD5Des1 sequences can be codon-optimized for expression in a particular host organism (see SEQ ID NO:45). 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.

[0192] EaD5Des1 could be codon-optimized for expression in Yarrowia lipolytica, as taught in PCT Publication No. WO 04/101757 and U.S. Pat. No. 7,125,672. In one embodiment, it may be desirable to modify a portion of the codons encoding EaD5Des1 (as set forth in SEQ ID NO:12) to enhance expression of the gene in a host organism including, but not limited to, a plant or plant part.

[0193] 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, based on the wildtype EaD5Des1 sequence. Accordingly, the instant invention relates to any codon-optimized delta-5 desaturase protein that is derived from the wildtype EaD5Des1 (i.e., encoded by SEQ ID NO:12).

Identification and Isolation of Homologs

[0194] Any of the instant desaturase sequences (i.e., EaD5Des1) or portions thereof may be used to search for delta-5 desaturase homologs in the same or other bacterial, algal, fungal, euglenoid 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.

[0195] Alternatively, any of the instant desaturase sequences or portions thereof may also be employed as hybridization reagents for the identification of delta-5 desaturase 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.

[0196] 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).

[0197] 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 from 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).

[0198] 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.

[0199] 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, euglenoid 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 et al., Proc. Acad. Sci. USA 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.

[0200] 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 and/or EPA 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.

[0201] 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 oligonucleotide 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.).

[0202] 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.

[0203] 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., PNAS USA 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 (Gibco/BRL, Gaithersburg, Md.), specific 3' or 5' cDNA fragments can be isolated (Ohara et al., PNAS USA 86:5673 (1989); Loh et al., Science 243:217 (1989)).

[0204] 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. As used herein, "domain" or "functional domain" refer to nucleic acid sequence(s) that are capable of eliciting a biological response in plants.

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

[0205] It is expected that introduction of chimeric genes encoding the delta-5 desaturases described herein (i.e., EaD5Des1 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 and/or ETA) to the desaturase enzymes described herein (e.g., EaD5Des1), such that the substrate is converted to the desired fatty acid product (i.e., ARA and/or EPA).

[0206] More specifically, it is an object of the present invention to provide a method for the production of ARA in a plant host cell (e.g. soybean), wherein the plant host cell comprises: [0207] (a) a recombinant construct encoding a delta-5 desaturase polypeptide selected from the group consisting of SEQ ID NO:13; and, [0208] (b) a source of DGLA; wherein the host plant 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.

[0209] In alternate embodiments of the present invention, the delta-5 desaturase may be used 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: [0210] (a) a recombinant construct encoding a delta-5 desaturase polypeptide selected from the group consisting of SEQ ID NO:13; and, [0211] (b) a source of ETA; wherein the host plant 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.

[0212] Alternatively, each delta-5 desaturase gene and its corresponding enzyme product described herein can be used indirectly for the production of various omega-6 and omega-3 PUFAs, including e.g., DGLA, ETA, ARA, EPA, DPA and/or DHA (see FIG. 1; see also PCT Publication No. WO 2004/101757). 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 (i.e., EaD5Des1, 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-8 desaturases, delta-15 desaturases, delta-9 desaturases, delta-12 desaturases, C.sub.14/16 elongases, C.sub.16/18 elongases, delta-9 elongases, delta-5 desaturases, delta-4 desaturases, C.sub.20/22 elongases) to result in higher levels of production of longer-chain omega-3/omega-6 fatty acids (e.g., ARA, EPA, DPA and DHA).

[0213] In preferred embodiments, the delta-5 desaturases of the present invention will minimally be expressed in conjunction with a delta-9 elongase and a delta-8 desaturases (e.g., a delta-8 desaturase or a codon-optimized delta-8 desaturase). The delta-5 desaturase could also be minimally expressed in conjunction with a delta-6 desaturase and C.sub.18/20 elongases. However, 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).

[0214] When the desired end product is SCI and/or JUP, then the delta-5 desaturase will be minimally expressed in conjunction with a delta-9 elongase in an organism which makes EDA and/or ERA, respectively.

[0215] The term "delta-6 desaturase/delta-6 elongase pathway" also refers to a biosynthetic pathway for production of long-chain PUFAs. This pathway, at a minimum, comprises a delta-6 desaturase and a delta-6 elongase, thereby enabling biosynthesis of DGLA and/or ETA from LA and ALA, respectively. With expression of other desaturases and elongases, ARA, EPA, DPA and DHA may also be synthesized. Occasionally, a delta-6 elongase may elongate fatty acids other than the intended fatty acid. For instance, delta-6 elongases generally convert GLA to DGLA but some delta-6 elongases may also convert unintended substrates such as LA or ALA to EDA or ETrA, respectively. In a delta-6 desaturase/delta-6 elongase pathway, EDA and ETrA would be considered "by-product fatty acids" as defined herein. Addition of a delta-8 desaturase to a delta-6 desaturase/delta-6 elongase pathway would provided a means to convert the "by-product fatty acids" EDA and ETrA back into the "intermediate fatty acids" (as defined previously) DGLA and ETA, respectively.

Plant Expression Systems, Cassettes and Vectors, and Transformation

[0216] 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 termination/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.

[0217] 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 Gy1 promoter, the beta subunit of beta conglycinin promoter, the P34/Gly Bd m 30K promoter, the albumin promoter, the Leg A1 promoter and the Leg A2 promoter.

[0218] 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 35S promoter (Atanassova et al., Plant Mol. Biol. 37:275-285 (1998); Battraw and Hall, Plant Mol. Biol. 15:527-538 (1990); Holtorf 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):12486-93 (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 et al., Plant Mol Biol. 37(2):275-85 (1989)).

[0219] 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., Dev. Genet. 10:112-122 (1989); Ellerstrom et al., Plant Mol. Biol. 32:1019-1027 (1996); Keddie et 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.

[0220] 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.

[0221] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J. et al., In Molecular Cloning: A Laboratory Manual; 2.sup.nd 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").

[0222] 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.

[0223] The recombinant constructs of the invention may be introduced into one plant cell; or, alternatively, each construct may be introduced into separate plant cells.

[0224] Expression in a plant cell may be accomplished in a transient or stable fashion as is described above.

[0225] 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.

[0226] Plant parts include 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.

[0227] 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.

[0228] 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.

[0229] 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.

[0230] 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 et 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)).

[0231] 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.

[0232] 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).

[0233] Examples of oilseed plants include, but are not limited to: soybean, Brassica species, sunflower, maize, cotton, flax and safflower.

[0234] 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.

[0235] Thus, in one embodiment this invention concerns an oilseed plant comprising: [0236] (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, [0237] (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.

[0238] 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.

[0239] 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.

[0240] In another aspect, this invention concerns a method for making long-chain PUFAs in a plant cell comprising: [0241] (a) transforming a plant cell with the recombinant construct of the invention; and, [0242] (b) selecting those transformed plant cells that make long-chain PUFAs.

[0243] In still another aspect, this invention concerns a method for producing at least one PUFA in a soybean cell comprising: [0244] (a) transforming a soybean cell with a first recombinant DNA construct comprising: [0245] (i) an isolated polynucleotide encoding a delta-5 desaturase polypeptide, operably linked to at least one regulatory sequence; and, [0246] (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; [0247] (b) regenerating a soybean plant from the transformed cell of step (a); and, [0248] (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 nontransformed soybean plant.

[0249] 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.

[0250] 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 2005/103253 (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 U.S. patent application Ser. No. 11/737,772 (filed Apr. 20, 2007; Attorney Docket No. BB-1566) discloses amino acid and nucleic acid sequences for a delta-8 desaturase enzyme from Pavlova lutheri (CCMP459). U.S. patent application Ser. No. 11/876,115 (filed Oct. 22, 2007; Attorney Docket No. BB-1574) discloses amino acid and nucleic acid 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

[0251] The delta-5 desaturase genes and gene products described herein (i.e., EaD5Des1, 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).

[0252] 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.

[0253] 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 nontransformed 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.

[0254] 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.

[0255] 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.

[0256] 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 bp of the 3' region of the Yarrowia lipolytica extracellular protease (XPR; GenBank Accession No. 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. Z50020) terminator; the Lip2 (GenBank Accession No. AJ012632) terminator; and the 3-oxoacyl-coA thiolase (OCT; GenBank Accession No. X69988) terminator.

[0257] 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.

[0258] 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.

[0259] 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 Lip1 gene locus (GenBank Accession No. Z50020) and/or the Lip2 gene locus (GenBank Accession No. AJ012632)].

[0260] 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.

[0261] 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.

[0262] 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)).

[0263] 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.

[0264] 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.

[0265] 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.

[0266] 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.

[0267] 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).

[0268] 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)).

[0269] 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.

[0270] 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. Microbiol., 66:4655 (2000)). Similarly, methods for transformation of Thraustochytriales microorganisms are disclosed in U.S. Pat. No. 7,001,772.

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

[0271] 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.

[0272] 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).

[0273] 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).

[0274] 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).

[0275] 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 plants 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 plants 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

[0276] 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.)).

[0277] Fermentation media may 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 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.

[0278] 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)).

[0279] Preferred growth media 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.

[0280] 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

[0281] 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)).

[0282] 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

[0283] 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.

[0284] Plant and microbial oils containing PUFAs that have been refined and/or 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.

[0285] 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.

[0286] 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).

[0287] 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

[0288] 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).

[0289] 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.

[0290] 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.

[0291] 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.

[0292] 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).

[0293] 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.

[0294] 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.

[0295] 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.

[0296] A snack food product comprises any of the above or below described food products.

[0297] A fried food product comprises any of the above or below described food products that has been fried.

[0298] A health food product is any food product that imparts a health benefit. Many oilseed-derived food products may be considered as health foods.

[0299] A beverage can be in a liquid or in a dry powdered form.

[0300] 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., Similac.RTM., Ensure.RTM., Jevity.RTM., and Alimentum.RTM. from Ross Products Division, Abbott Laboratories).

[0301] 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 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.

[0302] 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.

[0303] 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

[0304] 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

[0305] 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.

[0306] 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

[0307] The present invention is further defined in the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. 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. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0308] 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).

General Methods:

[0309] Transformation and Cultivation of Yarrowia lipolytica:

[0310] Yarrowia lipolytica strains with ATCC Accession Nos. #20362, #76982 and #90812 were purchased from the American Type Culture Collection (Rockville, Md.). Yarrowia lipolytica strains were typically grown at 28.degree. C. on YPD agar (1% yeast extract, 2% bactopeptone, 2% glucose, 2% agar).

[0311] Transformation of Yarrowia lipolytica was performed according to the method of Chen, D. C. et al. (Appl. Microbiol. 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 h. Several large loopfuls of cells were scraped from the plate and resuspended in 1 mL of transformation buffer, comprising: 2.25 mL of 50% PEG, average MW 3350; 0.125 mL of 2 M lithium 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 1004 of resuspended cells, and maintained at 39.degree. C. for 1 h 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.

[0312] 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% proline, 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).

[0313] 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, Detroit, Mich.) 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, Calif.) and 20 g/L agar.

Fatty Acid Analysis of Yarrowia lipolytica:

[0314] 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 (Roughan, 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.

[0315] 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 (1004 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 4004 hexane, the sample was vortexed and spun. The upper layer was removed and analyzed by GC as described above.

Example 1

[0316] Synthesis of a cDNA Library from Euglena anabaena UTEX 373

[0317] The present Example describes the synthesis of a cDNA library from Euglena anabaena UTEX 373. This work included the generation of RNA, synthesis of cDNA, and generation of a cDNA library.

Growth of Euglena anabaena UTEX 373 and Preparation of RNA

[0318] Euglena anabaena UTEX 373 was obtained from Dr. Richard Triemer's lab at Michigan State University (East Lansing, Mich.). Approximately 2 mL of culture was removed for lipid analysis and centrifuged at 1,800.times.g for 5 min. The pellet was washed once with water and re-centrifuged. The resulting pellet was dried for 5 min under vacuum, resuspended in 1004 of trimethylsulfonium hydroxide (TMSH) and incubated at room temperature for 15 min with shaking. After this step, 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 (Supelco Inc., Cat. No. 24152). The oven temperature was programmed to hold at 170.degree. C. for 1.0 min, increase to 240.degree. C. at 5.degree. C./min and then hold for an additional 1.0 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. Cat. No. U-99-A) and the resulting chromatogram is shown in FIG. 10. The presence of EDA, ERA, EPA and DHA in the fatty acid profile, with the absence of GLA and STA, suggested that Euglena anabaena uses the delta-9 elongase/delta-8 desaturase pathway for LC-PUFA biosynthesis and would be a good source for LC-PUFA biosynthetic genes such as, but not limited to, delta-5 desaturases.

[0319] The remaining 5 mL of an actively growing culture was transferred into 25 mL of AF-6 Medium (Watanabe & Hiroki, NIES-Collection List of Strains, 5.sup.th ed., National Institute for Environmental Studies, Tsukuba, 127 pp (2004)) in a 125 mL glass flask. Euglena anabaena cultures were grown at 22.degree. C. with a 16 h light, 8 h dark cycle for 2 weeks with very gentle agitation.

[0320] After 2 weeks, the culture (25 mL) was transferred to 100 mL of AF-6 medium in a 500 mL glass bottle and the culture was grown for 1 month as described above.

[0321] After this time, two 50 mL aliquots were transferred into two separate 500 mL glass bottles containing 250 mL of AF-6 medium and the cultures were grown for two months as described above (giving a total of .about.600 mL of culture). Next, the cultures were pelleted by centrifugation at 1,800.times.g for 10 min, washed once with water and re-centrifuged. Total RNA was extracted from one of the resulting pellets using the RNA STAT-60.TM. reagent (TEL-TEST, Inc., Friendswood, Tex.) and following the manufacturer's protocol provided (use 5 mL of reagent, dissolved RNA in 0.5 mL of water). In this way, 340 .mu.g of total RNA (680 .mu.g/mL) was obtained from the pellet. The remaining pellet was frozen in liquid nitrogen and stored at -80.degree. C. The mRNA was isolated from all 340 .mu.g of total RNA using the mRNA Purification Kit (Amersham Biosciences, Piscataway, N.J.) following the manufacturer's protocol provided. In this way, 9.0 .mu.g of mRNA was obtained.

Preparation of Euglena anabaena cDNA and Generation of cDNA Library eug1c

[0322] A cDNA library was generated using the Cloneminer.TM. cDNA Library Construction Kit (Cat. No. 18249-029, Invitrogen Corporation, Carlsbad, Calif.) and following the manufacturer's protocol provided (Version B, 25-0608). Using the non-radiolabeling method, cDNA was synthesized from 5.12 .mu.g of mRNA (described above) using the Biotin-attB2-Oligo(dT) primer. After synthesis of the first and second strand, the attB1 adapter was added, ligated and the cDNA was size fractionated using column chromatography. DNA from fractions were concentrated, recombined into pDONR.TM.222 and transformed into E. coli ElectroMAX.TM. DH10B.TM. T1 Phage-Resistant cells (Invitrogen Corporation). The Euglena anabaena library was named eug1c.

[0323] The cDNA library eug1c was plated onto LBKan plates (approx. 100,000 colonies), the colonies were scraped off and DNA was isolated using the QIAprep.RTM. Spin Miniprep Kit (Qiagen Inc., Valencia, Calif.) following the manufacturer's protocol. In this way, a plasmid DNA sub-library from eug1c was obtained.

Example 2

Isolation of the Full-Length Delta-5 Desaturases from Euglena anabaena UTEX 373

[0324] The present Example describes the identification of a cDNA (SEQ ID NO:1) encoding delta-5 desaturase from Euglena anabaena UTEX 373. This work included the generation of a probe derived from the Euglena gracilis delta-5 desaturase (EgD5; SEQ ID NO:2; which is described in U.S. Provisional Application No. 60/801,172 (filed May 17, 2006; Attorney Docket No. CL-3486)) and the hybridization of the probe to the cDNA library eug1c in order to identify delta-5 desaturase homologs from Euglena anabaena UTEX 373.

Generation of Construct pDMW367, Comprising EgD5:

[0325] Based on the cDNA sequence of the Euglena gracilis delta-5 desaturase (EgD5; SEQ ID NO:2) oligonucleotides YL794 and YL797 (SEQ ID NOs:3 and 4, respectively) were used as primers to amplify the first portion of EgD5 (FIG. 2A). Primer YL794 contained a NcoI site and primer YL797 contained a HindIII site. Then, primers YL796 and YL795 (SEQ ID NOs:5 and 6, respectively) were used as primers to amplify the second portion of EgD5. Primer YL796 contained a HindIII site, while primer YL797 contained a NotI site. The PCR reactions, using primer pairs YL794/YL797 or YL796/YL795, with Euglena gracilis cDNA (the generation of which is described in U.S. Provisional Application No. 60/801,172 (filed May 17, 2006; Attorney Docket No. CL-3486)) as template, were individually carried out in a 50 pt 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 7.degree. C. for 10 min. The individual PCR products were purified using a Qiagen PCR purification kit. The PCR product from the reaction amplified with primers YL794/797 was digested with NcoI and HindIII, while the PCR product from the reaction amplified with primers YL796/YL795 was digested with HindIII and NotI. The NcoI/HindIII and the HindIII NotI digested DNA fragments were purified following gel electrophoresis in 1% (w/v) agarose, and then directionally ligated with NcoI/NotI digested pZUF17 (FIG. 2B; SEQ ID NO:7; comprising a synthetic delta-17 desaturase gene ["D17st"] derived from S. diclina (U.S. Publication No. 2003/0196217 A1), codon-optimized for Yarrowia lipolytica (PCT Publication No. WO 2004/101757)). The product of this ligation was pDMW367 (FIG. 2C; SEQ ID NO:8), which thereby contained the following components:

TABLE-US-00004 TABLE 4 Components of Plasmid pDMW367 (SEQ ID NO: 8) RE Sites and Nucleotides Within Description Of Fragment And SEQ ID NO: 8 Chimeric Gene Components EcoR I/BsiW I FBAIN::EgD5::Pex20, comprising: (7416-1617) FBAIN: Yarrowia lipolytica FBAIN promoter (WO 2005/049805) EgD5: Euglena gracilis delta-5 desaturase (SEQ ID NO: 2 described herein) Pex20: Pex20 terminator sequence of Yarrowia Pex20 gene (GenBank Accession No. AF054613) 2707-1827 ColE1 plasmid origin of replication 3637-2777 ampicillin-resistance gene (Amp.sup.R) for selection in E. coli 4536-5840 Yarrowia autonomous replication sequence (ARS18; GenBank Accession No. A17608) 7373-5886 Yarrowia Ura 3 gene (GenBank Accession No. AJ306421)

[0326] 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.

Colony Lifts:

[0327] Approximately 17,000 clones of cDNA library eug1c were plated onto three large square (24 cm.times.24 cm) petri plates (Corning, Corning, N.Y.) each containing LB+50 .mu.g/mL kanamycin agar media. Cells were grown overnight at 37.degree. C. and plates were then cooled to room temperature.

[0328] Biodyne B 0.45 .mu.m membrane (Cat. No. 60207, Pall Corporation, Pensacola, Fla.) was trimmed to approximately 22 cm.times.22 cm and the membrane was carefully layed on top of the agar to avoid air bubbles. After incubation for 2 min at room temperature, the membrane was marked for orientation, lifted off with tweezers and placed colony-side up on filter paper soaked with 0.5 M sodium hydroxide and 1.5 M sodium chloride. After denaturation for 4 min, the sodium hydroxide was neutralized by placing the membrane on filter paper soaked with 0.5 M Tris-HCL (pH 7.5) and 1.5 M sodium chloride for 4 min. This step was repeated and the membrane was rinsed briefly in 2.times.SSC buffer (20.times.SSC is 3M sodium chloride, 0.3 M sodium citrate; pH 7.0) and air dried on filter paper.

Hybridization:

[0329] Membranes were pre-hybridized at 65.degree. C. in 200 mL hybridization solution for 2 h. Hybridization solution contained 6.times.SSPE (20.times.SSPE is 3 M sodium chloride, 0.2 M sodium phosphate, 20 mM EDTA; pH 7.4), 5.times.Denhardt's reagent (100.times.Denhardt's reagent is 2% (w/v) Ficoll, 2% (w/v) polyvinylpyrrolidone, 2% (w/v) acetylated bovine serum albumin), 0.5% sodium dodecyl sulfate (SDS), 100 .mu.g/mL sheared salmon sperm DNA and 5% dextran sulfate.

[0330] A DNA probe was made using an agarose gel purified NcoI/NotI DNA fragment, containing the Euglena gracilis delta-5 desaturase gene, from pDMW367 (SEQ ID NO:8) labeled with P.sup.32 dCTP using the RadPrime DNA Labeling System (Cat. No. 18428-011, Invitrogen, Carlsbad, Calif.) following the manufacture's instructions. Unincorporated P.sup.32 dCTP was separated using a NICK column (Cat. No. 17-0855-02, Amersham Biosciences, Piscataway, N.J.) following the manufacturer's instructions. The probe was denatured for 5 min at 100.degree. C., placed on ice for 3 min and half was added to the hybridization solution.

[0331] The membrane was hybridized with the probe overnight at 65.degree. C. with gentle shaking and then washed the following day twice with 2.times.SSC containing 0.5% SDS (5 min each) and twice with 0.2.times.SSC containing 0.1% SDS (15 min each). After washing, hyperfilm (Cat. No. RPN30K, Amersham Biosciences, Piscataway, N.J.) was exposed to the membrane overnight at -80.degree. C.

[0332] Based on alignment of plates with the exposed hyperfilm, positive colonies were picked using the blunt end of a Pasteur pipette into 1 mL of water and vortexed. Several dilutions were made and plated onto small round Petri dishes (82 mm) containing LB media plus 50 .mu.g/mL kanamycin to obtain around 100 well isolated colonies on a single plate. Lifts were done as described above except NytranN membrane circles (Cat, No. 10416116, Schleicher & Schuell, Keene, N.H.) were used and hybridization was carried out in 100 mL using the remaining radiolabeled probe. In this way, positive clones were confirmed.

[0333] Individual positive clones were grown at 37.degree. C. in LB+50 .mu.g/mL kanamycin liquid media and plasmid was purified using the QIAprep.RTM. Spin Miniprep Kit (Qiagen Inc.) following the manufacturer's protocol.

[0334] DNA inserts were end-sequenced in 384-well plates, using vector-primed M13F universal primer (SEQ ID NO:9), M13rev-28 primer (SEQ ID NO:10) and the poly(A) tail-primed WobbleT oligonucleotides, with 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 ABI 3700 automated sequencers. The WobbleT primer is an equimolar mix of 21 mer poly(T)A, poly(T)C, and poly(T)G, used to sequence the 3' end of cDNA clones.

[0335] Sequences were aligned and compared using Sequencher.TM. (Version 4.2, Gene Codes Corporation, Ann Arbor, Mich.) and in this way, it was determined that all all of the CDS in each cDNA were identical. A representative clone containing a cDNA (pLF119) is shown in SEQ ID NO:11 and the gene contained within the cDNA was called EaD5Des1. The coding sequence for EaD5Des1 is shown in SEQ ID NO:12. The corresponding amino acid sequence for EaD5Des1 is shown in SEQ ID NO:13.

Example 3

Primary Sequence Analysis of the Delta-5 Desaturase Sequence of Euglena anabaena UTEX 373 (EaD5Des1) and Comparison to the Delta-5 Desaturase Sequence of Euglena gracilis (EgD5)

[0336] The amino acid sequence for EaD5Des1 (SEQ ID NO:13) was evaluated by BLASTP (Basic Local Alignment Search Tool; Altschul 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 CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the last major release of the SWISS-PROT protein sequence database, EMBL and DDBJ databases) using default parameters with the filter turned off. For convenience, 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.

[0337] BLASTP analysis with EaD5Des1 yielded a pLog value of 76.52 (P value of 3e-77) versus the Thalassiosira pseudonana delta-8 fatty acid desaturase (TpsD8; SEQ ID NO:14) (NCBI Accession No. AAX14502(GI 60172920), locus AAX14502, CDS AY817152; Tonon et al., FEBS J. 272:3401-3412 (2005)) when compared to the "nr" database. Although identified as a delta-8 fatty acid desaturase in the NCBI database, AY817152 was identified as a delta-5 desaturase in Tonon et al. and the NCBI designation as a delta-8 fatty acid desaturase is likely an error. BLASTP analysis with EaD5Des1 also yielded a pLog value of 75.70 (P value of 2e-76) versus the Phaeodactylum tricornutum delta-5 fatty acid desaturase (SEQ ID NO:15) (NCBI Accession No. AAL92562(GI 19879687), locus AAL92562, CDS AY082392; Domergue et al., Eur. J. Biochem. 269:4105-4113 (2002)) when compared to the "nr" database.

[0338] The amino acid sequence for EaD5Des1 (SEQ ID NO:13) was compared to the Thalassiosira pseudonana delta-8 fatty acid desaturase (SEQ ID NO:14) and the Euglena gracilis delta-5 desaturase amino acid sequence (EgD5; SEQ ID NO:16; which is described in U.S. Provisional Application No. 60/801,172 (filed May 17, 2006; Attorney Docket No. CL-3486)) using BlastP, Clustal V and the Jotun Hein methods of sequence comparison. The % identity against the TpsD8 and EgD5 using each method is shown in Table 5 and Table 6, respectively.

[0339] Sequence percent identity calculations performed by the BlastP method are as described above. Sequence percent identity calculations were 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)) 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=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5 and GAP LENGTH PENALTY=10).

[0340] 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).

TABLE-US-00005 TABLE 5 Sequence Comparison of EaD5Des1 (SEQ ID NO: 13) to TpsD8 (SEQ ID NO: 14) % Identity to % Identity to % Identity to TpsD8 TpsD8 by the TpsD8 by the Desaturase by BLASTP Jotun Hein Method Clustal V Method EaD5Des1 37% 40.8% 30.8% (SEQ ID NO: 13)

TABLE-US-00006 TABLE 6 Sequence Comparison of EaD5Des1 (SEQ ID NO: 13) to EqD5 (SEQ ID NO: 16) % Identity to % Identity to % Identity to EgD5 EgD5 by the EgD5 by the Desaturase by BLASTP Jotun Hein Method Clustal V Method EaD5Des1 73% 72.4% 77.1% (SEQ ID NO: 13)

Example 4

Functional Analysis of the Euglena gracilis UTEX 373 Delta-5 Desaturase (EaD5Des1) in Yarrowia lipolytica

[0341] The present Example describes functional analysis of EaD5Des1 (SEQ ID NO:13) in Yarrowia lipolytica. This work included the following steps: (1) Construction of Gateway.RTM.-compatible Yarrowia expression vector pY159; (2) transfer of EaD5Des1 (SEQ ID NO:12) into pY159 to produce pY169; and, (3) comparison of lipid profiles within transformant organisms comprising pY169.

Construction of Gateway.RTM.-Compatible Yarrowia Expression Vector pY159

[0342] Plasmid pY5-30 (which was previously described in PCT Publication No. WO 2005/003310 (the contents of which are hereby incorporated by reference)), is a shuttle plasmid that can replicate both in E. coli and Yarrowia lipolytica. Plasmid pY5-30 contains the following: a Yarrowia autonomous replication sequence (ARS18); a ColE1 plasmid origin of replication; an ampicillin-resistance gene (Amp.sup.R), for selection in E. coli; a Yarrowia LEU2 gene, for selection in Yarrowia; and a chimeric TEF::GUS::XPR gene. Plasmid pDMW263 (SEQ ID NO:17) was created from pY5-30, by replacing the TEF promoter with the Yarrowia lipolytica FBAINm promoter (PCT Publication No. WO 2005/049805) using techniques well known to one skilled in the art. Briefly, this promoter refers to a modified promoter which is located in the 5' upstream untranslated region in front of the `ATG` translation initiation codon of the 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, wherein FBAINm has a 52 bp deletion between the ATG translation initiation codon and the intron of the FBAIN promoter (thereby including only 22 amino acids of the N-terminus) and a new translation consensus motif after the intron. Table 7 summarizes the components of pDMW263 (SEQ ID NO:17).

TABLE-US-00007 TABLE 7 Components of Plasmid pDMW263 (SEQ ID NO: 17) RE Sites and Nucleotides Within Description of Fragment and SEQ ID NO: 17 Chimeric Gene Components 4992-4296 ARS18 sequence (GenBank Accession No. A17608) SalI/SacII FBAINm::GUS::XPR, comprising: (8505-2014) FBAINm: FBAINm promoter (WO2005/049805) GUS: E. coli gene encoding .beta.-glucuronidase (Jefferson, R. A. Nature. 14: 342: 837-838 (1989) XPR: ~100 bp of the 3' region of the Yarrowia Xpr gene (GenBank Accession No. M17741) 6303-8505 Yarrowia Leu2 gene (GenBank Accession No. AF260230)

[0343] The NcoI/SalI DNA fragment from pDMW263 (SEQ ID NO:17), containing the Yarrowia lipolytica FBAINm promoter, was cloned into the NcoI/SalI DNA fragment of pDMW237 (SEQ ID NO:18), previously described in PCT Publication No. WO 2006/012325 (the contents of which are hereby incorporated by reference), containing a synthetic delta-9 elongase gene derived from Isochrysis galbana and codon-optimized for expression in Yarrowia lipolytica (IgD9eS), to produce pY115 (SEQ ID NO:19; FIG. 3). In FIG. 3, the modified FBAINm promoter is called FBA1+Intron. It is also FBA1+Intron in other figures, as well as YAR FBA1 PRO+Intron and these terms are used interchangeably with FBAINm.

[0344] The FBAINm promoter was amplified from plasmid pY115 (SEQ ID NO:19), using PCR with oligonucleotide primers oYFBA1 (SEQ ID NO:20) and oYFBA1-6 (SEQ ID NO:21). Primer oYFBA1 (SEQ ID NO:20) was designed to introduce an BglII site at the 5' end of the promoter and primer oYFBA1-6 (SEQ ID NO:21) was designed to introduce a NotI site at the 3' end of the promoter while removing the NcoI site and thus, the ATG start codon. The resulting PCR fragment was digested with BglII and NotI and cloned into the BglII/NotI fragment of pY115, containing the vector backbone, to form pY158 (SEQ ID NO:22).

[0345] Plasmid pY158 (SEQ ID NO:22) was digested with NotI and the resulting DNA ends were filled. After filling to form blunt ends, the DNA fragments were treated with calf intestinal alkaline phosphatase and separated using agarose gel electrophoresis. The 6992 bp fragment containing the Yarrowia lipolytica FBAINm promoter was excised from the agarose gel and purified using the QIAquick.RTM. Gel Extraction Kit (Qiagen Inc., Valencia, Calif.) following the manufacturer's protocol. The purified 6992 bp fragment was ligated with cassette rfA using the Gateway Vector Conversion System (Cat. No. 11823-029, Invitrogen Corporation) following the manufacturer's protocol to form Yarrowia lipolytica Gateway.RTM. destination vector pY159 (SEQ ID NO:23; FIG. 4).

Construction of Yarrowia Expression Vectors pY169

[0346] Using the Gateway.RTM. LR Clonase.TM. II enzyme mix (Cat. No. 11791-020, Invitrogen Corporation) and following the manufacturer's protocol, the cDNA insert from pLF119 (SEQ ID NO:11) was transferred to pY159 (SEQ ID NO:23) to form pY169 (SEQ ID NO:24, FIG. 5). In FIG. 5, EaD5Des1 is identified as EaD5-1 but they are identical.

Functional Analysis of EaD5Des1 in Yarrowia lipolytica

[0347] 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.

[0348] Strain Y2224 was transformed with pY169 (SEQ ID NO:24, FIG. 5) as described in the General Methods.

[0349] Single colonies of transformant Yarrowia lipolytica containing pY169 were grown in 3 mL minimal media lacking uracil supplemented with 0.2% tergitol at 30.degree. C. for 1 day. After this, 0.1 mL was transferred to 3 mL of the same medium supplemented with either ALA, EDA, ERA, DGLA, ETA, EPA, DPA 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 30 min. at 50.degree. C. after which 500 .mu.L of 1M sodium chloride and 100 .mu.L of heptane were added. After thorough mixing and centrifugation, fatty acid methyl esters (FAMEs) were analyzed by GC. 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 (Cat. 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. In the case of DPA feeding, GC analysis was carried out in a similar way except that the oven temperature was programmed to hold at 170.degree. C. for 1.0 min, increase to 240.degree. C. at 5.degree. C./min and then hold for an additional 1.0 min. Retention times were compared to those for methyl esters of standards commercially available (Nu-Chek Prep, Inc.).

[0350] The fatty acid profiles for Yarrowia lipolytica expressing pY169 and fed various substrates are shown in FIG. 6. Substrates (either LA--when no fatty acid fed, ALA, EDA, ERA, DGLA, ETA or DPA) were fed to assess delta-4 (DPA to DHA), delta-5 (DGLA to ARA, DTA to EPA, EDA to SCI, ERA to JUP), delta-6 (LA to GLA, ALA to STA), delta-8 (EDA to DGLA, ERA to ETA) or omega-3 (LA to ALA, EDA to ERA, DGLA to ETA) desaturase activities. Percent desaturation (% desat) was calculated by dividing the wt. % for substrate (either LA-when no fatty acid fed, ALA, EDA, ERA, DGLA, ETA or DPA) by the sum of the wt. % for the substrate (either LA--when no fatty acid fed, ALA, EDA, ERA, DGLA, ETA or DPA) and product (either GLA, STA, DGLA, ETA, ARA, EPA or DHA, respectively) and multiplying by 100 to express as a %, depending on which substrate was fed. In FIG. 6. shading indicates the substrates fed and products produced. Averages are indicated by Ave. followed by appropriate header. From the results in FIG. 6, it is clear that EaD5Des1 functions as a delta-5 desaturase with preference for DGLA and ETA over EDA and ERA. The ratio of desaturation of omega-6 substrate to omega-3 substrate (Ratio n-6/n-3) is calculated by dividing the Ave. % desat for either DGLA by ETA or EDA by ERA. In both cases, EaD5Des1 prefers n-6 substrates over n-3 substrates. The ratio of desaturation of the preferred substrate to that of the non-preferred substrate (Ratio Prod/By-Prod) is calculated by dividing the Ave. % desat for either DGLA by EDA or ETA by ERA. In both cases, EaD5Des1 has an approximately 3.5-fold preference for DGLA or ETA over EDA or ERA, respectively.

Example 5

Construction of Soybean Expression Vector pKR1153 for Co-Expression of the Euglena anabaena UTEX 373 Delta-5 Desaturase (EaD5Des1) with a Delta-9 Elongase Derived from Euglena gracilis (EgD9e) and a Delta-8 Desaturase Derived from Euglena gracilis (EgD8)

[0351] The present Example describes construction of a soybean vector for co-expression of EaD5Des1 with EgD9e (SEQ ID NO:25; which is described in U.S. application Ser. No. 11/601,563 (filed Nov. 16, 2006, which published May 24, 2007; Attorney Docket No. BB-1562) and EgD8 (SEQ ID NO:26; described as Eg5 in PCT Publication No. WO 2006/012325)

Euglena gracilis Delta-9 Elongase (EgD9e):

[0352] A clone from the Euglena cDNA library (eeg1c), called eeg1c.pk001.n5f, containing the Euglena gracilis delta-9 elongase (EgD9e; SEQ ID NO:25; which is described in U.S. application Ser. No. 11/601,563 (filed Nov. 16, 2006, which published May 24, 2007; Attorney Docket No. BB-1562) the contents of which are hereby incorporated by reference) was used as template to amplify EgD9e with oligonucleotide primers oEugEL1-1 (SEQ ID NO:27) and oEugEL1-2 (SEQ ID NO:28) using the VentR.RTM. DNA Polymerase (Cat. No. M0254S, 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:29).

[0353] A starting plasmid pKR72 (ATCC Accession No. PTA-6019; SEQ ID NO:30, 7085 bp 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 (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)) (35S/HPT/NOS3' cassette) for selection in plants such as soybean. pKR72 also contains a NotI restriction site, flanked by the promoter for the a' 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.

[0354] The AscI fragment from plasmid pKS102 (SEQ ID NO:31), previously described in PCT Publication No. WO 02/00905 (the contents of which are hereby incorporated by reference), containing the T7prom/hpt/T7term cassette and bacterial ori, was combined with the Asc1 fragment of plasmid pKR72 (SEQ ID NO:30), containing the .beta.con/NotI/Phas cassette to produce pKR197 (SEQ ID NO:32), previously described in PCT Publication No. WO 04/071467 (the contents of which are hereby incorporated by reference).

[0355] The gene for the Euglena gracilis delta-9 elongase was released from pKR906 (SEQ ID NO:29) by digestion with NotI and cloned into the NotI site of pKR197 to produce intermediate cloning vector pKR911 (SEQ ID NO:33).

Euglena gracilis Delta-8 Desaturase (EgD8):

[0356] Plasmid pKR680 (SEQ ID NO:34), 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:26; 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 (Kti/NotI/Kti3'Salb3' cassette).

[0357] Plasmid pKR680 (SEQ ID NO:34) was digested with BsiWI and the fragment containing EgD8 was cloned into the BsiWI site of pKR911 (SEQ ID NO:33) to produce pKR913 (SEQ ID NO:35).

Euglena anabaena UTEX 373 Delta-5 Desaturase (EaD5Des1):

[0358] In order to introduce NotI sites at the 5' and 3' ends of the coding sequence, EaD5Des1 was PCR amplified from pLF119 (SEQ ID NO:11) with oligonucleotide primers oEAd5-1-1 (SEQ ID NO:36) and oEAd5-1-2 (SEQ ID NO:37) using the Phusion.TM. High-Fidelity DNA Polymerase (Cat. No. F553S, Finnzymes Oy, Finland) 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 pKR1136 (SEQ ID NO:38).

[0359] Plasmid pKR767 (SEQ ID NO:39), 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:40, which is described in U.S. Pat. No. 6,075,183 and PCT Publication Nos. WO 2004/071467 and WO 2005/047479) flanked by the promoter for the soybean glycinin Gy1 gene and the pea leguminA2 3' transcription termination region (Gy1/MaD5/legA2 cassette; the construction of which is described in WO 2006/012325). Plasmid pKR974 (SEQ ID NO:41) is identical to pKR767 (SEQ ID NO:40) except the NotI fragment containing MaD5 has been replaced with a NotI fragment containing the Saprolegnia diclina delta-5 desaturase (SaD5; SEQ ID NO:42, which is described in PCT Publication No. WO 2004/071467. In addition, an MfeI site in the legA2 terminator of pKR974 (SEQ ID NO:41) 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:41) is 770 bp versus 766 bp for pKR767 (SEQ ID NO:40).

[0360] The gene for the Euglena anabaena delta-5 desaturase was released from pKR1136 (SEQ ID NO:38) by digestion with NotI and cloned into the NotI site of pKR974 (SEQ ID NO:41) to produce pKR1139 (SEQ ID NO:43).

[0361] Plasmid pKR1139 (SEQ ID NO:43) was digested with SbfI and the fragment containing the Euglena anabaena delta-5 desaturase was cloned into the SbfI site of pKR913 (SEQ ID NO:35) to produce pKR1153 (SEQ ID NO:44, FIG. 7). In this way, the Euglena anabaena delta-5 desaturase (EaD5Des1) could be co-expressed with the Euglena gracilis delta-8 desaturase (EgD8) and the Euglena gracilis delta-9 elongase (EgD9e) behind strong, seed-specific promoters. In FIG. 7, EaD5Des1, EgD8 and EgD9e are referred to as EA d5 DS, eug d8-sq5 and eug ell, respectively.

Example 6

Production and Model System Transformation of Somatic Soybean Embryo Cultures with Soybean Expression Vectors and Plant Regeneration Culture Conditions

[0362] 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/m2/s. Cultures are subcultured every 7 days to two weeks by inoculating approximately 35 mg of tissue into 35 mL of fresh liquid SB196 (the preferred subculture interval is every 7 days).

Soybean embryogenic suspension cultures are transformed with the 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:

[0363] Soybean cultures are initiated twice each month with 5-7 days between each initiation. Pods with immature seeds from available soybean plants are picked 45-55 days after planting. Seeds are removed from the pods 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. When cultures are being prepared for production transformation, cotyledons are transferred to plates containing SB1 medium (25-30 cotyledons per plate). Plates are wrapped with fiber tape and are maintained at 26.degree. C. with cool white fluorescent lights on 16:8 h day/night photoperiod at light intensity of 60-80 .mu.E/m2/s for eight weeks, with a media change after 4 weeks. When cultures are being prepared for model system experiments, cotyledons are transferred to plates containing SB199 medium (25-30 cotyledons per plate) for 2 weeks, and then transferred to SB1 for 2-4 weeks. Light and temperature conditions are the same as described above. After incubation on SB1 medium, secondary embryos are cut and placed into SB196 liquid media for 7 days.

Preparation of DNA for Bombardment:

[0364] Either an intact plasmid or a DNA plasmid fragment containing the genes of interest and the selectable marker gene are used for bombardment. Fragments from soybean expression plasmids are obtained by gel isolation of digested plasmids. In each case, 100 .mu.g of plasmid DNA is used in 0.5 mL of the specific enzyme mix described below. Plasmids are digested with AscI (100 units) in NEBuffer 4 (20 mM Tris-acetate, 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM dithiothreitol, pH 7.9), 100 .mu.g/mL BSA, and 5 mM beta-mercaptoethanol at 37.degree. C. for 1.5 hr. 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 manufacturer's protocol.

[0365] A 50 .mu.L aliquot of sterile distilled water containing 3 mg of gold particles (3 mg gold) is added to 30 .mu.L of a 10 ng/.mu.L DNA solution (either intact plasmid or DNA fragment prepared as described herein), 25 .mu.L 5M 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. The supernatant is removed, followed by a wash with 400 .mu.L 100% ethanol and another brief centrifugation. The 400 ul ethanol is removed and the pellet is resuspended in 40 .mu.L of 100% ethanol. Five .mu.L of DNA suspension is dispensed to each flying disk of the Biolistic PDS1000/HE instrument disk. Each 5 .mu.L aliquot contains approximately 0.375 mg gold per bombardment (e.g., per disk).

[0366] For model system transformations, the protocol is identical except for a few minor changes (ie, 1 mg of gold particles is added to 5 .mu.L of a 1 .mu.g/.mu.L DNA solution, 50 .mu.L of a 2.5M CaCl.sub.2 is used and the pellet is ultimately resuspended in 85 .mu.L of 100% ethanol thus providing 0.058 mg of gold particles per bombardment).

Tissue Preparation and Bombardment with DNA:

[0367] Approximately 150-200 mg of seven day old embryogenic suspension cultures is placed in an empty, sterile 60.times.15 mm petri dish and the dish is covered with plastic mesh. The chamber is evacuated to a vacuum of 27-28 inches of mercury, and tissue is bombarded one or two shots per plate with membrane rupture pressure set at 1100 PSI. Tissue is placed approximately 3.5 inches from the retaining/stopping screen. Model system transformation conditions are identical except 100-150 mg of embryogenic tissue is used, rupture pressure is set at 650 PSI and tissue is place approximately 2.5 inches from the retaining screen.

Selection of Transformed Embryos:

[0368] Transformed embryos are selected either using hygromycin (when the hygromycin B phosphotransferase (HPT) gene is used as the selectable marker) or chlorsulfuron (when the acetolactate synthase (ALS) gene is used as the selectable marker).

[0369] Following bombardment, the tissue is placed into fresh SB196 media and cultured as described above. Six to eight days post-bombardment, the SB196 is exchanged with fresh SB196 containing either 30 mg/L hygromycin or 100 ng/mL chlorsulfuron, depending on the selectable marker used. The selection media is refreshed weekly. Four to six weeks post-selection, green, transformed tissue is observed growing from untransformed, necrotic embryogenic clusters.

Embryo Maturation:

[0370] For production transformations, isolated, green tissue is removed and inoculated into multiwell plates to generate new, clonally propagated, transformed embryogenic suspension cultures. Transformed embryogenic clusters are cultured for four-six weeks in multiwell plates 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 one-two weeks and then subcultured to SB103 medium for 3-4 weeks to mature embryos. After maturation on plates in SB103, individual embryos are removed from the clusters, dried and screened for alterations in their fatty acid compositions as described in Example 7.

[0371] For model system transformations, embryos are 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 above, embryo clusters are removed to 35 mL of SB228 (SHaM liquid media) in a 250 mL Erlenmeyer flask. Tissue is 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/m2/s for 2 weeks as embryos mature. Embryos grown for 2 weeks in SHaM liquid media are equivalent in size and fatty acid content to embryos cultured on SB166/SB103 for 5-8 weeks.

[0372] After maturation in SHaM liquid media, individual embryos are removed from the clusters, dried and screened for alterations in their fatty acid compositions as described in Example 7.

Media Recipes:

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

TABLE-US-00008 [0373] 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-00009 [0374] 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)

TABLE-US-00010 [0375] 1 package MS salts (Gibco/BRL - Cat. No. 11117-066) 1 mL B5 vitamins 1000X stock 31.5 g glucose 2 mL 2,4-D (20 mg/L final concentration) pH 5.7 8 g TC agar

SB199 Solid Medium (Per Liter)

TABLE-US-00011 [0376] 1 package MS salts (Gibco/BRL - Cat. No. 11117-066) 1 mL B5 vitamins 1000X stock 30 g Sucrose 4 ml 2,4-D (40 mg/L final concentration) pH 7.0 2 gm Gelrite

SB 166 Solid Medium (Per Liter)

TABLE-US-00012 [0377] 1 package MS salts (Gibco/BRL - Cat. No. 11117-066) 1 mL B5 vitamins 1000X stock 60 g maltose 750 mg MgCl.sub.2 hexahydrate 5 g activated charcoal pH 5.7 2 g gelrite

SB 103 Solid Medium (Per Liter)

TABLE-US-00013 [0378] 1 package MS salts (Gibco/BRL - Cat. No. 11117-066) 1 mL B5 vitamins 1000X stock 60 g maltose 750 mg MgCl2 hexahydrate pH 5.7 2 g gelrite

SB 71-4 Solid Medium (Per Liter)

TABLE-US-00014 [0379] 1 bottle Gamborg's B5 salts w/sucrose (Gibco/BRL - Cat. No. 21153-036) pH 5.7 5 g TC agar

2,4-D Stock

TABLE-US-00015 [0380] Obtain premade from Phytotech Cat. No. D 295 - concentration 1 mg/mL

B5 Vitamins Stock (Per 100 mL)

TABLE-US-00016 [0381] Store aliquots at -20.degree. C. 10 g myo-inositol 100 mg nicotinic acid 100 mg pyridoxine HCl 1 g thiamine

If the solution does not dissolve quickly enough, apply a low level of heat via the hot stir plate.

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

TABLE-US-00017 [0382] 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 w/o glutamine.

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

TABLE-US-00018 [0383] (NH.sub.4)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-00019 [0384] H.sub.3BO.sub.3 (boric acid) 6.2 g MnSO.sub.4*H.sub.2O (manganese sulfate monohydrate) 16.9 g ZnSO4*7H20 (zinc sulfate heptahydrate) 8.6 g Na.sub.2MoO.sub.4*2H20 (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-00020 [0385] Na.sub.2EDTA* (sodium EDTA) 3.73 g FeSO.sub.4*7H.sub.20 (iron sulfate heptahydrate) 2.78 g *EDTA must be completely dissolved before adding iron. Bring to Volume

Solution is photosensitive. Bottle(s) should be wrapped in foil to omit light. Autoclave

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

TABLE-US-00021 [0386] 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-00022 [0387] 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-00023 [0388] 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.

Regeneration of Soybean Somatic Embryos into Plants:

[0389] In order to obtain whole plants from embryogenic suspension cultures, the tissue must be regenerated. Embryos are matured as described in above. After subculturing on medium SB103 for 3 weeks, individual embryos can be removed from the clusters and screened for alterations in their fatty acid compositions as described in Example 7. It should be noted that any detectable phenotype, resulting from the expression of the genes of interest, could 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.

[0390] 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 24-cell pack tray, covered with clear plastic dome. After 2 weeks the dome is removed and plants hardened off for a further week. If plantlets looked hardy they are transplanted to 10'' pot 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.

Example 7

Fatty Acid Analysis of Transgenic Somatic Soybean Embryos

[0391] 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 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, triacylglycerol 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.

[0392] A subset of soybean embryos for each event generated from either production transformation or model system transformation (as described in Example 6) are harvested in the following way. Embryos (5-10 embryos) from each event are picked into glass GC vials and fatty acid methyl esters are prepared by transesterification. For transesterification, 50 .mu.L of trimethylsulfonium hydroxide (TMSH) and 0.5 mL of hexane is 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) are separated and quantified using a Hewlett-Packard 6890 Gas Chromatograph fitted with an Omegawax 320 fused silica capillary column (Cat. No. 24152, Supelco Inc.). The oven temperature is 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.). Events having good phenotype can be re-analyzed by GC using identical conditions except the oven temperature is held at 150.degree. C. for 1 min and then increased to 240.degree. C. at 5.degree. C./min.

Example 8

Construction of Alternate Soybean Expression Vectors for Expression of Euglena anabaena UTEX 373 Delta-5 Desaturase (EaD5Des1)

[0393] 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 expression of EaD5Des1. Similarly, it may be desirable to express other PUFA genes (such as those described below in Table 10), for co-expression with the delta-5 desaturase of the present invention.

[0394] For instance, PCT Publication Nos. WO 2004/071467 and 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 Publication Nos. WO 2004/071467, WO 2005/047479 and 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 (such as those listed in, but not limited to, Table 8) and a transcription terminator (such as those listed in, but not limited to, Table 9) 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 7 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.

[0395] In addition, PCT Publication Nos. WO 2004/071467, WO 2005/047479 and 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-00024 TABLE 8 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 WO 2004/071467 glycinin Gy1 soybean 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 WO 2004/071467 BD30 (also called P34) soybean WO 2004/071467 legumin A2 pea Rerie et al., Mol. Gen. Genet. 225: 148-157 (1991)

TABLE-US-00025 TABLE 9 Transcription Terminators Transcription Terminator Organism Reference phaseolin 3' bean WO 2004/071467 kunitz trypsin inhibitor 3' soybean WO 2004/071467 BD30 (also called P34) 3' soybean WO 2004/071467 legumin A2 3' pea WO 2004/071467 albumin 2S 3' soybean WO 2004/071467

TABLE-US-00026 TABLE 10 PUFA Biosynthetic Pathway Genes Gene Organism Reference delta-6 desaturase Saprolegnia diclina WO 2002/081668 delta-6 desaturase Mortierella alpina U.S. Pat. No. 5,968,809 elongase Mortierella alpina 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 WO 2002/081668 delta-5 desaturase Peridinium sp. U.S. Provisional Application No. 60/801,119 delta-5 desaturase Euglena gracilis U.S. Provisional Application No. 60/801,172 delta-15 desaturase Fusarium WO 2005/047479 moniliforme delta-17 desaturase Saprolegnia diclina WO 2002/081668 elongase Thraustochytrium WO 2002/08401 aureum U.S. Pat. No. 6,677,145 elongase Pavlova sp. Pereira et al., Biochem. J. 384: 357-366 (2004) delta-4 desaturase Schizochytrium WO 2002/090493 aggregatum U.S. Pat. No. 7,045,683 delta-4 desaturase Isochrysis galbana WO 2002/090493 U.S. Pat. No. 7,045,683 delta-4 desaturase Thraustochytrium WO 2002/090493 aureum U.S. Pat. No. 7,045,683 delta-4 desaturase Euglena gracilis U.S. patent application No. 10/552,127 delta-9 elongase Isochrysis galbana WO 2002/077213 delta-9 elongase Euglena gracilis U.S. patent application No. 11/601,563 delta-9 elongase Eutreptiella sp. U.S. patent application No. CCMP389 11/601,564 delta-8 desaturase Euglena gracilis WO 2000/34439 U.S. Pat. No. 6,825,017 WO 2004/057001 WO 2006/012325 delta-8 desaturase Acanthamoeba Sayanova et al., FEBS Lett. castellanii 580: 1946-1952 (2006) delta-8 desaturase Pavlova salina WO 2005/103253 delta-8 desaturase Pavlova lutheri U.S. Provisional Application No. 60/795,810 delta-8 desaturase Tetruetreptia U.S. Provisional Application pomquetensis No. 60/853,563 CCMP1491 delta-8 desaturase Eutreptiella sp. U.S. Provisional Application CCMP389 No. 60/853,563 delta-8 desaturase Eutreptiella U.S. Provisional Application cf_gymnastica No. 60/853,563 CCMP1594

Example 9

Synthesis of a Codon-Optimized Delta-5 Desaturase Gene for Yarrowia lipolytica (EaD5S)

[0396] The codon usage of the delta-5 desaturase gene (EaD5) of Euglena anabaena was optimized for expression in Yarrowia lipolytica, in a manner similar to that described in PCT Publication No. WO 2004/101753. Specifically, a codon-optimized delta-5 desaturase gene (designated "EaD5S", SEQ ID NO:45) was designed based on the coding sequence of EaD5Des1 (SEQ ID NOs:12 and 13), 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, 183 bp of the 1362 bp coding region were modified (13.4%) and 174 codons were optimized (38.3%). The GC content was reduced from 57.6% within the wild type gene (i.e., EaD5Des1) to 54.6% within the synthetic gene (i.e., EaD5S). A NcoI site and NotI sites were incorporated around the translation initiation codon and after the stop codon of EaD5S (SEQ ID NO:45), respectively. FIGS. 8A, 8B and 8C show a comparison of the nucleotide sequences of EaD5Des1 (SEQ ID NO:12) and EaD5S (SEQ ID NO:45). The codon optimized EaD5S gene did not change any amino acid sequence of EaD5Des1 (SEQ ID NO:13). The designed EaD5S gene was synthesized by GenScript Corporation (Piscataway, N.J.) and cloned into pUC57 (GenBank Accession No. Y14837) to generate pEaD5S (SEQ ID NO:46; FIG. 9).

[0397] Based on the teachings herein concerning vector construction and suitable promoter and terminators for use in Yarrowia lipolytica, one of skill in the art will be able to construct additional plasmids suitable for expression of EaD5S (SEQ ID NO:45).

Example 10

Functional Analysis of the Euglena anabaena UTEX 373 Delta-5 Desaturase (EaD5Des1) Co-Expressed with a Delta-9 Elongase Derived from Euglena gracilis (EgD9e) a Delta-8 Desaturase Derived from Euglena gracilis (EgD8) and a Delta-17 Desaturase from Saprolegnia Diclina in Soy Somatic Embryos

[0398] The present example describes the transformation and expression in soybean somatic embryos of pKR1153 (SEQ ID NO:44; Example 5) comprising EaD5Des1, EgD9e and EgD8 along with pKR328 (described in PCT Publication No. WO 04/071467) comprising the Saprolegnia diclina delta-17 desaturase SdD17 under control of the annexin promoter and having a hygromycin resistance gene for selection in plants.

[0399] Soybean embryogenic suspension culture (cv. Jack) was transformed with pKR1153 (SEQ ID NO:44) and pKR328, and embryos were matured in soybean histodifferentiation and maturation liquid medium (SHaM liquid media; Schmidt et al., Cell Biology and Morphogenesis, 24:393 (2005)) as described in Example 6 and previously described in PCT Publication No. WO 2007/136877 (the contents of which are hereby incorporated by reference).

[0400] After maturation in SHaM liquid media, a subset of transformed soybean embryos (i.e., 5 embryos per event) were harvested and analyzed for fatty acid profiles by GC as described in Example 7 and herein.

[0401] In this way, approximately 30 events transformed with pKR1153 and pKR328 (Experiment MSE2140) were analyzed, and the ten events having the highest average correct delta-5 desaturase activities (average of the 5 embryos analyzed) are shown in FIG. 11.

[0402] In FIG. 11, 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 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.

[0403] The activity of the delta-5 desaturase is expressed as percent delta-5 desaturation ("% delta-5 desat"), calculated according to the following formula: ([product]/[substrate+product])*100. More specifically, the percent delta-5 desaturation was determined as: ([ARA+EPA]/[DGLA+ETA+ARA+EPA])*100.

Sequence CWU 1

1

4611524DNAEuglena anabaena 1acccagtgta cacctttgac agtcccttcg cccaggatgt caagcagagc gttcgggagg 60tcatgaaggg gcgcaactgg tacgccacgc ccggcttttg gctgcggacc gcgctgatca 120tcgcgtgcac tgccataggc gaatggtatt ggatcactac cggggcagtg atgtggggca 180tcttcaccgg gtacttccac agccagattg ggttggcgat tcaacacgat gcctctcacg 240gagccatcag caaaaagccc tgggtgaacg cctttttcgc ctacggcatc gacgccattg 300gatcctcccg ctggatctgg ctgcagtccc acattatgcg ccaccacacc tacaccaacc 360agcatggcct ggacctggac gctgcctcgg cggagccgtt cattttgttc cactcctacc 420cggcaacaaa tgcgtcacga aagtggtacc atcggttcca ggcgtggtac atgtacatcg 480ttttggggat gtatggtgtg tcgatggtgt acaatccgat gtacttgttc acgatgcagc 540acaacgacac aatcccagag gccacctctc ttagaccagg cagctttttc aaccggcagc 600gcgccttcgc cgtttccctc cgcctactgt tcatcttccg caacgccttc ctcccctggt 660acatcgcggg cgcctctccg ctgctcacca tcctgctggt gccaacggtc acaggcatct 720tcttgacatt tgtttttgtg ctgtcccata actttgaagg cgctgagcgg acccccgaaa 780agaactgcaa ggccaaaagg gccaaggagg ggaaggaggt ccgcgatgta gaggaggacc 840gggtggactg gtaccgggcg caggccgaga ccgcggcgac ctacgggggc agcgtcggga 900tgatgctgac cggcggtttg aacctgcaga tcgagcacca cttgttcccc cgcatgtcct 960cttggcacta ccccttcatc caagatacgg tgcgggaatg ttgcaagcgc catggcgtgc 1020gctacacata ctacccgacc atcctggaga atataatgtc cacgctccgc tacatgcaga 1080aggtgggcgt ggcccacaca attcaggatg cccaggaatt ctgagtgagt tcgatccgca 1140tcgacgtcta ccatttttga tgctgtctat tcctgttttc agtcacctcc agcattctca 1200tggctggtga ccactgcccc tctaacccat tgtgacacac cgccaaagac tttgcctctt 1260ttttttccct ttcttttgtc ctcggggtgc tttggccggt gtttactcgc cttgcagtcc 1320ccgcaaacga ccgacgttta agctccgttg ttgactgggc cgctcgtaaa cccatctgca 1380ggttgaggct cccatggaga attgtgatgg ctgattagga ggtggcgggg catacatgcc 1440tcgacactca aagccgggcg gcttctggat tcgaaaacgc aaatgggcgc tttggaaaaa 1500aaaaaaaaaa aaaaaaaaaa aaaa 152421347DNAEuglena gracilis 2atggctctca gtcttaccac agaacagctg ttagaacgcc ctgatttggt tgcgattgat 60ggcatcctct acgaccttga agggcttgcc aaagttcatc caggaggaga tttgattctc 120gcttctggtg cctctgatgc ctcccctctc ttttattcaa tgcatccata cgtcaaaccg 180gagaattcca aattgcttca acagttcgtc cgagggaagc atgaccgcac ctcgaaggac 240attgtctaca cgtatgattc tcccttcgca caagacgtta agcggacaat gcgcgaggtg 300atgaaaggga ggaactggta cgcaacccct ggcttctggc tgcgcaccgt tgggatcatc 360gccgtgacgg ccttttgcga gtggcactgg gctaccacgg ggatggtgct gtggggcctg 420ttgactggat tcatgcacat gcagatcggc ttatccatcc agcatgatgc gtcccacggg 480gccatcagca agaagccttg ggtcaacgcc ctcttcgcct acggcattga cgtcatcgga 540tcgtcccggt ggatttggct gcagtcgcac atcatgcggc accacaccta caccaaccag 600cacggcctcg acctggatgc ggagtcggca gagccgttcc tggtgttcca caactacccc 660gccgcaaaca ccgcccgaaa gtggttccac cgcttccaag cttggtacat gtaccttgtg 720ctgggggcat acggggtatc gctggtgtac aacccgctct acattttccg gatgcagcac 780aatgacacca tcccagagtc tgtcacggcc atgcgggaaa atggctttct gcggcgctac 840cgcacacttg cattcgtgat gcgagctttc ttcatcttcc ggaccgcatt cttgccctgg 900tacctcactg ggacctcatt gctgatcacc attcctctgg tgcccaccgc aactggtgcc 960ttcttgacgt tcttcttcat tttgtcccac aattttgatg gctccgaacg gatccccgac 1020aagaactgca aggttaagag atctgagaag gacgttgagg ctgaccaaat tgactggtat 1080cgggcgcagg tggagacgtc ctccacatac ggtggcccca tcgccatgtt cttcactggc 1140ggtctcaatt tccagatcga gcaccacctc tttccccgga tgtcgtcttg gcactacccc 1200ttcgtccagc aggcggtccg ggagtgttgc gaacgccatg gagtgcgata tgttttctac 1260cctaccatcg tcggcaacat catctccacc ctgaagtaca tgcataaggt gggtgtcgtc 1320cactgcgtga aggacgcaca ggattcc 1347332DNAArtificial SequenceEuglena gracilis delta-5 desaturase oligonucleotide YL794 3tttccatggc tctcagtctt accacagaac ag 32427DNAArtificial SequenceEuglena gracilis delta-5 desaturase oligonucleotide YL797 4gtacatgtac caagcttgga agcggtg 27527DNAArtificial SequenceEuglena gracilis delta-5 desaturase oligonucleotide YL796 5caccgcttcc aagcttggta catgtac 27639DNAArtificial SequenceEuglena gracilis delta-5 desaturase oligonucleotide YL795 6tttgcggccg cttaggaatc ctgtgcgtcc ttcacgcag 3978165DNAArtificial Sequenceplasmid pZUF17 7gtacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 60ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 120taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 180tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 240aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 300aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 360ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 420acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 480ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 540tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 600tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 660gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 720agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 780tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 840agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 900tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 960acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 1020tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 1080agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 1140tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 1200acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 1260tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 1320ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 1380agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 1440tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 1500acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 1560agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 1620actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 1680tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 1740gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 1800ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 1860tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 1920aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 1980tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 2040tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 2100gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 2160gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 2220acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 2280agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 2340ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 2400ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 2460taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 2520aacgcgaatt ttaacaaaat attaacgctt acaatttcca ttcgccattc aggctgcgca 2580actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 2640gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta 2700aaacgacggc cagtgaattg taatacgact cactataggg cgaattgggt accgggcccc 2760ccctcgaggt cgatggtgtc gataagcttg atatcgaatt catgtcacac aaaccgatct 2820tcgcctcaag gaaacctaat tctacatccg agagactgcc gagatccagt ctacactgat 2880taattttcgg gccaataatt taaaaaaatc gtgttatata atattatatg tattatatat 2940atacatcatg atgatactga cagtcatgtc ccattgctaa atagacagac tccatctgcc 3000gcctccaact gatgttctca atatttaagg ggtcatctcg cattgtttaa taataaacag 3060actccatcta ccgcctccaa atgatgttct caaaatatat tgtatgaact tatttttatt 3120acttagtatt attagacaac ttacttgctt tatgaaaaac acttcctatt taggaaacaa 3180tttataatgg cagttcgttc atttaacaat ttatgtagaa taaatgttat aaatgcgtat 3240gggaaatctt aaatatggat agcataaatg atatctgcat tgcctaattc gaaatcaaca 3300gcaacgaaaa aaatcccttg tacaacataa atagtcatcg agaaatatca actatcaaag 3360aacagctatt cacacgttac tattgagatt attattggac gagaatcaca cactcaactg 3420tctttctctc ttctagaaat acaggtacaa gtatgtacta ttctcattgt tcatacttct 3480agtcatttca tcccacatat tccttggatt tctctccaat gaatgacatt ctatcttgca 3540aattcaacaa ttataataag atataccaaa gtagcggtat agtggcaatc aaaaagcttc 3600tctggtgtgc ttctcgtatt tatttttatt ctaatgatcc attaaaggta tatatttatt 3660tcttgttata taatcctttt gtttattaca tgggctggat acataaaggt attttgattt 3720aattttttgc ttaaattcaa tcccccctcg ttcagtgtca actgtaatgg taggaaatta 3780ccatactttt gaagaagcaa aaaaaatgaa agaaaaaaaa aatcgtattt ccaggttaga 3840cgttccgcag aatctagaat gcggtatgcg gtacattgtt cttcgaacgt aaaagttgcg 3900ctccctgaga tattgtacat ttttgctttt acaagtacaa gtacatcgta caactatgta 3960ctactgttga tgcatccaca acagtttgtt ttgttttttt ttgttttttt tttttctaat 4020gattcattac cgctatgtat acctacttgt acttgtagta agccgggtta ttggcgttca 4080attaatcata gacttatgaa tctgcacggt gtgcgctgcg agttactttt agcttatgca 4140tgctacttgg gtgtaatatt gggatctgtt cggaaatcaa cggatgctca atcgatttcg 4200acagtaatta attaagtcat acacaagtca gctttcttcg agcctcatat aagtataagt 4260agttcaacgt attagcactg tacccagcat ctccgtatcg agaaacacaa caacatgccc 4320cattggacag atcatgcgga tacacaggtt gtgcagtatc atacatactc gatcagacag 4380gtcgtctgac catcatacaa gctgaacaag cgctccatac ttgcacgctc tctatataca 4440cagttaaatt acatatccat agtctaacct ctaacagtta atcttctggt aagcctccca 4500gccagccttc tggtatcgct tggcctcctc aataggatct cggttctggc cgtacagacc 4560tcggccgaca attatgatat ccgttccggt agacatgaca tcctcaacag ttcggtactg 4620ctgtccgaga gcgtctccct tgtcgtcaag acccaccccg ggggtcagaa taagccagtc 4680ctcagagtcg cccttaggtc ggttctgggc aatgaagcca accacaaact cggggtcgga 4740tcgggcaagc tcaatggtct gcttggagta ctcgccagtg gccagagagc ccttgcaaga 4800cagctcggcc agcatgagca gacctctggc cagcttctcg ttgggagagg ggactaggaa 4860ctccttgtac tgggagttct cgtagtcaga gacgtcctcc ttcttctgtt cagagacagt 4920ttcctcggca ccagctcgca ggccagcaat gattccggtt ccgggtacac cgtgggcgtt 4980ggtgatatcg gaccactcgg cgattcggtg acaccggtac tggtgcttga cagtgttgcc 5040aatatctgcg aactttctgt cctcgaacag gaagaaaccg tgcttaagag caagttcctt 5100gagggggagc acagtgccgg cgtaggtgaa gtcgtcaatg atgtcgatat gggttttgat 5160catgcacaca taaggtccga ccttatcggc aagctcaatg agctccttgg tggtggtaac 5220atccagagaa gcacacaggt tggttttctt ggctgccacg agcttgagca ctcgagcggc 5280aaaggcggac ttgtggacgt tagctcgagc ttcgtaggag ggcattttgg tggtgaagag 5340gagactgaaa taaatttagt ctgcagaact ttttatcgga accttatctg gggcagtgaa 5400gtatatgtta tggtaatagt tacgagttag ttgaacttat agatagactg gactatacgg 5460ctatcggtcc aaattagaaa gaacgtcaat ggctctctgg gcgtcgcctt tgccgacaaa 5520aatgtgatca tgatgaaagc cagcaatgac gttgcagctg atattgttgt cggccaaccg 5580cgccgaaaac gcagctgtca gacccacagc ctccaacgaa gaatgtatcg tcaaagtgat 5640ccaagcacac tcatagttgg agtcgtactc caaaggcggc aatgacgagt cagacagata 5700ctcgtcgact caggcgacga cggaattcct gcagcccatc tgcagaattc aggagagacc 5760gggttggcgg cgtatttgtg tcccaaaaaa cagccccaat tgccccggag aagacggcca 5820ggccgcctag atgacaaatt caacaactca cagctgactt tctgccattg ccactagggg 5880ggggcctttt tatatggcca agccaagctc tccacgtcgg ttgggctgca cccaacaata 5940aatgggtagg gttgcaccaa caaagggatg ggatgggggg tagaagatac gaggataacg 6000gggctcaatg gcacaaataa gaacgaatac tgccattaag actcgtgatc cagcgactga 6060caccattgca tcatctaagg gcctcaaaac tacctcggaa ctgctgcgct gatctggaca 6120ccacagaggt tccgagcact ttaggttgca ccaaatgtcc caccaggtgc aggcagaaaa 6180cgctggaaca gcgtgtacag tttgtcttaa caaaaagtga gggcgctgag gtcgagcagg 6240gtggtgtgac ttgttatagc ctttagagct gcgaaagcgc gtatggattt ggctcatcag 6300gccagattga gggtctgtgg acacatgtca tgttagtgta cttcaatcgc cccctggata 6360tagccccgac aataggccgt ggcctcattt ttttgccttc cgcacatttc cattgctcgg 6420tacccacacc ttgcttctcc tgcacttgcc aaccttaata ctggtttaca ttgaccaaca 6480tcttacaagc ggggggcttg tctagggtat atataaacag tggctctccc aatcggttgc 6540cagtctcttt tttcctttct ttccccacag attcgaaatc taaactacac atcacacaat 6600gcctgttact gacgtcctta agcgaaagtc cggtgtcatc gtcggcgacg atgtccgagc 6660cgtgagtatc cacgacaaga tcagtgtcga gacgacgcgt tttgtgtaat gacacaatcc 6720gaaagtcgct agcaacacac actctctaca caaactaacc cagctctcca tggctgagga 6780taagaccaag gtcgagttcc ctaccctgac tgagctgaag cactctatcc ctaacgcttg 6840ctttgagtcc aacctcggac tctcgctcta ctacactgcc cgagcgatct tcaacgcatc 6900tgcctctgct gctctgctct acgctgcccg atctactccc ttcattgccg ataacgttct 6960gctccacgct ctggtttgcg ccacctacat ctacgtgcag ggtgtcatct tctggggttt 7020ctttaccgtc ggtcacgact gtggtcactc tgccttctcc cgataccact ccgtcaactt 7080catcattggc tgcatcatgc actctgccat tctgactccc ttcgagtcct ggcgagtgac 7140ccaccgacac catcacaaga acactggcaa cattgataag gacgagatct tctaccctca 7200tcggtccgtc aaggacctcc aggacgtgcg acaatgggtc tacaccctcg gaggtgcttg 7260gtttgtctac ctgaaggtcg gatatgctcc tcgaaccatg tcccactttg acccctggga 7320ccctctcctg cttcgacgag cctccgctgt catcgtgtcc ctcggagtct gggctgcctt 7380cttcgctgcc tacgcctacc tcacatactc gctcggcttt gccgtcatgg gcctctacta 7440ctatgctcct ctctttgtct ttgcttcgtt cctcgtcatt actaccttct tgcatcacaa 7500cgacgaagct actccctggt acggtgactc ggagtggacc tacgtcaagg gcaacctgag 7560ctccgtcgac cgatcgtacg gagctttcgt ggacaacctg tctcaccaca ttggcaccca 7620ccaggtccat cacttgttcc ctatcattcc ccactacaag ctcaacgaag ccaccaagca 7680ctttgctgcc gcttaccctc acctcgtgag acgtaacgac gagcccatca ttactgcctt 7740cttcaagacc gctcacctct ttgtcaacta cggagctgtg cccgagactg ctcagatttt 7800caccctcaaa gagtctgccg ctgcagccaa ggccaagagc gactaagcgg ccgcaagtgt 7860ggatggggaa gtgagtgccc ggttctgtgt gcacaattgg caatccaaga tggatggatt 7920caacacaggg atatagcgag ctacgtggtg gtgcgaggat atagcaacgg atatttatgt 7980ttgacacttg agaatgtacg atacaagcac tgtccaagta caatactaaa catactgtac 8040atactcatac tcgtacccgg gcaacggttt cacttgagtg cagtggctag tgctcttact 8100cgtacagtgt gcaatactgc gtatcatagt ctttgatgta tatcgtattc attcatgtta 8160gttgc 816588438DNAArtificial Sequenceplasmid pDMW367 8catggctctc agtcttacca cagaacagct gttagaacgc cctgatttgg ttgcgattga 60tggcatcctc tacgaccttg aagggcttgc caaagttcat ccaggaggag atttgattct 120cgcttctggt gcctctgatg cctcccctct cttttattca atgcatccat acgtcaaacc 180ggagaattcc aaattgcttc aacagttcgt ccgagggaag catgaccgca cctcgaagga 240cattgtctac acgtatgatt ctcccttcgc acaagacgtt aagcggacaa tgcgcgaggt 300gatgaaaggg aggaactggt acgcaacccc tggcttctgg ctgcgcaccg ttgggatcat 360cgccgtgacg gccttttgcg agtggcactg ggctaccacg gggatggtgc tgtggggcct 420gttgactgga ttcatgcaca tgcagatcgg cttatccatc cagcatgatg cgtcccacgg 480ggccatcagc aagaagcctt gggtcaacgc cctcttcgcc tacggcattg acgtcatcgg 540atcgtcccgg tggatttggc tgcagtcgca catcatgcgg caccacacct acaccaacca 600gcacggcctc gacctggatg cggagtcggc agagccgttc ctggtgttcc acaactaccc 660cgccgcaaac accgcccgaa agtggttcca ccgcttccaa gcttggtaca tgtaccttgt 720gctgggggca tacggggtat cgctggtgta caacccgctc tacattttcc ggatgcagca 780caatgacacc atcccagagt ctgtcacggc catgcgggaa aatggctttc tgcggcgcta 840ccgcacactt gcattcgtga tgcgagcttt cttcatcttc cggaccgcat tcttgccctg 900gtacctcact gggacctcat tgctgatcac cattcctctg gtgcccaccg caactggtgc 960cttcttgacg ttcttcttca ttttgtccca caattttgat ggctccgaac ggatccccga 1020caagaactgc aaggttaaga gatctgagaa ggacgttgag gctgaccaaa ttgactggta 1080tcgggcgcag gtggagacgt cctccacata cggtggcccc atcgccatgt tcttcactgg 1140cggtctcaat ttccagatcg agcaccacct ctttccccgg atgtcgtctt ggcactaccc 1200cttcgtccag caggcggtcc gggagtgttg cgaacgccat ggagtgcgat atgttttcta 1260ccctaccatc gtcggcaaca tcatctccac cctgaagtac atgcataagg tgggtgtcgt 1320ccactgcgtg aaggacgcac aggattccta agcggccgca agtgtggatg gggaagtgag 1380tgcccggttc tgtgtgcaca attggcaatc caagatggat ggattcaaca cagggatata 1440gcgagctacg tggtggtgcg aggatatagc aacggatatt tatgtttgac acttgagaat 1500gtacgataca agcactgtcc aagtacaata ctaaacatac tgtacatact catactcgta 1560cccgggcaac ggtttcactt gagtgcagtg gctagtgctc ttactcgtac agtgtgcaat 1620actgcgtatc atagtctttg atgtatatcg tattcattca tgttagttgc gtacgagccg 1680gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt 1740tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg 1800gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg 1860actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 1920tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 1980aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 2040ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 2100aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 2160cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 2220cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 2280aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 2340cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 2400ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 2460ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 2520gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 2580agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 2640acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga 2700tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg 2760agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct 2820gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg 2880agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc 2940cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa 3000ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc 3060cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt 3120cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc 3180ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc

gatcgttgtc agaagtaagt 3240tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc 3300catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt 3360gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata 3420gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 3480tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag 3540catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa 3600aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt 3660attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 3720aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgcgccct 3780gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg 3840ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg 3900gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac 3960ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct 4020gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt 4080tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt 4140tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt 4200ttaacaaaat attaacgctt acaatttcca ttcgccattc aggctgcgca actgttggga 4260agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc 4320aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc 4380cagtgaattg taatacgact cactataggg cgaattgggt accgggcccc ccctcgaggt 4440cgatggtgtc gataagcttg atatcgaatt catgtcacac aaaccgatct tcgcctcaag 4500gaaacctaat tctacatccg agagactgcc gagatccagt ctacactgat taattttcgg 4560gccaataatt taaaaaaatc gtgttatata atattatatg tattatatat atacatcatg 4620atgatactga cagtcatgtc ccattgctaa atagacagac tccatctgcc gcctccaact 4680gatgttctca atatttaagg ggtcatctcg cattgtttaa taataaacag actccatcta 4740ccgcctccaa atgatgttct caaaatatat tgtatgaact tatttttatt acttagtatt 4800attagacaac ttacttgctt tatgaaaaac acttcctatt taggaaacaa tttataatgg 4860cagttcgttc atttaacaat ttatgtagaa taaatgttat aaatgcgtat gggaaatctt 4920aaatatggat agcataaatg atatctgcat tgcctaattc gaaatcaaca gcaacgaaaa 4980aaatcccttg tacaacataa atagtcatcg agaaatatca actatcaaag aacagctatt 5040cacacgttac tattgagatt attattggac gagaatcaca cactcaactg tctttctctc 5100ttctagaaat acaggtacaa gtatgtacta ttctcattgt tcatacttct agtcatttca 5160tcccacatat tccttggatt tctctccaat gaatgacatt ctatcttgca aattcaacaa 5220ttataataag atataccaaa gtagcggtat agtggcaatc aaaaagcttc tctggtgtgc 5280ttctcgtatt tatttttatt ctaatgatcc attaaaggta tatatttatt tcttgttata 5340taatcctttt gtttattaca tgggctggat acataaaggt attttgattt aattttttgc 5400ttaaattcaa tcccccctcg ttcagtgtca actgtaatgg taggaaatta ccatactttt 5460gaagaagcaa aaaaaatgaa agaaaaaaaa aatcgtattt ccaggttaga cgttccgcag 5520aatctagaat gcggtatgcg gtacattgtt cttcgaacgt aaaagttgcg ctccctgaga 5580tattgtacat ttttgctttt acaagtacaa gtacatcgta caactatgta ctactgttga 5640tgcatccaca acagtttgtt ttgttttttt ttgttttttt tttttctaat gattcattac 5700cgctatgtat acctacttgt acttgtagta agccgggtta ttggcgttca attaatcata 5760gacttatgaa tctgcacggt gtgcgctgcg agttactttt agcttatgca tgctacttgg 5820gtgtaatatt gggatctgtt cggaaatcaa cggatgctca atcgatttcg acagtaatta 5880attaagtcat acacaagtca gctttcttcg agcctcatat aagtataagt agttcaacgt 5940attagcactg tacccagcat ctccgtatcg agaaacacaa caacatgccc cattggacag 6000atcatgcgga tacacaggtt gtgcagtatc atacatactc gatcagacag gtcgtctgac 6060catcatacaa gctgaacaag cgctccatac ttgcacgctc tctatataca cagttaaatt 6120acatatccat agtctaacct ctaacagtta atcttctggt aagcctccca gccagccttc 6180tggtatcgct tggcctcctc aataggatct cggttctggc cgtacagacc tcggccgaca 6240attatgatat ccgttccggt agacatgaca tcctcaacag ttcggtactg ctgtccgaga 6300gcgtctccct tgtcgtcaag acccaccccg ggggtcagaa taagccagtc ctcagagtcg 6360cccttaggtc ggttctgggc aatgaagcca accacaaact cggggtcgga tcgggcaagc 6420tcaatggtct gcttggagta ctcgccagtg gccagagagc ccttgcaaga cagctcggcc 6480agcatgagca gacctctggc cagcttctcg ttgggagagg ggactaggaa ctccttgtac 6540tgggagttct cgtagtcaga gacgtcctcc ttcttctgtt cagagacagt ttcctcggca 6600ccagctcgca ggccagcaat gattccggtt ccgggtacac cgtgggcgtt ggtgatatcg 6660gaccactcgg cgattcggtg acaccggtac tggtgcttga cagtgttgcc aatatctgcg 6720aactttctgt cctcgaacag gaagaaaccg tgcttaagag caagttcctt gagggggagc 6780acagtgccgg cgtaggtgaa gtcgtcaatg atgtcgatat gggttttgat catgcacaca 6840taaggtccga ccttatcggc aagctcaatg agctccttgg tggtggtaac atccagagaa 6900gcacacaggt tggttttctt ggctgccacg agcttgagca ctcgagcggc aaaggcggac 6960ttgtggacgt tagctcgagc ttcgtaggag ggcattttgg tggtgaagag gagactgaaa 7020taaatttagt ctgcagaact ttttatcgga accttatctg gggcagtgaa gtatatgtta 7080tggtaatagt tacgagttag ttgaacttat agatagactg gactatacgg ctatcggtcc 7140aaattagaaa gaacgtcaat ggctctctgg gcgtcgcctt tgccgacaaa aatgtgatca 7200tgatgaaagc cagcaatgac gttgcagctg atattgttgt cggccaaccg cgccgaaaac 7260gcagctgtca gacccacagc ctccaacgaa gaatgtatcg tcaaagtgat ccaagcacac 7320tcatagttgg agtcgtactc caaaggcggc aatgacgagt cagacagata ctcgtcgact 7380caggcgacga cggaattcct gcagcccatc tgcagaattc aggagagacc gggttggcgg 7440cgtatttgtg tcccaaaaaa cagccccaat tgccccggag aagacggcca ggccgcctag 7500atgacaaatt caacaactca cagctgactt tctgccattg ccactagggg ggggcctttt 7560tatatggcca agccaagctc tccacgtcgg ttgggctgca cccaacaata aatgggtagg 7620gttgcaccaa caaagggatg ggatgggggg tagaagatac gaggataacg gggctcaatg 7680gcacaaataa gaacgaatac tgccattaag actcgtgatc cagcgactga caccattgca 7740tcatctaagg gcctcaaaac tacctcggaa ctgctgcgct gatctggaca ccacagaggt 7800tccgagcact ttaggttgca ccaaatgtcc caccaggtgc aggcagaaaa cgctggaaca 7860gcgtgtacag tttgtcttaa caaaaagtga gggcgctgag gtcgagcagg gtggtgtgac 7920ttgttatagc ctttagagct gcgaaagcgc gtatggattt ggctcatcag gccagattga 7980gggtctgtgg acacatgtca tgttagtgta cttcaatcgc cccctggata tagccccgac 8040aataggccgt ggcctcattt ttttgccttc cgcacatttc cattgctcgg tacccacacc 8100ttgcttctcc tgcacttgcc aaccttaata ctggtttaca ttgaccaaca tcttacaagc 8160ggggggcttg tctagggtat atataaacag tggctctccc aatcggttgc cagtctcttt 8220tttcctttct ttccccacag attcgaaatc taaactacac atcacacaat gcctgttact 8280gacgtcctta agcgaaagtc cggtgtcatc gtcggcgacg atgtccgagc cgtgagtatc 8340cacgacaaga tcagtgtcga gacgacgcgt tttgtgtaat gacacaatcc gaaagtcgct 8400agcaacacac actctctaca caaactaacc cagctctc 8438918DNAArtificial SequenceM13F universal primer 9tgtaaaacga cggccagt 181022DNAArtificial Sequenceprimer M13-28Rev 10gtaatacgac tcactatagg gc 22114276DNAArtificial Sequenceplasmid pLF119 11gtacaaagtt ggcattataa gaaagcattg cttatcaatt tgttgcaacg aacaggtcac 60tatcagtcaa aataaaatca ttatttgcca tccagctgat atcccctata gtgagtcgta 120ttacatggtc atagctgttt cctggcagct ctggcccgtg tctcaaaatc tctgatgtta 180cattgcacaa gataaaaata tatcatcatg ttagaaaaac tcatcgagca tcaaatgaaa 240ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa 300tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc 360gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt 420atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagcttatg 480catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc 540atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct 600gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc 660atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc 720ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt 780cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt 840ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa 900tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa 960atcagcatcc atgttggaat ttaatcgcgg cctcgagcaa gacgtttccc gttgaatatg 1020gctcatagat cttttctcca tcactgatag ggagtggtaa aataactcca tcaatgatag 1080agtgtcaaca acatgaccaa aatcccttaa cgtgagttac gcgtattaat tgcgttgcgc 1140tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa 1200cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg 1260ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 1320ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag 1380gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 1440gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 1500taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 1560accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc 1620tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 1680cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 1740agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 1800gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggttacac tagaagaaca 1860gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 1920tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 1980acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 2040cagggaacga cgcgtaccgc tagccaggaa gagtttgtag aaacgcaaaa aggccatccg 2100tcaggatggc cttctgctta gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc 2160accctccggg ccgttgcttc acaacgttca aatccgctcc cggcggattt gtcctactca 2220ggagagcgtt caccgacaaa caacagataa aacgaaaggc ccagtcttcc gactgagcct 2280ttcgttttat ttgatgcctg gcagttccct actctcgcgt taacgctagc atggatgttt 2340tcccagtcac gacgttgtaa aacgacggcc agtcttaagc tcgggcccca aataatgatt 2400ttattttgac tgatagtgac ctgttcgttg caacaaattg atgagcaatg cttttttata 2460atgccaactt tgtacaaaaa agttggattt tttttcggga tggccaccat ctctttgact 2520actgagcaac ttttagaaca cccagaactg gttgcaattg atggggtgtt gtacgatctc 2580ttcggactgg cgaaagtgca tccaggtggc aacctcattg aagccgccgg tgcctccgac 2640ggaaccgccc tgttctactc catgcaccct ggagtgaagc cagagaattc gaagctgctg 2700cagcaatttg cccgaggcaa acacgaacga agctcgaagg acccagtgta cacctttgac 2760agtcccttcg cccaggatgt caagcagagc gttcgggagg tcatgaaggg gcgcaactgg 2820tacgccacgc ccggcttttg gctgcggacc gcgctgatca tcgcgtgcac tgccataggc 2880gaatggtatt ggatcactac cggggcagtg atgtggggca tcttcaccgg gtacttccac 2940agccagattg ggttggcgat tcaacacgat gcctctcacg gagccatcag caaaaagccc 3000tgggtgaacg cctttttcgc ctacggcatc gacgccattg gatcctcccg ctggatctgg 3060ctgcagtccc acattatgcg ccaccacacc tacaccaacc agcatggcct ggacctggac 3120gctgcctcgg cggagccgtt cattttgttc cactcctacc cggcaacaaa tgcgtcacga 3180aagtggtacc atcggttcca ggcgtggtac atgtacatcg ttttggggat gtatggtgtg 3240tcgatggtgt acaatccgat gtacttgttc acgatgcagc acaacgacac aatcccagag 3300gccacctctc ttagaccagg cagctttttc aaccggcagc gcgccttcgc cgtttccctc 3360cgcctactgt tcatcttccg caacgccttc ctcccctggt acatcgcggg cgcctctccg 3420ctgctcacca tcctgctggt gccaacggtc acaggcatct tcttgacatt tgtttttgtg 3480ctgtcccata actttgaagg cgctgagcgg acccccgaaa agaactgcaa ggccaaaagg 3540gccaaggagg ggaaggaggt ccgcgatgta gaggaggacc gggtggactg gtaccgggcg 3600caggccgaga ccgcggcgac ctacgggggc agcgtcggga tgatgctgac cggcggtttg 3660aacctgcaga tcgagcacca cttgttcccc cgcatgtcct cttggcacta ccccttcatc 3720caagatacgg tgcgggaatg ttgcaagcgc catggcgtgc gctacacata ctacccgacc 3780atcctggaga atataatgtc cacgctccgc tacatgcaga aggtgggcgt ggcccacaca 3840attcaggatg cccaggaatt ctgagtgagt tcgatccgca tcgacgtcta ccatttttga 3900tgctgtctat tcctgttttc agtcacctcc agcattctca tggctggtga ccactgcccc 3960tctaacccat tgtgacacac cgccaaagac tttgcctctt ttttttccct ttcttttgtc 4020ctcggggtgc tttggccggt gtttactcgc cttgcagtcc ccgcaaacga ccgacgttta 4080agctccgttg ttgactgggc cgctcgtaaa cccatctgca ggttgaggct cccatggaga 4140attgtgatgg ctgattagga ggtggcgggg catacatgcc tcgacactca aagccgggcg 4200gcttctggat tcgaaaacgc aaatgggcgc tttggaaaaa aaaaaaaaaa aaaaaaaaaa 4260aaaacccaac tttctt 4276121362DNAEuglena anabaena 12atggccacca tctctttgac tactgagcaa cttttagaac acccagaact ggttgcaatt 60gatggggtgt tgtacgatct cttcggactg gcgaaagtgc atccaggtgg caacctcatt 120gaagccgccg gtgcctccga cggaaccgcc ctgttctact ccatgcaccc tggagtgaag 180ccagagaatt cgaagctgct gcagcaattt gcccgaggca aacacgaacg aagctcgaag 240gacccagtgt acacctttga cagtcccttc gcccaggatg tcaagcagag cgttcgggag 300gtcatgaagg ggcgcaactg gtacgccacg cccggctttt ggctgcggac cgcgctgatc 360atcgcgtgca ctgccatagg cgaatggtat tggatcacta ccggggcagt gatgtggggc 420atcttcaccg ggtacttcca cagccagatt gggttggcga ttcaacacga tgcctctcac 480ggagccatca gcaaaaagcc ctgggtgaac gcctttttcg cctacggcat cgacgccatt 540ggatcctccc gctggatctg gctgcagtcc cacattatgc gccaccacac ctacaccaac 600cagcatggcc tggacctgga cgctgcctcg gcggagccgt tcattttgtt ccactcctac 660ccggcaacaa atgcgtcacg aaagtggtac catcggttcc aggcgtggta catgtacatc 720gttttgggga tgtatggtgt gtcgatggtg tacaatccga tgtacttgtt cacgatgcag 780cacaacgaca caatcccaga ggccacctct cttagaccag gcagcttttt caaccggcag 840cgcgccttcg ccgtttccct ccgcctactg ttcatcttcc gcaacgcctt cctcccctgg 900tacatcgcgg gcgcctctcc gctgctcacc atcctgctgg tgccaacggt cacaggcatc 960ttcttgacat ttgtttttgt gctgtcccat aactttgaag gcgctgagcg gacccccgaa 1020aagaactgca aggccaaaag ggccaaggag gggaaggagg tccgcgatgt agaggaggac 1080cgggtggact ggtaccgggc gcaggccgag accgcggcga cctacggggg cagcgtcggg 1140atgatgctga ccggcggttt gaacctgcag atcgagcacc acttgttccc ccgcatgtcc 1200tcttggcact accccttcat ccaagatacg gtgcgggaat gttgcaagcg ccatggcgtg 1260cgctacacat actacccgac catcctggag aatataatgt ccacgctccg ctacatgcag 1320aaggtgggcg tggcccacac aattcaggat gcccaggaat tc 136213454PRTEuglena anabaena 13Met Ala Thr Ile Ser Leu Thr Thr Glu Gln Leu Leu Glu His Pro Glu 1 5 10 15 Leu Val Ala Ile Asp Gly Val Leu Tyr Asp Leu Phe Gly Leu Ala Lys 20 25 30 Val His Pro Gly Gly Asn Leu Ile Glu Ala Ala Gly Ala Ser Asp Gly 35 40 45 Thr Ala Leu Phe Tyr Ser Met His Pro Gly Val Lys Pro Glu Asn Ser 50 55 60 Lys Leu Leu Gln Gln Phe Ala Arg Gly Lys His Glu Arg Ser Ser Lys 65 70 75 80 Asp Pro Val Tyr Thr Phe Asp Ser Pro Phe Ala Gln Asp Val Lys Gln 85 90 95 Ser Val Arg Glu Val Met Lys Gly Arg Asn Trp Tyr Ala Thr Pro Gly 100 105 110 Phe Trp Leu Arg Thr Ala Leu Ile Ile Ala Cys Thr Ala Ile Gly Glu 115 120 125 Trp Tyr Trp Ile Thr Thr Gly Ala Val Met Trp Gly Ile Phe Thr Gly 130 135 140 Tyr Phe His Ser Gln Ile Gly Leu Ala Ile Gln His Asp Ala Ser His 145 150 155 160 Gly Ala Ile Ser Lys Lys Pro Trp Val Asn Ala Phe Phe Ala Tyr Gly 165 170 175 Ile Asp Ala Ile Gly Ser Ser Arg Trp Ile Trp Leu Gln Ser His Ile 180 185 190 Met Arg His His Thr Tyr Thr Asn Gln His Gly Leu Asp Leu Asp Ala 195 200 205 Ala Ser Ala Glu Pro Phe Ile Leu Phe His Ser Tyr Pro Ala Thr Asn 210 215 220 Ala Ser Arg Lys Trp Tyr His Arg Phe Gln Ala Trp Tyr Met Tyr Ile 225 230 235 240 Val Leu Gly Met Tyr Gly Val Ser Met Val Tyr Asn Pro Met Tyr Leu 245 250 255 Phe Thr Met Gln His Asn Asp Thr Ile Pro Glu Ala Thr Ser Leu Arg 260 265 270 Pro Gly Ser Phe Phe Asn Arg Gln Arg Ala Phe Ala Val Ser Leu Arg 275 280 285 Leu Leu Phe Ile Phe Arg Asn Ala Phe Leu Pro Trp Tyr Ile Ala Gly 290 295 300 Ala Ser Pro Leu Leu Thr Ile Leu Leu Val Pro Thr Val Thr Gly Ile 305 310 315 320 Phe Leu Thr Phe Val Phe Val Leu Ser His Asn Phe Glu Gly Ala Glu 325 330 335 Arg Thr Pro Glu Lys Asn Cys Lys Ala Lys Arg Ala Lys Glu Gly Lys 340 345 350 Glu Val Arg Asp Val Glu Glu Asp Arg Val Asp Trp Tyr Arg Ala Gln 355 360 365 Ala Glu Thr Ala Ala Thr Tyr Gly Gly Ser Val Gly Met Met Leu Thr 370 375 380 Gly Gly Leu Asn Leu Gln Ile Glu His His Leu Phe Pro Arg Met Ser 385 390 395 400 Ser Trp His Tyr Pro Phe Ile Gln Asp Thr Val Arg Glu Cys Cys Lys 405 410 415 Arg His Gly Val Arg Tyr Thr Tyr Tyr Pro Thr Ile Leu Glu Asn Ile 420 425 430 Met Ser Thr Leu Arg Tyr Met Gln Lys Val Gly Val Ala His Thr Ile 435 440 445 Gln Asp Ala Gln Glu Phe 450 14476PRTThalassiosira pseudonana 14Met 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 15469PRTPhaeodactylum tricornutum 15Met 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 16449PRTEuglena gracilis 16Met Ala Leu Ser Leu Thr Thr Glu Gln Leu Leu Glu Arg Pro Asp Leu 1 5 10 15 Val Ala Ile Asp Gly Ile Leu Tyr Asp Leu Glu Gly Leu Ala Lys Val 20 25 30 His Pro Gly Gly Asp Leu Ile Leu Ala Ser Gly Ala Ser Asp Ala Ser 35 40 45 Pro Leu Phe Tyr Ser Met His Pro Tyr Val Lys Pro Glu Asn Ser Lys 50 55 60 Leu Leu Gln Gln Phe Val Arg Gly Lys His Asp Arg Thr Ser Lys Asp 65 70 75 80 Ile Val Tyr Thr Tyr Asp Ser Pro Phe Ala Gln Asp Val Lys Arg Thr 85 90 95 Met Arg Glu Val Met Lys Gly Arg Asn Trp Tyr Ala Thr Pro Gly Phe 100 105 110 Trp Leu Arg Thr Val Gly Ile Ile Ala Val Thr Ala Phe Cys Glu Trp 115 120 125 His Trp Ala Thr Thr Gly Met Val Leu Trp Gly Leu Leu Thr Gly Phe 130 135 140 Met His Met Gln Ile Gly Leu Ser Ile Gln His Asp Ala Ser His Gly 145 150 155 160 Ala Ile Ser Lys Lys Pro Trp Val Asn Ala Leu Phe Ala Tyr Gly Ile 165 170 175 Asp Val Ile Gly Ser Ser Arg Trp Ile Trp Leu Gln Ser His Ile Met 180 185 190 Arg His His Thr Tyr Thr Asn Gln His Gly Leu Asp Leu Asp Ala Glu 195 200 205 Ser Ala Glu Pro Phe Leu Val Phe His Asn Tyr Pro Ala Ala Asn Thr 210 215 220 Ala Arg Lys Trp Phe His Arg Phe Gln Ala Trp Tyr Met Tyr Leu Val 225 230 235 240 Leu Gly Ala Tyr Gly Val Ser Leu Val Tyr Asn Pro Leu Tyr Ile Phe 245 250 255 Arg Met Gln His Asn Asp Thr Ile Pro Glu Ser Val Thr Ala Met Arg 260 265 270 Glu Asn Gly Phe Leu Arg Arg Tyr Arg Thr Leu Ala Phe Val Met Arg 275 280 285 Ala Phe Phe Ile Phe Arg Thr Ala Phe Leu Pro Trp Tyr Leu Thr Gly 290 295 300 Thr Ser Leu Leu Ile Thr Ile Pro Leu Val Pro Thr Ala Thr Gly Ala 305 310 315 320 Phe Leu Thr Phe Phe Phe Ile Leu Ser His Asn Phe Asp Gly Ser Glu 325 330 335 Arg Ile Pro Asp Lys Asn Cys Lys Val Lys Arg Ser Glu Lys Asp Val 340 345 350 Glu Ala Asp Gln Ile Asp Trp Tyr Arg Ala Gln Val Glu Thr Ser Ser 355 360 365 Thr Tyr Gly Gly Pro Ile Ala Met Phe Phe Thr Gly Gly Leu Asn Phe 370 375 380 Gln Ile Glu His His Leu Phe Pro Arg Met Ser Ser Trp His Tyr Pro 385 390 395 400 Phe Val Gln Gln Ala Val Arg Glu Cys Cys Glu Arg His Gly Val Arg 405 410 415 Tyr Val Phe Tyr Pro Thr Ile Val Gly Asn Ile Ile Ser Thr Leu Lys 420 425 430 Tyr Met His Lys Val Gly Val Val His Cys Val Lys Asp Ala Gln Asp 435 440 445 Ser 179472DNAArtificial Sequenceplasmid pDMW263 17catggcatgg atggtacgtc ctgtagaaac cccaacccgt gaaatcaaaa aactcgacgg 60cctgtgggca ttcagtctgg atcgcgaaaa ctgtggaatt gatcagcgtt ggtgggaaag 120cgcgttacaa gaaagccggg caattgctgt gccaggcagt tttaacgatc agttcgccga 180tgcagatatt cgtaattatg cgggcaacgt ctggtatcag cgcgaagtct ttataccgaa 240aggttgggca ggccagcgta tcgtgctgcg tttcgatgcg gtcactcatt acggcaaagt 300gtgggtcaat aatcaggaag tgatggagca tcagggcggc tatacgccat ttgaagccga 360tgtcacgccg tatgttattg ccgggaaaag tgtacgtatc accgtttgtg tgaacaacga 420actgaactgg cagactatcc cgccgggaat ggtgattacc gacgaaaacg gcaagaaaaa 480gcagtcttac ttccatgatt tctttaacta tgccgggatc catcgcagcg taatgctcta 540caccacgccg aacacctggg tggacgatat caccgtggtg acgcatgtcg cgcaagactg 600taaccacgcg tctgttgact ggcaggtggt ggccaatggt gatgtcagcg ttgaactgcg 660tgatgcggat caacaggtgg ttgcaactgg acaaggcact agcgggactt tgcaagtggt 720gaatccgcac ctctggcaac cgggtgaagg ttatctctat gaactgtgcg tcacagccaa 780aagccagaca gagtgtgata tctacccgct tcgcgtcggc atccggtcag tggcagtgaa 840gggcgaacag ttcctgatta accacaaacc gttctacttt actggctttg gtcgtcatga 900agatgcggac ttacgtggca aaggattcga taacgtgctg atggtgcacg accacgcatt 960aatggactgg attggggcca actcctaccg tacctcgcat tacccttacg ctgaagagat 1020gctcgactgg gcagatgaac atggcatcgt ggtgattgat gaaactgctg ctgtcggctt 1080taacctctct ttaggcattg gtttcgaagc gggcaacaag ccgaaagaac tgtacagcga 1140agaggcagtc aacggggaaa ctcagcaagc gcacttacag gcgattaaag agctgatagc 1200gcgtgacaaa aaccacccaa gcgtggtgat gtggagtatt gccaacgaac cggatacccg 1260tccgcaagtg cacgggaata tttcgccact ggcggaagca acgcgtaaac tcgacccgac 1320gcgtccgatc acctgcgtca atgtaatgtt ctgcgacgct cacaccgata ccatcagcga 1380tctctttgat gtgctgtgcc tgaaccgtta ttacggatgg tatgtccaaa gcggcgattt 1440ggaaacggca gagaaggtac tggaaaaaga acttctggcc tggcaggaga aactgcatca 1500gccgattatc atcaccgaat acggcgtgga tacgttagcc gggctgcact caatgtacac 1560cgacatgtgg agtgaagagt atcagtgtgc atggctggat atgtatcacc gcgtctttga 1620tcgcgtcagc gccgtcgtcg gtgaacaggt atggaatttc gccgattttg cgacctcgca 1680aggcatattg cgcgttggcg gtaacaagaa agggatcttc actcgcgacc gcaaaccgaa 1740gtcggcggct tttctgctgc aaaaacgctg gactggcatg aacttcggtg aaaaaccgca 1800gcagggaggc aaacaatgat taattaacta gagcggccgc caccgcggcc cgagattccg 1860gcctcttcgg ccgccaagcg acccgggtgg acgtctagag gtacctagca attaacagat 1920agtttgccgg tgataattct cttaacctcc cacactcctt tgacataacg atttatgtaa 1980cgaaactgaa atttgaccag atattgtgtc cgcggtggag ctccagcttt tgttcccttt 2040agtgagggtt aatttcgagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt 2100gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg 2160gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt 2220cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt 2280tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc 2340tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg 2400ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 2460ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 2520gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 2580gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 2640ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg 2700tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 2760gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 2820tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 2880tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc 2940tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 3000ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 3060ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 3120gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt 3180aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc 3240aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg 3300cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg 3360ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc 3420cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta 3480ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg 3540ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct 3600ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta 3660gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg 3720ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga 3780ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt 3840gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca 3900ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt 3960cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt 4020ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga 4080aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt 4140gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 4200gcacatttcc ccgaaaagtg ccacctgacg cgccctgtag cggcgcatta agcgcggcgg 4260gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 4320tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 4380gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 4440attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 4500cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 4560ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 4620aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa 4680tttccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc 4740gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc 4800agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattgtaat acgactcact 4860atagggcgaa ttgggtaccg ggccccccct cgaggtcgat ggtgtcgata agcttgatat 4920cgaattcatg tcacacaaac cgatcttcgc ctcaaggaaa cctaattcta catccgagag 4980actgccgaga tccagtctac actgattaat tttcgggcca ataatttaaa aaaatcgtgt 5040tatataatat tatatgtatt atatatatac atcatgatga tactgacagt catgtcccat 5100tgctaaatag acagactcca tctgccgcct ccaactgatg ttctcaatat ttaaggggtc 5160atctcgcatt gtttaataat aaacagactc catctaccgc ctccaaatga tgttctcaaa 5220atatattgta tgaacttatt tttattactt agtattatta gacaacttac ttgctttatg 5280aaaaacactt cctatttagg aaacaattta taatggcagt tcgttcattt aacaatttat 5340gtagaataaa tgttataaat gcgtatggga aatcttaaat atggatagca taaatgatat 5400ctgcattgcc taattcgaaa tcaacagcaa cgaaaaaaat cccttgtaca acataaatag 5460tcatcgagaa atatcaacta tcaaagaaca gctattcaca cgttactatt gagattatta 5520ttggacgaga atcacacact caactgtctt tctctcttct agaaatacag gtacaagtat 5580gtactattct cattgttcat acttctagtc atttcatccc acatattcct tggatttctc 5640tccaatgaat gacattctat cttgcaaatt caacaattat aataagatat accaaagtag 5700cggtatagtg gcaatcaaaa agcttctctg gtgtgcttct cgtatttatt tttattctaa 5760tgatccatta aaggtatata tttatttctt gttatataat ccttttgttt attacatggg 5820ctggatacat aaaggtattt tgatttaatt ttttgcttaa attcaatccc ccctcgttca 5880gtgtcaactg taatggtagg aaattaccat acttttgaag aagcaaaaaa aatgaaagaa 5940aaaaaaaatc gtatttccag gttagacgtt ccgcagaatc tagaatgcgg tatgcggtac 6000attgttcttc gaacgtaaaa gttgcgctcc ctgagatatt gtacattttt gcttttacaa 6060gtacaagtac atcgtacaac tatgtactac tgttgatgca tccacaacag tttgttttgt 6120ttttttttgt tttttttttt tctaatgatt cattaccgct atgtatacct acttgtactt 6180gtagtaagcc gggttattgg cgttcaatta atcatagact tatgaatctg cacggtgtgc 6240gctgcgagtt acttttagct tatgcatgct acttgggtgt aatattggga tctgttcgga 6300aatcaacgga tgctcaaccg atttcgacag taataatttg aatcgaatcg gagcctaaaa 6360tgaacccgag tatatctcat aaaattctcg gtgagaggtc tgtgactgtc agtacaaggt 6420gccttcatta tgccctcaac cttaccatac ctcactgaat gtagtgtacc tctaaaaatg 6480aaatacagtg ccaaaagcca aggcactgag ctcgtctaac ggacttgata tacaaccaat 6540taaaacaaat gaaaagaaat acagttcttt gtatcatttg taacaattac cctgtacaaa 6600ctaaggtatt gaaatcccac aatattccca aagtccaccc ctttccaaat tgtcatgcct 6660acaactcata taccaagcac

taacctacca aacaccacta aaaccccaca aaatatatct 6720taccgaatat acagtaacaa gctaccacca cactcgttgg gtgcagtcgc cagcttaaag 6780atatctatcc acatcagcca caactccctt cctttaataa accgactaca cccttggcta 6840ttgaggttat gagtgaatat actgtagaca agacactttc aagaagactg tttccaaaac 6900gtaccactgt cctccactac aaacacaccc aatctgcttc ttctagtcaa ggttgctaca 6960ccggtaaatt ataaatcatc atttcattag cagggcaggg ccctttttat agagtcttat 7020acactagcgg accctgccgg tagaccaacc cgcaggcgcg tcagtttgct ccttccatca 7080atgcgtcgta gaaacgactt actccttctt gagcagctcc ttgaccttgt tggcaacaag 7140tctccgacct cggaggtgga ggaagagcct ccgatatcgg cggtagtgat accagcctcg 7200acggactcct tgacggcagc ctcaacagcg tcaccggcgg gcttcatgtt aagagagaac 7260ttgagcatca tggcggcaga cagaatggtg gcaatggggt tgaccttctg cttgccgaga 7320tcgggggcag atccgtgaca gggctcgtac agaccgaacg cctcgttggt gtcgggcaga 7380gaagccagag aggcggaggg cagcagaccc agagaaccgg ggatgacgga ggcctcgtcg 7440gagatgatat cgccaaacat gttggtggtg atgatgatac cattcatctt ggagggctgc 7500ttgatgagga tcatggcggc cgagtcgatc agctggtggt tgagctcgag ctgggggaat 7560tcgtccttga ggactcgagt gacagtcttt cgccaaagtc gagaggaggc cagcacgttg 7620gccttgtcaa gagaccacac gggaagaggg gggttgtgct gaagggccag gaaggcggcc 7680attcgggcaa ttcgctcaac ctcaggaacg gagtaggtct cggtgtcgga agcgacgcca 7740gatccgtcat cctcctttcg ctctccaaag tagatacctc cgacgagctc tcggacaatg 7800atgaagtcgg tgccctcaac gtttcggatg ggggagagat cggcgagctt gggcgacagc 7860agctggcagg gtcgcaggtt ggcgtacagg ttcaggtcct ttcgcagctt gaggagaccc 7920tgctcgggtc gcacgtcggt tcgtccgtcg ggagtggtcc atacggtgtt ggcagcgcct 7980ccgacagcac cgagcataat agagtcagcc tttcggcaga tgtcgagagt agcgtcggtg 8040atgggctcgc cctccttctc aatggcagct cctccaatga gtcggtcctc aaacacaaac 8100tcggtgccgg aggcctcagc aacagacttg agcaccttga cggcctcggc aatcacctcg 8160gggccacaga agtcgccgcc gagaagaaca atcttcttgg agtcagtctt ggtcttctta 8220gtttcgggtt ccattgtgga tgtgtgtggt tgtatgtgtg atgtggtgtg tggagtgaaa 8280atctgtggct ggcaaacgct cttgtatata tacgcacttt tgcccgtgct atgtggaaga 8340ctaaacctcc gaagattgtg actcaggtag tgcggtatcg gctagggacc caaaccttgt 8400cgatgccgat agcgctatcg aacgtacccc agccggccgg gagtatgtcg gaggggacat 8460acgagatcgt caagggtttg tggccaactg gtaaataaat gatgtcgacg tttaaacagt 8520gtacgcagat ctactataga ggaacattta aattgccccg gagaagacgg ccaggccgcc 8580tagatgacaa attcaacaac tcacagctga ctttctgcca ttgccactag gggggggcct 8640ttttatatgg ccaagccaag ctctccacgt cggttgggct gcacccaaca ataaatgggt 8700agggttgcac caacaaaggg atgggatggg gggtagaaga tacgaggata acggggctca 8760atggcacaaa taagaacgaa tactgccatt aagactcgtg atccagcgac tgacaccatt 8820gcatcatcta agggcctcaa aactacctcg gaactgctgc gctgatctgg acaccacaga 8880ggttccgagc actttaggtt gcaccaaatg tcccaccagg tgcaggcaga aaacgctgga 8940acagcgtgta cagtttgtct taacaaaaag tgagggcgct gaggtcgagc agggtggtgt 9000gacttgttat agcctttaga gctgcgaaag cgcgtatgga tttggctcat caggccagat 9060tgagggtctg tggacacatg tcatgttagt gtacttcaat cgccccctgg atatagcccc 9120gacaataggc cgtggcctca tttttttgcc ttccgcacat ttccattgct cgatacccac 9180accttgcttc tcctgcactt gccaacctta atactggttt acattgacca acatcttaca 9240agcggggggc ttgtctaggg tatatataaa cagtggctct cccaatcggt tgccagtctc 9300ttttttcctt tctttcccca cagattcgaa atctaaacta cacatcacag aattccgagc 9360cgtgagtatc cacgacaaga tcagtgtcga gacgacgcgt tttgtgtaat gacacaatcc 9420gaaagtcgct agcaacacac actctctaca caaactaacc cagctctggt ac 9472187879DNAArtificial Sequenceplasmid pDMW237 18ggccgcaagt gtggatgggg aagtgagtgc ccggttctgt gtgcacaatt ggcaatccaa 60gatggatgga ttcaacacag ggatatagcg agctacgtgg tggtgcgagg atatagcaac 120ggatatttat gtttgacact tgagaatgta cgatacaagc actgtccaag tacaatacta 180aacatactgt acatactcat actcgtaccc gggcaacggt ttcacttgag tgcagtggct 240agtgctctta ctcgtacagt gtgcaatact gcgtatcata gtctttgatg tatatcgtat 300tcattcatgt tagttgcgta cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 360gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 420tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 480ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 540cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 600gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 660tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 720agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 780tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 840cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 900ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 960ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 1020ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 1080ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 1140cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 1200gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 1260atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 1320ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 1380gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 1440tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 1500ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 1560taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 1620gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 1680gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 1740ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 1800aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 1860gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 1920cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 1980actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 2040caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 2100gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 2160ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 2220caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 2280tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 2340gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 2400cccgaaaagt gccacctgac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg 2460ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct 2520tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc 2580ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg 2640atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt 2700ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg 2760tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc 2820tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttccattc 2880gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg 2940ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc 3000ccagtcacga cgttgtaaaa cgacggccag tgaattgtaa tacgactcac tatagggcga 3060attgggtacc gggccccccc tcgaggtcga tggtgtcgat aagcttgata tcgaattcat 3120gtcacacaaa ccgatcttcg cctcaaggaa acctaattct acatccgaga gactgccgag 3180atccagtcta cactgattaa ttttcgggcc aataatttaa aaaaatcgtg ttatataata 3240ttatatgtat tatatatata catcatgatg atactgacag tcatgtccca ttgctaaata 3300gacagactcc atctgccgcc tccaactgat gttctcaata tttaaggggt catctcgcat 3360tgtttaataa taaacagact ccatctaccg cctccaaatg atgttctcaa aatatattgt 3420atgaacttat ttttattact tagtattatt agacaactta cttgctttat gaaaaacact 3480tcctatttag gaaacaattt ataatggcag ttcgttcatt taacaattta tgtagaataa 3540atgttataaa tgcgtatggg aaatcttaaa tatggatagc ataaatgata tctgcattgc 3600ctaattcgaa atcaacagca acgaaaaaaa tcccttgtac aacataaata gtcatcgaga 3660aatatcaact atcaaagaac agctattcac acgttactat tgagattatt attggacgag 3720aatcacacac tcaactgtct ttctctcttc tagaaataca ggtacaagta tgtactattc 3780tcattgttca tacttctagt catttcatcc cacatattcc ttggatttct ctccaatgaa 3840tgacattcta tcttgcaaat tcaacaatta taataagata taccaaagta gcggtatagt 3900ggcaatcaaa aagcttctct ggtgtgcttc tcgtatttat ttttattcta atgatccatt 3960aaaggtatat atttatttct tgttatataa tccttttgtt tattacatgg gctggataca 4020taaaggtatt ttgatttaat tttttgctta aattcaatcc cccctcgttc agtgtcaact 4080gtaatggtag gaaattacca tacttttgaa gaagcaaaaa aaatgaaaga aaaaaaaaat 4140cgtatttcca ggttagacgt tccgcagaat ctagaatgcg gtatgcggta cattgttctt 4200cgaacgtaaa agttgcgctc cctgagatat tgtacatttt tgcttttaca agtacaagta 4260catcgtacaa ctatgtacta ctgttgatgc atccacaaca gtttgttttg tttttttttg 4320tttttttttt ttctaatgat tcattaccgc tatgtatacc tacttgtact tgtagtaagc 4380cgggttattg gcgttcaatt aatcatagac ttatgaatct gcacggtgtg cgctgcgagt 4440tacttttagc ttatgcatgc tacttgggtg taatattggg atctgttcgg aaatcaacgg 4500atgctcaatc gatttcgaca gtaattaatt aagtcataca caagtcagct ttcttcgagc 4560ctcatataag tataagtagt tcaacgtatt agcactgtac ccagcatctc cgtatcgaga 4620aacacaacaa catgccccat tggacagatc atgcggatac acaggttgtg cagtatcata 4680catactcgat cagacaggtc gtctgaccat catacaagct gaacaagcgc tccatacttg 4740cacgctctct atatacacag ttaaattaca tatccatagt ctaacctcta acagttaatc 4800ttctggtaag cctcccagcc agccttctgg tatcgcttgg cctcctcaat aggatctcgg 4860ttctggccgt acagacctcg gccgacaatt atgatatccg ttccggtaga catgacatcc 4920tcaacagttc ggtactgctg tccgagagcg tctcccttgt cgtcaagacc caccccgggg 4980gtcagaataa gccagtcctc agagtcgccc ttaggtcggt tctgggcaat gaagccaacc 5040acaaactcgg ggtcggatcg ggcaagctca atggtctgct tggagtactc gccagtggcc 5100agagagccct tgcaagacag ctcggccagc atgagcagac ctctggccag cttctcgttg 5160ggagagggga ctaggaactc cttgtactgg gagttctcgt agtcagagac gtcctccttc 5220ttctgttcag agacagtttc ctcggcacca gctcgcaggc cagcaatgat tccggttccg 5280ggtacaccgt gggcgttggt gatatcggac cactcggcga ttcggtgaca ccggtactgg 5340tgcttgacag tgttgccaat atctgcgaac tttctgtcct cgaacaggaa gaaaccgtgc 5400ttaagagcaa gttccttgag ggggagcaca gtgccggcgt aggtgaagtc gtcaatgatg 5460tcgatatggg ttttgatcat gcacacataa ggtccgacct tatcggcaag ctcaatgagc 5520tccttggtgg tggtaacatc cagagaagca cacaggttgg ttttcttggc tgccacgagc 5580ttgagcactc gagcggcaaa ggcggacttg tggacgttag ctcgagcttc gtaggagggc 5640attttggtgg tgaagaggag actgaaataa atttagtctg cagaactttt tatcggaacc 5700ttatctgggg cagtgaagta tatgttatgg taatagttac gagttagttg aacttataga 5760tagactggac tatacggcta tcggtccaaa ttagaaagaa cgtcaatggc tctctgggcg 5820tcgcctttgc cgacaaaaat gtgatcatga tgaaagccag caatgacgtt gcagctgata 5880ttgttgtcgg ccaaccgcgc cgaaaacgca gctgtcagac ccacagcctc caacgaagaa 5940tgtatcgtca aagtgatcca agcacactca tagttggagt cgtactccaa aggcggcaat 6000gacgagtcag acagatactc gtcgactcag gcgacgacgg aattcctgca gcccatctgc 6060agaattcagg agagaccggg ttggcggcgt atttgtgtcc caaaaaacag ccccaattgc 6120cccggagaag acggccaggc cgcctagatg acaaattcaa caactcacag ctgactttct 6180gccattgcca ctaggggggg gcctttttat atggccaagc caagctctcc acgtcggttg 6240ggctgcaccc aacaataaat gggtagggtt gcaccaacaa agggatggga tggggggtag 6300aagatacgag gataacgggg ctcaatggca caaataagaa cgaatactgc cattaagact 6360cgtgatccag cgactgacac cattgcatca tctaagggcc tcaaaactac ctcggaactg 6420ctgcgctgat ctggacacca cagaggttcc gagcacttta ggttgcacca aatgtcccac 6480caggtgcagg cagaaaacgc tggaacagcg tgtacagttt gtcttaacaa aaagtgaggg 6540cgctgaggtc gagcagggtg gtgtgacttg ttatagcctt tagagctgcg aaagcgcgta 6600tggatttggc tcatcaggcc agattgaggg tctgtggaca catgtcatgt tagtgtactt 6660caatcgcccc ctggatatag ccccgacaat aggccgtggc ctcatttttt tgccttccgc 6720acatttccat tgctcggtac ccacaccttg cttctcctgc acttgccaac cttaatactg 6780gtttacattg accaacatct tacaagcggg gggcttgtct agggtatata taaacagtgg 6840ctctcccaat cggttgccag tctctttttt cctttctttc cccacagatt cgaaatctaa 6900actacacatc acacaatgcc tgttactgac gtccttaagc gaaagtccgg tgtcatcgtc 6960ggcgacgatg tccgagccgt gagtatccac gacaagatca gtgtcgagac gacgcgtttt 7020gtgtaatgac acaatccgaa agtcgctagc aacacacact ctctacacaa actaacccag 7080ctctccatgg ctctggccaa cgacgctggc gagcgaatct gggctgccgt caccgatccc 7140gaaatcctca ttggcacctt ctcctacctg ctcctgaagc ctctcctgcg aaactctggt 7200ctcgtggacg agaagaaagg agcctaccga acctccatga tctggtacaa cgtcctcctg 7260gctctcttct ctgccctgtc cttctacgtg actgccaccg ctctcggctg ggactacggt 7320actggagcct ggctgcgaag acagaccggt gatactcccc agcctctctt tcagtgtccc 7380tctcctgtct gggactccaa gctgttcacc tggactgcca aggccttcta ctattctaag 7440tacgtggagt acctcgacac cgcttggctg gtcctcaagg gcaagcgagt gtcctttctg 7500caggccttcc atcactttgg agctccctgg gacgtctacc tcggcattcg actgcacaac 7560gagggtgtgt ggatcttcat gttctttaac tcgttcattc acaccatcat gtacacctac 7620tatggactga ctgccgctgg ctacaagttc aaggccaagc ctctgatcac tgccatgcag 7680atttgccagt tcgtcggtgg ctttctcctg gtctgggact acatcaacgt tccctgcttc 7740aactctgaca agggcaagct gttctcctgg gctttcaact acgcctacgt cggatctgtc 7800tttctcctgt tctgtcactt cttttaccag gacaacctgg ccaccaagaa atccgctaag 7860gctggtaagc agctttagc 7879197783DNAArtificial Sequenceplasmid pY115 19catggctctg gccaacgacg ctggcgagcg aatctgggct gccgtcaccg atcccgaaat 60cctcattggc accttctcct acctgctcct gaagcctctc ctgcgaaact ctggtctcgt 120ggacgagaag aaaggagcct accgaacctc catgatctgg tacaacgtcc tcctggctct 180cttctctgcc ctgtccttct acgtgactgc caccgctctc ggctgggact acggtactgg 240agcctggctg cgaagacaga ccggtgatac tccccagcct ctctttcagt gtccctctcc 300tgtctgggac tccaagctgt tcacctggac tgccaaggcc ttctactatt ctaagtacgt 360ggagtacctc gacaccgctt ggctggtcct caagggcaag cgagtgtcct ttctgcaggc 420cttccatcac tttggagctc cctgggacgt ctacctcggc attcgactgc acaacgaggg 480tgtgtggatc ttcatgttct ttaactcgtt cattcacacc atcatgtaca cctactatgg 540actgactgcc gctggctaca agttcaaggc caagcctctg atcactgcca tgcagatttg 600ccagttcgtc ggtggctttc tcctggtctg ggactacatc aacgttccct gcttcaactc 660tgacaagggc aagctgttct cctgggcttt caactacgcc tacgtcggat ctgtctttct 720cctgttctgt cacttctttt accaggacaa cctggccacc aagaaatccg ctaaggctgg 780taagcagctt tagcggccgc aagtgtggat ggggaagtga gtgcccggtt ctgtgtgcac 840aattggcaat ccaagatgga tggattcaac acagggatat agcgagctac gtggtggtgc 900gaggatatag caacggatat ttatgtttga cacttgagaa tgtacgatac aagcactgtc 960caagtacaat actaaacata ctgtacatac tcatactcgt acccgggcaa cggtttcact 1020tgagtgcagt ggctagtgct cttactcgta cagtgtgcaa tactgcgtat catagtcttt 1080gatgtatatc gtattcattc atgttagttg cgtacgagcc ggaagcataa agtgtaaagc 1140ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt 1200ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg 1260cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt 1320tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc 1380aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa 1440aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa 1500tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 1560ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 1620cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag 1680ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga 1740ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc 1800gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac 1860agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg 1920cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 1980aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 2040aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa 2100ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt 2160aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag 2220ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 2280agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc 2340cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa 2400ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca 2460gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa 2520cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt 2580cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc 2640ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact 2700catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc 2760tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg 2820ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct 2880catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc 2940cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag 3000cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 3060acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg 3120ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt 3180tccgcgcaca tttccccgaa aagtgccacc tgacgcgccc tgtagcggcg cattaagcgc 3240ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc 3300tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct 3360aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa 3420acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc 3480tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact 3540caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg 3600gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct 3660tacaatttcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc 3720tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta 3780acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtaatacgac 3840tcactatagg gcgaattggg taccgggccc cccctcgagg tcgatggtgt cgataagctt 3900gatatcgaat tcatgtcaca caaaccgatc ttcgcctcaa ggaaacctaa ttctacatcc 3960gagagactgc cgagatccag tctacactga ttaattttcg ggccaataat ttaaaaaaat 4020cgtgttatat aatattatat gtattatata tatacatcat gatgatactg acagtcatgt 4080cccattgcta aatagacaga ctccatctgc cgcctccaac tgatgttctc aatatttaag 4140gggtcatctc gcattgttta ataataaaca gactccatct accgcctcca aatgatgttc 4200tcaaaatata ttgtatgaac ttatttttat tacttagtat tattagacaa cttacttgct

4260ttatgaaaaa cacttcctat ttaggaaaca atttataatg gcagttcgtt catttaacaa 4320tttatgtaga ataaatgtta taaatgcgta tgggaaatct taaatatgga tagcataaat 4380gatatctgca ttgcctaatt cgaaatcaac agcaacgaaa aaaatccctt gtacaacata 4440aatagtcatc gagaaatatc aactatcaaa gaacagctat tcacacgtta ctattgagat 4500tattattgga cgagaatcac acactcaact gtctttctct cttctagaaa tacaggtaca 4560agtatgtact attctcattg ttcatacttc tagtcatttc atcccacata ttccttggat 4620ttctctccaa tgaatgacat tctatcttgc aaattcaaca attataataa gatataccaa 4680agtagcggta tagtggcaat caaaaagctt ctctggtgtg cttctcgtat ttatttttat 4740tctaatgatc cattaaaggt atatatttat ttcttgttat ataatccttt tgtttattac 4800atgggctgga tacataaagg tattttgatt taattttttg cttaaattca atcccccctc 4860gttcagtgtc aactgtaatg gtaggaaatt accatacttt tgaagaagca aaaaaaatga 4920aagaaaaaaa aaatcgtatt tccaggttag acgttccgca gaatctagaa tgcggtatgc 4980ggtacattgt tcttcgaacg taaaagttgc gctccctgag atattgtaca tttttgcttt 5040tacaagtaca agtacatcgt acaactatgt actactgttg atgcatccac aacagtttgt 5100tttgtttttt tttgtttttt ttttttctaa tgattcatta ccgctatgta tacctacttg 5160tacttgtagt aagccgggtt attggcgttc aattaatcat agacttatga atctgcacgg 5220tgtgcgctgc gagttacttt tagcttatgc atgctacttg ggtgtaatat tgggatctgt 5280tcggaaatca acggatgctc aatcgatttc gacagtaatt aattaagtca tacacaagtc 5340agctttcttc gagcctcata taagtataag tagttcaacg tattagcact gtacccagca 5400tctccgtatc gagaaacaca acaacatgcc ccattggaca gatcatgcgg atacacaggt 5460tgtgcagtat catacatact cgatcagaca ggtcgtctga ccatcataca agctgaacaa 5520gcgctccata cttgcacgct ctctatatac acagttaaat tacatatcca tagtctaacc 5580tctaacagtt aatcttctgg taagcctccc agccagcctt ctggtatcgc ttggcctcct 5640caataggatc tcggttctgg ccgtacagac ctcggccgac aattatgata tccgttccgg 5700tagacatgac atcctcaaca gttcggtact gctgtccgag agcgtctccc ttgtcgtcaa 5760gacccacccc gggggtcaga ataagccagt cctcagagtc gcccttaggt cggttctggg 5820caatgaagcc aaccacaaac tcggggtcgg atcgggcaag ctcaatggtc tgcttggagt 5880actcgccagt ggccagagag cccttgcaag acagctcggc cagcatgagc agacctctgg 5940ccagcttctc gttgggagag gggactagga actccttgta ctgggagttc tcgtagtcag 6000agacgtcctc cttcttctgt tcagagacag tttcctcggc accagctcgc aggccagcaa 6060tgattccggt tccgggtaca ccgtgggcgt tggtgatatc ggaccactcg gcgattcggt 6120gacaccggta ctggtgcttg acagtgttgc caatatctgc gaactttctg tcctcgaaca 6180ggaagaaacc gtgcttaaga gcaagttcct tgagggggag cacagtgccg gcgtaggtga 6240agtcgtcaat gatgtcgata tgggttttga tcatgcacac ataaggtccg accttatcgg 6300caagctcaat gagctccttg gtggtggtaa catccagaga agcacacagg ttggttttct 6360tggctgccac gagcttgagc actcgagcgg caaaggcgga cttgtggacg ttagctcgag 6420cttcgtagga gggcattttg gtggtgaaga ggagactgaa ataaatttag tctgcagaac 6480tttttatcgg aaccttatct ggggcagtga agtatatgtt atggtaatag ttacgagtta 6540gttgaactta tagatagact ggactatacg gctatcggtc caaattagaa agaacgtcaa 6600tggctctctg ggcgtcgcct ttgccgacaa aaatgtgatc atgatgaaag ccagcaatga 6660cgttgcagct gatattgttg tcggccaacc gcgccgaaaa cgcagctgtc agacccacag 6720cctccaacga agaatgtatc gtcaaagtga tccaagcaca ctcatagttg gagtcgtact 6780ccaaaggcgg caatgacgag tcagacagat actcgtcgac gtttaaacag tgtacgcaga 6840tctactatag aggaacattt aaattgcccc ggagaagacg gccaggccgc ctagatgaca 6900aattcaacaa ctcacagctg actttctgcc attgccacta ggggggggcc tttttatatg 6960gccaagccaa gctctccacg tcggttgggc tgcacccaac aataaatggg tagggttgca 7020ccaacaaagg gatgggatgg ggggtagaag atacgaggat aacggggctc aatggcacaa 7080ataagaacga atactgccat taagactcgt gatccagcga ctgacaccat tgcatcatct 7140aagggcctca aaactacctc ggaactgctg cgctgatctg gacaccacag aggttccgag 7200cactttaggt tgcaccaaat gtcccaccag gtgcaggcag aaaacgctgg aacagcgtgt 7260acagtttgtc ttaacaaaaa gtgagggcgc tgaggtcgag cagggtggtg tgacttgtta 7320tagcctttag agctgcgaaa gcgcgtatgg atttggctca tcaggccaga ttgagggtct 7380gtggacacat gtcatgttag tgtacttcaa tcgccccctg gatatagccc cgacaatagg 7440ccgtggcctc atttttttgc cttccgcaca tttccattgc tcgataccca caccttgctt 7500ctcctgcact tgccaacctt aatactggtt tacattgacc aacatcttac aagcgggggg 7560cttgtctagg gtatatataa acagtggctc tcccaatcgg ttgccagtct cttttttcct 7620ttctttcccc acagattcga aatctaaact acacatcaca gaattccgag ccgtgagtat 7680ccacgacaag atcagtgtcg agacgacgcg ttttgtgtaa tgacacaatc cgaaagtcgc 7740tagcaacaca cactctctac acaaactaac ccagctctgg tac 77832019DNAArtificial Sequenceoligonucleotide oYFBA1 20acgcagatct actatagag 192127DNAArtificial Sequenceoligonucleotide oYFBA1-6 21agcggccgct ggtaccagag ctgggtt 27226992DNAArtificial Sequenceplasmid pY158 22ggccgcaagt gtggatgggg aagtgagtgc ccggttctgt gtgcacaatt ggcaatccaa 60gatggatgga ttcaacacag ggatatagcg agctacgtgg tggtgcgagg atatagcaac 120ggatatttat gtttgacact tgagaatgta cgatacaagc actgtccaag tacaatacta 180aacatactgt acatactcat actcgtaccc ggcaacggtt tcacttgagt gcagtggcta 240gtgctcttac tcgtacagtg tgcaatactg cgtatcatag tctttgatgt atatcgtatt 300cattcatgtt agttgcgtac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 360agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 420gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 480gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 540ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 600aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 660ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 720gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 780cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 840gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 900tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 960cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 1020cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 1080gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc 1140agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 1200cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 1260tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 1320tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 1380ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 1440cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc 1500cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat 1560accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag 1620ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg 1680ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc 1740tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 1800acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg 1860tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 1920actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 1980ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 2040aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 2100ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 2160cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc 2220aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 2280actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag 2340cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc 2400ccgaaaagtg ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt 2460tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt 2520cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc 2580tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga 2640tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc 2700cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt 2760ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct 2820gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttccattcg 2880ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc 2940cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc 3000cagtcacgac gttgtaaaac gacggccagt gaattgtaat acgactcact atagggcgaa 3060ttgggtaccg ggccccccct cgaggtcgat ggtgtcgata agcttgatat cgaattcatg 3120tcacacaaac cgatcttcgc ctcaaggaaa cctaattcta catccgagag actgccgaga 3180tccagtctac actgattaat tttcgggcca ataatttaaa aaaatcgtgt tatataatat 3240tatatgtatt atatatatac atcatgatga tactgacagt catgtcccat tgctaaatag 3300acagactcca tctgccgcct ccaactgatg ttctcaatat ttaaggggtc atctcgcatt 3360gtttaataat aaacagactc catctaccgc ctccaaatga tgttctcaaa atatattgta 3420tgaacttatt tttattactt agtattatta gacaacttac ttgctttatg aaaaacactt 3480cctatttagg aaacaattta taatggcagt tcgttcattt aacaatttat gtagaataaa 3540tgttataaat gcgtatggga aatcttaaat atggatagca taaatgatat ctgcattgcc 3600taattcgaaa tcaacagcaa cgaaaaaaat cccttgtaca acataaatag tcatcgagaa 3660atatcaacta tcaaagaaca gctattcaca cgttactatt gagattatta ttggacgaga 3720atcacacact caactgtctt tctctcttct agaaatacag gtacaagtat gtactattct 3780cattgttcat acttctagtc atttcatccc acatattcct tggatttctc tccaatgaat 3840gacattctat cttgcaaatt caacaattat aataagatat accaaagtag cggtatagtg 3900gcaatcaaaa agcttctctg gtgtgcttct cgtatttatt tttattctaa tgatccatta 3960aaggtatata tttatttctt gttatataat ccttttgttt attacatggg ctggatacat 4020aaaggtattt tgatttaatt ttttgcttaa attcaatccc ccctcgttca gtgtcaactg 4080taatggtagg aaattaccat acttttgaag aagcaaaaaa aatgaaagaa aaaaaaaatc 4140gtatttccag gttagacgtt ccgcagaatc tagaatgcgg tatgcggtac attgttcttc 4200gaacgtaaaa gttgcgctcc ctgagatatt gtacattttt gcttttacaa gtacaagtac 4260atcgtacaac tatgtactac tgttgatgca tccacaacag tttgttttgt ttttttttgt 4320tttttttttt tctaatgatt cattaccgct atgtatacct acttgtactt gtagtaagcc 4380gggttattgg cgttcaatta atcatagact tatgaatctg cacggtgtgc gctgcgagtt 4440acttttagct tatgcatgct acttgggtgt aatattggga tctgttcgga aatcaacgga 4500tgctcaatcg atttcgacag taattaatta agtcatacac aagtcagctt tcttcgagcc 4560tcatataagt ataagtagtt caacgtatta gcactgtacc cagcatctcc gtatcgagaa 4620acacaacaac atgccccatt ggacagatca tgcggataca caggttgtgc agtatcatac 4680atactcgatc agacaggtcg tctgaccatc atacaagctg aacaagcgct ccatacttgc 4740acgctctcta tatacacagt taaattacat atccatagtc taacctctaa cagttaatct 4800tctggtaagc ctcccagcca gccttctggt atcgcttggc ctcctcaata ggatctcggt 4860tctggccgta cagacctcgg ccgacaatta tgatatccgt tccggtagac atgacatcct 4920caacagttcg gtactgctgt ccgagagcgt ctcccttgtc gtcaagaccc accccggggg 4980tcagaataag ccagtcctca gagtcgccct taggtcggtt ctgggcaatg aagccaacca 5040caaactcggg gtcggatcgg gcaagctcaa tggtctgctt ggagtactcg ccagtggcca 5100gagagccctt gcaagacagc tcggccagca tgagcagacc tctggccagc ttctcgttgg 5160gagaggggac taggaactcc ttgtactggg agttctcgta gtcagagacg tcctccttct 5220tctgttcaga gacagtttcc tcggcaccag ctcgcaggcc agcaatgatt ccggttccgg 5280gtacaccgtg ggcgttggtg atatcggacc actcggcgat tcggtgacac cggtactggt 5340gcttgacagt gttgccaata tctgcgaact ttctgtcctc gaacaggaag aaaccgtgct 5400taagagcaag ttccttgagg gggagcacag tgccggcgta ggtgaagtcg tcaatgatgt 5460cgatatgggt tttgatcatg cacacataag gtccgacctt atcggcaagc tcaatgagct 5520ccttggtggt ggtaacatcc agagaagcac acaggttggt tttcttggct gccacgagct 5580tgagcactcg agcggcaaag gcggacttgt ggacgttagc tcgagcttcg taggagggca 5640ttttggtggt gaagaggaga ctgaaataaa tttagtctgc agaacttttt atcggaacct 5700tatctggggc agtgaagtat atgttatggt aatagttacg agttagttga acttatagat 5760agactggact atacggctat cggtccaaat tagaaagaac gtcaatggct ctctgggcgt 5820cgcctttgcc gacaaaaatg tgatcatgat gaaagccagc aatgacgttg cagctgatat 5880tgttgtcggc caaccgcgcc gaaaacgcag ctgtcagacc cacagcctcc aacgaagaat 5940gtatcgtcaa agtgatccaa gcacactcat agttggagtc gtactccaaa ggcggcaatg 6000acgagtcaga cagatactcg tcgacgttta aacagtgtac gcagatctac tatagaggaa 6060catttaaatt gccccggaga agacggccag gccgcctaga tgacaaattc aacaactcac 6120agctgacttt ctgccattgc cactaggggg gggccttttt atatggccaa gccaagctct 6180ccacgtcggt tgggctgcac ccaacaataa atgggtaggg ttgcaccaac aaagggatgg 6240gatggggggt agaagatacg aggataacgg ggctcaatgg cacaaataag aacgaatact 6300gccattaaga ctcgtgatcc agcgactgac accattgcat catctaaggg cctcaaaact 6360acctcggaac tgctgcgctg atctggacac cacagaggtt ccgagcactt taggttgcac 6420caaatgtccc accaggtgca ggcagaaaac gctggaacag cgtgtacagt ttgtcttagc 6480aaaaagtgaa ggcgctgagg tcgagcaggg tggtgtgact tgttatagcc tttagagctg 6540cgaaagcgcg tatggatttg gctcatcagg ccagattgag ggtctgtgga cacatgtcat 6600gttagtgtac ttcaatcgcc ccctggatat agccccgaca ataggccgtg gcctcatttt 6660tttgccttcc gcacatttcc attgctcgat acccacacct tgcttctcct gcacttgcca 6720accttaatac tggtttacat tgaccaacat cttacaagcg gggggcttgt ctagggtata 6780tataaacagt ggctctccca atcggttgcc agtctctttt ttcctttctt tccccacaga 6840ttcgaaatct aaactacaca tcacagaatt ccgagccgtg agtatccacg acaagatcag 6900tgtcgagacg acgcgttttg tgtaatgaca caatccgaaa gtcgctagca acacacactc 6960tctacacaaa ctaacccagc tctggtacca gc 6992238707DNAArtificial Sequenceplasmid pY159 23ggccgcaagt gtggatgggg aagtgagtgc ccggttctgt gtgcacaatt ggcaatccaa 60gatggatgga ttcaacacag ggatatagcg agctacgtgg tggtgcgagg atatagcaac 120ggatatttat gtttgacact tgagaatgta cgatacaagc actgtccaag tacaatacta 180aacatactgt acatactcat actcgtaccc ggcaacggtt tcacttgagt gcagtggcta 240gtgctcttac tcgtacagtg tgcaatactg cgtatcatag tctttgatgt atatcgtatt 300cattcatgtt agttgcgtac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 360agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 420gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 480gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 540ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 600aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 660ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 720gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 780cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 840gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 900tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 960cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 1020cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 1080gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc 1140agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 1200cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 1260tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 1320tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 1380ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 1440cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc 1500cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat 1560accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag 1620ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg 1680ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc 1740tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 1800acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg 1860tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 1920actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 1980ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 2040aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 2100ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 2160cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc 2220aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 2280actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag 2340cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc 2400ccgaaaagtg ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt 2460tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt 2520cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc 2580tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga 2640tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc 2700cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt 2760ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct 2820gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttccattcg 2880ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc 2940cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc 3000cagtcacgac gttgtaaaac gacggccagt gaattgtaat acgactcact atagggcgaa 3060ttgggtaccg ggccccccct cgaggtcgat ggtgtcgata agcttgatat cgaattcatg 3120tcacacaaac cgatcttcgc ctcaaggaaa cctaattcta catccgagag actgccgaga 3180tccagtctac actgattaat tttcgggcca ataatttaaa aaaatcgtgt tatataatat 3240tatatgtatt atatatatac atcatgatga tactgacagt catgtcccat tgctaaatag 3300acagactcca tctgccgcct ccaactgatg ttctcaatat ttaaggggtc atctcgcatt 3360gtttaataat aaacagactc catctaccgc ctccaaatga tgttctcaaa atatattgta 3420tgaacttatt tttattactt agtattatta gacaacttac ttgctttatg aaaaacactt 3480cctatttagg aaacaattta taatggcagt tcgttcattt aacaatttat gtagaataaa 3540tgttataaat gcgtatggga aatcttaaat atggatagca taaatgatat ctgcattgcc 3600taattcgaaa tcaacagcaa cgaaaaaaat cccttgtaca acataaatag tcatcgagaa 3660atatcaacta tcaaagaaca gctattcaca cgttactatt gagattatta ttggacgaga 3720atcacacact caactgtctt tctctcttct agaaatacag gtacaagtat gtactattct 3780cattgttcat acttctagtc atttcatccc acatattcct tggatttctc tccaatgaat 3840gacattctat cttgcaaatt caacaattat aataagatat accaaagtag cggtatagtg 3900gcaatcaaaa agcttctctg gtgtgcttct cgtatttatt tttattctaa tgatccatta 3960aaggtatata tttatttctt gttatataat ccttttgttt attacatggg ctggatacat 4020aaaggtattt tgatttaatt ttttgcttaa attcaatccc ccctcgttca gtgtcaactg 4080taatggtagg aaattaccat acttttgaag aagcaaaaaa aatgaaagaa aaaaaaaatc 4140gtatttccag gttagacgtt ccgcagaatc tagaatgcgg

tatgcggtac attgttcttc 4200gaacgtaaaa gttgcgctcc ctgagatatt gtacattttt gcttttacaa gtacaagtac 4260atcgtacaac tatgtactac tgttgatgca tccacaacag tttgttttgt ttttttttgt 4320tttttttttt tctaatgatt cattaccgct atgtatacct acttgtactt gtagtaagcc 4380gggttattgg cgttcaatta atcatagact tatgaatctg cacggtgtgc gctgcgagtt 4440acttttagct tatgcatgct acttgggtgt aatattggga tctgttcgga aatcaacgga 4500tgctcaatcg atttcgacag taattaatta agtcatacac aagtcagctt tcttcgagcc 4560tcatataagt ataagtagtt caacgtatta gcactgtacc cagcatctcc gtatcgagaa 4620acacaacaac atgccccatt ggacagatca tgcggataca caggttgtgc agtatcatac 4680atactcgatc agacaggtcg tctgaccatc atacaagctg aacaagcgct ccatacttgc 4740acgctctcta tatacacagt taaattacat atccatagtc taacctctaa cagttaatct 4800tctggtaagc ctcccagcca gccttctggt atcgcttggc ctcctcaata ggatctcggt 4860tctggccgta cagacctcgg ccgacaatta tgatatccgt tccggtagac atgacatcct 4920caacagttcg gtactgctgt ccgagagcgt ctcccttgtc gtcaagaccc accccggggg 4980tcagaataag ccagtcctca gagtcgccct taggtcggtt ctgggcaatg aagccaacca 5040caaactcggg gtcggatcgg gcaagctcaa tggtctgctt ggagtactcg ccagtggcca 5100gagagccctt gcaagacagc tcggccagca tgagcagacc tctggccagc ttctcgttgg 5160gagaggggac taggaactcc ttgtactggg agttctcgta gtcagagacg tcctccttct 5220tctgttcaga gacagtttcc tcggcaccag ctcgcaggcc agcaatgatt ccggttccgg 5280gtacaccgtg ggcgttggtg atatcggacc actcggcgat tcggtgacac cggtactggt 5340gcttgacagt gttgccaata tctgcgaact ttctgtcctc gaacaggaag aaaccgtgct 5400taagagcaag ttccttgagg gggagcacag tgccggcgta ggtgaagtcg tcaatgatgt 5460cgatatgggt tttgatcatg cacacataag gtccgacctt atcggcaagc tcaatgagct 5520ccttggtggt ggtaacatcc agagaagcac acaggttggt tttcttggct gccacgagct 5580tgagcactcg agcggcaaag gcggacttgt ggacgttagc tcgagcttcg taggagggca 5640ttttggtggt gaagaggaga ctgaaataaa tttagtctgc agaacttttt atcggaacct 5700tatctggggc agtgaagtat atgttatggt aatagttacg agttagttga acttatagat 5760agactggact atacggctat cggtccaaat tagaaagaac gtcaatggct ctctgggcgt 5820cgcctttgcc gacaaaaatg tgatcatgat gaaagccagc aatgacgttg cagctgatat 5880tgttgtcggc caaccgcgcc gaaaacgcag ctgtcagacc cacagcctcc aacgaagaat 5940gtatcgtcaa agtgatccaa gcacactcat agttggagtc gtactccaaa ggcggcaatg 6000acgagtcaga cagatactcg tcgacgttta aacagtgtac gcagatctac tatagaggaa 6060catttaaatt gccccggaga agacggccag gccgcctaga tgacaaattc aacaactcac 6120agctgacttt ctgccattgc cactaggggg gggccttttt atatggccaa gccaagctct 6180ccacgtcggt tgggctgcac ccaacaataa atgggtaggg ttgcaccaac aaagggatgg 6240gatggggggt agaagatacg aggataacgg ggctcaatgg cacaaataag aacgaatact 6300gccattaaga ctcgtgatcc agcgactgac accattgcat catctaaggg cctcaaaact 6360acctcggaac tgctgcgctg atctggacac cacagaggtt ccgagcactt taggttgcac 6420caaatgtccc accaggtgca ggcagaaaac gctggaacag cgtgtacagt ttgtcttagc 6480aaaaagtgaa ggcgctgagg tcgagcaggg tggtgtgact tgttatagcc tttagagctg 6540cgaaagcgcg tatggatttg gctcatcagg ccagattgag ggtctgtgga cacatgtcat 6600gttagtgtac ttcaatcgcc ccctggatat agccccgaca ataggccgtg gcctcatttt 6660tttgccttcc gcacatttcc attgctcgat acccacacct tgcttctcct gcacttgcca 6720accttaatac tggtttacat tgaccaacat cttacaagcg gggggcttgt ctagggtata 6780tataaacagt ggctctccca atcggttgcc agtctctttt ttcctttctt tccccacaga 6840ttcgaaatct aaactacaca tcacagaatt ccgagccgtg agtatccacg acaagatcag 6900tgtcgagacg acgcgttttg tgtaatgaca caatccgaaa gtcgctagca acacacactc 6960tctacacaaa ctaacccagc tctggtacca gcggccatca caagtttgta caaaaaagct 7020gaacgagaaa cgtaaaatga tataaatatc aatatattaa attagatttt gcataaaaaa 7080cagactacat aatactgtaa aacacaacat atccagtcat attggcggcc gcattaggca 7140ccccaggctt tacactttat gcttccggct cgtataatgt gtggattttg agttaggatc 7200cgtcgagatt ttcaggagct aaggaagcta aaatggagaa aaaaatcact ggatatacca 7260ccgttgatat atcccaatgg catcgtaaag aacattttga ggcatttcag tcagttgctc 7320aatgtaccta taaccagacc gttcagctgg atattacggc ctttttaaag accgtaaaga 7380aaaataagca caagttttat ccggccttta ttcacattct tgcccgcctg atgaatgctc 7440atccggaatt ccgtatggca atgaaagacg gtgagctggt gatatgggat agtgttcacc 7500cttgttacac cgttttccat gagcaaactg aaacgttttc atcgctctgg agtgaatacc 7560acgacgattt ccggcagttt ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa 7620acctggccta tttccctaaa gggtttattg agaatatgtt tttcgtctca gccaatccct 7680gggtgagttt caccagtttt gatttaaacg tggccaatat ggacaacttc ttcgcccccg 7740ttttcaccat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg ctggcgattc 7800aggttcatca tgccgtttgt gatggcttcc atgtcggcag aatgcttaat gaattacaac 7860agtactgcga tgagtggcag ggcggggcgt aaacgcgtgg atccggctta ctaaaagcca 7920gataacagta tgcgtatttg cgcgctgatt tttgcggtat aagaatatat actgatatgt 7980atacccgaag tatgtcaaaa agaggtatgc tatgaagcag cgtattacag tgacagttga 8040cagcgacagc tatcagttgc tcaaggcata tatgatgtca atatctccgg tctggtaagc 8100acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac gctggaaagc ggaaaatcag 8160gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg gctcttttgc tgacgagaac 8220aggggctggt gaaatgcagt ttaaggttta cacctataaa agagagagcc gttatcgtct 8280gtttgtggat gtacagagtg atattattga cacgcccggg cgacggatgg tgatccccct 8340ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg tggtgcatat 8400cggggatgaa agctggcgca tgatgaccac cgatatggcc agtgtgccgg tctccgttat 8460cggggaagaa gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg ccattaacct 8520gatgttctgg ggaatataaa tgtcaggctc ccttatacac agccagtctg caggtcgacc 8580atagtgactg gatatgttgt gttttacagc attatgtagt ctgtttttta tgcaaaatct 8640aatttaatat attgatattt atatcatttt acgtttctcg ttcagctttc ttgtacaaag 8700tggtgat 8707248827DNAArtificial Sequenceplasmid pY169 24cttgtacaaa gtggtgatgg ccgcaagtgt ggatggggaa gtgagtgccc ggttctgtgt 60gcacaattgg caatccaaga tggatggatt caacacaggg atatagcgag ctacgtggtg 120gtgcgaggat atagcaacgg atatttatgt ttgacacttg agaatgtacg atacaagcac 180tgtccaagta caatactaaa catactgtac atactcatac tcgtacccgg caacggtttc 240acttgagtgc agtggctagt gctcttactc gtacagtgtg caatactgcg tatcatagtc 300tttgatgtat atcgtattca ttcatgttag ttgcgtacga gccggaagca taaagtgtaa 360agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc 420tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag 480aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 540cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 600atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 660taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 720aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 780tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 840gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 900cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 960cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 1020atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 1080tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat 1140ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 1200acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 1260aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 1320aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 1380tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 1440cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 1500catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 1560ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 1620aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 1680ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 1740caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 1800attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 1860agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 1920actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 1980ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 2040ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 2100gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 2160atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 2220cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 2280gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 2340gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 2400ggttccgcgc acatttcccc gaaaagtgcc acctgacgcg ccctgtagcg gcgcattaag 2460cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc 2520cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc 2580tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa 2640aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg 2700ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac 2760actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta 2820ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac 2880gcttacaatt tccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg 2940gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg 3000gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga attgtaatac 3060gactcactat agggcgaatt gggtaccggg ccccccctcg aggtcgatgg tgtcgataag 3120cttgatatcg aattcatgtc acacaaaccg atcttcgcct caaggaaacc taattctaca 3180tccgagagac tgccgagatc cagtctacac tgattaattt tcgggccaat aatttaaaaa 3240aatcgtgtta tataatatta tatgtattat atatatacat catgatgata ctgacagtca 3300tgtcccattg ctaaatagac agactccatc tgccgcctcc aactgatgtt ctcaatattt 3360aaggggtcat ctcgcattgt ttaataataa acagactcca tctaccgcct ccaaatgatg 3420ttctcaaaat atattgtatg aacttatttt tattacttag tattattaga caacttactt 3480gctttatgaa aaacacttcc tatttaggaa acaatttata atggcagttc gttcatttaa 3540caatttatgt agaataaatg ttataaatgc gtatgggaaa tcttaaatat ggatagcata 3600aatgatatct gcattgccta attcgaaatc aacagcaacg aaaaaaatcc cttgtacaac 3660ataaatagtc atcgagaaat atcaactatc aaagaacagc tattcacacg ttactattga 3720gattattatt ggacgagaat cacacactca actgtctttc tctcttctag aaatacaggt 3780acaagtatgt actattctca ttgttcatac ttctagtcat ttcatcccac atattccttg 3840gatttctctc caatgaatga cattctatct tgcaaattca acaattataa taagatatac 3900caaagtagcg gtatagtggc aatcaaaaag cttctctggt gtgcttctcg tatttatttt 3960tattctaatg atccattaaa ggtatatatt tatttcttgt tatataatcc ttttgtttat 4020tacatgggct ggatacataa aggtattttg atttaatttt ttgcttaaat tcaatccccc 4080ctcgttcagt gtcaactgta atggtaggaa attaccatac ttttgaagaa gcaaaaaaaa 4140tgaaagaaaa aaaaaatcgt atttccaggt tagacgttcc gcagaatcta gaatgcggta 4200tgcggtacat tgttcttcga acgtaaaagt tgcgctccct gagatattgt acatttttgc 4260ttttacaagt acaagtacat cgtacaacta tgtactactg ttgatgcatc cacaacagtt 4320tgttttgttt ttttttgttt tttttttttc taatgattca ttaccgctat gtatacctac 4380ttgtacttgt agtaagccgg gttattggcg ttcaattaat catagactta tgaatctgca 4440cggtgtgcgc tgcgagttac ttttagctta tgcatgctac ttgggtgtaa tattgggatc 4500tgttcggaaa tcaacggatg ctcaatcgat ttcgacagta attaattaag tcatacacaa 4560gtcagctttc ttcgagcctc atataagtat aagtagttca acgtattagc actgtaccca 4620gcatctccgt atcgagaaac acaacaacat gccccattgg acagatcatg cggatacaca 4680ggttgtgcag tatcatacat actcgatcag acaggtcgtc tgaccatcat acaagctgaa 4740caagcgctcc atacttgcac gctctctata tacacagtta aattacatat ccatagtcta 4800acctctaaca gttaatcttc tggtaagcct cccagccagc cttctggtat cgcttggcct 4860cctcaatagg atctcggttc tggccgtaca gacctcggcc gacaattatg atatccgttc 4920cggtagacat gacatcctca acagttcggt actgctgtcc gagagcgtct cccttgtcgt 4980caagacccac cccgggggtc agaataagcc agtcctcaga gtcgccctta ggtcggttct 5040gggcaatgaa gccaaccaca aactcggggt cggatcgggc aagctcaatg gtctgcttgg 5100agtactcgcc agtggccaga gagcccttgc aagacagctc ggccagcatg agcagacctc 5160tggccagctt ctcgttggga gaggggacta ggaactcctt gtactgggag ttctcgtagt 5220cagagacgtc ctccttcttc tgttcagaga cagtttcctc ggcaccagct cgcaggccag 5280caatgattcc ggttccgggt acaccgtggg cgttggtgat atcggaccac tcggcgattc 5340ggtgacaccg gtactggtgc ttgacagtgt tgccaatatc tgcgaacttt ctgtcctcga 5400acaggaagaa accgtgctta agagcaagtt ccttgagggg gagcacagtg ccggcgtagg 5460tgaagtcgtc aatgatgtcg atatgggttt tgatcatgca cacataaggt ccgaccttat 5520cggcaagctc aatgagctcc ttggtggtgg taacatccag agaagcacac aggttggttt 5580tcttggctgc cacgagcttg agcactcgag cggcaaaggc ggacttgtgg acgttagctc 5640gagcttcgta ggagggcatt ttggtggtga agaggagact gaaataaatt tagtctgcag 5700aactttttat cggaacctta tctggggcag tgaagtatat gttatggtaa tagttacgag 5760ttagttgaac ttatagatag actggactat acggctatcg gtccaaatta gaaagaacgt 5820caatggctct ctgggcgtcg cctttgccga caaaaatgtg atcatgatga aagccagcaa 5880tgacgttgca gctgatattg ttgtcggcca accgcgccga aaacgcagct gtcagaccca 5940cagcctccaa cgaagaatgt atcgtcaaag tgatccaagc acactcatag ttggagtcgt 6000actccaaagg cggcaatgac gagtcagaca gatactcgtc gacgtttaaa cagtgtacgc 6060agatctacta tagaggaaca tttaaattgc cccggagaag acggccaggc cgcctagatg 6120acaaattcaa caactcacag ctgactttct gccattgcca ctaggggggg gcctttttat 6180atggccaagc caagctctcc acgtcggttg ggctgcaccc aacaataaat gggtagggtt 6240gcaccaacaa agggatggga tggggggtag aagatacgag gataacgggg ctcaatggca 6300caaataagaa cgaatactgc cattaagact cgtgatccag cgactgacac cattgcatca 6360tctaagggcc tcaaaactac ctcggaactg ctgcgctgat ctggacacca cagaggttcc 6420gagcacttta ggttgcacca aatgtcccac caggtgcagg cagaaaacgc tggaacagcg 6480tgtacagttt gtcttagcaa aaagtgaagg cgctgaggtc gagcagggtg gtgtgacttg 6540ttatagcctt tagagctgcg aaagcgcgta tggatttggc tcatcaggcc agattgaggg 6600tctgtggaca catgtcatgt tagtgtactt caatcgcccc ctggatatag ccccgacaat 6660aggccgtggc ctcatttttt tgccttccgc acatttccat tgctcgatac ccacaccttg 6720cttctcctgc acttgccaac cttaatactg gtttacattg accaacatct tacaagcggg 6780gggcttgtct agggtatata taaacagtgg ctctcccaat cggttgccag tctctttttt 6840cctttctttc cccacagatt cgaaatctaa actacacatc acagaattcc gagccgtgag 6900tatccacgac aagatcagtg tcgagacgac gcgttttgtg taatgacaca atccgaaagt 6960cgctagcaac acacactctc tacacaaact aacccagctc tggtaccagc ggccatcaca 7020agtttgtaca aaaaagttgg attttttttc gggatggcca ccatctcttt gactactgag 7080caacttttag aacacccaga actggttgca attgatgggg tgttgtacga tctcttcgga 7140ctggcgaaag tgcatccagg tggcaacctc attgaagccg ccggtgcctc cgacggaacc 7200gccctgttct actccatgca ccctggagtg aagccagaga attcgaagct gctgcagcaa 7260tttgcccgag gcaaacacga acgaagctcg aaggacccag tgtacacctt tgacagtccc 7320ttcgcccagg atgtcaagca gagcgttcgg gaggtcatga aggggcgcaa ctggtacgcc 7380acgcccggct tttggctgcg gaccgcgctg atcatcgcgt gcactgccat aggcgaatgg 7440tattggatca ctaccggggc agtgatgtgg ggcatcttca ccgggtactt ccacagccag 7500attgggttgg cgattcaaca cgatgcctct cacggagcca tcagcaaaaa gccctgggtg 7560aacgcctttt tcgcctacgg catcgacgcc attggatcct cccgctggat ctggctgcag 7620tcccacatta tgcgccacca cacctacacc aaccagcatg gcctggacct ggacgctgcc 7680tcggcggagc cgttcatttt gttccactcc tacccggcaa caaatgcgtc acgaaagtgg 7740taccatcggt tccaggcgtg gtacatgtac atcgttttgg ggatgtatgg tgtgtcgatg 7800gtgtacaatc cgatgtactt gttcacgatg cagcacaacg acacaatccc agaggccacc 7860tctcttagac caggcagctt tttcaaccgg cagcgcgcct tcgccgtttc cctccgccta 7920ctgttcatct tccgcaacgc cttcctcccc tggtacatcg cgggcgcctc tccgctgctc 7980accatcctgc tggtgccaac ggtcacaggc atcttcttga catttgtttt tgtgctgtcc 8040cataactttg aaggcgctga gcggaccccc gaaaagaact gcaaggccaa aagggccaag 8100gaggggaagg aggtccgcga tgtagaggag gaccgggtgg actggtaccg ggcgcaggcc 8160gagaccgcgg cgacctacgg gggcagcgtc gggatgatgc tgaccggcgg tttgaacctg 8220cagatcgagc accacttgtt cccccgcatg tcctcttggc actacccctt catccaagat 8280acggtgcggg aatgttgcaa gcgccatggc gtgcgctaca catactaccc gaccatcctg 8340gagaatataa tgtccacgct ccgctacatg cagaaggtgg gcgtggccca cacaattcag 8400gatgcccagg aattctgagt gagttcgatc cgcatcgacg tctaccattt ttgatgctgt 8460ctattcctgt tttcagtcac ctccagcatt ctcatggctg gtgaccactg cccctctaac 8520ccattgtgac acaccgccaa agactttgcc tctttttttt ccctttcttt tgtcctcggg 8580gtgctttggc cggtgtttac tcgccttgca gtccccgcaa acgaccgacg tttaagctcc 8640gttgttgact gggccgctcg taaacccatc tgcaggttga ggctcccatg gagaattgtg 8700atggctgatt aggaggtggc ggggcataca tgcctcgaca ctcaaagccg ggcggcttct 8760ggattcgaaa acgcaaatgg gcgctttgga aaaaaaaaaa aaaaaaaaaa aaaaaaaacc 8820caacttt 882725774DNAEuglena gracilis 25atggaggtgg tgaatgaaat agtctcaatt gggcaggaag ttttacccaa agttgattat 60gcccaactct ggagtgatgc cagtcactgt gaggtgcttt acttgtccat cgcatttgtc 120atcttgaagt tcactcttgg cccccttggt ccaaaaggtc agtctcgtat gaagtttgtt 180ttcaccaatt acaaccttct catgtccatt tattcgttgg gatcattcct ctcaatggca 240tatgccatgt acaccatcgg tgttatgtct gacaactgcg agaaggcttt tgacaacaac 300gtcttcagga tcaccacgca gttgttctat ttgagcaagt tcctggagta tattgactcc 360ttctatttgc cactgatggg caagcctctg acctggttgc aattcttcca tcatttgggg 420gcaccgatgg atatgtggct gttctataat taccgaaatg aagctgtttg gatttttgtg 480ctgttgaatg gtttcatcca ctggatcatg tacggttatt attggaccag attgatcaag 540ctgaagttcc ccatgccaaa atccctgatt acatcaatgc agatcattca attcaatgtt 600ggtttctaca ttgtctggaa gtacaggaac attccctgtt atcgccaaga tgggatgagg 660atgtttggct ggttcttcaa ttacttttat gttggcacag tcttgtgttt gttcttgaat 720ttctatgtgc aaacgtatat cgtcaggaag cacaagggag ccaaaaagat tcag 774261263DNAEuglena gracilis 26atgaagtcaa agcgccaagc gcttcccctt acaattgatg gaacaacata tgatgtgtct 60gcctgggtca atttccaccc tggtggtgcg gaaattatag agaattacca aggaagggat 120gccactgatg ccttcatggt tatgcactct caagaagcct tcgacaagct caagcgcatg 180cccaaaatca atcccagttc tgagttgcca ccccaggctg cagtgaatga agctcaagag 240gatttccgga agctccgaga agagttgatc gcaactggca tgtttgatgc ctcccccctc 300tggtactcat acaaaatcag caccacactg ggccttggag tgctgggtta tttcctgatg 360gttcagtatc agatgtattt cattggggca gtgttgcttg ggatgcacta tcaacagatg 420ggctggcttt ctcatgacat ttgccaccac cagactttca agaaccggaa ctggaacaac 480ctcgtgggac tggtatttgg caatggtctg caaggttttt ccgtgacatg gtggaaggac 540agacacaatg cacatcattc ggcaaccaat gttcaagggc acgaccctga tattgacaac 600ctccccctct tagcctggtc tgaggatgac gtcacacggg cgtcaccgat ttcccgcaag 660ctcattcagt tccagcagta ctatttcttg gtcatctgta tcttgttgcg gttcatttgg

720tgtttccaga gcgtgttgac cgtgcgcagt ttgaaggaca gagataacca attctatcgc 780tctcagtata agaaggaggc cattggcctc gccctgcact ggaccttgaa gaccctgttc 840cacttattct ttatgcccag catcctcaca tcgctgttgg tgtttttcgt ttcggagctg 900gttggcggct tcggcattgc gatcgtggtg ttcatgaacc actacccact ggagaagatc 960ggggactcag tctgggatgg ccatggattc tcggttggcc agatccatga gaccatgaac 1020attcggcgag ggattatcac agattggttt ttcggaggct tgaattacca gattgagcac 1080catttgtggc cgaccctccc tcgccacaac ctgacagcgg ttagctacca ggtggaacag 1140ctgtgccaga agcacaacct gccgtatcgg aacccgctgc cccatgaagg gttggtcatc 1200ctgctgcgct atctggcggt gttcgcccgg atggcggaga agcaacccgc ggggaaggct 1260cta 12632730DNAArtificial SequenceEuglena gracilis elongase sense oligonucleotide oEugEL1-1 27agcggccgca ccatggaggt ggtgaatgaa 302830DNAArtificial SequenceEuglena gracilis elongase anti-sense oligonucleotide oEugEL1-2 28tgcggccgct cactgaatct ttttggctcc 30294311DNAArtificial Sequenceplasmid pKR906 29agcggccgca ccatggaggt ggtgaatgaa atagtctcaa ttgggcagga agttttaccc 60aaagttgatt atgcccaact ctggagtgat gccagtcact gtgaggtgct ttacttgtcc 120atcgcatttg tcatcttgaa gttcactctt ggcccccttg gtccaaaagg tcagtctcgt 180atgaagtttg ttttcaccaa ttacaacctt ctcatgtcca tttattcgtt gggatcattc 240ctctcaatgg catatgccat gtacaccatc ggtgttatgt ctgacaactg cgagaaggct 300tttgacaaca acgtcttcag gatcaccacg cagttgttct atttgagcaa gttcctggag 360tatattgact ccttctattt gccactgatg ggcaagcctc tgacctggtt gcaattcttc 420catcatttgg gggcaccgat ggatatgtgg ctgttctata attaccgaaa tgaagctgtt 480tggatttttg tgctgttgaa tggtttcatc cactggatca tgtacggtta ttattggacc 540agattgatca agctgaagtt ccccatgcca aaatccctga ttacatcaat gcagatcatt 600caattcaatg ttggtttcta cattgtctgg aagtacagga acattccctg ttatcgccaa 660gatgggatga ggatgtttgg ctggttcttc aattactttt atgttggcac agtcttgtgt 720ttgttcttga atttctatgt gcaaacgtat atcgtcagga agcacaaggg agccaaaaag 780attcagtgag cggccgcacc tgaattccag cacactggcg gccgttacta gtggatccga 840gctcggtacc aagcttgatg catagcttga gtattctaac gcgtcaccta aatagcttgg 900cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca 960acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca 1020cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 1080attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt 1140cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 1200caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 1260caaaaggcca gcaaaagccc aggaaccgta aaaaggccgc gttgctggcg tttttccata 1320ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 1380cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 1440ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 1500tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 1560gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 1620ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 1680ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 1740gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 1800aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 1860tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 1920ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 1980tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt agcacgtgtc 2040agtcctgctc ctcggccacg aagtgcacgc agttgccggc cgggtcgcgc agggcgaact 2100cccgccccca cggctgctcg ccgatctcgg tcatggccgg cccggaggcg tcccggaagt 2160tcgtggacac gacctccgac cactcggcgt acagctcgtc caggccgcgc acccacaccc 2220aggccagggt gttgtccggc accacctggt cctggaccgc gctgatgaac agggtcacgt 2280cgtcccggac cacaccggcg aagtcgtcct ccacgaagtc ccgggagaac ccgagccggt 2340cggtccagaa ctcgaccgct ccggcgacgt cgcgcgcggt gagcaccgga acggcactgg 2400tcaacttggc catggtggcc ctcctcacgt gctattattg aagcatttat cagggttatt 2460gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 2520gcacatttcc ccgaaaagtg ccacctgtat gcggtgtgaa ataccgcaca gatgcgtaag 2580gagaaaatac cgcatcagga aattgtaagc gttaataatt cagaagaact cgtcaagaag 2640gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg 2700gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg ctatgtcctg 2760atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc ggccattttc 2820caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct cgccgtcggg 2880catgctcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat gctcttcgtc 2940cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct cgatgcgatg 3000tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc 3060atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga gatcctgccc 3120cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt cgagcacagc 3180tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt cttgcagttc 3240attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct gcgctgacag 3300ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat agccgaatag 3360cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa 3420cgatcctcat cctgtctctt gatcagagct tgatcccctg cgccatcaga tccttggcgg 3480cgagaaagcc atccagttta ctttgcaggg cttcccaacc ttaccagagg gcgccccagc 3540tggcaattcc ggttcgcttg ctgtccataa aaccgcccag tctagctatc gccatgtaag 3600cccactgcaa gctacctgct ttctctttgc gcttgcgttt tcccttgtcc agatagccca 3660gtagctgaca ttcatccggg gtcagcaccg tttctgcgga ctggctttct acgtgaaaag 3720gatctaggtg aagatccttt ttgataatct catgcctgac atttatattc cccagaacat 3780caggttaatg gcgtttttga tgtcattttc gcggtggctg agatcagcca cttcttcccc 3840gataacggag accggcacac tggccatatc ggtggtcatc atgcgccagc tttcatcccc 3900gatatgcacc accgggtaaa gttcacggga gactttatct gacagcagac gtgcactggc 3960cagggggatc accatccgtc gccccggcgt gtcaataata tcactctgta catccacaaa 4020cagacgataa cggctctctc ttttataggt gtaaacctta aactgccgta cgtataggct 4080gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 4140agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg 4200ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tgggccctct 4260agatgcatgc tcgagcggcc gccagtgtga tggatatctg cagaattcag g 4311307085DNAArtificial Sequenceplasmid pKR72 30gtacggatcc gtcgacggcg cgcccgatca tccggatata gttcctcctt tcagcaaaaa 60acccctcaag acccgtttag aggccccaag gggttatgct agttattgct cagcggtggc 120agcagccaac tcagcttcct ttcgggcttt gttagcagcc ggatcgatcc aagctgtacc 180tcactattcc tttgccctcg gacgagtgct ggggcgtcgg tttccactat cggcgagtac 240ttctacacag ccatcggtcc agacggccgc gcttctgcgg gcgatttgtg tacgcccgac 300agtcccggct ccggatcgga cgattgcgtc gcatcgaccc tgcgcccaag ctgcatcatc 360gaaattgccg tcaaccaagc tctgatagag ttggtcaaga ccaatgcgga gcatatacgc 420ccggagccgc ggcgatcctg caagctccgg atgcctccgc tcgaagtagc gcgtctgctg 480ctccatacaa gccaaccacg gcctccagaa gaagatgttg gcgacctcgt attgggaatc 540cccgaacatc gcctcgctcc agtcaatgac cgctgttatg cggccattgt ccgtcaggac 600attgttggag ccgaaatccg cgtgcacgag gtgccggact tcggggcagt cctcggccca 660aagcatcagc tcatcgagag cctgcgcgac ggacgcactg acggtgtcgt ccatcacagt 720ttgccagtga tacacatggg gatcagcaat cgcgcatatg aaatcacgcc atgtagtgta 780ttgaccgatt ccttgcggtc cgaatgggcc gaacccgctc gtctggctaa gatcggccgc 840agcgatcgca tccatagcct ccgcgaccgg ctgcagaaca gcgggcagtt cggtttcagg 900caggtcttgc aacgtgacac cctgtgcacg gcgggagatg caataggtca ggctctcgct 960gaattcccca atgtcaagca cttccggaat cgggagcgcg gccgatgcaa agtgccgata 1020aacataacga tctttgtaga aaccatcggc gcagctattt acccgcagga catatccacg 1080ccctcctaca tcgaagctga aagcacgaga ttcttcgccc tccgagagct gcatcaggtc 1140ggagacgctg tcgaactttt cgatcagaaa cttctcgaca gacgtcgcgg tgagttcagg 1200cttttccatg ggtatatctc cttcttaaag ttaaacaaaa ttatttctag agggaaaccg 1260ttgtggtctc cctatagtga gtcgtattaa tttcgcggga tcgagatcga tccaattcca 1320atcccacaaa aatctgagct taacagcaca gttgctcctc tcagagcaga atcgggtatt 1380caacaccctc atatcaacta ctacgttgtg tataacggtc cacatgccgg tatatacgat 1440gactggggtt gtacaaaggc ggcaacaaac ggcgttcccg gagttgcaca caagaaattt 1500gccactatta cagaggcaag agcagcagct gacgcgtaca caacaagtca gcaaacagac 1560aggttgaact tcatccccaa aggagaagct caactcaagc ccaagagctt tgctaaggcc 1620ctaacaagcc caccaaagca aaaagcccac tggctcacgc taggaaccaa aaggcccagc 1680agtgatccag ccccaaaaga gatctccttt gccccggaga ttacaatgga cgatttcctc 1740tatctttacg atctaggaag gaagttcgaa ggtgaaggtg acgacactat gttcaccact 1800gataatgaga aggttagcct cttcaatttc agaaagaatg ctgacccaca gatggttaga 1860gaggcctacg cagcaggtct catcaagacg atctacccga gtaacaatct ccaggagatc 1920aaataccttc ccaagaaggt taaagatgca gtcaaaagat tcaggactaa ttgcatcaag 1980aacacagaga aagacatatt tctcaagatc agaagtacta ttccagtatg gacgattcaa 2040ggcttgcttc ataaaccaag gcaagtaata gagattggag tctctaaaaa ggtagttcct 2100actgaatcta aggccatgca tggagtctaa gattcaaatc gaggatctaa cagaactcgc 2160cgtgaagact ggcgaacagt tcatacagag tcttttacga ctcaatgaca agaagaaaat 2220cttcgtcaac atggtggagc acgacactct ggtctactcc aaaaatgtca aagatacagt 2280ctcagaagac caaagggcta ttgagacttt tcaacaaagg ataatttcgg gaaacctcct 2340cggattccat tgcccagcta tctgtcactt catcgaaagg acagtagaaa aggaaggtgg 2400ctcctacaaa tgccatcatt gcgataaagg aaaggctatc attcaagatg cctctgccga 2460cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag aagacgttcc 2520aaccacgtct tcaaagcaag tggattgatg tgacatctcc actgacgtaa gggatgacgc 2580acaatcccac tatccttcgc aagacccttc ctctatataa ggaagttcat ttcatttgga 2640gaggacacgc tcgagctcat ttctctatta cttcagccat aacaaaagaa ctcttttctc 2700ttcttattaa accatgaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat 2760cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc 2820tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg 2880tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga 2940agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca 3000gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc 3060ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt 3120cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga 3180tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca 3240ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca 3300cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga 3360ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag 3420gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct 3480tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca 3540gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat 3600cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt 3660ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg 3720tccgagggca aaggaatagt gaggtaccta aagaaggagt gcgtcgaagc agatcgttca 3780aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc 3840atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta 3900tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa 3960aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta 4020gatcgatgtc gaatcgatca acctgcatta atgaatcggc caacgcgcgg ggagaggcgg 4080tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 4140gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 4200ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4260ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 4320acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 4380tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4440ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc 4500ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4560ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4620actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 4680gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 4740tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4800caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 4860atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 4920acgttaaggg attttggtca tgacattaac ctataaaaat aggcgtatca cgaggccctt 4980tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac 5040ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc 5100gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag 5160agtgcaccat atggacatat tgtcgttaga acgcggctac aattaataca taaccttatg 5220tatcatacac atacgattta ggtgacacta tagaacggcg cgccaagctt gttgaaacat 5280ccctgaagtg tctcatttta ttttatttat tctttgctga taaaaaaata aaataaaaga 5340agctaagcac acggtcaacc attgctctac tgctaaaagg gttatgtgta gtgttttact 5400gcataaatta tgcagcaaac aagacaactc aaattaaaaa atttcctttg cttgtttttt 5460tgttgtctct gacttgactt tcttgtggaa gttggttgta taaggattgg gacaccattg 5520tccttcttaa tttaatttta ttctttgctg ataaaaaaaa aaatttcata tagtgttaaa 5580taataatttg ttaaataacc aaaaagtcaa atatgtttac tctcgtttaa ataattgaga 5640ttcgtccagc aaggctaaac gattgtatag atttatgaca atatttactt ttttatagat 5700aaatgttata ttataataaa tttatataca tatattatat gttatttatt attattttaa 5760atccttcaat attttatcaa accaactcat aatttttttt ttatctgtaa gaagcaataa 5820aattaaatag acccacttta aggatgatcc aacctttata cagagtaaga gagttcaaat 5880agtacccttt catatacata tcaactaaaa tattagaaat atcatggatc aaaccttata 5940aagacattaa ataagtggat aagtataata tataaatggg tagtatataa tatataaatg 6000gatacaaact tctctcttta taattgttat gtctccttaa catcctaata taatacataa 6060gtgggtaata tataatatat aaatggagac aaacttcttc cattataatt gttatgtctt 6120cttaacactt atgtctcgtt cacaatgcta aggttagaat tgtttagaaa gtcttatagt 6180acacatttgt ttttgtacta tttgaagcat tccataagcc gtcacgattc agatgattta 6240taataataag aggaaattta tcatagaaca ataaggtgca tagatagagt gttaatatat 6300cataacatcc tttgtttatt catagaagaa gtgagatgga gctcagttat tatactgtta 6360catggtcgga tacaatattc catgctctcc atgagctctt acacctacat gcattttagt 6420tcatacttgc ggccgcagta tatcttaaat tctttaatac ggtgtactag gatattgaac 6480tggttcttga tgatgaaaac ctgggccgag attgcagcta tttatagtca taggtcttgt 6540taacatgcat ggacatttgg ccacggggtg gcatgcagtt tgacgggtgt tgaaataaac 6600aaaaatgagg tggcggaaga gaatacgagt ttgaggttgg gttagaaaca acaaatgtga 6660gggctcatga tgggttgagt tggtgaatgt tttgggctgc tcgattgaca cctttgtgag 6720tacgtgttgt tgtgcatggc ttttggggtc cagttttttt ttcttgacgc ggcgatcctg 6780atcagctagt ggataagtga tgtccactgt gtgtgattgc gtttttgttt gaattttatg 6840aacttagaca ttgctatgca aaggatactc tcattgtgtt ttgtcttctt ttgttccttg 6900gctttttctt atgatccaag agactagtca gtgttgtggc attcgagact accaagatta 6960attatgatgg gggaaggata agtaactgat tagtacggac tgttaccaaa ttaattaata 7020agcggcaaat gaagggcatg gatcaaaagc ttggatctcc tgcaggatct ggccggccgg 7080atctc 7085312540DNAArtificial Sequenceplasmid KS102 31cgatcatccg gatatagttc ctcctttcag caaaaaaccc ctcaagaccc gtttagaggc 60cccaaggggt tatgctagtt attgctcagc ggtggcagca gccaactcag cttcctttcg 120ggctttgtta gcagccggat cgatccaagc tgtacctcac tattcctttg ccctcggacg 180agtgctgggg cgtcggtttc cactatcggc gagtacttct acacagccat cggtccagac 240ggccgcgctt ctgcgggcga tttgtgtacg cccgacagtc ccggctccgg atcggacgat 300tgcgtcgcat cgaccctgcg cccaagctgc atcatcgaaa ttgccgtcaa ccaagctctg 360atagagttgg tcaagaccaa tgcggagcat atacgcccgg agccgcggcg atcctgcaag 420ctccggatgc ctccgctcga agtagcgcgt ctgctgctcc atacaagcca accacggcct 480ccagaagaag atgttggcga cctcgtattg ggaatccccg aacatcgcct cgctccagtc 540aatgaccgct gttatgcggc cattgtccgt caggacattg ttggagccga aatccgcgtg 600cacgaggtgc cggacttcgg ggcagtcctc ggcccaaagc atcagctcat cgagagcctg 660cgcgacggac gcactgacgg tgtcgtccat cacagtttgc cagtgataca catggggatc 720agcaatcgcg catatgaaat cacgccatgt agtgtattga ccgattcctt gcggtccgaa 780tgggccgaac ccgctcgtct ggctaagatc ggccgcagcg atcgcatcca tagcctccgc 840gaccggctgc agaacagcgg gcagttcggt ttcaggcagg tcttgcaacg tgacaccctg 900tgcacggcgg gagatgcaat aggtcaggct ctcgctgaat tccccaatgt caagcacttc 960cggaatcggg agcgcggccg atgcaaagtg ccgataaaca taacgatctt tgtagaaacc 1020atcggcgcag ctatttaccc gcaggacata tccacgccct cctacatcga agctgaaagc 1080acgagattct tcgccctccg agagctgcat caggtcggag acgctgtcga acttttcgat 1140cagaaacttc tcgacagacg tcgcggtgag ttcaggcttt tccatgggta tatctccttc 1200ttaaagttaa acaaaattat ttctagaggg aaaccgttgt ggtctcccta tagtgagtcg 1260tattaatttc gcgggatcga gatctgatca acctgcatta atgaatcggc caacgcgcgg 1320ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 1380cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 1440cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 1500accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 1560acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 1620cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 1680acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt 1740atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 1800agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 1860acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 1920gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg 1980gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 2040gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 2100gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 2160acgaaaactc acgttaaggg attttggtca tgacattaac ctataaaaat aggcgtatca 2220cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc 2280tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg 2340gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga 2400ttgtactgag agtgcaccat atggacatat tgtcgttaga acgcggctac aattaataca 2460taaccttatg tatcatacac atacgattta ggtgacacta tagaacggcg cgccaagctt 2520ggatccgtcg acggcgcgcc

2540324359DNAArtificial Sequenceplasmid pKR197 32cgcgcccgat catccggata tagttcctcc tttcagcaaa aaacccctca agacccgttt 60agaggcccca aggggttatg ctagttattg ctcagcggtg gcagcagcca actcagcttc 120ctttcgggct ttgttagcag ccggatcgat ccaagctgta cctcactatt cctttgccct 180cggacgagtg ctggggcgtc ggtttccact atcggcgagt acttctacac agccatcggt 240ccagacggcc gcgcttctgc gggcgatttg tgtacgcccg acagtcccgg ctccggatcg 300gacgattgcg tcgcatcgac cctgcgccca agctgcatca tcgaaattgc cgtcaaccaa 360gctctgatag agttggtcaa gaccaatgcg gagcatatac gcccggagcc gcggcgatcc 420tgcaagctcc ggatgcctcc gctcgaagta gcgcgtctgc tgctccatac aagccaacca 480cggcctccag aagaagatgt tggcgacctc gtattgggaa tccccgaaca tcgcctcgct 540ccagtcaatg accgctgtta tgcggccatt gtccgtcagg acattgttgg agccgaaatc 600cgcgtgcacg aggtgccgga cttcggggca gtcctcggcc caaagcatca gctcatcgag 660agcctgcgcg acggacgcac tgacggtgtc gtccatcaca gtttgccagt gatacacatg 720gggatcagca atcgcgcata tgaaatcacg ccatgtagtg tattgaccga ttccttgcgg 780tccgaatggg ccgaacccgc tcgtctggct aagatcggcc gcagcgatcg catccatagc 840ctccgcgacc ggctgcagaa cagcgggcag ttcggtttca ggcaggtctt gcaacgtgac 900accctgtgca cggcgggaga tgcaataggt caggctctcg ctgaattccc caatgtcaag 960cacttccgga atcgggagcg cggccgatgc aaagtgccga taaacataac gatctttgta 1020gaaaccatcg gcgcagctat ttacccgcag gacatatcca cgccctccta catcgaagct 1080gaaagcacga gattcttcgc cctccgagag ctgcatcagg tcggagacgc tgtcgaactt 1140ttcgatcaga aacttctcga cagacgtcgc ggtgagttca ggcttttcca tgggtatatc 1200tccttcttaa agttaaacaa aattatttct agagggaaac cgttgtggtc tccctatagt 1260gagtcgtatt aatttcgcgg gatcgagatc tgatcaacct gcattaatga atcggccaac 1320gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 1380tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 1440tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 1500ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 1560agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 1620accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 1680ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct 1740gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 1800ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 1860gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 1920taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag 1980tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 2040gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 2100cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 2160agtggaacga aaactcacgt taagggattt tggtcatgac attaacctat aaaaataggc 2220gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca 2280tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc 2340gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag 2400agcagattgt actgagagtg caccatatgg acatattgtc gttagaacgc ggctacaatt 2460aatacataac cttatgtatc atacacatac gatttaggtg acactataga acggcgcgcc 2520aagcttgttg aaacatccct gaagtgtctc attttatttt atttattctt tgctgataaa 2580aaaataaaat aaaagaagct aagcacacgg tcaaccattg ctctactgct aaaagggtta 2640tgtgtagtgt tttactgcat aaattatgca gcaaacaaga caactcaaat taaaaaattt 2700cctttgcttg tttttttgtt gtctctgact tgactttctt gtggaagttg gttgtataag 2760gattgggaca ccattgtcct tcttaattta attttattct ttgctgataa aaaaaaaaat 2820ttcatatagt gttaaataat aatttgttaa ataaccaaaa agtcaaatat gtttactctc 2880gtttaaataa ttgagattcg tccagcaagg ctaaacgatt gtatagattt atgacaatat 2940ttactttttt atagataaat gttatattat aataaattta tatacatata ttatatgtta 3000tttattatta ttttaaatcc ttcaatattt tatcaaacca actcataatt ttttttttat 3060ctgtaagaag caataaaatt aaatagaccc actttaagga tgatccaacc tttatacaga 3120gtaagagagt tcaaatagta ccctttcata tacatatcaa ctaaaatatt agaaatatca 3180tggatcaaac cttataaaga cattaaataa gtggataagt ataatatata aatgggtagt 3240atataatata taaatggata caaacttctc tctttataat tgttatgtct ccttaacatc 3300ctaatataat acataagtgg gtaatatata atatataaat ggagacaaac ttcttccatt 3360ataattgtta tgtcttctta acacttatgt ctcgttcaca atgctaaggt tagaattgtt 3420tagaaagtct tatagtacac atttgttttt gtactatttg aagcattcca taagccgtca 3480cgattcagat gatttataat aataagagga aatttatcat agaacaataa ggtgcataga 3540tagagtgtta atatatcata acatcctttg tttattcata gaagaagtga gatggagctc 3600agttattata ctgttacatg gtcggataca atattccatg ctctccatga gctcttacac 3660ctacatgcat tttagttcat acttgcggcc gcagtatatc ttaaattctt taatacggtg 3720tactaggata ttgaactggt tcttgatgat gaaaacctgg gccgagattg cagctattta 3780tagtcatagg tcttgttaac atgcatggac atttggccac ggggtggcat gcagtttgac 3840gggtgttgaa ataaacaaaa atgaggtggc ggaagagaat acgagtttga ggttgggtta 3900gaaacaacaa atgtgagggc tcatgatggg ttgagttggt gaatgttttg ggctgctcga 3960ttgacacctt tgtgagtacg tgttgttgtg catggctttt ggggtccagt ttttttttct 4020tgacgcggcg atcctgatca gctagtggat aagtgatgtc cactgtgtgt gattgcgttt 4080ttgtttgaat tttatgaact tagacattgc tatgcaaagg atactctcat tgtgttttgt 4140cttcttttgt tccttggctt tttcttatga tccaagagac tagtcagtgt tgtggcattc 4200gagactacca agattaatta tgatggggga aggataagta actgattagt acggactgtt 4260accaaattaa ttaataagcg gcaaatgaag ggcatggatc aaaagcttgg atctcctgca 4320ggatctggcc ggccggatct cgtacggatc cgtcgacgg 4359335147DNAArtificial Sequenceplasmid pKR911 33ggccgcaagt atgaactaaa atgcatgtag gtgtaagagc tcatggagag catggaatat 60tgtatccgac catgtaacag tataataact gagctccatc tcacttcttc tatgaataaa 120caaaggatgt tatgatatat taacactcta tctatgcacc ttattgttct atgataaatt 180tcctcttatt attataaatc atctgaatcg tgacggctta tggaatgctt caaatagtac 240aaaaacaaat gtgtactata agactttcta aacaattcta accttagcat tgtgaacgag 300acataagtgt taagaagaca taacaattat aatggaagaa gtttgtctcc atttatatat 360tatatattac ccacttatgt attatattag gatgttaagg agacataaca attataaaga 420gagaagtttg tatccattta tatattatat actacccatt tatatattat acttatccac 480ttatttaatg tctttataag gtttgatcca tgatatttct aatattttag ttgatatgta 540tatgaaaggg tactatttga actctcttac tctgtataaa ggttggatca tccttaaagt 600gggtctattt aattttattg cttcttacag ataaaaaaaa aattatgagt tggtttgata 660aaatattgaa ggatttaaaa taataataaa taacatataa tatatgtata taaatttatt 720ataatataac atttatctat aaaaaagtaa atattgtcat aaatctatac aatcgtttag 780ccttgctgga cgaatctcaa ttatttaaac gagagtaaac atatttgact ttttggttat 840ttaacaaatt attatttaac actatatgaa attttttttt ttatcagcaa agaataaaat 900taaattaaga aggacaatgg tgtcccaatc cttatacaac caacttccac aagaaagtca 960agtcagagac aacaaaaaaa caagcaaagg aaatttttta atttgagttg tcttgtttgc 1020tgcataattt atgcagtaaa acactacaca taaccctttt agcagtagag caatggttga 1080ccgtgtgctt agcttctttt attttatttt tttatcagca aagaataaat aaaataaaat 1140gagacacttc agggatgttt caacaagctt ggcgcgccgt tctatagtgt cacctaaatc 1200gtatgtgtat gatacataag gttatgtatt aattgtagcc gcgttctaac gacaatatgt 1260ccatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac 1320acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca 1380gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga 1440aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgacca 1500aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag 1560gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac 1620cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa 1680ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc 1740accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag 1800tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac 1860cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc 1920gaacgaccta caccgaactg agatacctac agcgtgagca ttgagaaagc gccacgcttc 1980ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca 2040cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc 2100tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg 2160ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct 2220ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata 2280ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc 2340gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc aggttgatca 2400gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct 2460agaaataatt ttgtttaact ttaagaagga gatataccca tggaaaagcc tgaactcacc 2520gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag 2580ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc 2640ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt 2700gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg 2760acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa 2820ctgcccgctg ttctgcagcc ggtcgcggag gctatggatg cgatcgctgc ggccgatctt 2880agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg 2940cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac 3000gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac 3060tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac 3120aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac 3180gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc 3240tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc 3300cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct 3360tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca 3420caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat 3480agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagtgagg tacagcttgg 3540atcgatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag 3600caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa 3660ggaggaacta tatccggatg atcgggcgcg ccgtcgacgg atccgtacga gatccggccg 3720gccagatcct gcaggagatc caagcttttg atccatgccc ttcatttgcc gcttattaat 3780taatttggta acagtccgta ctaatcagtt acttatcctt cccccatcat aattaatctt 3840ggtagtctcg aatgccacaa cactgactag tctcttggat cataagaaaa agccaaggaa 3900caaaagaaga caaaacacaa tgagagtatc ctttgcatag caatgtctaa gttcataaaa 3960ttcaaacaaa aacgcaatca cacacagtgg acatcactta tccactagct gatcaggatc 4020gccgcgtcaa gaaaaaaaaa ctggacccca aaagccatgc acaacaacac gtactcacaa 4080aggtgtcaat cgagcagccc aaaacattca ccaactcaac ccatcatgag ccctcacatt 4140tgttgtttct aacccaacct caaactcgta ttctcttccg ccacctcatt tttgtttatt 4200tcaacacccg tcaaactgca tgccaccccg tggccaaatg tccatgcatg ttaacaagac 4260ctatgactat aaatagctgc aatctcggcc caggttttca tcatcaagaa ccagttcaat 4320atcctagtac accgtattaa agaatttaag atatactgcg gccgcaccat ggaggtggtg 4380aatgaaatag tctcaattgg gcaggaagtt ttacccaaag ttgattatgc ccaactctgg 4440agtgatgcca gtcactgtga ggtgctttac ttgtccatcg catttgtcat cttgaagttc 4500actcttggcc cccttggtcc aaaaggtcag tctcgtatga agtttgtttt caccaattac 4560aaccttctca tgtccattta ttcgttggga tcattcctct caatggcata tgccatgtac 4620accatcggtg ttatgtctga caactgcgag aaggcttttg acaacaacgt cttcaggatc 4680accacgcagt tgttctattt gagcaagttc ctggagtata ttgactcctt ctatttgcca 4740ctgatgggca agcctctgac ctggttgcaa ttcttccatc atttgggggc accgatggat 4800atgtggctgt tctataatta ccgaaatgaa gctgtttgga tttttgtgct gttgaatggt 4860ttcatccact ggatcatgta cggttattat tggaccagat tgatcaagct gaagttcccc 4920atgccaaaat ccctgattac atcaatgcag atcattcaat tcaatgttgg tttctacatt 4980gtctggaagt acaggaacat tccctgttat cgccaagatg ggatgaggat gtttggctgg 5040ttcttcaatt acttttatgt tggcacagtc ttgtgtttgt tcttgaattt ctatgtgcaa 5100acgtatatcg tcaggaagca caagggagcc aaaaagattc agtgagc 5147346559DNAArtificial Sequenceplasmid pKR680 34ggccgcgaca caagtgtgag agtactaaat aaatgctttg gttgtacgaa atcattacac 60taaataaaat aatcaaagct tatatatgcc ttccgctaag gccgaatgca aagaaattgg 120ttctttctcg ttatcttttg ccacttttac tagtacgtat taattactac ttaatcatct 180ttgtttacgg ctcattatat ccggtctaga ggatccaagg ccgcgaagtt aaaagcaatg 240ttgtcacttg tcgtactaac acatgatgtg atagtttatg ctagctagct ataacataag 300ctgtctctga gtgtgttgta tattaataaa gatcatcact ggtgaatggt gatcgtgtac 360gtaccctact tagtaggcaa tggaagcact tagagtgtgc tttgtgcatg gccttgcctc 420tgttttgaga cttttgtaat gttttcgagt ttaaatcttt gcctttgcgt acgtgggcgg 480atcccccggg ctgcaggaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc 540ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata 600gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc 660gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca 720ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac 780ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga 840ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac 900gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt 960agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct 1020aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat 1080attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg 1140cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg 1200aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc 1260ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat 1320gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact 1380attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca 1440tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact 1500tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg 1560atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg 1620agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg 1680aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg 1740caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag 1800ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc 1860gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga 1920tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat 1980atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc 2040tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 2100accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 2160gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 2220caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc 2280tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 2340ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 2400tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 2460gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 2520tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca 2580gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 2640gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 2700ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 2760ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta 2820ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag 2880tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga 2940ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg 3000caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg 3060ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc 3120atgattacgc caagcttgca tgcctgcagg tcgactcgac gtacgtcctc gaagagaagg 3180gttaataaca cattttttaa catttttaac acaaatttta gttatttaaa aatttattaa 3240aaaatttaaa ataagaagag gaactcttta aataaatcta acttacaaaa tttatgattt 3300ttaataagtt ttcaccaata aaaaatgtca taaaaatatg ttaaaaagta tattatcaat 3360attctcttta tgataaataa aaagaaaaaa aaaataaaag ttaagtgaaa atgagattga 3420agtgacttta ggtgtgtata aatatatcaa ccccgccaac aatttattta atccaaatat 3480attgaagtat attattccat agcctttatt tatttatata tttattatat aaaagcttta 3540tttgttctag gttgttcatg aaatattttt ttggttttat ctccgttgta agaaaatcat 3600gtgctttgtg tcgccactca ctattgcagc tttttcatgc attggtcaga ttgacggttg 3660attgtatttt tgttttttat ggttttgtgt tatgacttaa gtcttcatct ctttatctct 3720tcatcaggtt tgatggttac ctaatatggt ccatgggtac atgcatggtt aaattaggtg 3780gccaactttg ttgtgaacga tagaattttt tttatattaa gtaaactatt tttatattat 3840gaaataataa taaaaaaaat attttatcat tattaacaaa atcatattag ttaatttgtt 3900aactctataa taaaagaaat actgtaacat tcacattaca tggtaacatc tttccaccct 3960ttcatttgtt ttttgtttga tgactttttt tcttgtttaa atttatttcc cttcttttaa 4020atttggaata cattatcatc atatataaac taaaatacta aaaacaggat tacacaaatg 4080ataaataata acacaaatat ttataaatct agctgcaata tatttaaact agctatatcg 4140atattgtaaa ataaaactag ctgcattgat actgataaaa aaatatcatg tgctttctgg 4200actgatgatg cagtatactt ttgacattgc ctttatttta tttttcagaa aagctttctt 4260agttctgggt tcttcattat ttgtttccca tctccattgt gaattgaatc atttgcttcg 4320tgtcacaaat acaatttagn taggtacatg cattggtcag attcacggtt tattatgtca 4380tgacttaagt tcatggtagt acattacctg ccacgcatgc attatattgg ttagatttga 4440taggcaaatt tggttgtcaa caatataaat ataaataatg tttttatatt acgaaataac 4500agtgatcaaa acaaacagtt ttatctttat taacaagatt ttgtttttgt ttgatgacgt 4560tttttaatgt ttacgctttc ccccttcttt tgaatttaga acactttatc atcataaaat 4620caaatactaa aaaaattaca tatttcataa ataataacac aaatattttt aaaaaatctg 4680aaataataat gaacaatatt acatattatc acgaaaattc attaataaaa atattatata 4740aataaaatgt aatagtagtt atatgtagga aaaaagtact gcacgcataa tatatacaaa 4800aagattaaaa tgaactatta taaataataa cactaaatta atggtgaatc atatcaaaat 4860aatgaaaaag taaataaaat ttgtaattaa cttctatatg tattacacac acaaataata 4920aataatagta aaaaaaatta tgataaatat ttaccatctc ataagatatt taaaataatg 4980ataaaaatat agattatttt ttatgcaact agctagccaa aaagagaaca cgggtatata 5040taaaaagagt acctttaaat tctactgtac ttcctttatt cctgacgttt ttatatcaag 5100tggacatacg tgaagatttt aattatcagt ctaaatattt cattagcact taatactttt 5160ctgttttatt cctatcctat aagtagtccc gattctccca acattgctta ttcacacaac 5220taactaagaa agtcttccat agccccccaa gcggccgcgg gaattcgatt gaaatgaagt 5280caaagcgcca agcgcttccc cttacaattg atggaacaac atatgatgtg tctgcctggg 5340tcaatttcca ccctggtggt gcggaaatta tagagaatta ccaaggaagg gatgccactg

5400atgccttcat ggttatgcac tctcaagaag ccttcgacaa gctcaagcgc atgcccaaaa 5460tcaatcccag ttctgagttg ccaccccagg ctgcagtgaa tgaagctcaa gaggatttcc 5520ggaagctccg agaagagttg atcgcaactg gcatgtttga tgcctccccc ctctggtact 5580catacaaaat cagcaccaca ctgggccttg gagtgctggg ttatttcctg atggttcagt 5640atcagatgta tttcattggg gcagtgttgc ttgggatgca ctatcaacag atgggctggc 5700tttctcatga catttgccac caccagactt tcaagaaccg gaactggaac aacctcgtgg 5760gactggtatt tggcaatggt ctgcaaggtt tttccgtgac atggtggaag gacagacaca 5820atgcacatca ttcggcaacc aatgttcaag ggcacgaccc tgatattgac aacctccccc 5880tcttagcctg gtctgaggat gacgtcacac gggcgtcacc gatttcccgc aagctcattc 5940agttccagca gtactatttc ttggtcatct gtatcttgtt gcggttcatt tggtgtttcc 6000agagcgtgtt gaccgtgcgc agtttgaagg acagagataa ccaattctat cgctctcagt 6060ataagaagga ggccattggc ctcgccctgc actggacctt gaagaccctg ttccacttat 6120tctttatgcc cagcatcctc acatcgctgt tggtgttttt cgtttcggag ctggttggcg 6180gcttcggcat tgcgatcgtg gtgttcatga accactaccc actggagaag atcggggact 6240cagtctggga tggccatgga ttctcggttg gccagatcca tgagaccatg aacattcggc 6300gagggattat cacagattgg tttttcggag gcttgaatta ccagattgag caccatttgt 6360ggccgaccct ccctcgccac aacctgacag cggttagcta ccaggtggaa cagctgtgcc 6420agaagcacaa cctgccgtat cggaacccgc tgccccatga agggttggtc atcctgctgc 6480gctatctggc ggtgttcgcc cggatggcgg agaagcaacc cgcggggaag gctctataag 6540gaatcactag tgaattcgc 6559359014DNAArtificial Sequenceplasmid pKR913 35gtacgagatc cggccggcca gatcctgcag gagatccaag cttttgatcc atgcccttca 60tttgccgctt attaattaat ttggtaacag tccgtactaa tcagttactt atccttcccc 120catcataatt aatcttggta gtctcgaatg ccacaacact gactagtctc ttggatcata 180agaaaaagcc aaggaacaaa agaagacaaa acacaatgag agtatccttt gcatagcaat 240gtctaagttc ataaaattca aacaaaaacg caatcacaca cagtggacat cacttatcca 300ctagctgatc aggatcgccg cgtcaagaaa aaaaaactgg accccaaaag ccatgcacaa 360caacacgtac tcacaaaggt gtcaatcgag cagcccaaaa cattcaccaa ctcaacccat 420catgagccct cacatttgtt gtttctaacc caacctcaaa ctcgtattct cttccgccac 480ctcatttttg tttatttcaa cacccgtcaa actgcatgcc accccgtggc caaatgtcca 540tgcatgttaa caagacctat gactataaat agctgcaatc tcggcccagg ttttcatcat 600caagaaccag ttcaatatcc tagtacaccg tattaaagaa tttaagatat actgcggccg 660caccatggag gtggtgaatg aaatagtctc aattgggcag gaagttttac ccaaagttga 720ttatgcccaa ctctggagtg atgccagtca ctgtgaggtg ctttacttgt ccatcgcatt 780tgtcatcttg aagttcactc ttggccccct tggtccaaaa ggtcagtctc gtatgaagtt 840tgttttcacc aattacaacc ttctcatgtc catttattcg ttgggatcat tcctctcaat 900ggcatatgcc atgtacacca tcggtgttat gtctgacaac tgcgagaagg cttttgacaa 960caacgtcttc aggatcacca cgcagttgtt ctatttgagc aagttcctgg agtatattga 1020ctccttctat ttgccactga tgggcaagcc tctgacctgg ttgcaattct tccatcattt 1080gggggcaccg atggatatgt ggctgttcta taattaccga aatgaagctg tttggatttt 1140tgtgctgttg aatggtttca tccactggat catgtacggt tattattgga ccagattgat 1200caagctgaag ttccccatgc caaaatccct gattacatca atgcagatca ttcaattcaa 1260tgttggtttc tacattgtct ggaagtacag gaacattccc tgttatcgcc aagatgggat 1320gaggatgttt ggctggttct tcaattactt ttatgttggc acagtcttgt gtttgttctt 1380gaatttctat gtgcaaacgt atatcgtcag gaagcacaag ggagccaaaa agattcagtg 1440agcggccgca agtatgaact aaaatgcatg taggtgtaag agctcatgga gagcatggaa 1500tattgtatcc gaccatgtaa cagtataata actgagctcc atctcacttc ttctatgaat 1560aaacaaagga tgttatgata tattaacact ctatctatgc accttattgt tctatgataa 1620atttcctctt attattataa atcatctgaa tcgtgacggc ttatggaatg cttcaaatag 1680tacaaaaaca aatgtgtact ataagacttt ctaaacaatt ctaaccttag cattgtgaac 1740gagacataag tgttaagaag acataacaat tataatggaa gaagtttgtc tccatttata 1800tattatatat tacccactta tgtattatat taggatgtta aggagacata acaattataa 1860agagagaagt ttgtatccat ttatatatta tatactaccc atttatatat tatacttatc 1920cacttattta atgtctttat aaggtttgat ccatgatatt tctaatattt tagttgatat 1980gtatatgaaa gggtactatt tgaactctct tactctgtat aaaggttgga tcatccttaa 2040agtgggtcta tttaatttta ttgcttctta cagataaaaa aaaaattatg agttggtttg 2100ataaaatatt gaaggattta aaataataat aaataacata taatatatgt atataaattt 2160attataatat aacatttatc tataaaaaag taaatattgt cataaatcta tacaatcgtt 2220tagccttgct ggacgaatct caattattta aacgagagta aacatatttg actttttggt 2280tatttaacaa attattattt aacactatat gaaatttttt tttttatcag caaagaataa 2340aattaaatta agaaggacaa tggtgtccca atccttatac aaccaacttc cacaagaaag 2400tcaagtcaga gacaacaaaa aaacaagcaa aggaaatttt ttaatttgag ttgtcttgtt 2460tgctgcataa tttatgcagt aaaacactac acataaccct tttagcagta gagcaatggt 2520tgaccgtgtg cttagcttct tttattttat ttttttatca gcaaagaata aataaaataa 2580aatgagacac ttcagggatg tttcaacaag cttggcgcgc cgttctatag tgtcacctaa 2640atcgtatgtg tatgatacat aaggttatgt attaattgta gccgcgttct aacgacaata 2700tgtccatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc 2760gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 2820acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 2880cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga 2940ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 3000aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 3060caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 3120taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag 3180gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 3240cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 3300taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 3360agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gcattgagaa agcgccacgc 3420ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 3480gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 3540acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 3600acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 3660tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg 3720ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag 3780agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcaggttga 3840tcagatctcg atcccgcgaa attaatacga ctcactatag ggagaccaca acggtttccc 3900tctagaaata attttgttta actttaagaa ggagatatac ccatggaaaa gcctgaactc 3960accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg 4020cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat 4080gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac 4140tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc 4200ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc 4260gaactgcccg ctgttctgca gccggtcgcg gaggctatgg atgcgatcgc tgcggccgat 4320cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca 4380tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg 4440gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag 4500gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg 4560gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa 4620tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg 4680cgctacttcg agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg 4740ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca 4800gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt 4860acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc 4920gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaatagtg aggtacagct 4980tggatcgatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct 5040gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg 5100aaaggaggaa ctatatccgg atgatcgggc gcgccgtcga cggatccgta cgcaaaggca 5160aagatttaaa ctcgaaaaca ttacaaaagt ctcaaaacag aggcaaggcc atgcacaaag 5220cacactctaa gtgcttccat tgcctactaa gtagggtacg tacacgatca ccattcacca 5280gtgatgatct ttattaatat acaacacact cagagacagc ttatgttata gctagctagc 5340ataaactatc acatcatgtg ttagtacgac aagtgacaac attgctttta acttcgcggc 5400cttggatcct ctagaccgga tataatgagc cgtaaacaaa gatgattaag tagtaattaa 5460tacgtactag taaaagtggc aaaagataac gagaaagaac caatttcttt gcattcggcc 5520ttagcggaag gcatatataa gctttgatta ttttatttag tgtaatgatt tcgtacaacc 5580aaagcattta tttagtactc tcacacttgt gtcgcggccg cgaattcact agtgattcct 5640tatagagcct tccccgcggg ttgcttctcc gccatccggg cgaacaccgc cagatagcgc 5700agcaggatga ccaacccttc atggggcagc gggttccgat acggcaggtt gtgcttctgg 5760cacagctgtt ccacctggta gctaaccgct gtcaggttgt ggcgagggag ggtcggccac 5820aaatggtgct caatctggta attcaagcct ccgaaaaacc aatctgtgat aatccctcgc 5880cgaatgttca tggtctcatg gatctggcca accgagaatc catggccatc ccagactgag 5940tccccgatct tctccagtgg gtagtggttc atgaacacca cgatcgcaat gccgaagccg 6000ccaaccagct ccgaaacgaa aaacaccaac agcgatgtga ggatgctggg cataaagaat 6060aagtggaaca gggtcttcaa ggtccagtgc agggcgaggc caatggcctc cttcttatac 6120tgagagcgat agaattggtt atctctgtcc ttcaaactgc gcacggtcaa cacgctctgg 6180aaacaccaaa tgaaccgcaa caagatacag atgaccaaga aatagtactg ctggaactga 6240atgagcttgc gggaaatcgg tgacgcccgt gtgacgtcat cctcagacca ggctaagagg 6300gggaggttgt caatatcagg gtcgtgccct tgaacattgg ttgccgaatg atgtgcattg 6360tgtctgtcct tccaccatgt cacggaaaaa ccttgcagac cattgccaaa taccagtccc 6420acgaggttgt tccagttccg gttcttgaaa gtctggtggt ggcaaatgtc atgagaaagc 6480cagcccatct gttgatagtg catcccaagc aacactgccc caatgaaata catctgatac 6540tgaaccatca ggaaataacc cagcactcca aggcccagtg tggtgctgat tttgtatgag 6600taccagaggg gggaggcatc aaacatgcca gttgcgatca actcttctcg gagcttccgg 6660aaatcctctt gagcttcatt cactgcagcc tggggtggca actcagaact gggattgatt 6720ttgggcatgc gcttgagctt gtcgaaggct tcttgagagt gcataaccat gaaggcatca 6780gtggcatccc ttccttggta attctctata atttccgcac caccagggtg gaaattgacc 6840caggcagaca catcatatgt tgttccatca attgtaaggg gaagcgcttg gcgctttgac 6900ttcatttcaa tcgaattccc gcggccgctt ggggggctat ggaagacttt cttagttagt 6960tgtgtgaata agcaatgttg ggagaatcgg gactacttat aggataggaa taaaacagaa 7020aagtattaag tgctaatgaa atatttagac tgataattaa aatcttcacg tatgtccact 7080tgatataaaa acgtcaggaa taaaggaagt acagtagaat ttaaaggtac tctttttata 7140tatacccgtg ttctcttttt ggctagctag ttgcataaaa aataatctat atttttatca 7200ttattttaaa tatcttatga gatggtaaat atttatcata atttttttta ctattattta 7260ttatttgtgt gtgtaataca tatagaagtt aattacaaat tttatttact ttttcattat 7320tttgatatga ttcaccatta atttagtgtt attatttata atagttcatt ttaatctttt 7380tgtatatatt atgcgtgcag tacttttttc ctacatataa ctactattac attttattta 7440tataatattt ttattaatga attttcgtga taatatgtaa tattgttcat tattatttca 7500gattttttaa aaatatttgt gttattattt atgaaatatg taattttttt agtatttgat 7560tttatgatga taaagtgttc taaattcaaa agaaggggga aagcgtaaac attaaaaaac 7620gtcatcaaac aaaaacaaaa tcttgttaat aaagataaaa ctgtttgttt tgatcactgt 7680tatttcgtaa tataaaaaca ttatttatat ttatattgtt gacaaccaaa tttgcctatc 7740aaatctaacc aatataatgc atgcgtggca ggtaatgtac taccatgaac ttaagtcatg 7800acataataaa ccgtgaatct gaccaatgca tgtacctanc taaattgtat ttgtgacacg 7860aagcaaatga ttcaattcac aatggagatg ggaaacaaat aatgaagaac ccagaactaa 7920gaaagctttt ctgaaaaata aaataaaggc aatgtcaaaa gtatactgca tcatcagtcc 7980agaaagcaca tgatattttt ttatcagtat caatgcagct agttttattt tacaatatcg 8040atatagctag tttaaatata ttgcagctag atttataaat atttgtgtta ttatttatca 8100tttgtgtaat cctgttttta gtattttagt ttatatatga tgataatgta ttccaaattt 8160aaaagaaggg aaataaattt aaacaagaaa aaaagtcatc aaacaaaaaa caaatgaaag 8220ggtggaaaga tgttaccatg taatgtgaat gttacagtat ttcttttatt atagagttaa 8280caaattaact aatatgattt tgttaataat gataaaatat tttttttatt attatttcat 8340aatataaaaa tagtttactt aatataaaaa aaattctatc gttcacaaca aagttggcca 8400cctaatttaa ccatgcatgt acccatggac catattaggt aaccatcaaa cctgatgaag 8460agataaagag atgaagactt aagtcataac acaaaaccat aaaaaacaaa aatacaatca 8520accgtcaatc tgaccaatgc atgaaaaagc tgcaatagtg agtggcgaca caaagcacat 8580gattttctta caacggagat aaaaccaaaa aaatatttca tgaacaacct agaacaaata 8640aagcttttat ataataaata tataaataaa taaaggctat ggaataatat acttcaatat 8700atttggatta aataaattgt tggcggggtt gatatattta tacacaccta aagtcacttc 8760aatctcattt tcacttaact tttatttttt ttttcttttt atttatcata aagagaatat 8820tgataatata ctttttaaca tatttttatg acatttttta ttggtgaaaa cttattaaaa 8880atcataaatt ttgtaagtta gatttattta aagagttcct cttcttattt taaatttttt 8940aataaatttt taaataacta aaatttgtgt taaaaatgtt aaaaaatgtg ttattaaccc 9000ttctcttcga ggac 90143630DNAArtificial Sequenceoligonucleotide oEAd5-1-1 36agcggccgca ccatggccac catctctttg 303730DNAArtificial Sequenceolignonucleotide oEAd5-1-2 37tgcggccgct cagaattcct gggcatcctg 30384899DNAArtificial Sequenceplasmid pKR1136 38aattccagca cactggcggc cgttactagt ggatccgagc tcggtaccaa gcttgatgca 60tagcttgagt attctaacgc gtcacctaaa tagcttggcg taatcatggt catagctgtt 120tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa 180gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact 240gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc 300ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg 360ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 420cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaagcccag 480gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 540tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 600ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 660atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag 720gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 780tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 840cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 900cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt 960tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 1020cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 1080cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 1140gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 1200gatcctttta aattaaaaat gaagttttag cacgtgtcag tcctgctcct cggccacgaa 1260gtgcacgcag ttgccggccg ggtcgcgcag ggcgaactcc cgcccccacg gctgctcgcc 1320gatctcggtc atggccggcc cggaggcgtc ccggaagttc gtggacacga cctccgacca 1380ctcggcgtac agctcgtcca ggccgcgcac ccacacccag gccagggtgt tgtccggcac 1440cacctggtcc tggaccgcgc tgatgaacag ggtcacgtcg tcccggacca caccggcgaa 1500gtcgtcctcc acgaagtccc gggagaaccc gagccggtcg gtccagaact cgaccgctcc 1560ggcgacgtcg cgcgcggtga gcaccggaac ggcactggtc aacttggcca tggtggccct 1620cctcacgtgc tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 1680tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 1740acctgtatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggaaa 1800ttgtaagcgt taataattca gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc 1860gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc gccgccaagc 1920tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc cacacccagc 1980cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt cggcaagcag 2040gcatcgccat gggtcacgac gagatcctcg ccgtcgggca tgctcgcctt gagcctggcg 2100aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg atcgacaaga 2160ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg 2220caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat ggatactttc 2280tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc caatagcagc 2340cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg 2400gccagccacg atagccgcgc tgcctcgtct tgcagttcat tcagggcacc ggacaggtcg 2460gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc ggcatcagag 2520cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca agcggccgga 2580gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc tgtctcttga 2640tcagagcttg atcccctgcg ccatcagatc cttggcggcg agaaagccat ccagtttact 2700ttgcagggct tcccaacctt accagagggc gccccagctg gcaattccgg ttcgcttgct 2760gtccataaaa ccgcccagtc tagctatcgc catgtaagcc cactgcaagc tacctgcttt 2820ctctttgcgc ttgcgttttc ccttgtccag atagcccagt agctgacatt catccggggt 2880cagcaccgtt tctgcggact ggctttctac gtgaaaagga tctaggtgaa gatccttttt 2940gataatctca tgcctgacat ttatattccc cagaacatca ggttaatggc gtttttgatg 3000tcattttcgc ggtggctgag atcagccact tcttccccga taacggagac cggcacactg 3060gccatatcgg tggtcatcat gcgccagctt tcatccccga tatgcaccac cgggtaaagt 3120tcacgggaga ctttatctga cagcagacgt gcactggcca gggggatcac catccgtcgc 3180cccggcgtgt caataatatc actctgtaca tccacaaaca gacgataacg gctctctctt 3240ttataggtgt aaaccttaaa ctgccgtacg tataggctgc gcaactgttg ggaagggcga 3300tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga 3360ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa 3420ttgtaatacg actcactata gggcgaattg ggccctctag atgcatgctc gagcggccgc 3480cagtgtgatg gatatctgca gaattcagga gcggccgcac catggccacc atctctttga 3540ctactgagca acttttagaa cacccagaac tggttgcaat tgatggggtg ttgtacgatc 3600tcttcggact ggcgaaagtg catccaggtg gcaacctcat tgaagccgcc ggtgcctccg 3660acggaaccgc cctgttctac tccatgcacc ctggagtgaa gccagagaat tcgaagctgc 3720tgcagcaatt tgcccgaggc aaacacgaac gaagctcgaa ggacccagtg tacacctttg 3780acagtccctt cgcccaggat gtcaagcaga gcgttcggga ggtcatgaag gggcgcaact 3840ggtacgccac gcccggcttt tggctgcgga ccgcgctgat catcgcgtgc actgccatag 3900gcgaatggta ttggatcact accggggcag tgatgtgggg catcttcacc gggtacttcc 3960acagccagat tgggttggcg attcaacacg atgcctctca cggagccatc agcaaaaagc 4020cctgggtgaa cgcctttttc gcctacggca tcgacgccat tggatcctcc cgctggatct 4080ggctgcagtc ccacattatg cgccaccaca cctacaccaa ccagcatggc ctggacctgg 4140acgctgcctc ggcggagccg ttcattttgt tccactccta cccggcaaca aatgcgtcac 4200gaaagtggta ccatcggttc caggcgtggt acatgtacat cgttttgggg atgtatggtg 4260tgtcgatggt gtacaatccg atgtacttgt tcacgatgca gcacaacgac acaatcccag 4320aggccacctc tcttagacca ggcagctttt tcaaccggca gcgcgccttc gccgtttccc 4380tccgcctact gttcatcttc cgcaacgcct tcctcccctg gtacatcgcg ggcgcctctc 4440cgctgctcac catcctgctg gtgccaacgg tcacaggcat cttcttgaca

tttgtttttg 4500tgctgtccca taactttgaa ggcgctgagc ggacccccga aaagaactgc aaggccaaaa 4560gggccaagga ggggaaggag gtccgcgatg tagaggagga ccgggtggac tggtaccggg 4620cgcaggccga gaccgcggcg acctacgggg gcagcgtcgg gatgatgctg accggcggtt 4680tgaacctgca gatcgagcac cacttgttcc cccgcatgtc ctcttggcac taccccttca 4740tccaagatac ggtgcgggaa tgttgcaagc gccatggcgt gcgctacaca tactacccga 4800ccatcctgga gaatataatg tccacgctcc gctacatgca gaaggtgggc gtggcccaca 4860caattcagga tgcccaggaa ttctgagcgg ccgcacctg 4899395561DNAArtificial Sequenceplasmid pKR767 39catggtcaat caatgagacg ccaacttctt aatctattga gacctgcagg tctagaaggg 60cggatccccg ggtaccgagc tcgaattcac tggccgtcgt tttacaacgt cgtgactggg 120aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc 180gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg 240aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat 300ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc 360caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag 420ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg 480cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg 540tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 600ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 660aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 720tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 780atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 840agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 900tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 960tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 1020atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 1080ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 1140tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 1200acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 1260ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 1320aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 1380ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 1440cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 1500gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 1560actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 1620agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 1680cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 1740tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 1800agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 1860tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 1920acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 1980ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 2040gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 2100gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 2160gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 2220tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 2280caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 2340tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc 2400gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg 2460agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt 2520ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc 2580gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc 2640ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct 2700atgaccatga ttacgccaag cttgcatgcc tgcaggctag cctaagtacg tactcaaaat 2760gccaacaaat aaaaaaaaag ttgctttaat aatgccaaaa caaattaata aaacacttac 2820aacaccggat tttttttaat taaaatgtgc catttaggat aaatagttaa tatttttaat 2880aattatttaa aaagccgtat ctactaaaat gatttttatt tggttgaaaa tattaatatg 2940tttaaatcaa cacaatctat caaaattaaa ctaaaaaaaa aataagtgta cgtggttaac 3000attagtacag taatataaga ggaaaatgag aaattaagaa attgaaagcg agtctaattt 3060ttaaattatg aacctgcata tataaaagga aagaaagaat ccaggaagaa aagaaatgaa 3120accatgcatg gtcccctcgt catcacgagt ttctgccatt tgcaatagaa acactgaaac 3180acctttctct ttgtcactta attgagatgc cgaagccacc tcacaccatg aacttcatga 3240ggtgtagcac ccaaggcttc catagccatg catactgaag aatgtctcaa gctcagcacc 3300ctacttctgt gacgtgtccc tcattcacct tcctctcttc cctataaata accacgcctc 3360aggttctccg cttcacaact caaacattct ctccattggt ccttaaacac tcatcagtca 3420tcaccgcggc cgcatgggaa cggaccaagg aaaaaccttc acctgggaag agctggcggc 3480ccataacacc aaggacgacc tactcttggc catccgcggc agggtgtacg atgtcacaaa 3540gttcttgagc cgccatcctg gtggagtgga cactctcctg ctcggagctg gccgagatgt 3600tactccggtc tttgagatgt atcacgcgtt tggggctgca gatgccatta tgaagaagta 3660ctatgtcggt acactggtct cgaatgagct gcccatcttc ccggagccaa cggtgttcca 3720caaaaccatc aagacgagag tcgagggcta ctttacggat cggaacattg atcccaagaa 3780tagaccagag atctggggac gatacgctct tatctttgga tccttgatcg cttcctacta 3840cgcgcagctc tttgtgcctt tcgttgtcga acgcacatgg cttcaggtgg tgtttgcaat 3900catcatggga tttgcgtgcg cacaagtcgg actcaaccct cttcatgatg cgtctcactt 3960ttcagtgacc cacaacccca ctgtctggaa gattctggga gccacgcacg actttttcaa 4020cggagcatcg tacctggtgt ggatgtacca acatatgctc ggccatcacc cctacaccaa 4080cattgctgga gcagatcccg acgtgtcgac gtctgagccc gatgttcgtc gtatcaagcc 4140caaccaaaag tggtttgtca accacatcaa ccagcacatg tttgttcctt tcctgtacgg 4200actgctggcg ttcaaggtgc gcattcagga catcaacatt ttgtactttg tcaagaccaa 4260tgacgctatt cgtgtcaatc ccatctcgac atggcacact gtgatgttct ggggcggcaa 4320ggctttcttt gtctggtatc gcctgattgt tcccctgcag tatctgcccc tgggcaaggt 4380gctgctcttg ttcacggtcg cggacatggt gtcgtcttac tggctggcgc tgaccttcca 4440ggcgaaccac gttgttgagg aagttcagtg gccgttgcct gacgagaacg ggatcatcca 4500aaaggactgg gcagctatgc aggtcgagac tacgcaggat tacgcacacg attcgcacct 4560ctggaccagc atcactggca gcttgaacta ccaggctgtg caccatctgt tccccaacgt 4620gtcgcagcac cattatcccg atattctggc catcatcaag aacacctgca gcgagtacaa 4680ggttccatac cttgtcaagg atacgttttg gcaagcattt gcttcacatt tggagcactt 4740gcgtgttctt ggactccgtc ccaaggaaga gtaggcggcc gcatttcgca ccaaatcaat 4800gaaagtaata atgaaaagtc tgaataagaa tacttaggct tagatgcctt tgttacttgt 4860gtaaaataac ttgagtcatg tacctttggc ggaaacagaa taaataaaag gtgaaattcc 4920aatgctctat gtataagtta gtaatactta atgtgttcta cggttgtttc aatatcatca 4980aactctaatt gaaactttag aaccacaaat ctcaatcttt tcttaatgaa atgaaaaatc 5040ttaattgtac catgtttatg ttaaacacct tacaattggt tggagaggag gaccaaccga 5100tgggacaaca ttgggagaaa gagattcaat ggagatttgg ataggagaac aacattcttt 5160ttcacttcaa tacaagatga gtgcaacact aaggatatgt atgagacttt cagaagctac 5220gacaacatag atgagtgagg tggtgattcc tagcaagaaa gacattagag gaagccaaaa 5280tcgaacaagg aagacatcaa gggcaagaga caggaccatc catctcagga aaaggagctt 5340tgggatagtc cgagaagttg tacaagaaat tttttggagg gtgagtgatg cattgctggt 5400gactttaact caatcaaaat tgagaaagaa agaaaaggga gggggctcac atgtgaatag 5460aagggaaacg ggagaatttt acagttttga tctaatgggc atcccagcta gtggtaacat 5520attcaccatg tttaaccttc acgtacgtct agaggatccc c 5561401338DNAMortierella alpina 40atgggaacgg accaaggaaa aaccttcacc tgggaagagc tggcggccca taacaccaag 60gacgacctac tcttggccat ccgcggcagg gtgtacgatg tcacaaagtt cttgagccgc 120catcctggtg gagtggacac tctcctgctc ggagctggcc gagatgttac tccggtcttt 180gagatgtatc acgcgtttgg ggctgcagat gccattatga agaagtacta tgtcggtaca 240ctggtctcga atgagctgcc catcttcccg gagccaacgg tgttccacaa aaccatcaag 300acgagagtcg agggctactt tacggatcgg aacattgatc ccaagaatag accagagatc 360tggggacgat acgctcttat ctttggatcc ttgatcgctt cctactacgc gcagctcttt 420gtgcctttcg ttgtcgaacg cacatggctt caggtggtgt ttgcaatcat catgggattt 480gcgtgcgcac aagtcggact caaccctctt catgatgcgt ctcacttttc agtgacccac 540aaccccactg tctggaagat tctgggagcc acgcacgact ttttcaacgg agcatcgtac 600ctggtgtgga tgtaccaaca tatgctcggc catcacccct acaccaacat tgctggagca 660gatcccgacg tgtcgacgtc tgagcccgat gttcgtcgta tcaagcccaa ccaaaagtgg 720tttgtcaacc acatcaacca gcacatgttt gttcctttcc tgtacggact gctggcgttc 780aaggtgcgca ttcaggacat caacattttg tactttgtca agaccaatga cgctattcgt 840gtcaatccca tctcgacatg gcacactgtg atgttctggg gcggcaaggc tttctttgtc 900tggtatcgcc tgattgttcc cctgcagtat ctgcccctgg gcaaggtgct gctcttgttc 960acggtcgcgg acatggtgtc gtcttactgg ctggcgctga ccttccaggc gaaccacgtt 1020gttgaggaag ttcagtggcc gttgcctgac gagaacggga tcatccaaaa ggactgggca 1080gctatgcagg tcgagactac gcaggattac gcacacgatt cgcacctctg gaccagcatc 1140actggcagct tgaactacca ggctgtgcac catctgttcc ccaacgtgtc gcagcaccat 1200tatcccgata ttctggccat catcaagaac acctgcagcg agtacaaggt tccatacctt 1260gtcaaggata cgttttggca agcatttgct tcacatttgg agcacttgcg tgttcttgga 1320ctccgtccca aggaagag 1338415661DNAArtificial Sequenceplasmid pKR974 41gtacgtctag aggatccccc atggtcaatc aatgagacgc caacttctta atctattgag 60acctgcaggt ctagaagggc ggatccccgg gtaccgagct cgaattcact ggccgtcgtt 120ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat 180ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag 240ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc 300ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta 360agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg 420gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca 480ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt 540aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc 600ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa 660taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc 720cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa 780acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa 840ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg 900atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa 960gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc 1020acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc 1080atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta 1140accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag 1200ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca 1260acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata 1320gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc 1380tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca 1440ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca 1500actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg 1560taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 1620tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 1680gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 1740cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 1800gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 1860gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac 1920tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 1980ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 2040cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 2100gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 2160gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 2220gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 2280cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 2340tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc 2400cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 2460cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa 2520ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 2580tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 2640caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 2700tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcaggctagc 2760ctaagtacgt actcaaaatg ccaacaaata aaaaaaaagt tgctttaata atgccaaaac 2820aaattaataa aacacttaca acaccggatt ttttttaatt aaaatgtgcc atttaggata 2880aatagttaat atttttaata attatttaaa aagccgtatc tactaaaatg atttttattt 2940ggttgaaaat attaatatgt ttaaatcaac acaatctatc aaaattaaac taaaaaaaaa 3000ataagtgtac gtggttaaca ttagtacagt aatataagag gaaaatgaga aattaagaaa 3060ttgaaagcga gtctaatttt taaattatga acctgcatat ataaaaggaa agaaagaatc 3120caggaagaaa agaaatgaaa ccatgcatgg tcccctcgtc atcacgagtt tctgccattt 3180gcaatagaaa cactgaaaca cctttctctt tgtcacttaa ttgagatgcc gaagccacct 3240cacaccatga acttcatgag gtgtagcacc caaggcttcc atagccatgc atactgaaga 3300atgtctcaag ctcagcaccc tacttctgtg acgtgtccct cattcacctt cctctcttcc 3360ctataaataa ccacgcctca ggttctccgc ttcacaactc aaacattctc tccattggtc 3420cttaaacact catcagtcat caccgcggcc gccaattcat ggccccgcag acggagctcc 3480gccagcgcca cgccgccgtc gccgagacgc cggtggccgg caagaaggcc tttacatggc 3540aggaggtcgc gcagcacaac acggcggcct cggcctggat cattatccgc ggcaaggtct 3600acgacgtgac cgagtgggcc aacaagcacc ccggcggccg cgagatggtg ctgctgcacg 3660ccggtcgcga ggccaccgac acgttcgact cgtaccaccc gttcagcgac aaggccgagt 3720cgatcttgaa caagtatgag attggcacgt tcacgggccc gtccgagttt ccgaccttca 3780agccggacac gggcttctac aaggagtgcc gcaagcgcgt tggcgagtac ttcaagaaga 3840acaacctcca tccgcaggac ggcttcccgg gcctctggcg catgatggtc gtgtttgcgg 3900tcgccggcct cgccttgtac ggcatgcact tttcgactat ctttgcgctg cagctcgcgg 3960ccgcggcgct ctttggcgtc tgccaggcgc tgccgctgct ccacgtcatg cacgactcgt 4020cgcacgcgtc gtacaccaac atgccgttct tccattacgt cgtcggccgc tttgccatgg 4080actggtttgc cggcggctcg atggtgtcat ggctcaacca gcacgtcgtg ggccaccaca 4140tctacacgaa cgtcgcgggc tcggacccgg atcttccggt caacatggac ggcgacatcc 4200gccgcatcgt gaaccgccag gtgttccagc ccatgtacgc attccagcac atctaccttc 4260cgccgctcta tggcgtgctt ggcctcaagt tccgcatcca ggacttcacc gacacgttcg 4320gctcgcacac gaacggcccg atccgcgtca acccgcacgc gctctcgacg tggatggcca 4380tgatcagctc caagtcgttc tgggccttct accgcgtgta ccttccgctt gccgtgctcc 4440agatgcccat caagacgtac cttgcgatct tcttcctcgc cgagtttgtc acgggctggt 4500acctcgcgtt caacttccaa gtaagccatg tctcgaccga gtgcggctac ccatgcggcg 4560acgaggccaa gatggcgctc caggacgagt gggcagtctc gcaggtcaag acgtcggtcg 4620actacgccca tggctcgtgg atgacgacgt tccttgccgg cgcgctcaac taccaggtcg 4680tgcaccactt gttccccagc gtgtcgcagt accactaccc ggcgatcgcg cccatcatcg 4740tcgacgtctg caaggagtac aacatcaagt acgccatctt gccggacttt acggcggcgt 4800tcgttgccca cttgaagcac ctccgcaaca tgggccagca gggcatcgcc gccacgatcc 4860acatgggcta actcgagctc agctagatcg cggccgcatt tcgcaccaaa tcaatgaaag 4920taataatgaa aagtctgaat aagaatactt aggcttagat gcctttgtta cttgtgtaaa 4980ataacttgag tcatgtacct ttggcggaaa cagaataaat aaaaggtgaa attccaatgc 5040tctatgtata agttagtaat acttaatgtg ttctacggtt gtttcaatat catcaaactc 5100taattgaaac tttagaacca caaatctcaa tcttttctta atgaaatgaa aaatcttaat 5160tgtaccatgt ttatgttaaa caccttacaa ttaattggtt ggagaggagg accaaccgat 5220gggacaacat tgggagaaag agattcaatg gagatttgga taggagaaca acattctttt 5280tcacttcaat acaagatgag tgcaacacta aggatatgta tgagactttc agaagctacg 5340acaacataga tgagtgaggt ggtgattcct agcaagaaag acattagagg aagccaaaat 5400cgaacaagga agacatcaag ggcaagagac aggaccatcc atctcaggaa aaggagcttt 5460gggatagtcc gagaagttgt acaagaaatt ttttggaggg tgagtgatgc attgctggtg 5520actttaactc aatcaaaatt gagaaagaaa gaaaagggag ggggctcaca tgtgaataga 5580agggaaacgg gagaatttta cagttttgat ctaatgggca tcccagctag tggtaacata 5640ttcaccatgt ttaaccttca c 5661421413DNASaprolegnia diclina 42atggccccgc agacggagct ccgccagcgc cacgccgccg tcgccgagac gccggtggcc 60ggcaagaagg cctttacatg gcaggaggtc gcgcagcaca acacggcggc ctcggcctgg 120atcattatcc gcggcaaggt ctacgacgtg accgagtggg ccaacaagca ccccggcggc 180cgcgagatgg tgctgctgca cgccggtcgc gaggccaccg acacgttcga ctcgtaccac 240ccgttcagcg acaaggccga gtcgatcttg aacaagtatg agattggcac gttcacgggc 300ccgtccgagt ttccgacctt caagccggac acgggcttct acaaggagtg ccgcaagcgc 360gttggcgagt acttcaagaa gaacaacctc catccgcagg acggcttccc gggcctctgg 420cgcatgatgg tcgtgtttgc ggtcgccggc ctcgccttgt acggcatgca cttttcgact 480atctttgcgc tgcagctcgc ggccgcggcg ctctttggcg tctgccaggc gctgccgctg 540ctccacgtca tgcacgactc gtcgcacgcg tcgtacacca acatgccgtt cttccattac 600gtcgtcggcc gctttgccat ggactggttt gccggcggct cgatggtgtc atggctcaac 660cagcacgtcg tgggccacca catctacacg aacgtcgcgg gctcggaccc ggatcttccg 720gtcaacatgg acggcgacat ccgccgcatc gtgaaccgcc aggtgttcca gcccatgtac 780gcattccagc acatctacct tccgccgctc tatggcgtgc ttggcctcaa gttccgcatc 840caggacttca ccgacacgtt cggctcgcac acgaacggcc cgatccgcgt caacccgcac 900gcgctctcga cgtggatggc catgatcagc tccaagtcgt tctgggcctt ctaccgcgtg 960taccttccgc ttgccgtgct ccagatgccc atcaagacgt accttgcgat cttcttcctc 1020gccgagtttg tcacgggctg gtacctcgcg ttcaacttcc aagtaagcca tgtctcgacc 1080gagtgcggct acccatgcgg cgacgaggcc aagatggcgc tccaggacga gtgggcagtc 1140tcgcaggtca agacgtcggt cgactacgcc catggctcgt ggatgacgac gttccttgcc 1200ggcgcgctca actaccaggt cgtgcaccac ttgttcccca gcgtgtcgca gtaccactac 1260ccggcgatcg cgcccatcat cgtcgacgtc tgcaaggagt acaacatcaa gtacgccatc 1320ttgccggact ttacggcggc gttcgttgcc cacttgaagc acctccgcaa catgggccag 1380cagggcatcg ccgccacgat ccacatgggc taa 1413435592DNAArtificial Sequenceplasmid pKR1139 43ggccgcattt cgcaccaaat caatgaaagt aataatgaaa agtctgaata agaatactta 60ggcttagatg cctttgttac ttgtgtaaaa taacttgagt catgtacctt tggcggaaac 120agaataaata aaaggtgaaa ttccaatgct ctatgtataa gttagtaata cttaatgtgt 180tctacggttg tttcaatatc atcaaactct aattgaaact ttagaaccac aaatctcaat 240cttttcttaa tgaaatgaaa aatcttaatt gtaccatgtt tatgttaaac accttacaat 300taattggttg gagaggagga ccaaccgatg ggacaacatt

gggagaaaga gattcaatgg 360agatttggat aggagaacaa cattcttttt cacttcaata caagatgagt gcaacactaa 420ggatatgtat gagactttca gaagctacga caacatagat gagtgaggtg gtgattccta 480gcaagaaaga cattagagga agccaaaatc gaacaaggaa gacatcaagg gcaagagaca 540ggaccatcca tctcaggaaa aggagctttg ggatagtccg agaagttgta caagaaattt 600tttggagggt gagtgatgca ttgctggtga ctttaactca atcaaaattg agaaagaaag 660aaaagggagg gggctcacat gtgaatagaa gggaaacggg agaattttac agttttgatc 720taatgggcat cccagctagt ggtaacatat tcaccatgtt taaccttcac gtacgtctag 780aggatccccc atggtcaatc aatgagacgc caacttctta atctattgag acctgcaggt 840ctagaagggc ggatccccgg gtaccgagct cgaattcact ggccgtcgtt ttacaacgtc 900gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg 960ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc 1020tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 1080accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc 1140gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 1200acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 1260cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga 1320taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta 1380tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat 1440aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc 1500ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga 1560aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca 1620acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt 1680ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg 1740gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc 1800atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata 1860acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt 1920tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag 1980ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca 2040aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg 2100aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg 2160ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag 2220atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg 2280aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag 2340accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga 2400tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt 2460tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc 2520tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc 2580cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac 2640caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac 2700cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt 2760cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct 2820gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat 2880acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt 2940atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg 3000cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt 3060gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt 3120tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg 3180tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg 3240agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc 3300ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg 3360gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac 3420actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag 3480gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcaggctagc ctaagtacgt 3540actcaaaatg ccaacaaata aaaaaaaagt tgctttaata atgccaaaac aaattaataa 3600aacacttaca acaccggatt ttttttaatt aaaatgtgcc atttaggata aatagttaat 3660atttttaata attatttaaa aagccgtatc tactaaaatg atttttattt ggttgaaaat 3720attaatatgt ttaaatcaac acaatctatc aaaattaaac taaaaaaaaa ataagtgtac 3780gtggttaaca ttagtacagt aatataagag gaaaatgaga aattaagaaa ttgaaagcga 3840gtctaatttt taaattatga acctgcatat ataaaaggaa agaaagaatc caggaagaaa 3900agaaatgaaa ccatgcatgg tcccctcgtc atcacgagtt tctgccattt gcaatagaaa 3960cactgaaaca cctttctctt tgtcacttaa ttgagatgcc gaagccacct cacaccatga 4020acttcatgag gtgtagcacc caaggcttcc atagccatgc atactgaaga atgtctcaag 4080ctcagcaccc tacttctgtg acgtgtccct cattcacctt cctctcttcc ctataaataa 4140ccacgcctca ggttctccgc ttcacaactc aaacattctc tccattggtc cttaaacact 4200catcagtcat caccgcggcc gcaccatggc caccatctct ttgactactg agcaactttt 4260agaacaccca gaactggttg caattgatgg ggtgttgtac gatctcttcg gactggcgaa 4320agtgcatcca ggtggcaacc tcattgaagc cgccggtgcc tccgacggaa ccgccctgtt 4380ctactccatg caccctggag tgaagccaga gaattcgaag ctgctgcagc aatttgcccg 4440aggcaaacac gaacgaagct cgaaggaccc agtgtacacc tttgacagtc ccttcgccca 4500ggatgtcaag cagagcgttc gggaggtcat gaaggggcgc aactggtacg ccacgcccgg 4560cttttggctg cggaccgcgc tgatcatcgc gtgcactgcc ataggcgaat ggtattggat 4620cactaccggg gcagtgatgt ggggcatctt caccgggtac ttccacagcc agattgggtt 4680ggcgattcaa cacgatgcct ctcacggagc catcagcaaa aagccctggg tgaacgcctt 4740tttcgcctac ggcatcgacg ccattggatc ctcccgctgg atctggctgc agtcccacat 4800tatgcgccac cacacctaca ccaaccagca tggcctggac ctggacgctg cctcggcgga 4860gccgttcatt ttgttccact cctacccggc aacaaatgcg tcacgaaagt ggtaccatcg 4920gttccaggcg tggtacatgt acatcgtttt ggggatgtat ggtgtgtcga tggtgtacaa 4980tccgatgtac ttgttcacga tgcagcacaa cgacacaatc ccagaggcca cctctcttag 5040accaggcagc tttttcaacc ggcagcgcgc cttcgccgtt tccctccgcc tactgttcat 5100cttccgcaac gccttcctcc cctggtacat cgcgggcgcc tctccgctgc tcaccatcct 5160gctggtgcca acggtcacag gcatcttctt gacatttgtt tttgtgctgt cccataactt 5220tgaaggcgct gagcggaccc ccgaaaagaa ctgcaaggcc aaaagggcca aggaggggaa 5280ggaggtccgc gatgtagagg aggaccgggt ggactggtac cgggcgcagg ccgagaccgc 5340ggcgacctac gggggcagcg tcgggatgat gctgaccggc ggtttgaacc tgcagatcga 5400gcaccacttg ttcccccgca tgtcctcttg gcactacccc ttcatccaag atacggtgcg 5460ggaatgttgc aagcgccatg gcgtgcgcta cacatactac ccgaccatcc tggagaatat 5520aatgtccacg ctccgctaca tgcagaaggt gggcgtggcc cacacaattc aggatgccca 5580ggaattctga gc 55924411920DNAArtificial Sequenceplasmid pKR1153 44ggagatccaa gcttttgatc catgcccttc atttgccgct tattaattaa tttggtaaca 60gtccgtacta atcagttact tatccttccc ccatcataat taatcttggt agtctcgaat 120gccacaacac tgactagtct cttggatcat aagaaaaagc caaggaacaa aagaagacaa 180aacacaatga gagtatcctt tgcatagcaa tgtctaagtt cataaaattc aaacaaaaac 240gcaatcacac acagtggaca tcacttatcc actagctgat caggatcgcc gcgtcaagaa 300aaaaaaactg gaccccaaaa gccatgcaca acaacacgta ctcacaaagg tgtcaatcga 360gcagcccaaa acattcacca actcaaccca tcatgagccc tcacatttgt tgtttctaac 420ccaacctcaa actcgtattc tcttccgcca cctcattttt gtttatttca acacccgtca 480aactgcatgc caccccgtgg ccaaatgtcc atgcatgtta acaagaccta tgactataaa 540tagctgcaat ctcggcccag gttttcatca tcaagaacca gttcaatatc ctagtacacc 600gtattaaaga atttaagata tactgcggcc gcaccatgga ggtggtgaat gaaatagtct 660caattgggca ggaagtttta cccaaagttg attatgccca actctggagt gatgccagtc 720actgtgaggt gctttacttg tccatcgcat ttgtcatctt gaagttcact cttggccccc 780ttggtccaaa aggtcagtct cgtatgaagt ttgttttcac caattacaac cttctcatgt 840ccatttattc gttgggatca ttcctctcaa tggcatatgc catgtacacc atcggtgtta 900tgtctgacaa ctgcgagaag gcttttgaca acaacgtctt caggatcacc acgcagttgt 960tctatttgag caagttcctg gagtatattg actccttcta tttgccactg atgggcaagc 1020ctctgacctg gttgcaattc ttccatcatt tgggggcacc gatggatatg tggctgttct 1080ataattaccg aaatgaagct gtttggattt ttgtgctgtt gaatggtttc atccactgga 1140tcatgtacgg ttattattgg accagattga tcaagctgaa gttccccatg ccaaaatccc 1200tgattacatc aatgcagatc attcaattca atgttggttt ctacattgtc tggaagtaca 1260ggaacattcc ctgttatcgc caagatggga tgaggatgtt tggctggttc ttcaattact 1320tttatgttgg cacagtcttg tgtttgttct tgaatttcta tgtgcaaacg tatatcgtca 1380ggaagcacaa gggagccaaa aagattcagt gagcggccgc aagtatgaac taaaatgcat 1440gtaggtgtaa gagctcatgg agagcatgga atattgtatc cgaccatgta acagtataat 1500aactgagctc catctcactt cttctatgaa taaacaaagg atgttatgat atattaacac 1560tctatctatg caccttattg ttctatgata aatttcctct tattattata aatcatctga 1620atcgtgacgg cttatggaat gcttcaaata gtacaaaaac aaatgtgtac tataagactt 1680tctaaacaat tctaacctta gcattgtgaa cgagacataa gtgttaagaa gacataacaa 1740ttataatgga agaagtttgt ctccatttat atattatata ttacccactt atgtattata 1800ttaggatgtt aaggagacat aacaattata aagagagaag tttgtatcca tttatatatt 1860atatactacc catttatata ttatacttat ccacttattt aatgtcttta taaggtttga 1920tccatgatat ttctaatatt ttagttgata tgtatatgaa agggtactat ttgaactctc 1980ttactctgta taaaggttgg atcatcctta aagtgggtct atttaatttt attgcttctt 2040acagataaaa aaaaaattat gagttggttt gataaaatat tgaaggattt aaaataataa 2100taaataacat ataatatatg tatataaatt tattataata taacatttat ctataaaaaa 2160gtaaatattg tcataaatct atacaatcgt ttagccttgc tggacgaatc tcaattattt 2220aaacgagagt aaacatattt gactttttgg ttatttaaca aattattatt taacactata 2280tgaaattttt ttttttatca gcaaagaata aaattaaatt aagaaggaca atggtgtccc 2340aatccttata caaccaactt ccacaagaaa gtcaagtcag agacaacaaa aaaacaagca 2400aaggaaattt tttaatttga gttgtcttgt ttgctgcata atttatgcag taaaacacta 2460cacataaccc ttttagcagt agagcaatgg ttgaccgtgt gcttagcttc ttttatttta 2520tttttttatc agcaaagaat aaataaaata aaatgagaca cttcagggat gtttcaacaa 2580gcttggcgcg ccgttctata gtgtcaccta aatcgtatgt gtatgataca taaggttatg 2640tattaattgt agccgcgttc taacgacaat atgtccatat ggtgcactct cagtacaatc 2700tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc 2760tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc 2820tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg 2880atacgcctat ttttataggt taatgtcatg accaaaatcc cttaacgtga gttttcgttc 2940cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 3000cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 3060gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 3120aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 3180cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 3240tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 3300acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 3360ctacagcgtg agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 3420ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 3480tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 3540tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 3600ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg 3660gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag 3720cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc 3780gcgcgttggc cgattcatta atgcaggttg atcagatctc gatcccgcga aattaatacg 3840actcactata gggagaccac aacggtttcc ctctagaaat aattttgttt aactttaaga 3900aggagatata cccatggaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat 3960cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc 4020tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg 4080tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga 4140agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca 4200gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc 4260ggaggctatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt 4320cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga 4380tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca 4440ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca 4500cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga 4560ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag 4620gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct 4680tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca 4740gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat 4800cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt 4860ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg 4920tccgagggca aaggaatagt gaggtacagc ttggatcgat ccggctgcta acaaagcccg 4980aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc 5040ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gatgatcggg 5100cgcgccgtcg acggatccgt acgcaaaggc aaagatttaa actcgaaaac attacaaaag 5160tctcaaaaca gaggcaaggc catgcacaaa gcacactcta agtgcttcca ttgcctacta 5220agtagggtac gtacacgatc accattcacc agtgatgatc tttattaata tacaacacac 5280tcagagacag cttatgttat agctagctag cataaactat cacatcatgt gttagtacga 5340caagtgacaa cattgctttt aacttcgcgg ccttggatcc tctagaccgg atataatgag 5400ccgtaaacaa agatgattaa gtagtaatta atacgtacta gtaaaagtgg caaaagataa 5460cgagaaagaa ccaatttctt tgcattcggc cttagcggaa ggcatatata agctttgatt 5520attttattta gtgtaatgat ttcgtacaac caaagcattt atttagtact ctcacacttg 5580tgtcgcggcc gcgaattcac tagtgattcc ttatagagcc ttccccgcgg gttgcttctc 5640cgccatccgg gcgaacaccg ccagatagcg cagcaggatg accaaccctt catggggcag 5700cgggttccga tacggcaggt tgtgcttctg gcacagctgt tccacctggt agctaaccgc 5760tgtcaggttg tggcgaggga gggtcggcca caaatggtgc tcaatctggt aattcaagcc 5820tccgaaaaac caatctgtga taatccctcg ccgaatgttc atggtctcat ggatctggcc 5880aaccgagaat ccatggccat cccagactga gtccccgatc ttctccagtg ggtagtggtt 5940catgaacacc acgatcgcaa tgccgaagcc gccaaccagc tccgaaacga aaaacaccaa 6000cagcgatgtg aggatgctgg gcataaagaa taagtggaac agggtcttca aggtccagtg 6060cagggcgagg ccaatggcct ccttcttata ctgagagcga tagaattggt tatctctgtc 6120cttcaaactg cgcacggtca acacgctctg gaaacaccaa atgaaccgca acaagataca 6180gatgaccaag aaatagtact gctggaactg aatgagcttg cgggaaatcg gtgacgcccg 6240tgtgacgtca tcctcagacc aggctaagag ggggaggttg tcaatatcag ggtcgtgccc 6300ttgaacattg gttgccgaat gatgtgcatt gtgtctgtcc ttccaccatg tcacggaaaa 6360accttgcaga ccattgccaa ataccagtcc cacgaggttg ttccagttcc ggttcttgaa 6420agtctggtgg tggcaaatgt catgagaaag ccagcccatc tgttgatagt gcatcccaag 6480caacactgcc ccaatgaaat acatctgata ctgaaccatc aggaaataac ccagcactcc 6540aaggcccagt gtggtgctga ttttgtatga gtaccagagg ggggaggcat caaacatgcc 6600agttgcgatc aactcttctc ggagcttccg gaaatcctct tgagcttcat tcactgcagc 6660ctggggtggc aactcagaac tgggattgat tttgggcatg cgcttgagct tgtcgaaggc 6720ttcttgagag tgcataacca tgaaggcatc agtggcatcc cttccttggt aattctctat 6780aatttccgca ccaccagggt ggaaattgac ccaggcagac acatcatatg ttgttccatc 6840aattgtaagg ggaagcgctt ggcgctttga cttcatttca atcgaattcc cgcggccgct 6900tggggggcta tggaagactt tcttagttag ttgtgtgaat aagcaatgtt gggagaatcg 6960ggactactta taggatagga ataaaacaga aaagtattaa gtgctaatga aatatttaga 7020ctgataatta aaatcttcac gtatgtccac ttgatataaa aacgtcagga ataaaggaag 7080tacagtagaa tttaaaggta ctctttttat atatacccgt gttctctttt tggctagcta 7140gttgcataaa aaataatcta tatttttatc attattttaa atatcttatg agatggtaaa 7200tatttatcat aatttttttt actattattt attatttgtg tgtgtaatac atatagaagt 7260taattacaaa ttttatttac tttttcatta ttttgatatg attcaccatt aatttagtgt 7320tattatttat aatagttcat tttaatcttt ttgtatatat tatgcgtgca gtactttttt 7380cctacatata actactatta cattttattt atataatatt tttattaatg aattttcgtg 7440ataatatgta atattgttca ttattatttc agatttttta aaaatatttg tgttattatt 7500tatgaaatat gtaatttttt tagtatttga ttttatgatg ataaagtgtt ctaaattcaa 7560aagaaggggg aaagcgtaaa cattaaaaaa cgtcatcaaa caaaaacaaa atcttgttaa 7620taaagataaa actgtttgtt ttgatcactg ttatttcgta atataaaaac attatttata 7680tttatattgt tgacaaccaa atttgcctat caaatctaac caatataatg catgcgtggc 7740aggtaatgta ctaccatgaa cttaagtcat gacataataa accgtgaatc tgaccaatgc 7800atgtacctan ctaaattgta tttgtgacac gaagcaaatg attcaattca caatggagat 7860gggaaacaaa taatgaagaa cccagaacta agaaagcttt tctgaaaaat aaaataaagg 7920caatgtcaaa agtatactgc atcatcagtc cagaaagcac atgatatttt tttatcagta 7980tcaatgcagc tagttttatt ttacaatatc gatatagcta gtttaaatat attgcagcta 8040gatttataaa tatttgtgtt attatttatc atttgtgtaa tcctgttttt agtattttag 8100tttatatatg atgataatgt attccaaatt taaaagaagg gaaataaatt taaacaagaa 8160aaaaagtcat caaacaaaaa acaaatgaaa gggtggaaag atgttaccat gtaatgtgaa 8220tgttacagta tttcttttat tatagagtta acaaattaac taatatgatt ttgttaataa 8280tgataaaata ttttttttat tattatttca taatataaaa atagtttact taatataaaa 8340aaaattctat cgttcacaac aaagttggcc acctaattta accatgcatg tacccatgga 8400ccatattagg taaccatcaa acctgatgaa gagataaaga gatgaagact taagtcataa 8460cacaaaacca taaaaaacaa aaatacaatc aaccgtcaat ctgaccaatg catgaaaaag 8520ctgcaatagt gagtggcgac acaaagcaca tgattttctt acaacggaga taaaaccaaa 8580aaaatatttc atgaacaacc tagaacaaat aaagctttta tataataaat atataaataa 8640ataaaggcta tggaataata tacttcaata tatttggatt aaataaattg ttggcggggt 8700tgatatattt atacacacct aaagtcactt caatctcatt ttcacttaac ttttattttt 8760tttttctttt tatttatcat aaagagaata ttgataatat actttttaac atatttttat 8820gacatttttt attggtgaaa acttattaaa aatcataaat tttgtaagtt agatttattt 8880aaagagttcc tcttcttatt ttaaattttt taataaattt ttaaataact aaaatttgtg 8940ttaaaaatgt taaaaaatgt gttattaacc cttctcttcg aggacgtacg agatccggcc 9000ggccagatcc tgcaggtctc aatagattaa gaagttggcg tctcattgat tgaccatggg 9060ggatcctcta gacgtacgtg aaggttaaac atggtgaata tgttaccact agctgggatg 9120cccattagat caaaactgta aaattctccc gtttcccttc tattcacatg tgagccccct 9180cccttttctt tctttctcaa ttttgattga gttaaagtca ccagcaatgc atcactcacc 9240ctccaaaaaa tttcttgtac aacttctcgg actatcccaa agctcctttt cctgagatgg 9300atggtcctgt ctcttgccct tgatgtcttc cttgttcgat tttggcttcc tctaatgtct 9360ttcttgctag gaatcaccac ctcactcatc tatgttgtcg tagcttctga aagtctcata 9420catatcctta gtgttgcact catcttgtat tgaagtgaaa aagaatgttg ttctcctatc 9480caaatctcca ttgaatctct ttctcccaat gttgtcccat cggttggtcc tcctctccaa 9540ccaattaatt gtaaggtgtt taacataaac atggtacaat taagattttt catttcatta 9600agaaaagatt gagatttgtg gttctaaagt ttcaattaga gtttgatgat attgaaacaa 9660ccgtagaaca cattaagtat tactaactta tacatagagc attggaattt caccttttat

9720ttattctgtt tccgccaaag gtacatgact caagttattt tacacaagta acaaaggcat 9780ctaagcctaa gtattcttat tcagactttt cattattact ttcattgatt tggtgcgaaa 9840tgcggccgct cagaattcct gggcatcctg aattgtgtgg gccacgccca ccttctgcat 9900gtagcggagc gtggacatta tattctccag gatggtcggg tagtatgtgt agcgcacgcc 9960atggcgcttg caacattccc gcaccgtatc ttggatgaag gggtagtgcc aagaggacat 10020gcgggggaac aagtggtgct cgatctgcag gttcaaaccg ccggtcagca tcatcccgac 10080gctgcccccg taggtcgccg cggtctcggc ctgcgcccgg taccagtcca cccggtcctc 10140ctctacatcg cggacctcct tcccctcctt ggcccttttg gccttgcagt tcttttcggg 10200ggtccgctca gcgccttcaa agttatggga cagcacaaaa acaaatgtca agaagatgcc 10260tgtgaccgtt ggcaccagca ggatggtgag cagcggagag gcgcccgcga tgtaccaggg 10320gaggaaggcg ttgcggaaga tgaacagtag gcggagggaa acggcgaagg cgcgctgccg 10380gttgaaaaag ctgcctggtc taagagaggt ggcctctggg attgtgtcgt tgtgctgcat 10440cgtgaacaag tacatcggat tgtacaccat cgacacacca tacatcccca aaacgatgta 10500catgtaccac gcctggaacc gatggtacca ctttcgtgac gcatttgttg ccgggtagga 10560gtggaacaaa atgaacggct ccgccgaggc agcgtccagg tccaggccat gctggttggt 10620gtaggtgtgg tggcgcataa tgtgggactg cagccagatc cagcgggagg atccaatggc 10680gtcgatgccg taggcgaaaa aggcgttcac ccagggcttt ttgctgatgg ctccgtgaga 10740ggcatcgtgt tgaatcgcca acccaatctg gctgtggaag tacccggtga agatgcccca 10800catcactgcc ccggtagtga tccaatacca ttcgcctatg gcagtgcacg cgatgatcag 10860cgcggtccgc agccaaaagc cgggcgtggc gtaccagttg cgccccttca tgacctcccg 10920aacgctctgc ttgacatcct gggcgaaggg actgtcaaag gtgtacactg ggtccttcga 10980gcttcgttcg tgtttgcctc gggcaaattg ctgcagcagc ttcgaattct ctggcttcac 11040tccagggtgc atggagtaga acagggcggt tccgtcggag gcaccggcgg cttcaatgag 11100gttgccacct ggatgcactt tcgccagtcc gaagagatcg tacaacaccc catcaattgc 11160aaccagttct gggtgttcta aaagttgctc agtagtcaaa gagatggtgg ccatggtgcg 11220gccgcggtga tgactgatga gtgtttaagg accaatggag agaatgtttg agttgtgaag 11280cggagaacct gaggcgtggt tatttatagg gaagagagga aggtgaatga gggacacgtc 11340acagaagtag ggtgctgagc ttgagacatt cttcagtatg catggctatg gaagccttgg 11400gtgctacacc tcatgaagtt catggtgtga ggtggcttcg gcatctcaat taagtgacaa 11460agagaaaggt gtttcagtgt ttctattgca aatggcagaa actcgtgatg acgaggggac 11520catgcatggt ttcatttctt ttcttcctgg attctttctt tccttttata tatgcaggtt 11580cataatttaa aaattagact cgctttcaat ttcttaattt ctcattttcc tcttatatta 11640ctgtactaat gttaaccacg tacacttatt ttttttttag tttaattttg atagattgtg 11700ttgatttaaa catattaata ttttcaacca aataaaaatc attttagtag atacggcttt 11760ttaaataatt attaaaaata ttaactattt atcctaaatg gcacatttta attaaaaaaa 11820atccggtgtt gtaagtgttt tattaatttg ttttggcatt attaaagcaa cttttttttt 11880atttgttggc attttgagta cgtacttagg ctagcctgca 11920451362DNAArtificial Sequencecodon-optimized Euglena anabaena delta-5 desaturase 45atggccacca tctccctgac taccgagcag ctcctggaac accccgagct cgttgccatc 60gacggagtcc tgtacgatct cttcggtctg gccaaggtgc atccaggagg caacctcatc 120gaagctgccg gtgcatccga cggaaccgct ctgttctact ccatgcatcc tggagtcaag 180ccagagaact cgaagcttct gcagcaattt gcccgaggca agcacgaacg aagctccaag 240gatcccgtgt acaccttcga ctctcccttt gctcaggacg tcaagcagtc cgttcgagag 300gtcatgaagg gtcgaaactg gtacgccact cctggcttct ggctgagaac cgcactcatc 360atcgcttgta ctgccattgg cgagtggtac tggatcacaa ccggagcagt gatgtggggt 420atctttactg gatacttcca ctcgcagatt ggcttggcca ttcaacacga tgcttctcac 480ggagccatca gcaaaaagcc ctgggtcaac gcctttttcg cttatggcat cgacgccatt 540ggttcctctc gttggatctg gctgcagtcc cacattatgc gacatcacac ttacaccaac 600cagcatggcc tcgacctgga tgctgcctcg gcagagccgt tcatcttgtt ccactcctat 660cctgctacca acgcctctcg aaagtggtac caccgatttc aggcgtggta catgtacatc 720gttctgggaa tgtatggtgt ctcgatggtg tacaatccca tgtacctctt cacaatgcag 780cacaacgaca ccattcccga ggccacttct ctcagaccag gcagcttttt caatcggcag 840cgagctttcg ccgtttccct tcgactgctc ttcatcttcc gaaacgcctt tcttccctgg 900tacattgctg gtgcctctcc tctgctcacc attcttctgg tgcccacggt cacaggcatc 960ttcctcacct ttgtgttcgt tctgtcccat aacttcgagg gagccgaacg gaccccagag 1020aagaactgca aggccaaacg agctaaggaa ggcaaggagg tcagagacgt ggaagaggat 1080cgagtcgact ggtaccgagc acaggccgag actgctgcca cctacggtgg cagcgtggga 1140atgatgctta caggcggtct caacctgcag atcgagcatc acttgtttcc ccgaatgtcc 1200tcttggcact atcccttcat tcaagacacc gttcgggagt gttgcaagcg acatggcgtc 1260cgttacacat actatcctac cattctcgag aacatcatgt ccactcttcg atacatgcag 1320aaggtgggtg ttgctcacac cattcaggat gcccaggagt tc 1362463983DNAArtificial Sequenceplasmid pEaD5S 46tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa 420tgcatctaga tccatggtca agcgacccgc tctgcctctc accgtggacg gtgtcaccta 480cgacgtttct gcctggctca accaccatcc cggaggtgcc gacattatcg agaactaccg 540aggtcgggat gctaccgacg tcttcatggt tatgcactcc gagaacgccg tgtccaaact 600cagacgaatg cccatcatgg aaccttcctc tcccctgact ccaacacctc ccaagccaaa 660ctccgacgaa cctcaggagg atttccgaaa gctgcgagac gagctcattg ctgcaggcat 720gttcgatgcc tctcccatgt ggtacgctta caagaccctg tcgactctcg gactgggtgt 780ccttgccgtg ctgttgatga cccagtggca ctggtacctg gttggtgcta tcgtcctcgg 840cattcacttt caacagatgg gatggctctc gcacgacatt tgccatcacc agctgttcaa 900ggaccgatcc atcaacaatg ccattggcct gctcttcgga aacgtgcttc agggcttttc 960tgtcacttgg tggaaggacc gacacaacgc tcatcactcc gccaccaacg tgcagggtca 1020cgatcccgac atcgacaacc tgcctctcct ggcgtggtcc aaggaggacg tcgagcgagc 1080tggcccgttt tctcgacgga tgatcaagta ccaacagtat tacttctttt tcatctgtgc 1140ccttctgcga ttcatctggt gctttcagtc cattcatact gccacgggtc tcaaggatcg 1200aagcaatcag tactatcgaa gacagtacga gaaggagtcc gtcggtctgg cactccactg 1260gggtctcaag gccttgttct actatttcta catgccctcg tttctcaccg gactcatggt 1320gttctttgtc tccgagctgc ttggtggctt cggaattgcc atcgttgtct tcatgaacca 1380ctaccctctg gagaagattc aggactccgt gtgggatggt catggcttct gtgctggaca 1440gattcacgag accatgaacg ttcagcgagg cctcgtcaca gactggtttt tcggtggcct 1500caactaccag atcgaacatc acctgtggcc tactcttccc agacacaacc tcaccgctgc 1560ctccatcaaa gtggagcagc tgtgcaagaa gcacaacctg ccctaccgat ctcctcccat 1620gctcgaaggt gtcggcattc ttatctccta cctgggcacc ttcgctcgaa tggttgccaa 1680ggcagacaag gcctaagcgg ccgcatcgga tcccgggccc gtcgactgca gaggcctgca 1740tgcaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac 1800aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt 1860gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc 1920gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg 1980ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 2040atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 2100gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 2160gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 2220gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 2280gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 2340aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 2400ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 2460taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 2520tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 2580gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt 2640taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 2700tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 2760tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 2820ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 2880taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 2940tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 3000cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 3060gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 3120cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 3180ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac 3240aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 3300atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 3360tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 3420gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 3480aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat 3540acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 3600ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 3660tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 3720aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 3780catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 3840atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 3900aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag 3960gcgtatcacg aggccctttc gtc 3983

Claims

1. (canceled)

2. An isolated polypeptide comprising an amino acid sequence having at least 85% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:13.

3. The isolated polypeptide of claim 2, wherein the amino acid sequence has at least 90% sequence identity, based on the Clustal V method of alignment when compared to SEQ ID NO:13.

4. The isolated polypeptide of claim 3, wherein the amino acid sequence has at least 95% sequence identity, based on the Clustal V method of alignment when compared to SEQ ID NO:13.

5-38. (canceled)