Delta-8 Desaturases And Their Use In Making Polyunsaturated Fatty Acids

Abstract

Isolated nucleic acid fragments and recombinant constructs comprising such fragments encoding delta-8 desaturases along with a method of making long-chain polyunsaturated fatty acids (PUFAs) using these delta-8 desaturases in plants and oleaginous yeast.

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 60/853,563, filed Oct. 23, 2006, the entire contents of which are herein incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention is in the field of biotechnology. More specifically, this invention pertains to polynucleotide sequences encoding delta-8 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 a-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. 6). 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). Based on the role delta-8 desaturase enzymes play in the synthesis of e.g., ARA, EPA and DHA, there has been considerable effort to identify and characterize these enzymes. Most efforts thus far have focused 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.

[0006] 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 Provisional Application No. 60/795,810 filed Apr. 28, 2006 (Attorney Docket No. BB-1566) discloses amino acid and nucleic acid sequences for a delta-8 desaturase enzyme from Pavlova lutheri (CCMP459).

[0007] Based on the utility of expressing delta-8 desaturases in conjunction with delta-9 elongases, there has also been considerable effort to identify and characterize delta-9 elongases from various sources. 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. Provisional Application No. 60/739,989 filed Nov. 23, 2005 (Attorney Docket No. BB-1562), discloses a delta-9 elongase from Eulgena gracilis.

[0008] 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).

[0009] 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

[0010] The present invention concerns an isolated polynucleotide comprising: [0011] (a) a nucleotide sequence encoding a polypeptide having delta-8 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:47, SEQ ID NO:49 or SEQ ID NO:57; [0012] (b) a nucleotide sequence encoding a polypeptide having delta-8 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:92, SEQ ID NO:93 or SEQ ID NO:62; [0013] (c) a nucleotide sequence encoding a polypeptide having delta-8 desaturase activity, wherein the nucleotide sequence hybridizes under stringent conditions to a nucleotide sequence as set forth in SEQ ID NO:92, SEQ ID NO:93 or SEQ ID NO:62; or [0014] (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.

[0015] 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.

[0016] In a third embodiment, the invention concerns a cell comprising in its genome the recombinant DNA construct of the invention. Such cells can be plant cells or yeast cells.

[0017] In a fourth embodiment, the invention concerns a method for transforming a cell, comprising transforming a cell with a recombinant construct of the invention or an isolated polynucleotide of the invention and selecting those cells transformed with the recombinant construct or the isolated polynucleotide.

[0018] 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.

[0019] In a sixth embodiment, the invention concerns a method for making long-chain polyunsaturated fatty acids in a plant cell comprising: [0020] (a) transforming a cell with the recombinant construct of the invention; and [0021] (b) selecting those transformed cells that make long-chain polyunsaturated fatty acids.

[0022] In a seventh embodiment, the invention concerns a method for producing at least one polyunsaturated fatty acid in an oilseed plant cell comprising: [0023] (a) transforming an oilseed plant cell with a first recombinant DNA construct comprising an isolated polynucleotide encoding at least one delta-8 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; [0024] (b) regenerating an oilseed plant from the transformed cell of step (a); and [0025] (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.

[0026] 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.

[0027] In a ninth embodiment, the invention concerns an oilseed plant comprising: [0028] (a) a first recombinant DNA construct comprising an isolated polynucleotide encoding at least one delta-8 desaturase polypeptide, operably linked to at least one regulatory sequence; and [0029] (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.

[0030] 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 product is lecithin.

[0031] 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.

[0032] In an eleventh embodiment, the invention concerns a method for making long-chain polyunsaturated fatty acids in a cell having a reduced level of by-product fatty acids, said method comprising: [0033] (a) transforming a host cell with at least one recombinant DNA construct comprising an isolated polynucleotide encoding at least two delta-8 desaturases operably linked to at least one regulatory sequence; and [0034] (b) selecting those transformed host cells obtained having a reduced level of by-product fatty acids, when compared to the level of such metabolic by-product fatty acids in a transformed host cell having at least one recombinant DNA construct comprising an isolated polynucleotide encoding one delta-8 desaturase operably linked to a regulatory sequence.

[0035] In a twelfth 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 the yeast expression vector pY126.

[0039] FIG. 2 is the soybean expression vector pKR1013.

[0040] FIG. 3 is the soybean expression vector pKR1014.

[0041] FIG. 4 is the soybean expression vector pKR1005.

[0042] FIG. 5 are the lipid profiles of somatic soybean embryos expressing the Tetruetreptia pomquetensis CCMP1491 delta-8 desaturase and the Isochrysis galbana delta-9 elongase (see Example 10).

[0043] FIG. 6 is a representative omega-3 and omega-6 fatty acid pathway providing for the conversion of myristic acid through various intermediates to DHA.

[0044] FIGS. 7A and 7B show a Clustal V alignment of the delta-8 desaturases from Tetruetreptia pomquetensis CCMP1491 (SEQ ID NO:57), Eutreptiella sp. CCMP389 (SEQ ID NO:47), Eutreptiella cf.sub.--gymnastica CCMP1594 (SEQ ID NO:49), Euglena gracilis (SEQ ID NO:98; NCBI Accession No. AAD45877 (GI 5639724)) and Euglena gracilis (SEQ ID NO:112).

[0045] FIG. 8 is a schematic of the Yarrowia lipolytica expression vector pFBAIn-MOD1.

[0046] FIG. 9 is a schematic of the construct pZKLeuN-29E3.

[0047] FIG. 10 are the lipid profiles of somatic soybean embryos expressing Tetruetreptia pomquetensis CCMP1491 (TpomD8) and Euglena gracilis delta-9 elongase (EgD9e) for the top 5 events (see Example 12).

[0048] FIG. 11 is the soybean expression vector pKR973.

[0049] FIG. 12 is the soybean expression vector pKR1084.

[0050] FIG. 13 is the soybean expression vector pKR1123

[0051] FIG. 14 shows the lipid profiles of somatic soybean embryos expressing E1594D8 and EgD9e for the top 5 events. Fatty acids are identified as 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic acid), LA, GLA, ALA, EDA, DGLA, ERA and ETA; and, fatty acid compositions listed in FIG. 14 are expressed as a weight percent (wt. %) of total fatty acids. The activity of E1594D8 is expressed as percent desaturation (% desat), calculated according to the following formula: ([product]/[substrate+product])*100. The combined percent desaturation for EDA and ERA is shown as "C20% delta-8 desat". The individual omega-6 delta-8 desaturation ("EDA % delta-8 desat.") was calculated as: ([DGLA]/[DGLA+EDA])*100. Similarly, the individual omega-3 delta-8 desaturation ("ERA % delta-8 desat.") was calculated as: ([ETA]/[ETA+ERA])*100. The ratio of delta-8 desaturation for omega-6 versus omega-3 substrates ("ratio [EDA/ERA] % desat.") was obtained by dividing the EDA % delta-8 desaturation by the ERA % delta-8 desaturation.

[0052] FIG. 15 is the soybean expression vector pKR1122.

[0053] FIG. 16 shows the lipid profiles of somatic soybean embryos expressing E389D8 and EgD9e for the top 5 events.

[0054] FIG. 17 is the Arabidopsis Binary Expression pKR1022R.

[0055] FIG. 18 shows the lipid profiles of T2 bulk seed for 22 events where wild-type-Arabidopsis was transformed with pKR1022R (SEQ ID NO:141).

[0056] FIG. 19 shows the average fatty acid profiles (average of 10 embryos per event) of soybean embryos transformed with the AscI fragments of pKR1005 (SEQ ID NO:90; FIG. 4) and pKR973 (SEQ ID NO:125; FIG. 11), for the 10 events having the highest amounts of delta-8 desaturation products. Fatty acids are identified as 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic acid), LA, GLA, ALA, EDA, SCI, DGLA, ARA, ERA, JUP, ETA, EPA and DPA; and are expressed as a weight percent (wt. %) of total fatty acids. Fatty acids listed as "others" include: 18:2 (5,9), STA, 20:0, 20:1(11), 20:2 (7,11) or 20:2 (8,11), and DHA. The total wt. % of fatty acids containing a delta-8 double bond is expressed as C20 delta-8 desat (DGLA+ARA+ETA+EPA+DPA) and the delta-8 desaturase activity is expressed as percent desaturation (C20% delta-8 desat), calculated according to the following formula: ([DGLA+ETA]/[DGLA+ETA+EDA+ERA])*100.

[0057] FIG. 20 shows the fatty acid profiles for ten individual T1 seeds from 2 plants from event AFS 4882-4-6 (plant #4882-4-6-1 & #4882-4-6-2) having some of the highest amounts of total delta-8 desaturation products

[0058] FIG. 21 shows the average fatty acid profiles (average of 10 embryos per event) of soybean embryos transformed with the AscI fragments of pKR1005 (SEQ ID NO:90; FIG. 4) and pKR1084 (SEQ ID NO:129; FIG. 12), for the 10 events having the highest amounts of delta-8 desaturation products. Fatty acids are identified as 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic acid), LA, GLA, ALA, EDA, SCI, DGLA, ARA, ERA, JUP, ETA, EPA and DPA; and, fatty acid compositions are expressed as a weight percent (wt. %) of total fatty acids. Fatty acids listed as "others" include: 18:2 (5,9), STA, 20:0, 20:1(11), 20:2 (7,11) or 20:2 (8,11), and DHA. The total wt. % of fatty acids containing a delta-8 double bond is expressed as C20 delta-8 desat (DGLA+ARA+ETA+EPA+DPA) and the delta-8 desaturase activity is expressed as percent desaturation (C20% delta-8 desat), calculated according to the following formula: ([DGLA+ETA]/[DGLA+ETA+EDA+ERA)]*100.

[0059] FIG. 22 shows the fatty acid profiles for individual T1 seeds from 2 plants from event AFS 5003-1-8 (plant #5003-1-8-1 & #5003-1-8-2) having some of the highest amounts of total delta-8 desaturation products.

[0060] 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).

[0061] A Sequence Listing is provided herewith on Compact Disk. The contents of the Compact Disk containing the Sequence Listing are hereby incorporated by reference in compliance with 37 C.F.R. .sctn.1.52(e).

[0062] SEQ ID NOs:1-11 are the nucleotide sequences of primers D8F1, D8F2, D8F3, D8F4, D8F5, D8F6, D8F7, D8F8, D8F9, D8R1 and D8R2, respectively.

[0063] SEQ ID NO:12 is the amino acid sequence of primers D8F1 and D8F4.

[0064] SEQ ID NO:13 is the amino acid sequence of primers D8F2, D8F3, D8F5 and D8F6.

[0065] SEQ ID NO:14 is the amino acid sequence of primers D8F7, D8F8 and D8F9.

[0066] SEQ ID NO:15 is the amino acid sequence of primers D8R1 and D8R2.

[0067] SEQ ID NO:16 is the partial nucleotide sequence of the delta-8 desaturase from Tetruetreptia pomquetensis CCMP1491 using the primer combination D8F4/D8R1 (see Example 1).

[0068] SEQ ID NO:17 is the partial nucleotide sequence of the delta-8 desaturase from Eutreptiella sp. CCMP389 using the primer combination D8F4/D8R1 (see Example 1).

[0069] SEQ ID NO:18 is the partial nucleotide sequence of the delta-8 desaturase from Eutreptiella cf.sub.--gymnastica CCMP1594 using the primer combination

[0070] D8F4/D8R1 (see Example 1).

[0071] SEQ ID NO:19 is the nucleotide sequence of the SMART IV oligonucleotide.

[0072] SEQ ID NOs:20-24 are the nucleotide sequences of primers 389D8-3-1, 389D8-3-2, 389D8-5-1, 389D8-5-2 and 389D8-5-3, respectively.

[0073] SEQ ID NOs:25-29 are the nucleotide sequences of primers ED8-5-1, ED8-5-2, ED8-5-3, ED8-3-1 and ED8-3-2, respectively.

[0074] SEQ ID NO:30 is the nucleotide sequence of CDSIII/3' PCR primer.

[0075] SEQ ID NO:31 is the nucleotide sequence of the Adaptor Primer from Invitrogen 3'-RACE kit.

[0076] SEQ ID NOs:32-36 are the nucleotide sequences of primers 1594D8-3-1, 1594D8-3-2, 1594D8-5-1, 1594D8-5-2 and 1594D8-5-3, respectively.

[0077] SEQ ID NO:37 is the nucleotide sequence of the GenomeWalker adaptor (see also SEQ ID NO:111).

[0078] SEQ ID NOs:38 and 39 are the nucleotide sequences of primer AP1 and AP2, respectively.

[0079] SEQ ID NO:40 is nucleotide sequence of pCR2.1-TOPO.

[0080] SEQ ID NO:41 is the 5'-region nucleotide sequence of the delta-8 desaturase from Eutreptiella cf.sub.--gymnastica CCMP1594 (see Example 2).

[0081] SEQ ID NO:42 is the 5'-region nucleotide sequence of the delta-8 desaturase from Eutreptiella sp. CCMP389 (see Example 2).

[0082] SEQ ID NO:43 is the 3'-region nucleotide sequence of the delta-8 desaturase from Eutreptiella sp. CCMP389 (see Example 2).

[0083] SEQ ID NO:44 is a 3'-region nucleotide sequence of the delta-8 desaturase from Eutreptiella cf.sub.--gymnastica CCMP1594 (1594D8-3'A) (see Example 2).

[0084] SEQ ID NO:45 is a 3'-region nucleotide sequence of the delta-8 desaturase from Eutreptiella cf.sub.--gymnastica CCMP1594 (1594D8-3'B) (see Example 2).

[0085] SEQ ID NO:46 is the nucleotide sequence of the delta-8 desaturase from Eutreptiella sp. CCMP389 (1963 by contig).

[0086] SEQ ID NO:47 is the amino acid sequence of the delta-8 desaturase from Eutreptiella sp. CCMP389 (coding region of SEQ ID NO:46 and SEQ ID NO:92).

[0087] SEQ ID NO:48 is the nucleotide sequence of the delta-8 desaturase from Eutreptiella cf.sub.--gymnastica CCMP1594 (2063 by contig).

[0088] SEQ ID NO:49 is the amino acid sequence of the delta-8 desaturase from Eutreptiella cf.sub.--gymnastica CCMP1594 (coding region of SEQ ID NO:48 and SEQ ID NO:93).

[0089] SEQ ID NO:50 is the nucleotide sequence of the TOPO linker.

[0090] SEQ ID NO:51 is the nucleotide sequence of the LinkAmp primer 1.

[0091] SEQ ID NO:52 is the nucleotide sequence of the LinkAmp primer 2.

[0092] SEQ ID NO:53 is the 5'-region nucleotide sequence of the delta-8 desaturase from Tetruetreptia pomquetensis CCMP1491 (see Example 3).

[0093] SEQ ID NO:54 is the nucleotide sequence of primer AUAP.

[0094] SEQ ID NO:55 is the 3'-region nucleotide sequence of the delta-8 desaturase from Tetruetreptia pomquetensis CCMP1491 (see Example 3).

[0095] SEQ ID NO:56 is the nucleotide sequence of the delta-8 desaturase from Tetruetreptia pomquetensis CCMP1491 (2233 by contig).

[0096] SEQ ID NO:57 is the amino acid sequence of the delta-8 desaturase from Tetruetreptia pomquetensis CCMP1491 (coding region of SEQ ID NO:56 and SEQ ID NO:62).

[0097] SEQ ID NOs:58 and 59 are the nucleotide sequences of TpomNot-5 and TpomNot-3, respectively.

[0098] SEQ ID NO:60 is the nucleotide sequence of primer T7.

[0099] SEQ ID NO:61 is the nucleotide sequence of primer M13-28Rev.

[0100] SEQ ID NO:62 is the nucleotide sequence of the coding sequence of Tetruetreptia pomquetensis CCMP1491 delta-8 desaturase.

[0101] SEQ ID NO:63 is the nucleotide sequence of pLF114-10.

[0102] SEQ ID NO:64 is the nucleotide sequence of pY-75.

[0103] SEQ ID NO:65 is the nucleotide sequence of pY126 (see FIG. 1).

[0104] SEQ ID NO:66 is the nucleotide sequence of pKR123r.

[0105] SEQ ID NO:67 is the nucleotide sequence of pKR1007.

[0106] SEQ ID NO:68 is the nucleotide sequence of pKR607.

[0107] SEQ ID NO:69 is the nucleotide sequence of pKR1013 (see FIG. 2).

[0108] SEQ ID NO:70 is the nucleotide sequence of the coding sequence of the Isochrysis galbana delta-9 elongase (NCBI Accession No. AAL37626 (GI 17226123), locus AAL37626, CDS AF390174; Qi et al., FEBS Lett. 510(3):159-165 (2002)).

[0109] SEQ ID NO:71 is the sequence of a portion of the cDNA insert from Euglena gracilis clone eeg1c.pk001.n5.f (5' end of cDNA insert).

[0110] SEQ ID NO:72 is the sequence of a portion of the cDNA insert from clone eeg1c.pk001.n5.f (3' end of cDNA insert).

[0111] SEQ ID NO:73 is the sequence of clone eeg1c.pk001.n5.f (5' and 3' sequences were aligned).

[0112] SEQ ID NO:74 is the Euglena gracilis delta-9 elongase coding sequence from the cDNA in clone eeg1c.pk001.n5.f.

[0113] SEQ ID NO:75 is the amino acid sequence of the Euglena gracilis delta-9 elongase from clone eeg1c.pk001.n5.f (coding region of SEQ ID NO:74).

[0114] SEQ ID NO:76 is the amino acid sequence of the long-chain PUFA elongation enzyme (delta-9 elongase) from Isochrysis galbana (NCBI Accession No. AAL37626 (GI 17226123), locus AAL37626, CDS AF390174) (designated "IgD9e").

[0115] SEQ ID NOs:77 and 78 are the nucleotide sequences of oligonucleotide primers oEugEL1-1 and oEugEL1-2, respectively.

[0116] SEQ ID NO:79 is the nucleotide sequence of pKR906.

[0117] SEQ ID NO:80 is the nucleotide sequence of pKR72 (ATCC Accession No. PTA-6019).

[0118] SEQ ID NO:81 is the nucleotide sequence of pK912.

[0119] SEQ ID NO:82 is the nucleotide sequence of pKR1014 (see FIG. 3).

[0120] SEQ ID NO:83 is the nucleotide sequence of pKR271.

[0121] SEQ ID NO:84 is the nucleotide sequence of pKR226.

[0122] SEQ ID NO:85 is the nucleotide sequence of pKR886r.

[0123] SEQ ID NOs:86 and 87 are the nucleotide sequences of oligonucleotide primers oCon1 and oCon2, respectively.

[0124] SEQ ID NO:88 is the nucleotide sequence of pKR179.

[0125] SEQ ID NO:89 is the nucleotide sequence of pKR1002.

[0126] SEQ ID NO:90 is the nucleotide sequence of pKR1005 (see FIG. 4).

[0127] SEQ ID NO:91 is the nucleotide sequence of the M13F universal primer.

[0128] SEQ ID NO:92 is the nucleotide sequence of the coding sequence of Eutreptiella sp. CCMP389 delta-8 desaturase.

[0129] SEQ ID NO:93 is the nucleotide sequence of the coding sequence of Eutreptiella cf.sub.--gymnastica CCMP1594 delta-8 desaturase.

[0130] SEQ ID NO:94 is the nucleotide sequence of Yarrowia lipolytica expression vector pFBAIn-MOD1.

[0131] SEQ ID NO:95 is the nucleotide sequence of Yarrowia lipolytica expression vector pFBAIn-389D8.

[0132] SEQ ID NO:96 is the nucleotide sequence of Yarrowia lipolytica expression vector pFBAIn-1594D8.

[0133] SEQ ID NO:97 is the nucleotide sequence of Yarrowia lipolytica expression vector pFBAIn-1491D8.

[0134] SEQ ID NO:98 is the amino acid sequence of the Euglena gracilis delta-8 fatty acid desaturase gene (NCBI Accession No. AAD45877 (GI 5639724)). SEQ ID NO:98 is the amino acid sequence encoded by nucleotides 14-1273 of NCBI Accession No. AF139720 (GI 5639723). This delta-8 fatty acid desaturase has been shown to be non-functional.

[0135] SEQ ID NOs:99 and 100 are the nucleotide sequences of primers 389D8-F and 389D8-R, respectively.

[0136] SEQ ID NOs:101 and 102 are the nucleotide sequences of primers 1491D8-F and 1491 D8-R, respectively.

[0137] SEQ ID NOs:103 and 104 are the nucleotide sequences of primers 1594D8-F and 1594D8-R, respectively.

[0138] SEQ ID NO:105 is the 5' PCR primer used in Example 1.

[0139] SEQ ID NO:106 is the nucleotide sequence of plasmid pZKLeuN-29E3 (see FIG. 9).

[0140] SEQ ID NO:107 is the nucleotide sequence of a synthetic delta-9 elongase (initially from Euglena gracilis--see SEQ ID NO:74) codon-optimized for Yarrowia lipolytica; see also U.S. Patent Application No. 60/739,989, filed Nov. 23, 2005 (Attorney Docket No. BB-1562) (designated "EgD9E" or "EgD9S")

[0141] SEQ ID NO:108 is the nucleotide sequence of the LoxP sequence from Escherichia coli.

[0142] SEQ ID NO:109 is the nucleotide sequence of a synthetic C.sub.16/18 elongase (initially from M. alpina) codon-optimized for Yarrowia lipolytica; see also U.S. patent application Ser. No. 11/253,882, filed Oct. 19, 2005.

[0143] SEQ ID NO:110 is the nucleotide sequence of a synthetic delta-9 elongase (initially from Isochrysis galbana) codon-optimized for Yarrowia lipolytica (designated "IgD9eS").

[0144] SEQ ID NO:111 is the nucleotide sequence of the GenomeWalker adaptor (see also SEQ ID NO:37).

[0145] SEQ ID NO:112 is the amino acid sequence of Euglena gracilis delta-8 desaturase (SEQ ID NO:2 of U.S. Publication No. 20050287652).

[0146] SEQ ID NO:113 is the nucleotide sequence of pKR132.

[0147] SEQ ID NO:114 is the nucleotide sequence of pKR953.

[0148] SEQ ID NO:115 is the nucleotide sequence of pKR287.

[0149] SEQ ID NO:116 is the nucleotide sequence of Mortierella alpina delta-5 desaturase (which is described in U.S. Pat. No. 6,075,183).

[0150] SEQ ID NO:117 is the nucleotide sequence of pKR277.

[0151] SEQ ID NO:118 is the nucleotide sequence of pKR952.

[0152] SEQ ID NO:119 is the nucleotide sequence of pKR457.

[0153] SEQ ID NO:120 is the nucleotide sequence of the modified Kti-NotI-Kti3'Salb3'cassette.

[0154] SEQ ID NO:121 is the nucleotide sequence of the Pavlova lutheri Delta-8 Desaturase codon sequence described in U.S. Provisional Application No. 60/795,810 and U.S. patent application Ser. No. 11/737,772.

[0155] SEQ ID NO:122 is the nucleotide sequence of oligonucleotide primer PvDES5'Not-1.

[0156] SEQ ID NO:123 is the nucleotide sequence of oligonucleotide primer PvDES3'Not-1.

[0157] SEQ ID NO:124 is the nucleotide sequence of pKR970.

[0158] SEQ ID NO:125 is the nucleotide sequence of pKR973.

[0159] SEQ ID NO:126 is the nucleotide sequence of pKS129.

[0160] SEQ ID NO:127 is the nucleotide sequence of pKR606.

[0161] SEQ ID NO:128 is the nucleotide sequence of pKR804.

[0162] SEQ ID NO:129 is the nucleotide sequence of pKR1084.

[0163] SEQ ID NO:130 is the nucleotide sequence of pKR908.

[0164] SEQ ID NO:131 is the nucleotide sequence of pKR1118

[0165] SEQ ID NO:132 is the nucleotide sequence of pKR1120.

[0166] SEQ ID NO:133 is the nucleotide sequence of pKR1123

[0167] SEQ ID NO:134 is the nucleotide sequence of pKR1117.

[0168] SEQ ID NO:135 is the nucleotide sequence of pKR1119.

[0169] SEQ ID NO:136 is the nucleotide sequence of pKR1122.

[0170] SEQ ID NO:137 is the nucleotide sequence of pKR393.

[0171] SEQ ID NO:138 is the nucleotide sequence of pKR407.

[0172] SEQ ID NO:139 is the nucleotide sequence of pKR1018.

[0173] SEQ ID NO:140 is the nucleotide sequence of pKR1020R.

[0174] SEQ ID NO:141 is the nucleotide sequence of pKR1022R.

DETAILED DESCRIPTION OF THE INVENTION

[0175] The disclosure of each reference set forth herein is hereby incorporated by reference in its entirety.

[0176] 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.

[0177] The present invention relates to delta-8 desaturase enzymes and nucleic acid for encoding the same isolated from Tetruetreptia pomquetensis CCMP1491, Eutreptiella sp. CCMP389 and Eutreptiella cf.sub.--gymnastica CCMP1594 delta-8. These are useful for, inter alia, for the manipulation of biochemical pathways for the production of healthful PUFAs. Thus, the subject invention finds many applications. 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).

[0178] 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.

[0179] In the context of this disclosure, a number of terms and abbreviations are used. The following definitions are provided.

[0180] "Open reading frame" is abbreviated ORF.

[0181] "Polymerase chain reaction" is abbreviated PCR.

[0182] "American Type Culture Collection" is abbreviated ATCC.

[0183] "Polyunsaturated fatty acid(s)" is abbreviated PUFA(s).

[0184] "Triacylglycerols" are abbreviated TAGs.

[0185] 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.

[0186] 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.

[0187] 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

[0188] 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.

[0189] 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.

[0190] 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. 6, 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.

[0191] 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.

[0192] 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.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] "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.

[0197] 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.

[0198] "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-8 desaturases that will desaturate a fatty acid between the eighth and ninth carbon atom numbered from the carboxyl-terminal end of the molecule and that can, for example, catalyze the conversion of EDA to DGLA and/or ETrA to ETA. Other useful fatty acid desaturases include, for example: (1) delta-5 desaturases that catalyze the conversion of DGLA to ARA and/or ETA to EPA; (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.

[0199] For the purposes herein, the term "TpomD8" refers to a delta-8 desaturase enzyme (SEQ ID NO:57) isolated from Tetruetreptia pomquetensis CCMP1491, encoded by SEQ ID NO:62 herein. The term "E389D8" refers to a delta-8 desaturase enzyme (SEQ ID NO:47) isolated from Eutreptiella sp. CCMP389, encoded by SEQ ID NO:92 herein. Likewise, the term "E1594D8" refers to a delta-8 desaturase enzyme (SEQ ID NO:49) isolated from Eutreptiella cf.sub.--gymnastica CCMP1594, encoded by SEQ ID NO:93 herein.

[0200] Similarly, the term "EgD8" refers to a delta-8 desaturase enzyme (SEQ ID NO:112) 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).

[0201] 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.

[0202] For the purposes herein, an enzyme catalyzing the first condensation reaction (i.e., conversion of malonyl-CoA and long-chain acyl-CvoA 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). Similarily, 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.

[0203] For the purposes herein, the term "IgD9e" refers to a delta-9 elongase (SEQ ID NO:76; NCBI Accession No. AAL37626 [GI 17226123], locus AAL37626, CDS AF390174; GenBank Accession No. AF390174) isolated from Isochrysis galbana, encoded by SEQ ID NO:70. In contrast, the term "IgD9eS" refers to a synthetic (codon-optimized) delta-9 elongase (SEQ ID NO:110) derived from the DNA sequence of the Isochrysis galbana delta-9 elongase (SEQ ID NO:70) which can be used for expression in Yarrowia lipolytica.

[0204] Similarly for the purposes herein, the term "EgD9e" refers to a delta-9 elongase (SEQ ID NO:75) isolated from Euglena gracilis, encoded by SEQ ID NO:74 (see Example 11 herein).

[0205] 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.

[0206] 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.

[0207] 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.

[0208] 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.

[0209] 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.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] 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.

[0214] "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.

[0215] 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.

[0216] 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.

[0217] 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.

[0218] "BLASTN method of alignment" is an algorithm provided by the National Center for Biotechnology Information (NCBI) to compare nucleotide sequences using default parameters.

[0219] 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.

[0220] "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.

[0221] 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.

[0222] 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.

[0223] 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.

[0224] "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.

[0225] "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).

[0226] "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)).

[0227] "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).

[0228] "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.

[0229] 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.

[0230] 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.

[0231] "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".

[0232] 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.

[0233] 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.)

[0234] 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.

[0235] 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]).

[0236] 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 ac ell, 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).

[0237] "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.

[0238] "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.

[0239] 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.

[0240] "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)).

[0241] 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.). 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.

[0242] 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: Eutreptiella and Tetruetreptia.

[0243] 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.

[0244] "Progeny" comprises any subsequent generation of a plant.

An Overview: Microbial Biosynthesis of Fatty Acids and Triacylglycerols

[0245] 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. 6).

[0246] 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

[0247] 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. 6 and as described below, there are often multiple alternate pathways for production of a specific long chain omega-3/omega-6 fatty acid.

[0248] 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.

[0249] 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.

[0250] 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.

[0251] 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).

[0252] 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.

[0253] 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-8 Desaturases

[0254] In the present invention, nucleotide sequences encoding delta-8 desaturases have been isolated from Tetruetreptia pomquetensis CCMP1491 (designated herein as "TpomD8"), Eutreptiella sp. CCMP389 (designated herein as "E389D8") and Eutreptiella cf.sub.--gymnastica CCMP1594 (designated herein as "E1594D8").

[0255] Thus, the present invention concerns an isolated polynucleotide comprising: [0256] (a) a nucleotide sequence encoding a polypeptide having delta-8 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:47 [E389D8], SEQ ID NO:49 [E1594D8] or SEQ ID NO:57 [TpomD8]; [0257] (b) a nucleotide sequence encoding a polypeptide having delta-8 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:92 [E389D8], SEQ ID NO:93 [E1594D8] or SEQ ID NO:62 [TpomD8]; or, [0258] (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.

[0259] In still another aspect, this invention concerns an isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having delta-8 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:92, SEQ ID NO:93 or SEQ ID NO:62.

[0260] In alternate embodiments, the instant E389D8, E1594D8 or TpomD8 desaturase sequences can be codon-optimized for expression in a particular host organism. As is well known in the art, this can be a useful means to further optimize the expression of the enzyme in the alternate host, since use of host-preferred codons can substantially enhance the expression of the foreign gene encoding the polypeptide. In general, host-preferred codons can be determined within a particular host species of interest by examining codon usage in proteins (preferably those expressed in the largest amount) and determining which codons are used with highest frequency. Then, the coding sequence for a polypeptide of interest having e.g., desaturase activity can be synthesized in whole or in part using the codons preferred in the host species.

[0261] In one embodiment of the invention herein, E389D8, E1594D8 and/or TpomD8 could be codon-optimized for expression in Yarrowia lipolytica, as taught in PCT Publication No. WO 04/101757. In alternate embodiments, it may be desirable to modify a portion of the codons encoding E389D8, E1594D8 and/or TpomD8 (as set forth in SEQ ID NOs:92, 93 and 62, respectively) to enhance expression of the gene in a host organism including, but not limited to, a plant or plant part.

[0262] One skilled in the art would be able to use the teachings herein to create various other codon-optimized delta-8 desaturase proteins suitable for optimal expression in alternate hosts, based on the wildtype E389D8, E1594D8 and/or TpomD8 sequences. Accordingly, the instant invention relates to any codon-optimized delta-8 desaturase protein that is derived from the wildtype E389D8 (i.e., encoded by SEQ ID NO:47), the wildtype E1594D8 (i.e., encoded by SEQ ID NO:49) or the wildtype TpomD8 (i.e., encoded by SEQ ID NO:57).

Identification and Isolation of Homologs

[0263] Any of the instant desaturase sequences (i.e., E389D8, E1594D8 or TpomD8) or portions thereof may be used to search for delta-8 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.

[0264] Alternatively, any of the instant desaturase sequences or portions thereof may also be employed as hybridization reagents for the identification of delta-8 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.

[0265] 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).

[0266] 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).

[0267] 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.

[0268] In additional embodiments, any of the delta-8 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.

[0269] For example, genes encoding similar proteins or polypeptides to the delta-8 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 DGLA and/or ETA 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.

[0270] 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.).

[0271] 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.

[0272] 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)).

[0273] In other embodiments, any of the delta-8 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-8 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

[0274] It is expected that introduction of chimeric genes encoding the delta-8 desaturases described herein (i.e., E389D8, E1594D8, TpomD8 or other mutant enzymes, codon-optimized enzymes or homologs thereof), under the control of the appropriate promoters will result in increased production of DGLA and/or ETA 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., EDA and/or ETrA) to the desaturase enzymes described herein (e.g., E389D8, E1594D8, TpomD8), such that the substrate is converted to the desired fatty acid product (i.e., DGLA and/or ETA).

[0275] More specifically, it is an object of the present invention to provide a method for the production of DGLA in a host cell (e.g., oleaginous yeast, soybean), wherein the host cell comprises: [0276] (a) a recombinant construct encoding a delta-8 desaturase polypeptide selected from the group consisting of SEQ ID NO:47, SEQ ID NO:49 and SEQ ID NO:57; and, [0277] (b) a source of EDA; wherein the host cell is grown under conditions such that the delta-8 desaturase is expressed and the EDA is converted to DGLA, and wherein the DGLA is optionally recovered.

[0278] In alternate embodiments of the present invention, the delta-8 desaturase may be used for the use of the enzyme for the conversion of ETrA to ETA. Accordingly the invention provides a method for the production of ETA, wherein the host cell comprises: [0279] (a) a recombinant construct encoding a delta-8 desaturase polypeptide selected from the group consisting of SEQ ID NO:47, SEQ ID NO:49 and SEQ ID NO:57; and, [0280] (b) a source of ETrA; wherein the host cell is grown under conditions such that the delta-8 desaturase is expressed and the ETrA is converted to ETA, and wherein the ETA is optionally recovered.

[0281] Alternatively, each delta-8 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 (FIG. 6; see 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-8 desaturases described herein (i.e., E389D8, E1594D8, TpomD8, 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).

[0282] In preferred embodiments, the delta-8 desaturases of the present invention will minimally be expressed in conjunction with a delta-9 elongase (e.g., a delta-9 elongase as set forth in SEQ ID NO:75 or a codon-optimized delta-9 elongase as set forth in SEQ ID NO:110). 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).

[0283] At times, it may be desirable to minimize by-product fatty acids. The relative abundance of by-product fatty acids could be decreased by increasing total delta-8 desaturase activity. One approach to minimize by-product fatty acids would be to express more than one delta-8 desaturase (i.e., the same or different delta-8 desaturase). For instance, the presence of sciadonic acid (SCI) and/or juniperonic acid (JUP) [commonly found in the seed lipids of gymnosperms (Wolff et al., Lipids 35(1):1-22 (2000)), such as those in the Pinaceae family (pine)] might be considered by-product fatty acids of a delta-6 desaturase/delta-6 elongase pathway or delta-9-elongase/delta-8 desaturase pathway. Although these fatty acids are considered to have various health-enhancing properties themselves (Nakane et al., Biol. Pharm. Bull. 23: 758-761 (2000)), their presence as by-product fatty acids in an engineered PUFA pathway, such as in an oilseed crop, may not be desirable depending on the application.

[0284] 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 below. 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 below) DGLA and ETA, respectively.

Plant Expression Systems, Cassettes and Vectors, and Transformation

[0285] In one embodiment, this invention concerns a recombinant construct comprising any one of the delta-8 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.

[0286] The origin of the promoter chosen to drive expression of the delta-8 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.

[0287] 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)).

[0288] 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.

[0289] Following identification of an appropriate promoter suitable for expression of a specific delta-8 desaturase coding sequence, the promoter is then operably linked in a sense orientation using conventional means well known to those skilled in the art.

[0290] 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, New York, 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").

[0291] 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.

[0292] The recombinant constructs of the invention may be introduced into one plant cell; or, alternatively, each construct may be introduced into separate plant cells.

[0293] Expression in a plant cell may be accomplished in a transient or stable fashion as is described above.

[0294] 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.

[0295] 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.

[0296] 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.

[0297] 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 Claim 8.

[0298] Also of interest is a method for producing a transformed plant comprising transforming a plant cell with the delta-8 desaturase polynucleotides of the instant invention and regenerating a plant from the transformed plant cell.

[0299] 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 bombardment (McCabe, D. E. et. al., Bio/Technology 6:923 (1988); Christou et al., Plant Physiol. 87:671-674 (1988)).

[0300] 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.

[0301] 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: N.Y. (1989); Maliga et al., Methods in Plant Molecular Biology, Cold Spring Harbor: N.Y. (1995); Birren et al., Genome Analysis: Detecting Genes, Vol. 1, Cold Spring Harbor: N.Y. (1998); Birren et al., Genome Analysis: Analyzing DNA, Vol.2, Cold Spring Harbor: N.Y. (1998); Plant Molecular Biology: A Laboratory Manual, eds. Clark, Springer: NY (1997).

[0302] Examples of oilseed plants include, but are not limited to: soybean, Brassica species, sunflower, maize, cotton, flax and safflower.

[0303] Examples of PUFAs having at least twenty carbon atoms and five or more carbon-carbon double bonds include, but are not limited to, omega-3 fatty acids such as EPA, DPA and DHA. Seeds obtained from such plants are also within the scope of this invention as well as oil obtained from such seeds.

[0304] Thus, in one embodiment this invention concerns an oilseed plant comprising: [0305] (a) a first recombinant DNA construct comprising an isolated polynucleotide encoding a delta-8 desaturase polypeptide, operably linked to at least one regulatory sequence; and, [0306] (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.

[0307] Such 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.

[0308] 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.

[0309] In another aspect, this invention concerns a method for making long-chain PUFAs in a plant cell comprising: [0310] (a) transforming a cell with the recombinant construct of the invention; and, [0311] (b) selecting those transformed cells that make long-chain PUFAs.

[0312] In still another aspect, this invention concerns a method for producing at least one PUFA in a soybean cell comprising: [0313] (a) transforming a soybean cell with a first recombinant DNA construct comprising: [0314] (i) an isolated polynucleotide encoding a delta-8 desaturase polypeptide, operably linked to at least one regulatory sequence; and, [0315] (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; [0316] (b) regenerating a soybean plant from the transformed cell of step (a); and, [0317] (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.

[0318] 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) as set forth in SEQ ID NO:76 or the delta-9 elongase isolated or derived from Euglena gracilis as set forth in SEQ ID NO:75.

Microbial Expression Systems, Cassettes and Vectors, and Transformation

[0319] The delta-8 elongase genes and gene products described herein (i.e., E389D8, E1594D8, TpomD8, 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).

[0320] 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.

[0321] Vectors or DNA cassettes useful for the transformation of suitable microbial host cells are well known in the art. The specific choice of sequences present in the construct is dependent upon the desired expression products (supra), the nature of the host cell and the proposed means of separating transformed cells versus non-transformed cells. Typically, however, the vector or cassette contains sequences directing transcription and translation of the relevant gene(s), a selectable marker and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5' of the gene that controls transcriptional initiation (e.g., a promoter) and a region 3' of the DNA fragment that controls transcriptional termination (i.e., a terminator). It is most preferred when both control regions are derived from genes from the transformed microbial host cell, although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a production host.

[0322] Initiation control regions or promoters which are useful to drive expression of the instant delta-8 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.

[0323] 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.

[0324] The termination region can be derived from the 3' region of the gene from which the initiation region was obtained or from a different gene. A large number of termination regions are known and function satisfactorily in a variety of hosts (when utilized both in the same and different genera and species from where they were derived). The termination region usually is selected more as a matter of convenience rather than because of any particular property. Preferably, when the microbial host is a yeast cell, the termination region is derived from a yeast gene (particularly Saccharomyces, Schizosaccharomyces, Candida, Yarrowia or Kluyveromyces). The 3'-regions of mammalian genes encoding .gamma.-interferon and .alpha.-2 interferon are also known to function in yeast. Termination control regions may also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary; however, it is most preferred if included. Although not intended to be limiting, termination regions useful in the disclosure herein include: .about.100 by of the 3' region of the Yarrowia lipolytica extracellular protease (XPR; GenBank Accession No. M17741); the acyl-coA oxidase (Aco3: GenBank Accession No. AJ001301 and No. CAA04661; Pox3: GenBank Accession No. XP 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.

[0325] 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-8 desaturases described herein.

[0326] 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.

[0327] In the present invention, the preferred method of expressing genes in Yarrowia lipolytica is by integration of linear DNA into the genome of the host; and, integration into multiple locations within the genome can be particularly useful when high level expression of genes are desired [e.g., in the Ura3 locus (GenBank Accession No. AJ306421), the Leu2 gene locus (GenBank Accession No. AF260230), the Lys5 gene (GenBank Accession No. M34929), the Aco2 gene locus (GenBank Accession No. AJ001300), the Pox3 gene locus (Pox3: GenBank Accession No. XP.sub.--503244; or, Aco3: GenBank Accession No. AJ001301), the delta-12 desaturase gene locus (PCT Publication No. WO2004/104167), the Lip1 gene locus (GenBank Accession No. Z50020) and/or the Lip2 gene locus (GenBank Accession No. AJ012632)].

[0328] 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.

[0329] 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.

[0330] 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)).

[0331] 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. 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.

[0332] The transformed host cell can be identified by various selection techniques, as described in PCT Publication Nos. WO2004/101757 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.

[0333] Following transformation, substrates suitable for the instant delta-8 desaturases (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.

[0334] 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.

[0335] 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).

[0336] 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)).

[0337] 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.

[0338] 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-8 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.

[0339] Based on the teachings described above, in one embodiment this invention is drawn to a method of producing either DGLA or ETA, respectively, comprising: [0340] (a) providing an oleaginous yeast comprising: [0341] (i) a first recombinant DNA construct comprising an isolated polynucleotide encoding a delta-8 desaturase polypeptide, operably linked to at least one regulatory sequence; and, [0342] (ii) a source of desaturase substrate consisting of either EDA or ETrA, respectively; and, [0343] (b) growing the yeast of step (a) in the presence of a suitable fermentable carbon source wherein the gene encoding the delta-8 desaturase polypeptide is expressed and EDA is converted to DGLA or ETrA is converted to ETA, respectively; and, [0344] (c) optionally recovering the DGLA or ETA, respectively, of step (b). Substrate feeding may be required.

[0345] Of course, since naturally produced PUFAs in oleaginous yeast are limited to 18:2 fatty acids (i.e., LA), and less commonly, 18:3 fatty acids (i.e., ALA), in more preferred embodiments of the present invention the oleaginous yeast will be genetically engineered to express multiple enzymes necessary for long-chain PUFA biosynthesis (thereby enabling production of e.g., ARA, EPA, DPA and DHA), in addition to the delta-8 desaturases described herein.

[0346] Specifically, in one embodiment this invention concerns an oleaginous yeast comprising: [0347] (a) a first recombinant DNA construct comprising an isolated polynucleotide encoding a delta-8 desaturase polypeptide, operably linked to at least one regulatory sequence; and, [0348] (b) at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of: a delta-4 desaturase, a delta-5 desaturase, delta-6 desaturase, a delta-9 desaturase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a delta-9 elongase, a C.sub.14/16 elongase, a C.sub.16/18 elongase, a C.sub.18/20 elongase and a C.sub.20/22 elongase.

[0349] In particularly 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 IgD9eS) as set forth in SEQ ID NO:76 or the delta-9 elongase isolated or derived from Euglena gracilis as set forth in SEQ ID NO:75.

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

[0350] 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.

[0351] 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).

[0352] 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).

[0353] 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).

[0354] Within the context of the present invention, it may be useful to modulate the expression of the fatty acid biosynthetic pathway by any one of the strategies described above. For example, the present invention provides methods whereby genes encoding key enzymes in the delta-9 elongase/delta-8 desaturase biosynthetic pathway are introduced into oleaginous yeasts for the production of omega-3 and/or omega-6 fatty acids. It will be particularly useful to express the present the delta-8 desaturase genes in oleaginous yeasts that do not naturally possess omega-3 and/or omega-6 fatty acid biosynthetic pathways and coordinate the expression of these genes, to maximize production of preferred PUFA products using various means for metabolic engineering of the host organism.

Microbial Fermentation Processes for PUFA Production

[0355] 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.)).

[0356] Fermentation media in the present invention must contain a suitable carbon source. Suitable carbon sources are taught in PCT Publication No. WO 2004/101757. Although it is contemplated that the source of carbon utilized in the present invention may encompass a wide variety of carbon-containing sources, preferred carbon sources are sugars, glycerol, and/or fatty acids. Most preferred is glucose and/or fatty acids containing between 10-22 carbons.

[0357] 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)).

[0358] Preferred growth media in the present invention are common commercially prepared media, such as Yeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.). Other defined or synthetic growth media may also be used and the appropriate medium for growth of the transformant host cells will be known by one skilled in the art of microbiology or fermentation science. A suitable pH range for the fermentation is typically between about pH 4.0 to pH 8.0, wherein pH 5.5 to pH 7.5 is preferred as the range for the initial growth conditions. The fermentation may be conducted under aerobic or anaerobic conditions, wherein microaerobic conditions are preferred.

[0359] 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

[0360] 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)).

[0361] 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

[0362] 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

[0363] 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.

[0364] 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.

[0365] 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.

[0366] 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).

[0367] 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-Containinq Oils for Use in Foodstuffs

[0368] 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).

[0369] 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.

[0370] 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.

[0371] 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.

[0372] 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).

[0373] 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.

[0374] 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.

[0375] 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.

[0376] A snack food product comprises any of the above or below described food products.

[0377] A fried food product comprises any of the above or below described food products that has been fried.

[0378] A health food product is any food product that imparts a health benefit. Many oilseed-derived food products may be considered as health foods.

[0379] A beverage can be in a liquid or in a dry powdered form.

[0380] 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 from Ross Products Division, Abbott Laboratories).

[0381] 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.

[0382] 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.

[0383] 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-Containinq Oils for Use in Health Food Products and Pharmaceuticals

[0384] 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

[0385] 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.

[0386] 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

[0387] 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.

General Methods

[0388] Standard recombinant DNA and molecular cloning techniques used in the Examples are well known in the art and are described by: (1) Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989) (Maniatis); (2) T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1984); and (3) Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987).

[0389] Materials and methods suitable for the maintenance and growth of microbial cultures are well known in the art. Techniques suitable for use in the following examples may be found as set out in Manual of Methods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, Eds), American Society for Microbiology: Washington, D.C. (1994)); or by Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, 2nd ed., Sinauer Associates: Sunderland, Mass. (1989). All reagents, restriction enzymes and materials used for the growth and maintenance of microbial cells were obtained from Aldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.), unless otherwise specified.

[0390] General molecular cloning was performed according to standard methods (Sambrook et al., supra). DNA sequence was generated on an ABI Automatic sequencer using dye terminator technology (U.S. Pat. No. 5,366,860; EP 272,007) using a combination of vector and insert-specific primers. Sequence editing was performed in Sequencher (Gene Codes Corporation, Ann Arbor, Mich.). All sequences represent coverage at least two times in both directions. Comparisons of genetic sequences were accomplished using DNASTAR software (DNA Star, Inc.).

[0391] The meaning of abbreviations is as follows: "sec" means second(s), "min" means minute(s), "h" means hour(s), "d" means day(s), ".mu.l" means microliter(s), "mL" means milliliter(s), "L" means liter(s), ".mu.M" means micromolar, "mM" means millimolar, "M" means molar, "mmol" means millimole(s), ".mu.mole" mean micromole(s), "g" means gram(s), ".mu.g" means microgram(s), "ng" means nanogram(s), "U" means unit(s), "bp" means base pair(s) and "kB" means kilobase(s).

Transformation and Cultivation of Yarrowia lipolytica:

[0392] Yarrowia lipolytica strains with ATCC Accession Nos. 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).

[0393] Transformation of Yarrowia lipolytica was performed according to the method of Chen 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 containing: 2.25 mL of 50% PEG, average MW 3350; 0.125 mL of 2 M Li acetate, pH 6.0; 0.125 mL of 2 M DTT; and 50 .mu.g sheared salmon sperm DNA. Then, approximately 500 ng of linearized plasmid DNA was incubated in 100 .mu.L of resuspended cells, and maintained at 39.degree. C. for 1 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.

[0394] 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 leucine and/or uracil were added as appropriate to a final concentration of 0.01% (thereby producing "MMLe" and "MMU" selection media, each prepared with 20 g/L agar).

[0395] 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 lipolvtica:

[0396] 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.

[0397] For direct base transesterification, Yarrowia culture (3 mL) was harvested, washed once in distilled water, and dried under vacuum in a Speed-Vac for 5-10 min. Sodium methoxide (100 .mu.L of 1%) was added to the sample, and then the sample was vortexed and rocked for 20 min. After adding 3 drops of 1 M NaCl and 400 .mu.L hexane, the sample was vortexed and spun. The upper layer was removed and analyzed by GC as described above.

Example 1

Identification of Delta-8 Desaturase Enzyme Homologs from Tetruetreptia Pomquetensis CCMP1491, Eutreptiella sp. CCMP389 and Eutreptiella cf gymnastica CCMP1594

[0398] The present Example describes the identification of cDNA fragments (SEQ ID NOs:16, 17 and 18) encoding portions of delta-8 desaturases from Tetruetreptia pomquetensis CCMP1491, Eutreptiella sp. CCMP389 and Eutreptiella cf.sub.--gymnastica CCMP1594, respectively. This work included the generation of genomic DNA and RNA, synthesis of cDNA, and then the identification of portions of the genes encoding delta-8 desaturase, by use of primers derived from the Euglena gracilis delta-8 desaturase.

Preparation of Euglenoid RNA and Genomic DNA

[0399] Tetruetreptia pomquetensis CCMP1491, Eutreptiella sp. CCMP389 and Eutreptiella cf.sub.--gymnastica CCMP1594 cells (each from 1 liter of culture) were purchased from the Provasoli-Guillard National Center for Culture of Marine Phytoplakton (CCMP) (Bigelow Laboratory for Ocean Sciences, West Boothbay Harbor, Me.). Total RNA and genomic DNA were isolated from each strain using the trizol reagent (Invitrogen, Carlsbad, Calif.), according to the manufacturer's protocol. Cell pellet from each strain was individually resuspended in 0.75 mL of trizol reagent, mixed with 0.5 mL of 0.5 mm glass beads, and homogenized in a Biospec mini beadbeater (Bartlesville, Okla.) at the highest setting for 3 min. The mixtures were centrifuged in an Eppendorf centrifuge for 30 sec at 14,000 rpm to remove debri and glass beads. Supernatant from each sample was extracted with 150 .mu.L of 24:1 chloroform:isoamy alcohol. The upper aqueous phase was used for RNA isolation and lower organic phase for DNA isolation.

[0400] For RNA isolation, the aqueous phase from each sample was mixed with 0.375 mL of isopropyl alcohol and allowed to incubate at room temperature for 5 min. Precipitated RNA was collected by centrifugation at 8,000 rpm and 4.degree. C. for 5 min. The pellet was washed once with 0.7 mL of 80% ethanol and air dried. Thus, 360 .mu.g of total RNA was obtained from Eutreptiella sp. CCMP389, 95 .mu.g from Tetruetreptia pomquetensis CCMP1491 and 720 .mu.g from Eutreptiella cf.sub.--gymnastica CCMP1594.

[0401] For genomic DNA isolation, the lower organic phase of each sample was mixed with 75 .mu.L of ethanol and incubated at room temperature for 5 min. The samples were then centrifuged at 5,000 rpm for 2 min in an Eppendorf centrifuge. Each pellet was washed with 0.75 mL of 0.1 M sodium citrate:10% ethanol twice. Each time, samples were incubated for 15 min at room temperature in the wash solution, followed by centrifugation at 5,000 rpm for 5 min at 4.degree. C. in an Eppendorf centrifuge. The pellet was air dried and re-dissolved in 300 .mu.L of 8 mM NaOH. The pH of each sample was adjusted to 7.5 with 1 M HEPES. Each sample was then further purified with the Qiagen PCR purification kit according to the manufacturer's protocol. In this way, 40 .mu.g of genomic DNA was isolated from Eutreptiella sp. CCMP389, 15 .mu.g from Tetruetreptia pomquetensis CCMP1491 and 45 .mu.g from Eutreptiella cf.sub.--gymnastica CCMP1594.

Preparation of Euglenoid cDNA

[0402] Total RNA (1.2 .mu.g from Eutreptiella sp. CCMP389 and 2.4 .mu.g from Eutreptiella cf.sub.--gymnastica CCMP1594) was used as template to synthesize double stranded cDNA. The Creator.TM. SMART.TM. cDNA Library Construction Kit from BD Bioscience Clontech (Palo Alto, Calif.) was used. Each total RNA sample (1 .mu.L) was mixed individually with 1 .mu.L of SMART IV oligonucleotide (SEQ ID NO:19), 1 .mu.L CDSIII/3' PCR primer (SEQ ID NO:30) and 2 .mu.L of water. The mixtures were heated to 75.degree. C. for 5 min and then cooled on ice for 5 min. To each sample were added 2 .mu.L of 5.times. first strand buffer, 1 .mu.L 20 mM DTT, 1 .mu.L of dNTP mix (10 mM each of dATP, dCTP, dGTP and dTTP) and 1 .mu.L of PowerScript reverse transcriptase. The samples were incubated at 42.degree. C. for 1 h. The resulting first strand cDNAs were then used as template for amplification. Each reaction mixture contained 2 .mu.L of the above first strand cDNA sample, 80 .mu.L of water, 10 .mu.L of 10.times. Advantage 2 PCR buffer, 2 .mu.L 50.times. dNTP mix (10 mM each of dATP, dCTP, dGTP and dTTP), 2 .mu.L of 5' PCR primer (SEQ ID NO:105), 2 .mu.L CDSIII/3' PCR primer (SEQ ID NO:30) and 2 .mu.L 50.times. Advantage 2 polymerase mix. PCR amplification was performed using the following conditions: 95.degree. C. for 1 min, followed by 20 cycles of 95.degree. C. for 10 sec and 68.degree. C. for 6 min. Amplification products were purified with Qiagen PCR purification kits according to the manufacturer's protocol. Purified products were eluted with 50 .mu.L of water.

[0403] For Tetruetreptia pomquetensis CCMP1491, 0.95 .mu.g of total RNA in 1 .mu.L was used as template. The procedure used to synthesize cDNA was the same as above except that CDSIII/3' PCR primer (SEQ ID NO:30) was replaced with the Adaptor Primer from Invitrogen 3'-RACE kit (SEQ ID NO:31).

Identification of cDNA Fragments Encoding Partial Putative Delta-8 Desaturases

[0404] Each of the above three cDNA samples were used as template for degenerate PCR using primers based on the amino acid sequence of the Euglena gracilis delta-8 fatty acid desaturase (EgD8; SEQ ID NO:112). The 9 forward and 2 reverse primers used are shown in Table 4:

TABLE-US-00004 TABLE 4 Degenerate Oligonucleotides Used to Amplify Portions of the Delta-8 Desaturase Genes From Eutreptiella sp. CCMP389, Eutreptiella cf_qymnastica CCMP1594 and Tetruetreptia pomquetensis CCMP1491 Pri- Amino Acid mer Nucleotide Sequence Sequence D8F1 GAYGCNACNGAYGCNTTCATG DATDAFM (SEQ ID NO: 1) (SEQ ID NO: 12) D8F2 GAYGCNACNGAYGCNGTTATG DATDAVM (SEQ ID NO: 2) (SEQ ID NO: 13) D8F3 GAYGCNACNGAYGCNGTGATG DATDAVM (SEQ ID NO: 3) (SEQ ID NO: 13) D8F4 GAYGCNACNGAYGCNTTTATG DATDAFM (SEQ ID NO: 4) (SEQ ID NO: 12) D8F5 GAYGCNACNGAYGCNGTAATG DATDAVM (SEQ ID NO: 5) (SEQ ID NO: 13) D8F6 GAYGCNACNGAYGCNGTGATG DATDAVM (SEQ ID NO: 6) (SEQ ID NO: 13) D8F7 TNGGNTGGTTRGGNGAYGA GWLGD(D/E) (SEQ ID NO: 7) (SEQ ID NO: 14) D8F8 TNGGNTGGCTRGGNGAYGA GWLGD(D/E) (SEQ ID NO: 8) (SEQ ID NO: 14) D8F9 TNGGNTGGCTYGGNGAYGA GWLGD(D/E) (SEQ ID NO: 9) (SEQ ID NO: 14) D8R1 TGRTGYTCDATYTGRTARTT NYQIEH (SEQ ID NO: 10) (SEQ ID NO: 15) D8R2 TGRTGYTCDATYTGCATRTT NYQIEH (SEQ ID NO: 11) (SEQ ID NO: 15)

[0405] A total of 18 reactions were set up for each cDNA sample, using all the possible combinations of the 9 forward and 2 reverse primers. The reaction mixture contained 1 .mu.L of cDNA, 1 .mu.L each of the forward and reverse primers (20 .mu.M), 22 .mu.L water and 25 .mu.L of TaKaRa ExTaq 2.times. premix (TaKaRa Bio, Mountain View, Calif.). PCR amplification was performed using the following conditions: 94.degree. C. for 1 min, 30 cycles of 94.degree. C. for 30 sec, 55.degree. C. for 30 sec, and 72.degree. C. for 1 min, followed by 7 min at 72.degree. C.

[0406] Agarose gel analysis of the PCR products showed that, with several primer combinations, a .about.1 kb fragment was amplified from each cDNA sample. The fragments from the primer combination D8F4/D8R1 were cloned into pCR2.1-TOPO (SEQ ID NO:40) and sequenced to afford partial sequences of the putative delta-8 desaturases from Tetruetreptia pomquetensis CCMP1491 (SEQ ID NO:16; 977 bp), Eutreptiella sp. CCMP389 (SEQ ID NO:17; 968 bp) and Eutreptiella cf.sub.--gymnastica CCMP1594 (SEQ ID NO:18; 968 bp).

Example 2

Isolation of the Full-Length Delta-8 Desaturases from Eutreptiella sp. CCMP389 and Eutreptiella cf.sub.--gymnastica CCMP1594

[0407] Primers were designed (see Table 5), based on the partial sequences of the putative delta-8 desaturases from Eutreptiella sp. CCMP389 (SEQ ID NO:17) and Eutreptiella cf.sub.--gymnastica CCMP1594 (SEQ ID NO:18), to isolate the 5' and 3' ends of each gene from cDNA and genomic DNA samples.

TABLE-US-00005 TABLE 5 Primers Used to Clone the Full-Length Delta-8 Desaturas Genes From Eutreptiella sp. CCMP389 and Eutreptiella cf_gymnastica CCMP1594 SEQ Primer ID Organism Name Primer Sequence NO: Eutreptiella 389D8-3-1 CAACGCCAGTACGCAAAGGAG 20 sp. CCMP389 389D8-3-2 CTCTGCATTGGATTCTGAAAGG 21 389D8-5-1 AATCATGTCCTTTCGAAGCTTG 22 389D8-5-2 GTCCTCAGCAACCTCGTCGTTG 23 389D8-5-3 CTTGGGGCTTCGTGGCGAAGTG 24 Eutreptiella 1594D8-3-1 GAGCGTTTTCTTGTTCTGTTAC 32 cf_gymnastica 1594D8-3-2 CGTTTTTCCTTATCTCGGAGTG 33 CCMP1594 1594D8-5-1 GATTTGTACACATAAAACAGAG 34 1594D8-5-2 ACCCTTCTCAACCATACTGTTG 35 1594D8-5-3 CTTGGGAGTAAGTGGTGAAGAG 36

Isolation of the 5'-End Sequences of the Eutreptiella sp. CCMP389 and Eutreptiella cf.sub.--gymnastica CCMP1594 Delta-8 Desaturase Genes

[0408] The full 5'-end sequences of the putative delta-8 desaturases from Eutreptiella sp. CCMP389 and Eutreptiella cf.sub.--gymnastica CCMP1594 were obtained by genome walking using the Universal GenomeWalker.TM. kit (BD Biosciences Clonetech, Palo Alto, Calif.) following the manufacturer's protocol (Prot #PT3042-1). First, genomic DNA from Eutreptiella sp. CCMP389 and Eutreptiella cf.sub.--gymnastica CCMP1594 were digested with DraI, EcoRV, PvuII and StuI individually as described in the manufacturer's protocol. Genomic DNA (2 .mu.g) was used for each digestion. Digested DNA samples were purified with a Qiagen enzyme reaction clean-up kit according to the manufacturer's protocol. Each sample was eluted with 20 .mu.L of water.

[0409] The digested genomic DNA samples were ligated with the GenomeWalker adaptor (SEQ ID NO:37 and SEQ ID NO:111). Specifically, 4 .mu.L each of the digested DNA was mixed with 1.9 .mu.L of 25 .mu.M GenomeWalker adaptor (SEQ ID NO:37 and SEQ ID NO:111), 1.6 .mu.L of 10.times. ligation buffer and 0.5 .mu.L of T4 DNA ligase. The reaction was carried out overnight at 16.degree. C. After heating at 70.degree. C. for 5 min, 72 .mu.L of 10 mM Tris, 1 mM EDTA, pH 7.4 buffer was added to each reaction mixture. These reaction mixtures were then used as templates for PCR amplification.

[0410] For the first round of PCR, primers 389D8-5-1 (SEQ ID NO:22) and Universal GenomeWalker.TM. primer AP1 (SEQ ID NO:38) from the kit were used to amplify from Eutreptiella sp. CCMP389 samples, while primers 1594D8-5-1 (SEQ ID NO:34) and AP1 (SEQ ID NO:38) were used for Eutreptiella cf.sub.--gymnastica CCMP1594 samples. Each reaction mixture contained 1 .mu.L of each primer at 10 .mu.M, 2 .mu.L of the purified ligation products as template, 21 .mu.L water and 25 .mu.L of TaKaRa ExTaq 2.times. premix. The PCR reaction conditions used were as follows: 94.degree. C. for 30 sec, 30 cycles of 94.degree. C. for 20 sec, 55.degree. C. for 20 sec, and 72.degree. C. for 2 min, followed by 5 min at 72.degree. C.

[0411] The PCR products were diluted 1:100, and 1 .mu.L of each diluted PCR product was used as template for a second round of PCR using primers 389D8-5-3 (SEQ ID NO:24) and Universal GenomeWalker.TM. primer AP2 (SEQ ID NO:39) for Eutreptiella sp. CCMP389 samples, and primers 1594D8-5-3 (SEQ ID NO:36) and Universal GenomeWalker.TM. primer AP2 (SEQ ID NO:39) for Eutreptiella cf.sub.--gymnastica CCMP1594 samples. Amplification was conducted as described above.

[0412] The second-round PCR products were purified by Qiagen PCR purification kit, cloned into pCR2.1-TOPO (SEQ ID NO:40) and sequenced. A 694 by PCR fragment generated from Eutreptiella cf.sub.--gymnastica CCMP1594 samples and a 648 by fragment generated from Eutreptiella sp. CCMP389 samples were shown to contain the 5' end of the putative delta-8 desaturase genes, including parts of the non-translated region (SEQ ID NO:41 and SEQ ID NO:42, respectively).

Isolation of the 3'-End Sequences of the Eutreptiella sp. CCMP389 and Eutreptiella cf.sub.--gymnastica CCMP1594 Delta-8 Desaturase Genes

[0413] The full 3'-end sequences of the putative delta-8 desaturases from Eutreptiella sp. CCMP389 and Eutreptiella cf.sub.--gymnastica CCMP1594 were obtained by PCR amplification using cDNA samples as templates.

[0414] 389D8-3-1 (SEQ ID NO:19) and CDSIII/3' PCR primer (SEQ ID NO:30; supplied with the Creator.TM. SMART.TM. cDNA Library Construction Kit of Example 1) were used as primers for first round amplification, using Eutreptiella sp. CCMP389 cDNA as template. 1594D8-3-1 (SEQ ID NO:32) and CDSIII/3' PCR primer (SEQ ID NO:30) were used as primers for amplification with Eutreptiella cf.sub.--gymnastica CCMP1594 cDNA as template. The reaction mixtures contained: 1 .mu.L of each primer (10 .mu.M), 1 .mu.L of cDNA from Example 1, 22 .mu.L water and 25 .mu.L TaKaRa ExTaq 2.times. premix. The PCR reaction conditions used were as follows: 94.degree. C. for 90 sec, 30 cycles of 94.degree. C. for 30 sec, 55.degree. C. for 30 sec, and 72.degree. C. for 30 sec, followed by 5 min at 72.degree. C.

[0415] The PCR product was diluted 1:50, and 1 .mu.L of the diluted product was used as template for a second round of PCR using either 389D8-3-2 (SEQ ID NO:21) or 1594D8-3-2 (SEQ ID NO:33) with the CDSIII/3' PCR primer (SEQ ID NO:30) under the conditions described above. The second-round PCR products were purified with Qiagen PCR purification kit, cloned into pCR2.1-TOPO (SEQ ID NO:40) and sequenced. A fragment amplified from Eutreptiella sp. CCMP389 cDNA was shown to contain the 3'-end of the cDNA of putative delta-8 desaturase, including the polyA tail (SEQ ID NO:43; 717 bp).

[0416] Two different fragments were obtained and shown to contain the 3' end of the delta-8 desaturase from Eutreptiella cf.sub.--gymnastica CCMP1594. One of them, 1594D8-3'A (SEQ ID NO:44), was 1164 by long and contained a long 3' untranslated region of 760 by and a polyA tail. The other, 1594D8-3'B (SEQ ID NO:45), was 435 by long and had a short 3' untranslated region of 30 bp. The sequences of the coding region of both fragments were the same.

Assembly of the Full-Length Sequences of the Eutreptiella sp. CCMP389 and Eutreptiella cf.sub.--gymnastica CCMP1594 Delta-8 Desaturase Genes

[0417] Assembly of the 5' genomic region, the original partial cDNA sequence and the 3'-cDNA sequence resulted in the complete sequence of the delta-8 desaturases from Eutreptiella sp. CCMP389 and Eutreptiella cf.sub.--gymnastica CCMP1594 (SEQ ID NO:46 (1963 bp) and SEQ ID NO:48 (2063 bp), respectively; each sequence also contained untranslated 5' and 3' ends). Each coding region is 1254 by long and each encodes a peptide of 417 amino acids (SEQ ID NO:47 and SEQ ID NO:49, respectively). SEQ ID NO:92 is the nucleotide sequence of the coding sequence of Eutreptiella sp. CCMP389 delta-8 desaturase (designated herein as "E389D8"), while SEQ ID NO:93 is the nucleotide sequence of the coding sequence of Eutreptiella cf.sub.--gymnastica CCMP1594 delta-8 desaturase (designated herein as "E1594D8").

Example 3

Isolation of the Full-Length Delta-8 Desaturase from Tetruetreptia pomquetensis CCMP1491

[0418] Primers were designed (see Table 6), based on the partial sequence of the putative delta-8 desaturase from Tetruetreptia pomquetensis CCMP1491 (SEQ ID NO:16), to isolate the 5' and 3' end of the gene from cDNA and genomic DNA samples.

TABLE-US-00006 TABLE 6 Primers Used to Clone the Full-Length Delta-8 Desaturase Gene From Tetruetreptia pomquetensis CCMP1491 Primer Name Primer Sequence SEQ ID NO: ED8-5-1 CTCGAACATACCCTTGGAGATG 25 ED8-5-2 CCCGCAACTTGCGGAAATCCTC 26 ED8-5-3 GGGCTCATCACGCTTAGGCTTG 27 ED8-3-1 CACTTTCTATTGCAGTGCCATG 28 ED8-3-2 CTTTGCCACCGGTTTGGGATGC 29

Isolation of the 5'-End Sequence of the Tetruetreptia pomquetensis CCMP1491 Delta-8 Desaturase Gene

[0419] The Invitrogen TOPO walker kit was used for isolating the 5' end of the putative delta-8 desaturase gene from Tetruetreptia pomquetensis CCMP1491, following the manufacturer's protocol. Genomic DNA (0.3 .mu.g) from Tetruetreptia pomquetensis CCMP1491 (see Example 1) was digested with ApaI. The reaction mixture contained 10 .mu.L genomic DNA (.about.0.3 .mu.g), 4 .mu.L of 10.times. restriction buffer, 2 .mu.L restriction enzyme (ApaI or KpnI) and 24 .mu.L water. The reaction was carried out at 37.degree. C. for 2 h. Then, 50 .mu.L of water, 6 .mu.L of dephosphorylation buffer and 4 .mu.L of kit-supplied CIP were added to the mixture, and the reaction was allowed to continue for 1 h at 37.degree. C. The reaction mixture was then purified with Qiagen reaction purification kit according to the manufacturer's protocol. DNA was eluted in 40 .mu.L of water.

[0420] For primer extension, 15 .mu.L of the purified DNA was mixed with 2 .mu.L of 10.times. PCR buffer (Invitrogen Corporation), 1 .mu.L of 2.5 mM each dNTPs, 1 .mu.L of primer ED8-5-1 (SEQ ID NO:25) (20 .mu.M) and 1 .mu.L of Advantage 2 cDNA polymerase mix (BD Biosciences Clonetech, Palo Alto, Calif.). The PCR reaction conditions used were as follows: 94.degree. C. for 4 min, 56.degree. C. for 1 min, and 72.degree. C. for 20 min. The primer extension reaction product (8 .mu.L) was then used as substrate for TOPO linker in a mixture additionally comprising 1 .mu.L TOPO linker (SEQ ID NO:50) and 1 .mu.L 10.times.PCR buffer (Invitrogen Corporation). The mixture was incubated at 37.degree. C. for 10 min and used directly as PCR template.

[0421] PCR amplification of the 5' end was carried out in a 50 .mu.L reaction mix that contained 2 .mu.L of TOPO linked genomic DNA, 1 .mu.L of primer ED8-5-2 (SEQ ID NO:26) (10 .mu.M), 1 .mu.L of LinkAmp primer 1 (SEQ ID NO:51) (10 .mu.M), 21 .mu.L water and 25 .mu.L TaKaRa ExTaq 2.times. premix. The PCR reaction conditions used were as follows: 94.degree. C. for 90 sec, 30 cycles of 94.degree. C. for 30 sec, 55.degree. C. for 30 sec, and 72.degree. C. for 2 min, followed by 7 min at 72.degree. C. The PCR product was diluted 1:50, and 1 .mu.L of the diluted product was used as template for a second round of PCR under the same conditions, except that primers ED8-5-3 (SEQ ID NO:27) and LinkAmp primer 2 (SEQ ID NO:52) replaced ED8-5-2 (SEQ ID NO:26) and LinkAmp primer 1 (SEQ ID NO:51).

[0422] A .about.600 by PCR product was purified with a Qiagen PCR purification kit, cloned into pCR2.1-TOPO (SEQ ID NO:40) and sequenced. Comparison of the partial delta-8 desaturase sequence of SEQ ID NO:16 with the 5' extension product (SEQ ID NO:53; 601 bp) showed that SEQ ID NO:53 extended upstream of the `ATG` initiation codon of the delta-8 desaturase.

Isolation of the 3'-End Sequence of the Tetruetreptia pomquetensis CCMP1491 Delta-8 Desaturase Gene

[0423] The full 3'-end sequence of the putative delta-8 desaturase from Tetruetreptia pomquetensis CCMP1491 was obtained by PCR amplification using a cDNA sample as template. Specifically, primers ED8-3-1 (SEQ ID NO:28) and AUAP (SEQ ID NO:54; supplied in Invitrogen's 3'-RACE kit) were used as primers. The reaction mixture contained 1 .mu.L of each primer (10 .mu.M), 1 .mu.L of Tetruetreptia pomquetensis CCMP1491 cDNA from Example 1, 22 .mu.L water and 25 .mu.L TaKaRa ExTaq 2.times. premix. The PCR reaction conditions used were as follows: 94.degree. C. for 90 sec, 30 cycles of 94.degree. C. for 30 sec, 55.degree. C. for 30 sec, and 72.degree. C. for 30 sec, followed by 5 min at 72.degree. C.

[0424] The PCR product was diluted 1:50, and 1 .mu.L of the diluted product was used as template for a second round of PCR using ED8-3-2 (SEQ ID NO:29) and AUAP (SEQ ID NO:54) as primers under the same conditions as described above. The second round PCR generated a .about.1 kb fragment, which was purified with Qiagen PCR purification kit, cloned into pCR2.1-TOPO (SEQ ID NO:40) and sequenced. The result of sequence analysis showed that this fragment (SEQ ID NO:55; 1028 bp) contained the 3' end of the putative delta-8 desaturase, including the polyA tail.

Assembly of the Full-Length Sequence of the Tetruetreptia pomquetensis CCMP1491 Delta-8 Desaturase Gene

[0425] Assembly of the 5' genomic region, the original partial cDNA fragment and 3'-cDNA fragment resulted in the complete sequence of the delta-8 desaturase from Tetruetreptia pomquetensis CCMP1491, plus 358 by of 5' untranslated region and 612 by of 3' untranslated region (SEQ ID NO:56; 2233 bp). The coding region is 1263 by long and encodes a protein of 420 amino acids (SEQ ID NO:57). SEQ ID NO:62 is the nucleotide sequence of the coding sequence of Tetruetreptia pomquetensis CCMP1491 delta-8 desaturase (designated herein as "TpomD8").

Example 4

Comparison of the Delta-8 Desaturase Sequences of Tetruetreptia pomquetensis CCMP1491, Eutreptiella sp. CCMP389 and Eutreptiella cf.sub.--gymnastica CCMP1594 to a Delta-8 Desaturase Sequence of Euglena gracilis

[0426] The delta-8 desaturase sequences of Tetruetreptia pomquetensis CCMP1491 (i.e., TpomD8), Eutreptiella sp. CCMP389 (i.e., E389D8) and Eutreptiella cf.sub.--gymnastica CCMP1594 (i.e., E1594D8) were analyzed for similarity to all publicly available protein sequences contained in the "nr" database provided by the NCBI. 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. TpomD8, E389D8 and E1594D8 each shared the greatest identity and similarity with the delta-8 desaturase of Euglena gracilis set forth as SEQ ID NO:98 (corresponding to NCBI Accession No. AAD45877 (GI 5639724)).

[0427] The delta-8 desaturase sequences of Tetruetreptia pomquetensis CCMP1491 (i.e., TpomD8), Eutreptiella sp. CCMP389 (i.e., E389D8) and Eutreptiella cf.sub.--gymnastica CCMP1594 (i.e., E1594D8) were also analyzed for similarity to the Euglena gracilis delta-8 desaturase (SEQ ID NO:112 of the instant application) in 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)).

[0428] FIGS. 7A and 7B show a Clustal V alignment of the delta-8 desaturases from Tetruetreptia pomquetensis CCMP1491 (SEQ ID NO:57), Eutreptiella sp. CCMP389 (SEQ ID NO:47), Eutreptiella cf.sub.--gymnastica CCMP1594 (SEQ ID NO:49), Euglena gracilis (SEQ ID NO:98; NCBI Accession No. AAD45877 (GI 5639724)) and Euglena gracilis (SEQ ID NO:112). SEQ ID NO:57 has 70.5%, 71.7%, 57.5% and 61.8% identity to SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:98 and SEQ ID NO:112, respectively. SEQ ID NO:47 has 83.0%, 58.3% and 63% identity to SEQ ID NO:49, SEQ ID NO:98 and SEQ ID NO:112, respectively. SEQ ID NO:49 has 58.0% and 62.7% identity to SEQ ID NO:98 and SEQ ID NO:112, respectively. SEQ ID NO:98 has 95% identity to SEQ ID NO:112.

[0429] More specifically, TpomD8, E389D8 and E1594D8 were evaluated by BLASTP, yielding a pLog value versus EgD8 (SEQ ID NO:112). Then, the % identity of TpomD8, E389D8 and E1594D8 was determined with respect to EgD8 using the Jotun Hein method. Sequence percent identity calculations performed by the Jotun Hein method (Hein, J. J., Meth. Enz. 183:626-645 (1990)) were done using the MegAlign.TM. v6.1 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.) with the default parameters for pairwise alignment (KTUPLE=2). As discussed above, the % identity of TpomD8, E389D8 and E1594D8 was determined with respect to EgD8 using the Clustal V method. Sequence percent identity calculations performed by the Clustal V method (Higgins, D. G. and Sharp, P. M., Comput. Appl. Biosci. 5:151-153 (1989); Higgins et al., Comput. Appl. Biosci. 8:189-191 (1992)) were done using the MegAlign.TM. v6.1 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., 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). These results are summarized in Table 7.

TABLE-US-00007 TABLE 7 Sequence Comparison of TpomD8, E389D8 and E1594D8 to EgD8 (SEQ ID NO: 112) % Identity % Identity to EgD8 to EgD8 by the by the pLog value versus Jotun Hein Clustal V Desaturase EgD8 by BLASTP Method Method TpomD8 155 63.5% 61.8% (SEQ ID NO: 57) (E value of 1e-155) E389D8 164 63.3% 63.0% (SEQ ID NO: 47) (E value of 1e-164) E1594D8 163 64.2% 62.7% (SEQ ID NO: 49) (E value of 1e-163) BLAST scores and probabilities indicate that the nucleic acid fragments set forth in SEQ ID NO: 57, SEQ ID NO: 47 and SEQ ID NO: 49 each encode an entire delta-8 desaturase.

Example 5

Functional Analysis of the Tetruetreptia pomquetensis CCMP1491 Delta-8 Desaturase (TpomD8) in Saccharomyces cerevisiae

[0430] The present Example describes functional analysis of TpomD8 in Saccharomyces cerevisiae. This work included the following steps: (1) cloning of TpomD8 from a Tetruetreptia pomquetensis CCMP1491 cDNA library; (2) cloning of TpomD8 into yeast expression vector pY-75 to produce pY126; and, (3) comparison of lipid profiles within transformant organisms comprising pY-75 and pY126, after substrate feeding.

Cloning TpomD8 from a cDNA Library

[0431] Tetruetreptia pomquetensis CCMP1491 cDNA (1 .mu.L; synthesized as described in Example 1) was combined with 50 pmol of TpomNot-5 (SEQ ID NO:58), 50 pmol of TpomNot-3 (SEQ ID NO:59), 1 .mu.L of PCR nucleotide mix (10 mM, Promega, Madison, Wis.), 5 .mu.L of 10.times.PCR buffer (Invitrogen Corporation), 1.5 .mu.L of MgCl.sub.2 (50 mM, Invitrogen Corporation), 0.5 .mu.L of Taq polymerase (Invitrogen Corporation) and water to 50 .mu.L. The reaction conditions were 94.degree. C. for 3 min followed by 35 cycles of 94.degree. C. for 45 sec, 55.degree. C. for 45 sec and 72.degree. C. for 1 min. The PCR was finished at 72.degree. C. for 7 min and then held at 4.degree. C. The PCR reaction was analyzed by agarose gel electrophoresis on 5 .mu.L and a DNA band with molecular weight around 1.3 kb was observed.

[0432] The remaining 45 .mu.L of product was separated by agarose gel electrophoresis and the DNA purified using the Zymoclean.TM. Gel DNA Recovery Kit (Zymo Research, Orange, Calif.) following the manufacturer's protocol. The resulting DNA was cloned into the pGEM.RTM.-T Easy Vector (Promega) following the manufacturer's protocol. Multiple clones were sequenced using the T7 (SEQ ID NO:60) and M13-28Rev (SEQ ID NO:61) oligonucleotides to verify that the TpomD8 sequence was identical to the previously deduced coding sequence of Example 3 (i.e., SEQ ID NOs:62 and 57). Clone pLF114-10 (SEQ ID NO:63) was chosen for further expression studies.

Construction of Plasmids pY-75 (Control) and pY126, Comprising TpomD8

[0433] The yeast episomal plasmid (YEp)-type vector pRS425 (Christianson et al., Gene 110:119-122 (1992)) contains sequences from the Saccharomyces cerevisiae 2.mu. endogenous plasmid, a LEU2 selectable marker and sequences based on the backbone of a multifunctional phagemid, pBluescript II SK(+). The Saccharomyces cerevisiae strong, constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter was cloned between the SacII and Spel sites of pRS425 in the same way as described by Jia et al. (Physiol. Genom. 3:83-92 (2000)) to produce pGPD-425. A NotI site was introduced into the BamHI site of pGPD-425, thus giving a NotI site flanked by BamHI sites, and this plasmid was called pY-75 (SEQ ID NO:64), which was previously described in PCT Publication No. WO 2006/012325 (published Feb. 2, 2006; the contents of which are hereby incorporated by reference).

[0434] TpomD8 was released from pLF114-10 (supra) by digestion with NotI and cloned into the NotI site of pY75 to produce pY126 (SEQ ID NO:65; FIG. 1).

Functional Analysis of TpomD8

[0435] Expression plasmids pY75 (control) and pY126 were transformed into Saccharomyces cerevisiae INVSC1 (Invitrogen Corporation) using standard lithium acetate transformation procedures. Transformants were selected on DOBA media supplemented with CSM-leu (Qbiogene, Carlsbad, Calif.). Transformants were evaluated for delta-8 desaturase activities in the following way. Transformants from each plate were inoculated into 2 mL of DOB medium supplemented with CSM-leu (Qbiogene) and 0.2% tergitol. Cells were grown for 1 day at 30.degree. C. after which 0.1 mL was transferred to 3 mL of the same medium supplemented with either EDA [20:2(11,14)] or ETrA [20:3(11,14,17)] to 0.175 mM. These cells 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 1 M sodium chloride and 100 .mu.L of heptane were added. After thorough mixing and centrifugation, fatty acid methyl esters (FAMEs) were analyzed by GC as described in Example 10.

[0436] Results for 3 individual clones of pY126 (i.e., clones 6, 7 and 10) as well as the vector control are shown in Table 8. Fatty acid compositions are expressed as a weight percent of total fatty acids. The activity of the delta-8 desaturase is expressed as "percent desaturation", where % Desat. was calculated according to the following formula: ([product]/[substrate+product])*100.

TABLE-US-00008 TABLE 8 Comparison of Lipid Profiles of Yeast Expressing TpomD8 Fatty % Vector Acid 16:0 16:1 18:0 18:1 EDA DGLA ETrA ETA Desat. pY75 EDA 13.3 37.4 4.0 34.2 11.1 0.0 0.0 0.0 0.0 pY126-6 EDA 14.4 38.6 4.0 32.9 10.0 0.2 0.0 0.0 1.5 pY126-7 EDA 13.6 36.3 4.4 34.3 11.2 0.2 0.0 0.0 1.9 pY126-10 EDA 11.7 37.9 3.9 34.5 11.5 0.4 0.1 0.0 3.5 pY75 ETrA 11.8 33.5 3.1 24.3 0.1 0.0 27.2 0.0 0.1 pY126-6 ETrA 13.4 35.3 3.4 25.3 0.1 0.0 22.3 0.2 1.0 pY126-7 ETrA 12.2 32.8 3.4 24.8 0.1 0.0 26.2 0.4 1.6 pY126-10 ETrA 11.1 29.5 3.4 25.0 0.1 0.0 30.0 0.9 2.9 When feeding the cells EDA, the product of the TpomD8 delta-8 desaturation is DGLA; in constrast, substrate feeding with ETrA results in production of ETA by TpomD8 desaturation.

Example 6

Generation of Yarrowia lipolytica Strain Y4001 to Produce about 17% EDA of Total Lipids

[0437] The present Example describes the construction of strain Y4001, derived from Yarrowia lipolytica ATCC #20362, capable of producing 17% EDA (C20:2) relative to the total lipids. The strain was engineered to test functional expression of TpomD8, E389D8 and E1594D8; specifically, it was necessary to construct a host strain capable of producing the delta-8 desaturase substrate, EDA.

[0438] The development of strain Y4001 required the construction of strain Y2224 (a FOA resistant mutant from an autonomous mutation of the Ura3 gene of wildtype Yarrowia strain ATCC #20362).

Generation of Strain Y2224

[0439] 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.

Generation of Strain Y4001 to Produce 17% EDA of Total Lipids

[0440] Strain Y4001 was created via integration of construct pZKLeuN-29E3 (FIG. 9; comprising four chimeric genes--a delta-12 desaturase, a C.sub.16/18 elongase and two delta-9 elongases) into the Leu2 loci of Y2224 strain to thereby enable production of EDA.

[0441] Construct pZKLeuN-29E3 (FIG. 9) contained the components shown in Table 9.

TABLE-US-00009 TABLE 9 Description of Plasmid pZKLeuN-29E3 (SEQ ID NO: 106) RE Sites And Nucleotides Within SEQ ID Description Of Fragment And NO: 106 Chimeric Gene Components BsiW I/Asc I 795 by 3' part of Yarrowia Leu2 gene (GenBank (7797-7002) Accession No. AF260230) Sph I/Pac I 703 by 5' part of Yarrowia Leu2 gene (GenBank (4302-3591) Accession No. AF260230) Swa I/BsiW I GPD::F.D12::Pex20, comprising: (10500-7797) GPD: Yarrowia lipolytica GPD promoter (WO 2005/003310) F.D12: Fusarium moniliforme delta-12 desaturase gene (WO 2005/047485) Pex20: Pex20 terminator sequence from Yarrowia Pex20 gene (GenBank Accession No. AF054613) Bgl II/Swa I Exp pro::EgD9E::Lip1, comprising: (12526-10500) Exp pro: Yarrowia lipolytica export protein (EXP1) promoter (WO 2006/052870 and U.S. Patent Application No. 11/265,761) EgD9E: (same as EgD9S, see infra): codon-optimized delta-9 elongase gene (SEQ ID NO: 107), derived from Euglena gracilis (SEQ ID NOs: 74 and 75 (see also U.S. Provisional Application No. 60/739,989) Lip1: Lip1 terminator sequence from Yarrowia Lip1 gene (GenBank Accession No. Z50020) Pme I/Cla I FBAINm::EgD9S::Lip2, comprising: (12544-1) FBAINm: Yarrowia lipolytica FBAINm promoter (WO 2005/049805) EgD9S: codon-optimized delta-9 elongase gene (SEQ ID NO: 107), derived from Euglena gracilis (SEQ ID NOs: 74 and 75 (see also U.S. Provisional Application No. 60/739,989) Lip2: Lip2 terminator sequence from Yarrowia Lip2 gene (GenBank Accession No. AJ012632) Cla I/EcoR I LoxP::Ura3::LoxP, comprising: (1-1736) LoxP sequence (SEQ ID NO: 108) Yarrowia Ura3 gene (GenBank Accession No. AJ306421) LoxP sequence (SEQ ID NO: 108) EcoR I/Pac I NT::ME3S::Pex16, comprising: (1736-3591) NT: Yarrowia lipolytica YAT1 promoter (Patent Publication No. U.S. 2006/0094102-A1) ME3S: codon-optimized C.sub.16/18 elongase gene (SEQ ID NO: 109), derived from M. alpina (see U.S. Patent Application No. 11/253,882 and also WO 2006/052870) Pex16: Pex16 terminator sequence of Yarrowia Pex 16 gene (GenBank Accession No. U75433)

[0442] Plasmid pZKLeuN-29E3 was digested with Asc I/Sph I, and then used for transformation of Y. lipolytica strain Y2224 (i.e., ATCC #20362 Ura3-) according to the General Methods. The transformant cells were plated onto MMLeu media plates and maintained at 3.degree. C. for 2 to 3 days. The colonies were picked and streaked onto MM and MMLeu selection plates. The colonies that could grow on MMLeu plates but not on MM plates were selected as Leu-strains. Single colonies of Leu-strains were then inoculated into liquid MMLeu at 30.degree. C. and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.

[0443] GC analyses showed the presence of EDA in the transformants containing the 4 chimeric genes of pZKLeuN-29E3, but not in the Yarrowia Y2224 control strain. Most of the selected 36 Leu-strains produced about 12 to 16.9% EDA of total lipids. There were 3 strains (i.e., strains #11, #30 and #34) that produced about 17.4%, 17% and 17.5% EDA of total lipids; they were designated as strains Y4001, Y4002 and Y4003, respectively.

Example 7

Functional Analysis of the Tetruetreptia pomquetensis CCMP1491 (TpomD8), Eutreptiella sp. CCMP389 (E389D8) and Eutreptiella cf.sub.--gymnastica CCMP1594 (E1594D8) Delta-8 Desaturases in Yarrowia lipolytica Strain Y4001

[0444] The present Example describes functional analysis of TpomD8, E389D8 and E1594D8 in Yarrowia lipolytica strain Y4001. This work included the following steps: (1) cloning of E389D8 from a Eutreptiella sp. CCMP389 cDNA library, E1594D8 from a Eutreptiella cf.sub.--gymnastica CCMP1594 cDNA library, and TpomD8 from a Tetruetreptia pomquetensis CCMP1491 cDNA library; (2) cloning of E389D8, E1594D8 and TpomD8 into yeast expression vector pFBAIn-MOD1 (SEQ ID NO:94); and, (3) comparison of lipid profiles within transformant organisms of Yarrowia lipolytica strain Y4001 that were additionally comprising each desaturase.

Cloning E389D8, E1594D8 and TpomD8 from cDNA Libraries

[0445] The Phusion polymerase from New England Biolab was used for amplification of E389D8 and E1594D8 cDNAs. Primers 389D8-F (SEQ ID NO:99) and 389D8-R (SEQ ID NO:100) were used for amplification of E389D8; in contrast, primers 1594D8-F (SEQ ID NO:103) and 1594D8-R (SEQ ID NO:104) were used for amplification of E1584D8. Each reaction mixture contained 1 .mu.L each of 20 .mu.M forward and reverse primers, 1 .mu.L cDNA, 10 .mu.L 5.times.PCR buffer, 1 .mu.L dNTP mix (10 mM each), 35 .mu.L water and 1 .mu.L Phusion polymerase. The PCR reaction conditions used were as follows: 98.degree. C. for 1 min, 30 cycles of 98.degree. C. for 10 sec, 55.degree. C. for 10 sec, and 72.degree. C. for 40 sec, followed by 5 min at 72.degree. C. The PCR product was digested with NcoI and NotI, and cloned into pFBAIn-MOD1 (SEQ ID NO:94) predigested with the same enzymes. The resulting plasmids were named pFBAIn-389D8 (SEQ ID NO:95) and pFBAIn-1594D8 (SEQ ID NO:96).

[0446] For amplification of TpomD8, the TaKaRa ExTaq 2.times. premix was used for PCR instead of the Phusion polymerase. The reaction mixture contained 1 .mu.L of Tetruetreptia pomquetensis CCMP1491 cDNA, 1 .mu.L each of 20 .mu.M primers 1491 D8-F (SEQ ID NO:101) and 1491D8-R (SEQ ID NO:102), 22 .mu.L water and 25 .mu.L ExTaq premix. The PCR reaction conditions used were as follows: 94.degree. C. for 30 sec, 30 cycles of 94.degree. C. for 20 sec, 55.degree. C. for 20 sec, and 72.degree. C. for 1 min 30 sec., followed by 7 min at 72.degree. C. The PCR products were cloned into pCR2.1-TOPO (SEQ ID NO:40) and sequenced. One clone with the correct sequence was digested with NcoI and NotI, and the 1.3 kb fragment containing TpomD8 was excised from agarose gel and purified with Qiagen gel purification kit. The purified fragment was then cloned into pFBAIn-MOD1 (SEQ ID NO:94; see FIG. 8) pre-digested with NcoI and NotI. The resulting plasmid was named pFBAIn-1491D8 (SEQ ID NO:97). Construct pFBAIn-MOD1 (SEQ ID NO:94; FIG. 8) contained the components shown in Table 10.

TABLE-US-00010 TABLE 10 Components of Plasmid pFBAIN-MOD1 (SEQ ID NO: 94) RE Sites and Nucleotides Within SEQ ID NO: 94 Description of Fragment and Chimeric Gene Components BglII-BsiWI FBAIN promoter:: PEX20 terminator region, comprising: (6278-539) FBAIN: Yarrowia lipolytica FBAIN promoter (WO 2005/049805) Stuffer DNA fragment derived from pDNR-LIB _PEX20_terminator sequence of Yarrowia PEX20 gene (GenBank Accession No. AF054613.) PacI-BglII Y. lipolytica URA3 (GenBank Accession No. AJ306421) (4768-6278) (3361-4725) ARS18, (GenBank Accession No. A17608) (2702-3102) f1 origin (1662-2522) AmpR gene (for selection in media containing ampicilin) (712-1592) ColE1 E. coli origin of replication

[0447] Functional Analysis of TpomD8, E389D8 and E1594D8 Plasmids pFBAIn-389D8 (SEQ ID NO:95), pFBAIn-1491D8 (SEQ ID NO:97), and pFBAIn-1594D8 (SEQ ID NO:96) were transformed into Yarrowia lipolytica strain Y4001 according to the General Methods.

[0448] The cells were plated onto MM plates (lacking uracil) and maintained at 30.degree. C. for 2 to 3 days. Single colonies of transformants were then patched onto fresh MM plates (lacking uracil) and allowed to grow at 30.degree. C. for 1 day. After this step, cells were scraped off the patches and transferred into 1.5 mL microfuge tubes. They were transesterified as described in the General Methods. FAMEs from cells containing each plasmid were analyzed by GC.

[0449] Lipid profiles of the transformant cells are shown below in Table 11. Fatty acids are identified as 16:0 (palmitic acid), 16:1 (palmitoleic acid), 18:0 (stearic acid), 18:1 (oleic acid), 18:2 (linoleic acid), 20:1 (eicosenoic acid), 20:2 (eicosadiencoic acid) and DGLA (20:3; dihomo-.gamma.-linolenic acid); and the composition of each is presented as a % of the total fatty acids.

[0450] The conversion efficiency was measured according to the following formula: ([product]/[substrate+product])*100, where `product` includes the immediate product and all products in the pathway derived from it. As shown in Table 11, the results demonstrated that each delta-8 desaturase was able to convert EDA (20:2) to DGLA (20:3); this confirmed that TpomD8, E389D8 and E1594D8 indeed were delta-8 desaturases. The substrate conversion efficiency for E389D8 and E1594D8 was about 6%, and for that of TpomD8 was 2.89%. Although not included within the data herein, expression of pFBAIN-MOD (control) in strain Y4001 under comparable conditions resulted in c.a. 0% C20:2 (on average), wherein the conversion efficiency was c.a. 0% Son average).

TABLE-US-00011 TABLE 11 Comparison of Lipid Profiles of Yarrowia lipolytica Expressing TpomD8, E389D8 and E1594D8 Plasmid Conv. (Desaturase) C16:0 C16:1 C18:0 C18:1 C18:2 C20:1 C20:2 DGLA efficiency % pFBAIn-389D8 11.71 7.74 2.06 13.89 40.93 0.58 14.34 0.89 5.84 (E389D8) pFBAIn-389D8 11.64 7.74 2.06 14.57 39.95 0.57 14.69 0.98 6.25 (E389D8) PFBAIn-1491D8 11.68 7.91 2.01 14.16 40.27 0.54 14.81 0.44 2.89 (TpomD8) PFBAIn-1594D8 12.03 7.71 2.3 15.18 38.95 0.57 14.97 0.9 5.67 (E1594D8)

Example 8

Construction of Soybean Expression Vector pKR1013 for Co-Expression of the Tetruetreptia pomquetensis CCMP1491 Delta-8 Desaturase (TpomD8) with a Delta-9 Elonqase Derived from Isochrysis Galbana (IqD9eS)

[0451] The present Example describes construction of a soybean vector for co-expression of TpomD8 with IgD9eS (a synthetic delta-9 elongase derived from Isochrysis galbana and codon-optimized for expression in Yarrowia lipolytica). As demonstrated in Examples 9 and 10 (infra), high concentrations of DGLA and/or ETA could readily be produced via expression of this vector in soybean.

[0452] Vector pKR123r (SEQ ID NO:66), which was previously described in PCT Publication No. WO 2004/071467 (published Aug. 26, 2004; the contents of which are hereby incorporated by reference), contains a NotI site flanked by the Kunitz soybean Trypsin Inhibitor (KTi3) 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 (KTi3/NotI/KTi3' cassette). TpomD8 (SEQ ID NO:57) was released from pLF114-10 (SEQ ID NO:63; Example 5) by digestion with NotI and cloned into the NotI site of pKR123r to produce pKR1007 (SEQ ID NO:67).

[0453] Plasmid pKR607 (SEQ ID NO:68), previously described in PCT Publication No. WO 2006/012325 (the contents of which are hereby incorporated by reference), contained a chimeric construct comprising the .alpha.' subunit of .beta.-conglycinin ("BCON Pro"; Beachy et al., EMBO J. 4:3047-3053 (1985)), IgD9eS (identified as "IG syel1" on FIG. 2 herein) and the 3' transcription termination region of the phaseolin gene (Doyle et al., J. Biol. Chem. 261:9228-9238 (1986)). The synthesis of IgDeS is similarly described in PCT Publication No. WO 2006/012325. Briefly, the codon usage of the delta-9 elongase gene of Isochrysis galbana was optimized for expression in Yarrowia lipolytica, in a manner similar to that described in PCT Publication No. WO 2004/101753. Thus, a codon-optimized delta-9 elongase gene (designated "IgD9eS", SEQ ID NO:110) was designed based on the coding sequence of IgD9e (SEQ ID NO:70) 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, 127 by of the 792 by coding region were modified (16.0%), and 122 codons were optimized. None of the modifications in the codon-optimized gene changed the amino acid sequence of the encoded protein (SEQ ID NO:76).

[0454] Plasmid pKR1007 (SEQ ID NO:67) was digested with PstI and the fragment containing TpomD8 was cloned into the SbfI site of plasmid pKR607 (SEQ ID NO:68) to produce pKR1013 (SEQ ID NO:69). In this way, TpomD8 is co-expressed with IgD9eS behind strong, seed-specific promoters. A schematic depiction of pKR1013 is shown in FIG. 2.

Example 9

Transformation of Somatic Soybean Embryo Cultures with Soybean Expression Vector pKR1013, for Co-Expression of TpomD8 And IgD9eS

Culture Conditions:

[0455] Soybean embryogenic suspension cultures (cv. Jack) were maintained in 35 mL liquid medium SB196 (described 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 were 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).

[0456] Soybean embryogenic suspension cultures were 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:

[0457] Soybean cultures were initiated twice each month with 5-7 days between each initiation. Pods with immature seeds from available soybean plants 45-55 days after planting were picked, removed from their shells and placed into a sterilized magenta box. The soybean seeds were 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 were rinsed using 2 1-liter bottles of sterile distilled water and those less than 4 mm were placed on individual microscope slides. The small end of the seed was cut and the cotyledons pressed out of the seed coat. Cotyledons were transferred to plates containing SB199 medium (25-30 cotyledons per plate) for 2 weeks, then transferred to SB1 for 2-4 weeks. Plates were wrapped with fiber tape. After this time secondary embryos were cut and placed into SB196 liquid media for 7 days.

Preparation of DNA for Bombardment:

[0458] Either an intact plasmid or a DNA plasmid fragment containing the genes of interest and the selectable marker gene were used for bombardment. Fragments from soybean expression plasmids pKR1013 (see Example 8) were obtained by gel isolation of digested plasmids. In each case, 100 .mu.g of plasmid DNA was used in 0.5 mL of the specific enzyme mix described below. Plasmids were 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 were separated by gel electrophoresis on 1% SeaPlaque GTG agarose (BioWhitaker Molecular Applications) and the DNA fragments containing gene cassettes were cut from the agarose gel. DNA was purified from the agarose using the GELase digesting enzyme following the manufacturer's protocol.

[0459] A 50 .mu.L aliquot of sterile distilled water containing 1 mg of gold particles was added to 5 .mu.L of a 1 .mu.g/.mu.L DNA solution (either intact plasmid or DNA fragment prepared as described above), 50 .mu.L 2.5M CaCl.sub.2 and 20 .mu.L of 0.1 M spermidine. The mixture was pulsed 5 times on level 4 of a vortex shaker and spun for 5 sec in a bench microfuge. After a wash with 150 .mu.L of 100% ethanol, the pellet was suspended by sonication in 85 .mu.L of 100% ethanol. Five .mu.L of DNA suspension was dispensed to each flying disk of the Biolistic PDS1000/HE instrument disk. Each 5 .mu.L aliquot contained approximately 0.058 mg gold particles per bombardment (i.e., per disk).

Tissue Preparation and Bombardment with DNA:

[0460] Approximately 100-150 mg of 7 day old embryonic suspension cultures were placed in an empty, sterile 60.times.15 mm petri dish and the dish was placed inside of an empty 150.times.25 mm Petri dish. Tissue was bombarded 1 shot per plate with membrane rupture pressure set at 650 PSI and the chamber was evacuated to a vacuum of 27-28 inches of mercury. Tissue was placed approximately 2.5 inches from the retaining /stopping screen.

Selection of Transformed Embryos:

[0461] Transformed embryos were selected using hygromycin as the selectable marker. Specifically, following bombardment, the tissue was placed into fresh SB196 media and cultured as described above. Six to eight days post-bombardment, the SB196 is exchanged with fresh SB196 containing 30 mg/L hygromycin. The selection media was refreshed weekly. Four to six weeks post-selection, green, transformed tissue was observed growing from untransformed, necrotic embryogenic clusters. Isolated, green tissue was removed and inoculated into multiwell plates to generate new, clonally propagated, transformed embryogenic suspension cultures.

Embryo Maturation:

[0462] Transformed embryogenic clusterswere cultured for one-three weeks at 26.degree. C. in SB196 under cool white fluorescent (Phillips cool white Econowatt F40/CW/RS/EW) and Agro (Phillips F40 Agro) bulbs (40 watt) on a 16:8 hr photoperiod with light intensity of 90-120 .mu.E/m.sup.2 s. After this time embryo clusters were removed to a solid agar media, SB166, for 1 week and then subcultured to medium SB103 for 3 weeks. Alternatively, embryo clusters were removed from SB196 media to 35 mL of SB228 (described infra) (SHaM liquid media; Schmidt et al., Cell Biology and Morphogenesis 24:393 (2005)) in a 250 mL Erlenmeyer flask for 2-3 weeks. Tissue cultured in SB228 was maintained 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. After maturation on plates in SB103 or in flasks on SB228 media, individual embryos were removed from the clusters, dried and screened for alterations in their fatty acid compositions as described supra. Media Recipes:

TABLE-US-00012 SB 196 - FN Lite Liquid Proliferation Medium (per liter) MS FeEDTA - 100x Stock 1 10 mL MS Sulfate - 100x Stock 2 10 mL FN Lite Halides - 100x Stock 3 10 mL FN Lite P, B, Mo - 100x Stock 4 10 mL B5 vitamins (1 mL/L) 1.0 mL 2,4-D (10 mg/L final concentration) 1.0 mL KNO.sub.3 2.83 gm (NH.sub.4).sub.2SO.sub.4 0.463 gm asparagine 1.0 gm sucrose (1%) 10 gm pH 5.8

TABLE-US-00013 FN Lite Stock Solutions 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)

[0463] 1 package MS salts (Gibco/BRL--Cat. No. 11117-066)

[0464] 1 mL B5 vitamins 1000.times. stock

[0465] 31.5 g glucose

[0466] 2 mL 2,4-D (20 mg/L final concentration)

[0467] pH 5.7

[0468] 8 g TC agar

SB199 Solid Medium (Per Liter)

[0469] 1 package MS salts (Gibco/BRL--Cat. No. 11117-066)

[0470] 1 mL B5 vitamins 1000.times. stock

[0471] 30 g Sucrose

[0472] 4 mL 2,4-D (40 mg/L final concentration)

[0473] pH 7.0

[0474] 2 g Gelrite

SB 166 Solid Medium (Per Liter)

[0475] 1 package MS salts (Gibco/BRL--Cat. No. 11117-066)

[0476] 1 mL B5 vitamins 1000.times. stock

[0477] 60 g maltose

[0478] 750 mg MgCl.sub.2 hexahydrate

[0479] 5 g activated charcoal

[0480] pH 5.7

[0481] 2 g gelrite

SB 103 Solid Medium (Per Liter)

[0482] 1 package MS salts (Gibco/BRL--Cat. No. 11117-066)

[0483] 1 mL B5 vitamins 1000.times. stock

[0484] 60 g maltose

[0485] 750 mg MgCl2 hexahydrate

[0486] pH 5.7

[0487] 2 g gelrite

SB 71-4 Solid Medium (Per Liter)

[0488] 1 bottle Gamborg's B5 salts w/ sucrose (Gibco/BRL--Cat. No. 21153-036)

[0489] pH 5.7

[0490] 5 g TC agar

2,4-D Stock

[0491] Obtain Premade from Phytotech Cat. No. D 295--Concentration 1 mg/mL

B5 Vitamins Stock (Per 100 mL)

[0492] Store aliquots at -20.degree. C.

[0493] 10 g myo-inositol

[0494] 100 mg nicotinic acid

[0495] 100 mg pyridoxine HCl

[0496] 1 g thiamine

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

TABLE-US-00014 SB 228 - Soybean Histodifferentiation & Maturation (SHaM) (per liter) 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

[0497] Adjust volume to 900 mL [0498] pH 5.8 [0499] Autoclave [0500] Add to cooled media (.ltoreq.30.degree. C.):

TABLE-US-00015 [0500] *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.

TABLE-US-00016 FN-lite Macro for SHAM 10X - Stock #1 (per liter) (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

[0501] Bring to volume [0502] Autoclave

TABLE-US-00017 [0502] MS Micro 1000X - Stock #2 (per 1 liter) 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

[0503] Bring to volume [0504] Autoclave

TABLE-US-00018 [0504] FeEDTA 100X - Stock #3 (per liter) 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.

[0505] Bring to Volume [0506] Solution is photosensitive. Bottle(s) should be wrapped in foil to omit light. [0507] Autoclave

TABLE-US-00019 [0507] Ca 100X - Stock #4 (per liter) CaCl.sub.2*2H.sub.20 (calcium chloride dihydrate) 44 g

[0508] Bring to Volume [0509] Autoclave

TABLE-US-00020 [0509] B5 Vitamin 1000X - Stock #5 (per liter) Thiamine*HCl 10 g Nicotinic Acid 1 g Pyridoxine*HCl 1 g Myo-Inositol 100 g

[0510] Bring to Volume [0511] Store frozen

TABLE-US-00021 [0511] 4% Glutamine - Stock #6 (per liter) 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.

[0512] Bring to Volume [0513] Filter Sterilize [0514] Store frozen*

[0515] *Note: Warm thawed stock in 31.degree. C. bath to fully dissolve crystals.

Example 10

Functional Analysis of the Tetruetreptia pomquetensis CCMP1491 Delta-8 Desaturase (TpomD8) and the Isochrysis galbana Delta-9 Elongase (IgD9eS) in Somatic Soybean Embryos Transformed with Soybean Expression Vector pKR1013

[0516] 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 ''-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.

Fatty Acid Analysis of Transgenic Somatic Soybean Embryos Expressing pKR1013

[0517] Individual single, matured, somatic soybean embryos that had been transformed with pKR1013 (as described in Example 9 transformants were matured on SHaM liquid media) were picked into glass GC vials, frozen at minus 80.degree. C., freeze dried overnight and fatty acid methyl esters were prepared by transesterification. For transesterification, 50 .mu.L of trimethylsulfonium hydroxide (TMSH) and 0.5 mL of hexane were added to the embryos in glass vials and incubated for 30 min at room temperature while shaking. Fatty acid methyl esters (5 .mu.L injected from hexane layer) were separated and quantified using a Hewlett-Packard 6890 Gas Chromatograph fitted with an Omegawax 320 fused silica capillary column (Catalog #24152, Supelco Inc.). The oven temperature was programmed to hold at 220.degree. C. for 2.6 min, increase to 240.degree. C. at 20.degree. C./min and then hold for an additional 2.4 min. Carrier gas was supplied by a Whatman hydrogen generator. Retention times were compared to those for methyl esters of standards commercially available (Nu-Chek Prep, Inc.). Routinely, 5-10 embryos per event were analyzed by GC, using the methodology described above.

[0518] Embryo fatty acid profiles for each event (6 embryos each) containing pKR1013 were obtained and the lipid profiles of somatic soybean embryos expressing TpomD8 and IgD9eS for the top 5 events are shown in FIG. 5. Fatty acids are identified as 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic acid), LA, GLA, ALA, EDA, DGLA, ERA and ETA; and, fatty acid compositions listed in FIG. 5 are expressed as a weight percent (wt. %) of total fatty acids. The activity of TpomD8 is expressed as percent desaturation (% desat), calculated according to the following formula: ([product]/[substrate+product])*100.

[0519] More specifically, the combined percent desaturation for EDA and ERA is shown as "C20% delta-8 desat", determined as: ([DGLA+ETA]/[DGLA+ETA+EDA+ERA])*100. This is also referred to as the overall % desaturation. The individual omega-6 delta-8 desaturation ("EDA % delta-8 desat.") was calculated as: ([DGLA]/[DGLA+EDA])*100. Similarly, the individual omega-3 delta-8 desaturation ("ERA % delta-8 desat.") was calculated as: ([ETA]/[ETA+ERA])*100. The ratio of delta-8 desaturation for omega-6 versus omega-3 substrates ("ratio [EDA/ERA]% desat.") was obtained by dividing the EDA % delta-8 desaturation by the ERA % delta-8 desaturation.

[0520] In summary of FIG. 5, TpomD8 worked in soybean to convert both EDA and ERA to DGLA and ETA, respectively. The line with the highest average DGLA content (i.e., 1974-5-6) had embryos with an average DGLA content of 12.9% and an average ETA content of 2.9%. The highest DGLA and ETA content for an individual embryo from this line was 14.6% and 3.4%, respectively. The highest average overall % desaturation was 50.7% with the highest overall % desaturation for an individual embryo being 55.5%. When broken down into % desaturation for the omega-6 and omega-3 substrates, the highest average % desaturation was 48.3% and 65.0% for EDA and ERA, respectively. The highest % desaturation for an individual embryo was 52.9% and 72.7% for EDA and ERA, respectively. In this example, TpomD8 had a preference for ERA over EDA, with the average desaturation ratio ranging from 0.6 to 0.8. No GLA was found to accumulate in the embryos.

Example 11

cDNA Synthesis and PCR of Euglena gracilis Delta-9 Elonqase

[0521] The present Example, disclosed in U.S. Provisional Application No. 60/739,989 (filed Nov. 23, 2005, having Attorney Docket No. BB-1562), describes the isolation of a delta-9 elongase from Euglena gracilis ("EgD9e"; SEQ ID NOs:74 and 75). The isolation of this gene allowed co-expression of EgD9e and the delta-8 desaturases of the present invention, to thereby permit expression of the delta-9 elongase/delta-8 desaturase pathway leading to accumulation of DGLA and/or ETA from LA and/or ALA, respectively.

Euglena gracilis Growth Conditions, Lipid Profile and mRNA Isolation

[0522] Euglena gracilis was obtained from Dr. Richard Triemer's lab at Michigan State University (East Lansing, Mich.). From 10 mL of actively growing culture, a 1 mL aliquot was transferred into 250 mL of Euglena gracilis (Eg) Medium in a 500 mL glass bottle. Eg medium was made by combining 1 g of sodium acetate, 1 g of beef extract (U126-01, Difco Laboratories, Detroit, Mich.), 2 g of Bacto.RTM. tryptone (0123-17-3, Difco Laboratories), 2 g of Bacto.RTM. yeast extract (0127-17-9, Difco Laboratories) in 970 mL of water. After filter sterilizing, 30 mL of soil-water supernatant (15-3790, Carolina Biological Supply Company, Burlington, N.C.) was aseptically added to give the final Eg medium. Euglena gracilis cultures were grown at 23.degree. C. with a 16 h light, 8 h dark cycle for 2 weeks with no agitation.

[0523] After 2 weeks, 10 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 100 .mu.L of trimethylsulfonium hydroxide (TMSH) and incubated at room temperature for 15 min with shaking. After this, 0.5 mL of hexane was added and the vials were incubated for 15 min at room temperature with shaking. Fatty acid methyl esters (5 .mu.L injected from hexane layer) were separated and quantified using a Hewlett-Packard 6890 Gas Chromatograph fitted with an Omegawax 320 fused silica capillary column (Supelco Inc., Catalog No. 24152). The oven temperature was programmed to hold at 220.degree. C. for 2.7 min, increase to 240.degree. C. at 20.degree. C./min and then hold for an additional 2.3 min. Carrier gas was supplied by a Whatman hydrogen generator. Retention times were compared to those for methyl esters of standards commercially available (Nu-Chek Prep, Inc. Catalog No. U-99-A).

[0524] The remaining 2 week culture (240 mL) was pelleted by centrifugation at 1,800.times.g for 10 min, washed once with water and re-centrifuged. Total RNA was extracted from the resulting pellet 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, 1 mg of total RNA (2 mg/mL) was obtained from the pellet. The mRNA was isolated from 1 mg of total RNA using the mRNA Purification Kit (Amersham Biosciences, Piscataway, N.J.) following the manufacturer's protocol provided. In this way, 85 .mu.g of mRNA was obtained.

Euglena gracilis cDNA Synthesis, Library Construction and Sequencing

[0525] A cDNA library was generated using the Cloneminer.TM. cDNA Library Construction Kit (Catalog 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 3.2 .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 7 and 8 (size ranging from .about.800-1500 bp) were concentrated, recombined into pDONR.TM.222 and transformed into E. coli ElectroMAX.TM. DH10B.TM. T1 Phage-Resistant cells (Invitrogen Corporation). The Euglena gracilis library was named eeg1c.

[0526] For sequencing, clones first were recovered from archived glycerol cultures grown/frozen in 384-well freezing media plates, and replicated with a sterile 384 pin replicator (Genetix, Boston, Mass.) in 384-well microtiter plates containing LB+75 .mu.g/mL Kanamycin (replicated plates). Plasmids then were isolated, using the Templiphi DNA sequencing template amplification kit method (Amersham Biosciences) following the manufacturer's protocol. Briefly, the Templiphi method uses bacteriophage .phi.29 DNA polymerase to amplify circular single-stranded or double-stranded DNA by isothermal rolling circle amplification (Dean et al., Genome Res., 11:1095-1099 (2001); Nelson et al., Biotechniques, 32:S44-S47 (2002)). After growing 20 h at 37.degree. C., cells from the replicated plate were added to 5 .mu.L of dilution buffer and denatured at 95.degree. C. for 3 min to partially lyse cells and release the denatured template. Templiphi premix (5 .mu.L) was then added to each sample and the resulting reaction mixture was incubated at 30.degree. C. for 16 h, then at 65.degree. C. for 10 min to inactivate the .phi.29 DNA polymerase activity. DNA quantification with the PicoGreen.RTM. dsDNA Quantitation Reagent (Molecular Probes) was performed after diluting the amplified samples 1:3 in distilled water.

[0527] The amplified products then were denatured at 95.degree. C. for 10 min and end-sequenced in 384-well plates, using the M13F universal primer (SEQ ID NO:91), and the ABI BigDye version 3.1 Prism Sequencing Kit. For the sequencing reaction, 100-200 ng of templates and 6.4 pmol of primers 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 3730.times.1 automated sequencers.

Identification of Long-Chain Polyunsaturated Fatty Acid Elongation Enzyme Homologs from Euglena gracilis cDNA Library eeg1c

[0528] cDNA clones encoding long-chain polyunsaturated fatty acid elongation enzyme homologs (i.e., LC-PUFA ELO homologs or delta-9 elongases) were identified by conducting BLAST (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). The Euglena gracilis cDNA sequences obtained above were analyzed for similarity to all publicly available DNA sequences contained in the "nr" database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). The DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the "nr" database using the BLASTX algorithm (Gish and States, Nat. Genet., 3:266-272 (1993)) provided by the NCBI. 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 (as described in Example 4).

[0529] The BLASTX search using the nucleotide sequences from clone eeg1c.pk001.n5.f revealed similarity of the protein encoded by the cDNA to the long-chain PUFA elongation enzyme from Isochrysis galbana (IgD9e; SEQ ID NO:76) (GenBank Accession No. AAL37626 (GI 17226123), locus AAL37626, CDS AF390174; Qi et al., FEBS Lett. 510(3):159-165 (2002)). The sequence of a portion of the cDNA insert from clone eeg1c.pk001.n5.f is shown in SEQ ID NO:71 (5' end of cDNA insert).

[0530] Additional sequence was obtained from the 3' end of the cDNA insert of eeg1c.pk001.n5.1 as described above, but using the poly(A) tail-primed WobbleT oligonucleotides. Briefly, 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. The 3' end sequence is shown in SEQ ID NO:72.

[0531] Both the 5' and 3' sequences were aligned using Sequencher.sup.TM (Version 4.2, Gene Codes Corporation, Ann Arbor, Mich.) and the resulting sequence for the cDNA is shown in SEQ ID NO:73 (1201 bp). Sequence for the coding sequence from the cDNA in eeg1c.pk001.n5.f and the corresponding deduced amino acid sequence is shown in SEQ ID NO:74 (777 bp) and SEQ ID NO:75 (258 amino acids), respectively.

[0532] The amino acid sequence set forth in SEQ ID NO:75 was evaluated by BLASTP, yielding a pLog value of 38.70 (E value of 2e-39) versus the Isochrysis galbana sequence (SEQ ID NO:76). The Euglena gracilis delta-9 elongase is 39.4% identical to IgD9e using the Jotun Hein method (as described in Example 4); similarly, the Euglena gracilis delta-9 elongase is 31.8% identical to IgD9e using the Clustal V method (as described in Example 4). BLAST scores and probabilities indicate that the nucleic acid fragment described herein as SEQ ID NO:75 encodes an entire Euglena gracilis delta-9 elongase (designated herein as "EgD9e").

Example 12

Construction of Soybean Expression Vector pKR1014 for Co-Expression of the Tetruetreptia pomquetensis CCMP1491 Delta-8 Desaturase (TpomD8) with the Delta-9 Elonqase from Euglena qracilis (EqD9e)

[0533] The present Example describes construction of a soybean vector for co-expression of TpomD8 with EgD9e.

[0534] EgD9e (SEQ ID NOs:74 and 75; Example 11) was amplified with oligonucleotide primers oEugEL1-1 (SEQ ID NO:77) and oEugEL1-2 (SEQ ID NO:78) using the VentR.RTM. DNA Polymerase (Catalog No. MO254S, New England Biolabs Inc., Beverly, Mass.) following the manufacturer's protocol. The resulting DNA fragment was cloned into the pCR-Blunt.RTM. cloning vector using the Zero Blunt.RTM. PCR Cloning Kit (Invitrogen Corporation), following the manufacturer's protocol, to produce pKR906 (SEQ ID NO:79).

[0535] A starting plasmid pKR72 (ATCC Accession No. PTA-6019; SEQ ID NO:80, 7085 by sequence), a derivative of pKS123 which was previously described in PCT Publication No. WO 02/008269 (the contents of which are hereby incorporated by reference), contains the hygromycin B phosphotransferase gene (HPT) (Gritz, L. and Davies, J., Gene 25:179-188 (1983)), flanked by the T7 promoter and transcription terminator (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 .alpha.' subunit of .beta.-conglycinin (Beachy et al., EMBO J. 4:3047-3053 (1985)) and the 3' transcription termination region of the phaseolin gene (Doyle et al., J. Biol. Chem. 261:9228-9238 (1986)), thus allowing for strong tissue-specific expression in the seeds of soybean of genes cloned into the NotI site.

[0536] EgD9e was released from pKR906 by digestion with NotI and cloned into the NotI site of pKR72 to produce pKR912 (SEQ ID NO:81). In some instances, pKR912 is referred to as pKR1010 but they are identical.

[0537] Plasmid pKR1007 (in Example 8, SEQ ID NO:67) was digested with PstI and the fragment containing the Tetruetreptia pomquetensis delta-8 desaturase was cloned into the SbfI site of pKR912 (SEQ ID NO:81), to give pKR1014 (SEQ ID NO:82). In this way, the Tetruetreptia pornquetensis delta-8 desaturase is co-expressed with the Isochrysis galbana delta-9 elongase behind strong, seed-specific promoters. A schematic depiction of pKR1014 is shown in FIG. 3.

[0538] Plasmid pKR1014 was transformed into soybean embryogenic suspension cultures as described in Example 9 and embryos co-expressing of TpomD8 and EgD9e were analyzed as described in Example 10.

[0539] Embryo fatty acid profiles for each event (6 embryos each) containing pKR1014 were obtained and the lipid profiles of somatic soybean embryos expressing TpomD8 and EgD9e for the top 5 events are shown in FIG. 10. Fatty acids are identified as 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic acid), LA, GLA, ALA, EDA, DGLA, ERA and ETA; and, fatty acid compositions listed in FIG. 10 are expressed as a weight percent (wt. %) of total fatty acids. The activity of TpomD8 is expressed as percent desaturation (% desat), calculated according to the following formula: ([product]/[substrate+product])*100.

[0540] More specifically, the combined percent desaturation for EDA and ERA is shown as "C20% delta-8 desat", determined as: ([DGLA+ETA]/[DGLA+ETA+EDA+ERA])*100. This is also referred to as the overall % desaturation. The individual omega-6 delta-8 desaturation ("EDA % delta-8 desat.") was calculated as: ([DGLA]/[DGLA+EDA])*100. Similarly, the individual omega-3 delta-8 desaturation ("ERA % delta-8 desat.") was calculated as: ([ETA]/[ETA+ERA])*100. The ratio of delta-8 desaturation for omega-6 versus omega-3 substrates ("ratio [EDA/ERA]% desat.") was obtained by dividing the EDA % delta-8 desaturation by the ERA % delta-8 desaturation.

[0541] In summary of FIG. 10, TpomD8 worked in soybean to convert both EDA and ERA to DGLA and ETA, respectively. The line with the highest average DGLA content (i.e., 2024-3-9) had embryos with an average DGLA content of 14.8% and an average ETA content of 3.8%. The highest DGLA and ETA content for an individual embryo from this line was 16.0% and 3.9%, respectively. The highest average overall % desaturation was 60.9% with the highest overall % desaturation for an individual embryo being 68.7%. When broken down into % desaturation for the omega-6 and omega-3 substrates, the highest average % desaturation was 59.1% and 73.9% for EDA and ERA, respectively. The highest % desaturation for an individual embryo was 66.7% and 80.9% for EDA and ERA, respectively. In this example, TpomD8 had a preference for ERA over EDA, with the average desaturation ratio ranging from 0.8 to 0.9. No GLA was found to accumulate in the embryos.

Example 13

Construction of Soybean Expression Vector pKR1005 for Co-Expression of the Tetruetreptia pomauetensis CCMP1491 Delta-8 Desaturase (TpomD8) with the Delta-17 Desaturase from Saprolegnia diclina (SdD17)

[0542] The present Example describes construction of a soybean vector for co-expression of TpomD8 with SdD17.

[0543] The PstI fragment, containing the Ann/Sdd17/BD30 cassette from pKR271 (SEQ ID NO:83; which is described in PCT Publication No. WO 2004/071467 and the contents of which are hereby incorporated by reference), was cloned into the SbfI site of pKR226 (SEQ ID NO:84, which is also described in PCT Publication No. WO 2004/071467) to produce vector pKR886r (SEQ ID NO:85). In this way, the Saprolegnia diclina delta-17 desaturase (SdD17) was cloned behind the annexin promoter which is strong and seed specific.

[0544] The .beta.con/NotI/Phas3' cassette in plasmid pKR72 (SEQ ID NO:80, having ATCC Accession No. PTA-6019) was amplified using oligonucleotide primers oCon-1 (SEQ ID NO:86) and oCon-2 (SEQ ID NO:87) using the VentR.RTM. DNA Polymerase (Catalog No. M0254S, New England Biolabs Inc., Beverly, Mass.) following the manufacturer's protocol. The resulting DNA fragment was digested with XbaI and cloned into the XbaI site of pUC19, to produce pKR179 (SEQ ID NO:88).

[0545] TpomD8 was released from plasmid pLF114-10 (SEQ ID NO:63, Example 5) by digestion with NotI and was cloned into the NotI site of plasmid pKR179 (SEQ ID NO:88) to produce pKR1002 (SEQ ID NO:89).

[0546] Vector pKR1002 was digested with PstI and the fragment containing TpomD8 was cloned into the SbfI site of pKR886r (SEQ ID NO:85) to produce pKR1005 (SEQ ID NO:90). A schematic depiction of pKR1005 is shown in FIG. 4.

[0547] One skilled in the art will recognize that pKR1005 could be readily transformed into soybean embryogenic suspension cultures (as described in Example 9) and co-expression of TpomD8 and SdD17 could analyzed (as described in Example 10).

Example 14

Construction of Alternate Soybean Expression Vectors for Expression of Tetruetreptia pomquetensis CCMP1491 (TpomD8), Eutreptiella sp. CCMP389 (E389D8) and/or Eutreptiella cf.sub.--gymnastica CCMP1594 (E1594D8) Delta-8 Desaturases

[0548] 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 TpomD8, E389D8 and/or E1594D8. Similarly, it may be desirable to express other PUFA genes (such as those described below in Table 14), for co-expression with any of the delta-8 desaturases of the present invention.

[0549] 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 12) and a transcription terminator (such as those listed in, but not limited to, Table 13) 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 14 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.

[0550] 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-00022 TABLE 12 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-00023 TABLE 13 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-00024 TABLE 14 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-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 delta-9 elongase Isochrysis galbana WO 2002/077213 delta-9 elongase Euglena gracilis U.S. Provisional Application No. 60/739,989 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

Example 15

Chlorsulfuron Selection (ALS) and Plant Regeneration Chlorsulfuron (ALS) Selection:

[0551] Following bombardment, the tissue is divided between 2 flasks with fresh SB196 media and cultured as described in Example 9. Six to seven days post-bombardment, the SB196 is exchanged with fresh SB196 containing selection agent of 100 ng/mL chlorsulfuron (chlorsulfuron stock is 1 mg/mL in 0.01 N ammonium hydroxide). The selection media is refreshed weekly. Four to six weeks post selection, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated, green tissue is removed and inoculated into multiwell plates containing SB196 to generate new, clonally propagated, transformed embryogenic suspension cultures.

Regeneration of Soybean Somatic Embryos into Plants:

[0552] In order to obtain whole plants from embryogenic suspension cultures, the tissue must be regenerated. Embyros are matured as described in Example 9. 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 10. 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.

[0553] 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 are 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, are chipped and are analyzed for fatty acids as described in Example 10 above.

Example 16

Construction of Soybean Expression Vector pKR973 for Co-Expression of the Pavlova lutheri Delta-8 Desaturase (PavD8) with the Euglena gracilis Delta-9 Elongase (EgD9e) and the Mortierella alpina Delta-5 Desaturase (MaD5)

[0554] Euglena gracilis delta-9 elongase (EgD9e):

[0555] Plasmid pKR906 (SEQ ID NO:79, Example 12) was digested with NotI and the fragment containing the Euglena gracilis delta-9 elongase was cloned into plasmid pKR132 (SEQ ID NO:113; which is described in PCT Publication No. WO 2004/071467) to produce pKR953 (SEQ ID NO:114).

Mortierella alpina Delta-5 Desaturase (MaD5):

[0556] Vector pKR287 (SEQ ID NO:115; which is described in PCT Publication No. WO 2004/071467, published Aug. 26, 2004; the contents of which are hereby incorporated by reference), contains the Mortierella alpina delta-5 desaturase (MaD5; SEQ ID NO:116, which is described in U.S. Pat. No. 6,075,183 and PCT Publication Nos. WO 2004/071467 and WO 2005/047479, the contents of which are hereby incorporated by reference), flanked by the soybean glycinin Gy1 promoter and the pea leguminA2 3' termination region (Gy1/MaD5/legA2 cassette). Vector pKR287 was digested with SbfI/BsiWI and the fragment containing the Gy1/MaD5/legA2 cassette was cloned into the SbfI/BsiWI fragment of pKR277 (SEQ ID NO:117; which is described in PCT Publication No. WO 2004/071467, the contents of which are hereby incorporated by reference) to produce pK952 (SEQ ID NO:118).

[0557] Vector pKR457 (SEQ ID NO:119), which was previously described in PCT Publication No. WO 2005/047479 (the contents of which are hereby incorporated by reference), contains a NotI site 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). Through a number of sub-cloning steps, sequences containing Asp718 restriction sites were added to the 5' and 3' ends of the Kti/NotI/Kti3'Salb3' cassette to produce SEQ ID NO:120.

Pavlova lutheri Delta-8 Desaturase (PavD8):

[0558] Pavlova lutheri (CCMP459) was obtained from the Culture of Marine Phytoplankton (CCMP, West Boothbay Harbor, Me.) and grown in 250 mL flasks containing 50 mL of F/2-Si medium (made using F/2 Family Medium Kit-KIT20F2 and Filtered Seqwater-SEA2 from COMP) at 26.degree. C. with shaking at 150 rpm. Cultures were transferred to new medium on a weekly basis using 1:4 (old culture:new medium) dilution.

[0559] Cultures from 28 flasks (1400 mL) were combined, cells were pelleted by centrifugation at 1,800.times.g for 10 min, washed once with water and re-centrifuged. Total RNA was extracted from the resulting pellet using the RNA STAT-60.TM. reagent (TEL-TEST, Inc., Friendswood, Tex.) and following the manufacturer's protocol provided. In this way, 2.6 mg of total RNA (2.6 mg/mL) was obtained from the pellet. The mRNA was isolated from 1.25 mg of total RNA using the mRNA Purification Kit (Amersham Biosciences, Piscataway, N.J.) following the manufacturer's protocol provided. In this way, 112 .mu.g of mRNA was obtained.

[0560] cDNA was synthesized from 224 ng of mRNA using the SuperScript.TM. First-Strand Synthesis System for RT-PCR Kit (Invitrogen.TM. Life Technologies, Carlsbad, Calif.) with the provided oligo(dT) primer according to the manufacturer's protocol. After RNase H treatment as per the protocol, the Pavlova lutheri delta-8 desaturase (PavD8; SEQ ID NO:121; which is described in U.S. Provisional Application No. 60/795,810 (filed Apr. 28, 2006) and U.S. patent application Ser. No. 11/737,772 (filed Apr. 20, 2007) the contents of which are hereby incorporated by reference) was amplified from the resulting cDNA with oligonucleotide primers PvDES5'Not-1 (SEQ ID NO:122) and PvDES3'Not-1 (SEQ ID NO:123) using the conditions described below.

[0561] cDNA (2 .mu.L) from the reaction described above was combined with 50 pmol of PvDES5'Not-1 (SEQ ID NO:122), 50 pmol of PvDES3'Not-1 (SEQ ID NO:123), 1 .mu.L of PCR nucleotide mix (10 mM, Promega, Madison, Wis.), 5 .mu.L of 10.times.PCR buffer (Invitrogen Corporation), 1.5 .mu.L of MgCl.sub.2 (50 mM, Invitrogen Corporation), 0.5 .mu.L of Taq polymerase (Invitrogen Corporation) and water to 50 .mu.L. The reaction conditions were 94.degree. C. for 3 min followed by 35 cycles of 94.degree. C. for 45 sec, 55.degree. C. for 45 sec and 72.degree. C. for 1 min. The PCR was finished at 72.degree. C. for 7 min and then held at 4.degree. C. The PCR reaction was analyzed by agarose gel electrophoresis on 5 .mu.L and a DNA band with molecular weight around 1.3 kb was observed. The remaining 45 .mu.L of product was separated by agarose gel electrophoresis and the DNA purified using the Zymoclean.TM. Gel DNA Recovery Kit (Zymo Research, Orange, Calif.) following the manufacturer's protocol.

[0562] The PavD8, flanked by NotI sites, was cloned into the NotI site of the modified Kti/NotI/Kti3'Salb3' cassette (SEQ ID NO:120), and then the DNA fragment was digested with Asp718 and cloned into the SbfI site of pKR952 (SEQ ID NO:118) to produce pKR970 (SEQ ID NO:124).

[0563] Plasmid pKR953 (SEQ ID NO:114) was digested with PstI and the fragment containing the Euglena gracilis delta-9 elongase was cloned into the SbfI site of pKR970 (SEQ ID NO:124) to produce pKR973 (SEQ ID NO:125, FIG. 11). In this way, the Pavlova lutheri delta-8 desaturase could be co-expressed with the Mortierella alpina delta-5 desaturase and the Euglena gracilis delta-9 elongase behind strong, seed-specific promoters.

Example 17

Construction of Soybean Expression Vector pKR1084 for Co-Expression of the Euglena gracilis Delta-9 Elonqase (EqD9e) with the Mortierella alpina Delta-5 Desaturase (MaD5)

[0564] The NotI fragment of pKS129 (SEQ ID NO:126; which is described in PCT Publication No. WO 04/071467), containing the MaD5 (SEQ ID NO:116; Example 16) was cloned into the NotI site of pKR457 (SEQ ID NO:119; Example 16), to give pKR606 (SEQ ID NO:127).

[0565] Vector pKR606 (SEQ ID NO:127) was digested with BsiWI and after filling to blunt the ends, the fragment containing the Gy1/MaD5/legA2 cassette was cloned into the filled NgoMI site of pKR277 (SEQ ID NO:117; Example 16) to produce pKR804 (SEQ ID NO:128).

[0566] Plasmid pKR953 (SEQ ID NO:114; Example 16) was digested with PstI and the fragment containing the EgD9e was cloned into the SbfI site of pKR804 (SEQ ID NO:128) to give pKR1084 (SEQ ID NO:129; FIG. 12).

[0567] In this way, the Mortierella alpina delta-5 desaturase (MaD5) was expressed with the Euglena gracilis delta-9 elongase (EgD9e) behind strong, seed-specific promoters.

Example 18

Construction of Soybean Expression Vector pKR1123 for Co-Expression of the Eutreptiella cf.sub.--qymnastica CCMP1594 delta-8 Desaturase (E1594D8) with the Delta-9 Elonqase from Euglena gracilis (EqD9e)

[0568] The present Example describes construction of a soybean vector for co-expression of E1594D8 with EgD9e and expression of these genes in somatic embryos.

[0569] An intermediate plasmid pKR908 (SEQ ID NO:130) allows the cloning of DNA fragments into an NcoI/XbaI site and thus add a flanking NotI site 5' to the NcoI site.

[0570] The NcoI/XbaI fragment of pFBAIn-1594D8 (SEQ ID NO:96; Example 7), containing E1594D8 and where a NotI site is already present just 5' to the XbaI site, was cloned into the NcoI/XbaI sites of pKR908 (SEQ ID NO:130) to produce pKR1118 (SEQ ID NO:131) and where E1594D8 is now flanked by NotI sites at the 5' and 3' ends.

[0571] E1594D8 was released from pKR1118 (SEQ ID NO:131) by digestion with NotI and cloned into the NotI site of pKR123r (SEQ ID NO:66; Example 8) to produce pKR1120 (SEQ ID NO:132).

[0572] Plasmid pKR1120 (SEQ ID NO:132) was digested with SbfI and the fragment containing E1594D8 was cloned into the SbfI site of pKR912 (SEQ ID NO:81; Example 12), to give pKR1123 (SEQ ID NO:133). In this way, the Eutreptiella cf gymnastica CCMP1594 delta-8 desaturase is co-expressed with the Euglena gracilis delta-9 elongase behind strong, seed-specific promoters. A schematic depiction of pKR1123 is shown in FIG. 13.

[0573] Plasmid pKR1123 was transformed into soybean embryogenic suspension cultures as described in Example 9 and embryos co-expressing E1594D8 and EgD9e were analyzed as described in Example 10.

[0574] Embryo fatty acid profiles for each event (6 embryos each) containing pKR1123 were obtained and the lipid profiles of somatic soybean embryos expressing E1594D8 and EgD9e for the top 5 events are shown in FIG. 14. Fatty acids are identified as 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic acid), LA, GLA, ALA, EDA, DGLA, ERA and ETA; and, fatty acid compositions listed in FIG. 14 are expressed as a weight percent (wt. %) of total fatty acids. The activity of E1594D8 is expressed as percent desaturation (% desat), calculated according to the following formula: ([product]/[substrate+product])*100.

[0575] More specifically, the combined percent desaturation for EDA and ERA is shown as "C20% delta-8 desat", determined as: ([DGLA+ETA]/[DGL+ETA+EDA+ERA])*100. This is also referred to as the overall % desaturation. The individual omega-6 delta-8 desaturation ("EDA % delta-8 desat.") was calculated as: ([DGLA]/[DGLA+EDA])*100. Similarly, the individual omega-3 delta-8 desaturation ("ERA % delta-8 desat.") was calculated as: ([ETA]/[ETA+ERA])*100. The ratio of delta-8 desaturation for omega-6 versus omega-3 substrates ("ratio [EDA/ERA]% desat.") was obtained by dividing the EDA % delta-8 desaturation by the ERA % delta-8 desaturation.

[0576] In summary of FIG. 14, E1594D8 worked in soybean to convert both EDA and ERA to DGLA and ETA, respectively. The line with the highest average DGLA content (i.e., 2108-6-6) had embryos with an average DGLA content of 13.6% and an average ETA content of 3.9%. The highest DGLA and ETA content for an individual embryo from this line was 17.7% and 4.7%, respectively. The highest average overall % desaturation was 66.4% (2108-5-2) with the highest overall desaturation for an individual embryo being 71.3%. When broken down into desaturation for the omega-6 and omega-3 substrates, the highest average desaturation was 61.6% and 82.0% for EDA and ERA, respectively. The highest desaturation for an individual embryo from this event was 62.5% and 82.2% for EDA and ERA, respectively. In this example, E1594D8 had a preference for ERA over EDA, with the average desaturation ratio ranging from 0.6-0.8. No GLA was found to accumulate in the embryos.

Example 19

Construction of Soybean Expression Vector pKR1122 for Co-Expression of the Eutreptiella sp. CCMP389 Delta-8 Desaturase (E389D8) with the Delta-9 Elonqase from Euglena gracilis (EqD9e)

[0577] The present Example describes construction of a soybean vector for co-expression of E389D8 with EgD9e and expression of these genes in somatic embryos.

[0578] The NcoI/XbaI fragment of pFBAIn-389D8 (SEQ ID NO:95; Example 7), containing E389D8 and where a NotI site is already present just 5' to the XbaI site, was cloned into the NcoI/XbaI sites of pKR908 (SEQ ID NO:130) to produce pKR1117 (SEQ ID NO:134) and where E389D8 is now flanked by NotI sites at the 5' and 3' ends.

[0579] E389D8 was released from pKR1117 (SEQ ID NO:134) by digestion with NotI and cloned into the NotI site of pKR123r (SEQ ID NO:66; Example 8) to produce pKR1119 (SEQ ID NO:135).

[0580] Plasmid pKR1119 (SEQ ID NO:135) was digested with SbfI and the fragment containing E389D8 was cloned into the SbfI site of pKR912 (SEQ ID NO:81; Example 12), to give pKR1122 (SEQ ID NO:136). In this way, the Eutreptiella sp. CCMP389 delta-8 desaturase is co-expressed with the Euglena gracilis delta-9 elongase behind strong, seed-specific promoters. A schematic depiction of pKR1122 is shown in FIG. 15.

[0581] Plasmid pKR1122 was transformed into soybean embryogenic suspension cultures as described in Example 9 and embryos co-expressing E389D8 and EgD9e were analyzed as described in Example 10.

[0582] Embryo fatty acid profiles for each event (6 embryos each) containing pKR1122 were obtained and the lipid profiles of somatic soybean embryos expressing E389D8 and EgD9e for the top 5 events are shown in FIG. 16. Fatty acids are identified as 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic acid), LA, GLA, ALA, EDA, DGLA, ERA and ETA; and, fatty acid compositions listed in FIG. 16 are expressed as a weight percent (wt. %) of total fatty acids. The activity of E389D8 is expressed as percent desaturation (% desat), calculated according to the following formula: ([product]/[substrate+product])*100.

[0583] More specifically, the combined percent desaturation for EDA and ERA is shown as "C20% delta-8 desat", determined as: ([DGLA+ETA]/[DGLA+ETA+EDA+ERA])*100. This is also referred to as the overall % desaturation. The individual omega-6 delta-8 desaturation ("EDA % delta-8 desat.") was calculated as: ([DGLA]/[DGLA+EDA])*100. Similarly, the individual omega-3 delta-8 desaturation ("ERA % delta-8 desat.") was calculated as: ([ETA]/[ETA+ERA])*100. The ratio of delta-8 desaturation for omega-6 versus omega-3 substrates ("ratio [EDA/ERA]% desat.") was obtained by dividing the EDA % delta-8 desaturation by the ERA % delta-8 desaturation.

[0584] In summary of FIG. 16, E389D8 worked in soybean to convert both EDA and ERA to DGLA and ETA, respectively. The line with the highest average DGLA content (i.e., 2107-4-14) had embryos with an average DGLA content of 16.1% and an average ETA content of 5.2%. The highest DGLA and ETA content for an individual embryo from this line was 16.1% and 6.0%, respectively. The highest average overall % desaturation was 68.5% (2107-4-14) with the highest overall desaturation for an individual embryo being 68.6%. When broken down into desaturation for the omega-6 and omega-3 substrates, the highest average % desaturation was 64.0% and 81.7% for EDA and ERA, respectively. The highest % desaturation for an individual embryo from this event was 68.6% and 83.4% for EDA and ERA, respectively. In this example, E389D8 had a preference for ERA over EDA, with the average desaturation ratio ranging from 0.6-0.8. No GLA was found to accumulate in the embryos.

Example 20

Construction of Arabidopsis Binary Expression Vector pKR1022R for Co-Expression of the Tetruetreptia pomquetensis CCMP1491 Delta-8 Desaturase (TpomD8) with the Delta-9 Elongase from Euglena gracilis (EgD9e)

[0585] The Gy1/Pavelo/legA2 cassette was released from plasmid pKR336 (described in PCT Publication Nos. WO 04/071467; the contents of which are hereby incorporated by reference) by digestion with PstI/BamHI and cloned into the PstI/BamHi site of pKR268 (described in PCT Publication Nos. WO 04/071467) to produce pKR393 (SEQ ID NO:137).

[0586] The Pavelo gene was released from pKR393 (SEQ ID NO:137) by digestion with NotI and the vector was re-ligated to from pKR407 (SEQ ID NO:138). Vector pLF114-10 (SEQ ID NO:63; Example 5) was digested with NotI and the fragment containing TpomD8 was cloned into the NotI site of pKR407 (SEQ ID NO:138) to produce pKR1018 (SEQ ID NO:139).

[0587] The PstI fragment of pKR1018 (SEQ ID NO:139), containing the TpomD8 was cloned into the SbfI fragment of pKR911 (previously described in WO2007/061845 published on May 31, 2007 the contents of which are hereby incorporated by reference) to produce pKR102OR (SEQ ID NO:140). The AscI fragment of pKR102OR (SEQ ID NO:140), containing EgD9e and TpomD8 was cloned into the AscI site of pKR92 (which was previously described in WO2007/061845 published on May 31, 2007 to give pKR1022R (SEQ ID NO:141). A schematic depiction of pKR1022R is shown in FIG. 17. In this way, EgD9e was expressed in Arabidopsis under control of the soybean beta-conglycinin promoter and TpomD8 was expressed under contol of the soybean glycinin Gy1 promoter. The soybean beta-conglycinin promoter and Gy1 promoter function as a strong, seed-specific promoters in Arabidopsis.

Example 21

Transformation of Arabidopsis

[0588] Transformed Arabidopsis plants were created by whole plant Agrobacterium transformation. Binary vector pKR1022R (SEQ ID NO:141) was transformed into Agrobacterium tumefaciens NTL4 (Luo et al., Molecular Plant-Microbe Interactions 14(1):98-103 (2001)) by electroporation. Briefly, 1 .mu.g plasmid DNA was mixed with 100 .mu.L of electro-competent cells on ice. The cell suspension was transferred to a 100 .mu.L electro oration curette (1 mm gap width) and electro orated using a BIORAD electro orator set to 1 kV, 400.OMEGA. and 25 .mu.F. Cells were transferred to 1 mL LB medium and incubated for 2 h at 30.degree. C. Cells were plated onto LB medium containing 50 .mu.g/mL kanamycin. Plates were incubated at 30.degree. C. for 60 h. Recombinant agrobacterium cultures (500 mL LB, 50 .mu.g/mL kanamycin) were inoculated from single colonies of transformed Agrobacterium cells and grown at 30.degree. C. for 60 h.

[0589] Cells were harvested by centrifugation (5000.times.g, 10 min) and resuspended in 1 L of 5% (W/V) sucrose containing 0.05% (V/V) Silwet L-77 (OSI Specialties, Inc). Arabidopsis plants were grown in soil at a density of 10 plants per 100 cm.sup.2 pot in metromix 360 soil mixture for 4 weeks (22.degree. C., 16 h light/8 h dark, 100 .mu.E m.sup.-2s.sup.-1). At early bolting, Arabidopsis plants were dipped into the Agrobacterium suspension. Two days later, the same plants were dipped again with the same Agrobacterium strain in sucrose/Silwet. Plants were grown for three to four weeks under standard plant growth conditions described above and plant material was harvested and dried for one week at ambient temperatures in paper bags. Seeds were harvested using a 0.425 mm mesh brass sieve.

[0590] Cleaned Arabidopsis seeds (2 grams, corresponding to about 100,000 seeds) were sterilized by washes in 45 mL of 80% ethanol, 0.01% triton X-100, followed by 45 mL of 30% (VN) household bleach in water, 0.01% triton X-100 and finally by repeated rinsing in sterile water. Aliquots of 20,000 seeds were transferred to square plates (20.times.20 cm) containing 150 mL of sterile plant growth medium comprised of 0.5.times.MS salts, 1.0% (WN) sucrose, 0.05 MES/KOH (pH 5.8), 200 .mu.g/mL timentin, and 50 .mu.g/mL kanamycin solidified with 10 g/L agar. Homogeneous dispersion of the seed on the medium was facilitated by mixing the aqueous seed suspension with an equal volume of melted plant growth medium. Plates were incubated under standard growth conditions for fourteen days. Kanamycin-resistant seedlings were transferred to soil and grown to maturity as described above. T2 seed was obtained from these individual transformants.

Example 22

Functional Analysis of the Tetruetreptia pomquetensis CCMP1491 Delta-8 Desaturase (TpomD8) Co-Expressed with the Delta-9 Elongase from Euglena gracilis (EgD9e) in Arabidopsis Seed Transformed with Arabidopsis Expression Vector pKR1022R

[0591] Wild-type Arabidopsis thaliana (Columbia ecotype) were transformed with pKR1022R (SEQ ID NO:141) as described in Example 21 and segregating T2 seed was obtained from a number of individual events for each. Bulk T2 seed lipid profiles for each event were obtained by transesterification with TMSH as described in Example 10 with the following modificiations. For each event, a small scoopful of seeds (approximately 25-50 seed each scoopful) was crushed in 50 .mu.L of TMSH in a 1.5 mL eppendorf tube. After shaking in TMSH for 15 min., 400 .mu.L of heptane was added and the tubes were vortexed well, shaken for an additional 15 min and centrifuged at 13,000.times.g for 1 min. After shaking, the heptane layer was removed into glass GC vials and the fatty acid methyl esters were analyzed as described in Example 10.

[0592] Bulk T2 seed fatty acid profiles were obtained for 22 events where wild-type Arabidopsis was transformed with pKR1022R (SEQ ID NO:141). The lipid profiles of T2 bulk seed for the 22 wild-type-transformed events is shown in FIG. 18. Fatty acids are identified as 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic acid), LA, ALA, 20:0 (arachidic acid), 20:1 (eicosenoic acid), EDA, DGLA, ERA and ETA; and, fatty acid compositions listed in FIG. 18 are expressed as a weight percent (wt. %) of total fatty acids.

Example 23

Functional Analysis of the Tetruetreptia Domauetensis CCMP1491 Delta-8 Desaturase (TpomD8) Co-Expressed with the Saproleania diclina Delta-17 Desaturase (SdD17), the Euglena gracilis Delta-9 Elonqase (EqD9e), the Pavlova lutheri delta-8 Desaturase (PavD8) and the Mortierella alpina Delta-5 Desaturase (MaD5) in Soybean Embryos and Seed Transformed with Soybean Expression Vectors pKR1005 and pKR973

[0593] The present Example describes the expression of an EPA biosynthetic pathway using a delta-9 elongase (EgD9e), a delta-5 desaturase (MaD5) and a delta-17 desaturase (SdD17) co-expressed with two delta-8 desaturases (TpomD8 & PavD8).

[0594] Soybean embryogenic suspension culture (cv. Jack) was transformed with the AscI fragments of pKR1005 (SEQ ID NO:90; FIG. 4) and pKR973 (SEQ ID NO:125; FIG. 11), as described in in Example 9. Embryos were matured as described in Example 14 and a subset of soybean embryos generated from each event (ten embryos per event) were harvested, picked into glass GC vials, fatty acid methyl esters (FAMEs) were prepared by transesterification and analyzed by GC as described in Example 10. Retention times were compared to those for methyl esters of standards commercially available (Nu-Chek Prep, Inc.).

[0595] In this way, 373 events transformed with pKR1005 (SEQ ID NO:90; FIG. 4) and pKR973 (SEQ ID NO:125; FIG. 11) (experiment called Heal 17) were analyzed. From the 373 events analyzed, 319 were identified that produced delta-8 desaturation products (i.e. DGLA, ARA, ETA, EPA, DPA, DHA) in at least one embryo out of ten analyzed at a relative abundance greater than 1.0% of the total fatty acids. Of these, 140 were identified that produced delta-8 desaturation products at a relative abundance greater than 10.0% of the total fatty acids, 61 were identified that produced delta-8 desaturation products at a relative abundance greater than 20.0% of the total fatty acids and 20 were identified that produced delta-8 desaturation products at a relative abundance greater than 30.0% of the total fatty acids, in at least one embryo out of ten analyzed.

[0596] The average fatty acid profiles (average of 10 embryos per event) for the ten events having the highest amounts of delta-8 desaturation products are shown in FIG. 19. Fatty acids are identified as 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic acid), LA, GLA, ALA, EDA, SCI, DGLA, ARA, ERA, JUP, ETA, EPA and DPA; and, fatty acid compositions listed in FIG. 19 are expressed as a weight percent (wt. %) of total fatty acids. For FIG. 19, fatty acids listed as "others" include: 18:2 (5,9), STA, 20:0, 20:1(11), 20:2 (7,11) or 20:2 (8,11), and DHA. Each of these fatty acids is present at a relative abundance of less than 1.6% of the total fatty acids. The total wt. % of fatty acids containing a delta-8 double bond is expressed as C20 delta-8 desat (DGLA+ARA+ETA+EPA+DPA) and the delta-8 desaturase activity is expressed as percent desaturation (C20% delta-8 desat), calculated according to the following formula: ([DGLA+ETA]/[DGLA+ETA+EDA+ERA])*100.

[0597] In summary of FIG. 19, TpomD8 and PavD8 functioned in soybean to convert both EDA and ERA to DGLA and ETA, respectively. Additionally, the activity of the delta-5 desaturase also functioned to convert the DGLA and ETA produced to ARA and EPA, respectively. In events such as AFS 4881-6-5 & 4881-4-5, delta-5 desaturase is somewhate limiting and DGLA and ETA are high while in others (e.g. AFS 4829-6-5 & AFS 4885-1-2), delta-5 desaturase activity is strong and the delta-8 desaturated products are further converted to ARA and EPA, respectively. Further, the presence of the delta-17 desaturase also functioned to convert DGLA and ARA to ETA and EPA, respectively. In events such as AFS 4880-8-8, the delta-17 desaturase is somewhat limiting while in others (e.g. AFS 4881-6-5 & AFS 4829-6-5), delta-17 desaturase activity is strong with DGLA and ARA being efficiently converted to ETA and EPA, respectively. The individual embryo with the highest total delta-8 desaturated products came from event AFS 4881-6-5,with as high as 43% of total fatty acids. The average concentration of delta-8 desaturated products from the top ten events was 27.7% of the total fatty acids.

[0598] The fatty acid profiles for ten individual T1 seeds from 2 plants from event AFS 4882-4-6 (plant #4882-4-6-1 & #4882-4-6-2) having some of the highest amounts of total delta-8 desaturation products are shown in FIG. 20. Fatty acids are identified as 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic acid), 18:2 (5,9), LA, GLA, ALA, 20:1 (11), EDA, SCI, DGLA, ARA, ERA, JUP, ETA, EPA and DPA; and, fatty acid compositions listed in FIG. 20 are expressed as a weight percent (wt. %) of total fatty acids. For FIG. 20, fatty acids listed as "others" include: STA, 20:0, 20:2 (7,11) or 20:2 (8,11), and DHA. Each of these fatty acids is present at a relative abundance of less than 1.0% of the total fatty acids. The total wt. % of fatty acids containing a delta-8 double bond is expressed as C20 delta-8 desat (DGLA+ARA+ETA+EPA+DPA) and the delta-8 desaturase activity is expressed as percent desaturation (C20% delta-8 desat), calculated according to the following formula: ([DGLA+ETA]/[DGLA+ETA+EDA+ERA])*100.

[0599] In summary of FIG. 20, TpomD8 and PavD8 worked in soybean seed to convert both EDA and ERA to DGLA and ETA, respectively. Fatty acid compositions in T1 seed are similar to those in embryos. The T1 seed is segregating as expected with some wild-type present.

Example 24

Functional Analysis of the Tetruetreptia pomquetensis CCMP1491 Delta-8 Desaturase (TpomD8) Co-Expressed with the Saproleqnia diclina Delta-17 Desaturase (SdD17), the Euglena gracilis Delta-9 Elonqase (EqD9e) and the Mortierella alpina Delta-5 Desaturase (MaD5) in Soybean Embryos and Seed Transformed with Soybean Expression Vectors pKR1005 and pKR1084

[0600] The present Example describes the expression of an EPA biosynthetic pathway using a delta-9 elongase (EgD9e), a delta-5 desaturase (MaD5) and a delta-17 desaturase (SdD17) co-expressed with one delta-8 desaturases (TpomD8). Soybean embryogenic suspension culture (cv. Jack) was transformed with the AscI fragments of pKR1005 (SEQ ID NO:90; FIG. 4) and pKR1084 (SEQ ID NO:129; FIG. 12), as described in in Example 9. Embryos were matured as described in Example 14 and a subset of soybean embryos generated from each event (ten embryos per event) were harvested, picked into glass GC vials, fatty acid methyl esters (FAMEs) were prepared by transesterification and analyzed by GC as described in Example 10. Retention times were compared to those for methyl esters of standards commercially available (Nu-Chek Prep, Inc.).

[0601] In this way, 182 events transformed with pKR1005 (SEQ ID NO:90; FIG. 4) and pKR1084 (SEQ ID NO:129; FIG. 11) (experiment called Heal21) were analyzed. From the 182 events analyzed, 172 were identified that produced delta-8 desaturation products (i.e. DGLA, ARA, ETA, EPA, DPA, DHA) in at least one embryo out of ten analyzed at a relative abundance greater than 1.0% of the total fatty acids. Of these, 103 were identified that produced delta-8 desaturation products at a relative abundance greater than 10.0% of the total fatty acids, 59 were identified that produced delta-8 desaturation products at a relative abundance greater than 20.0% of the total fatty acids and 9 were identified that produced delta-8 desaturation products at a relative abundance greater than 30.0% of the total fatty acids, in at least one embryo out of ten analyzed.

[0602] The average fatty acid profiles (average of 10 embryos per event) for the ten events having the highest amounts of delta-8 desaturation products are shown in FIG. 21. Fatty acids are identified as 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic acid), LA, GLA, ALA, EDA, SCI, DGLA, ARA, ERA, JUP, ETA, EPA and DPA; and, fatty acid compositions listed in FIG. 21 are expressed as a weight percent (wt. %) of total fatty acids. For FIG. 21, fatty acids listed as "others" include: 18:2 (5,9), STA, 20:0, 20:1(11), 20:2 (7,11) or 20:2 (8,11), and DHA. Each of these fatty acids is present at a relative abundance of less than 2.0% of the total fatty acids. The total wt. % of fatty acids containing a delta-8 double bond is expressed as C20 delta-8 desat (DGLA+ARA+ETA+EPA+DPA) and the delta-8 desaturase activity is expressed as percent desaturation (C20% delta-8 desat), calculated according to the following formula: ([DGLA+ETA]/[DGLA+ETA+EDA+ERA])*100.

[0603] As similar to that seen for the Heal17 embryos in Example 23, the Tpom delta-8 functioned alone to convert EDA and ERA to DGLA and ETA, respectively. Downsteam products also varied depending on the expression of the delta-5 desaturase and delta-17 desaturase activities. But, while the range of delta-8 desaturated products for the Heal17 embryos, expressing 2 delta-8 desaturases, ranged from 25.5-33.7% of total fatty acids, those for the Heal21 embryos expressing only the single TpomD8 ranged from 18.4-22.7. The average delta-8 desaturated products for Heal17 and Heal21 embryos was 27.7% and 20.2%, respectively. With the decrease in overall delta-8 desaturase activity in Heal21 embryos compared to Heal17 embryos, EDA and ERA levels also increased from an aveage of 3.3% EDA and 1.2% ERA to 5.2% EDA and 2.0% ERA, respectively. An increase in the amounts of the fatty acid by-products, SCI and JUP, in Heal21 embryos compared to Heal17 embryos from 0% SCI and 0.6% JUP to 0.4% SCI and 2.3% JUP, respectively, was also observed.

[0604] The fatty acid profiles for individual T1 seeds from 2 plants from event AFS 5003-1-8 (plant #5003-1-8-1 & #5003-1-8-2) having some of the highest amounts of total delta-8 desaturation products are shown in FIG. 22. Fatty acids are identified as 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic acid), 18:2 (5,9), LA, GLA, ALA, 20:1 (11), EDA, SCI, DGLA, ARA, ERA, JUP, ETA, EPA and DPA; and, fatty acid compositions listed in FIG. 22 are expressed as a weight percent (wt. %) of total fatty acids. For FIG. 22, fatty acids listed as "others" include: STA, 20:0, 20:2 (7,11) or 20:2 (8,11), and DHA. Each of these fatty acids is present at a relative abundance of less than 1.0% of the total fatty acids. The total wt. % of fatty acids containing a delta-8 double bond is expressed as C20 delta-8 desat (DGLA+ARA+ETA+EPA+DPA) and the delta-8 desaturase activity is expressed as percent desaturation (C20% delta-8 desat), calculated according to the following formula: ([DGLA+ETA]/[DGLA+ETA+EDA+ERA])*100.

[0605] In summary of FIG. 22, TpomD8 worked in soybean seed to convert both EDA and ERA to DGLA and ETA, respectively. Fatty acid compositions in T1 seed are similar to those in embryos. The T1 seed is segregating as expected with some wild-type present.

Sequence CWU 1

1

141121DNAArtificial Sequenceprimer D8F1 1gaygcnacng aygcnttcat g 21221DNAArtificial Sequenceprimer D8F2 2gaygcnacng aygcngttat g 21321DNAArtificial Sequenceprimer D8F3 3gaygcnacng aygcngtgat g 21421DNAArtificial Sequenceprimer D8F4 4gaygcnacng aygcntttat g 21521DNAArtificial Sequenceprimer D8F5 5gaygcnacng aygcngtaat g 21621DNAArtificial Sequenceprimer D8F6 6gaygcnacng aygcngtgat g 21719DNAArtificial Sequenceprimer D8F7 7tnggntggtt rggngayga 19819DNAArtificial Sequenceprimer D8F8 8tnggntggct rggngayga 19919DNAArtificial Sequenceprimer D8F9 9tnggntggct yggngayga 191020DNAArtificial Sequenceprimer D8R1 10tgrtgytcda tytgrtartt 201120DNAArtificial Sequenceprimer D8R2 11tgrtgytcda tytgcatrtt 20127PRTArtificial Sequenceprimers D8F1 and D8F4 12Asp Ala Thr Asp Ala Phe Met1 5137PRTArtificial Sequenceprimers D8F2, D8F3, D8F5 and D8F6 13Asp Ala Thr Asp Ala Val Met1 5146PRTArtificial Sequenceprimers D8F7, D8F8 and D8F9 14Gly Trp Leu Gly Asp Xaa1 5156PRTArtificial Sequenceprimers D8R1 and D8R2 15Asn Tyr Gln Ile Glu His1 516977DNATetruetreptia pomquetensis CCMP1491 16gatgctacgg atgcgtttat ggtgatgcac tctcaagaag ccgtcgccaa gttgaagaga 60atgcctgtta tggagccttc ctctcctgac acacctgttg cacccaagcc taagcgtgat 120gagccccagg aggatttccg caagttgcgg gaggaattca tctccaaggg tatgttcgag 180acgagtttcc tttggtattt ttacaagact tcaactaccg tcggtttgat ggtcctttcc 240atcttgatga ccgtgtacac gaattggtat ttcaccgctg ctttggttct tggcgtgtgc 300taccaacagc taggctggtt gtcccacgac tattgccatc accaggtttt cacgaaccgc 360aagattaacg acgctttcgg tctctttttc ggtaacgtga tgcagggata ctcacagact 420tggtggaagg ataggcacaa tggtcaccat gccgccacca atgtggtcgg ccatgaccca 480gatactgata acctccccat cctggcttgg tctcccgaag atgtcaagag ggctactcct 540tcgactcgga atctcatcaa gtaccagcag tactacttca ttcccaccat tgcatccctt 600aggttcatct ggcgcctcca atccatcggc ggcgtcatgt cctacaagag cgaggagagg 660aacctgtact acaagcgccg gtacactaag gaggcgattg gtctggccct cccttgggtg 720ctcaaggcca ctttctattg cagtgccatg cctagctttg ccaccggttt gggatgcttc 780ttgatctccg agctgctcgg aggatttggc attgccatcg ttgtgtttct gaatcactat 840cctttggaca aggttgagga gactgtctgg gatgagcacg ggttcagcgc cagccagatc 900cacgagacgt tgaacattaa gcccggcctt ctcaccgatt gggtctttgg tggtctcaac 960taccagatcg aacacca 97717968DNAEutreptiella sp. CCMP389 17gacgctacsg aygcwtttat ggtcatgcac tcccacgatg cgttgaacaa gctgaagcgc 60atgcctgtga tggagcccac ttcgccacga agccccaaga ctcccaacga cgaggttgct 120gaggacttcc gcaagcttcg aaaggacatg attgcaaaag gcatgttcaa cgcatcccct 180ctcttctacg tgtacaaaag tgcgaccaca gtagccctgg gcgccctggc tattctgatg 240gtgatgcacc tgcagtggta ctacatccca gccattttgt tgggactttg ctaccagcag 300ctggggtggt tggcacacga ttactgccac catcaggtgt tctctaaccg ggcgtacaac 360aattttgctg gacttgtatt cggcaatgtg atgcaaggat actccgrgac ttggtggaag 420gacaggcaca acggccatca cgccgccacg aacgtgcaag ggcacgatcc cgacatcgac 480racctcccgg tgttggcctg gtccccggag gacgtcaaaa acgccggtcc cacgacgcgg 540aagctcatca agtggcaaca atactatttc ctccccacca tcgcaaccct ccgattcatc 600tggtgcttcc agagcattct ggcggttatg gcatacaaga cagatgcaag gaatatatat 660taccaacgcc agtacgcaaa ggaggccgtg gggctggctc tgcattggat tctgaaaggg 720gtrttcatgt tctgttacat gcccggcata ctgacgggct tggccttctt cctcatcycg 780gagtgcctgg gcgggtttgg gattgccatt gtcgtgttct tgaatcacta cccattggag 840aaggtggagg agtccgtgtg ggacagccac gggttttgcg cggggcagat ccacacgacg 900atgaacatcc aacgcggggt catcgttgac tggttctttg gaggcctgaa ctaccaaatc 960gaacacca 96818968DNAEutreptiella cf_gymnastica CCMP1594 18gacgcyacsg acgcrtttat ggtcatgcat tcacagcagg cgctcaacaa gttgaagcgg 60atgcctgtta tggagccctc ttcaccactt actcccaaga gcccaagtga cgacatttcc 120kaggatttcc gcaagctccg caacagtatg gttgagaagg gtatgttcaa cgcgtcccct 180ctgttttatg tgtacaaatc actgaccact gtcgcccttg gcgccgtggg tgttctcatg 240gttatgtacc tgcagtggta ctacgtttca gccatgtttt tgggactttg ctaccaacag 300ctgggttggg tggcgcatga ctacgcgcat caccaggttt tcacgaaccg tgattatggc 360aatcttggtg ggcttttctt tggcracgtt ctccaaggat attctttgac ttggtggaag 420gacaggcaca acggccatca cgccgccaca aacgtgcaag gacatgaccc cgacattgat 480aatctccccg ttttggcttg gtcgccagag gacgtcaaga atgccggacc tggaacccgc 540aatatcatca agtaccagca gtattatttc ctccctacca tcgccatcct tcggttcatc 600tggtgtttcc aaagcattct gggggtgatg tcatacaaga cagactccra gaatctctat 660tacaaacggc agtaccggag agaggcagcc ggtctggcgc tgcactggat tctgaagagc 720gttttcttgt tctgttacat gccaagtttc ctcactggcc tggcgttttt ccttatctcg 780gagtgtctgg gcggctttgg gatcgcgatt gtggtgtttt tgaaccacta tccgctggat 840aaggttgagg aatccgtttg ggatggtcac ggtttctgtg ctgggcagat cctcacaacc 900atgaacatcc aacgcggact catcactgac tggttctttg gaggtttgaa ytaccaaatc 960gaacacca 9681939DNAArtificial SequenceSMART IV oligonucleotide 19aagcagtggt atcaacgcag agtggccatt acggccggg 392021DNAArtificial Sequenceprimer 389D8-3-1 20caacgccagt acgcaaagga g 212122DNAArtificial Sequenceprimer 389D8-3-2 21ctctgcattg gattctgaaa gg 222222DNAArtificial Sequenceprimer 389D8-5-1 22aatcatgtcc tttcgaagct tg 222322DNAArtificial Sequenceprimer 389D8-5-2 23gtcctcagca acctcgtcgt tg 222422DNAArtificial Sequenceprimer 389D8-5-3 24cttggggctt cgtggcgaag tg 222522DNAArtificial Sequenceprimer ED8-5-1 25ctcgaacata cccttggaga tg 222622DNAArtificial Sequenceprimer ED8-5-2 26cccgcaactt gcggaaatcc tc 222722DNAArtificial Sequenceprimer ED8-5-3 27gggctcatca cgcttaggct tg 222822DNAArtificial Sequenceprimer ED8-3-1 28cactttctat tgcagtgcca tg 222922DNAArtificial Sequenceprimer ED8-3-2 29ctttgccacc ggtttgggat gc 223059DNAArtificial SequenceCDSIII/3' PCR primer 30attctagagg ccgaggcggc cgacatgttt tttttttttt tttttttttt tttttttvn 593137DNAArtificial SequenceAdaptor Primer from Invitrogen 3'-RACE kit 31ggccacgcgt cgactagtac tttttttttt ttttttt 373222DNAArtificial Sequenceprimer 1594D8-3-1 32gagcgttttc ttgttctgtt ac 223322DNAArtificial Sequenceprimer 1594D8-3-2 33cgtttttcct tatctcggag tg 223422DNAArtificial Sequenceprimer 1594D8-5-1 34gatttgtaca cataaaacag ag 223522DNAArtificial Sequenceprimer 1594D8-5-2 35acccttctca accatactgt tg 223622DNAArtificial Sequenceprimer 1594D8-5-3 36cttgggagta agtggtgaag ag 223748DNAArtificial SequenceGenomeWalker adaptor-1 37gtaatacgac tcactatagg gcacgcgtgg tcgacggccc gggctggt 483822DNAArtificial Sequenceprimer AP1 38gtaatacgac tcactatagg gc 223919DNAArtificial Sequenceprimer AP2 39actatagggc acgcgtggt 19403931DNAArtificial Sequenceplasmid pCR2.1-TOPO 40aagggcgaat tctgcagata tccatcacac tggcggccgc tcgagcatgc atctagaggg 60cccaattcgc cctatagtga gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt 120gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc 180agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 240aatggcgaat ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 300gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 360cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 420ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 480cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 540tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 600cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 660aacaaaaatt taacgcgaat tttaacaaaa ttcagggcgc aagggctgct aaaggaagcg 720gaacacgtag aaagccagtc cgcagaaacg gtgctgaccc cggatgaatg tcagctactg 780ggctatctgg acaagggaaa acgcaagcgc aaagagaaag caggtagctt gcagtgggct 840tacatggcga tagctagact gggcggtttt atggacagca agcgaaccgg aattgccagc 900tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc 960gccaaggatc tgatggcgca ggggatcaag atctgatcaa gagacaggat gaggatcgtt 1020tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct 1080attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct 1140gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga 1200actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc 1260tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg 1320gcaggatctc ctgtcatccc accttgctcc tgccgagaaa gtatccatca tggctgatgc 1380aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca 1440tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga 1500cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc 1560cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga 1620aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca 1680ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg 1740cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct 1800tcttgacgag ttcttctgaa ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc 1860gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg 1920gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat 1980ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc 2040acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa 2100ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa 2160aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt 2220gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct 2280tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat 2340gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg 2400cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg 2460atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt 2520attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg 2580ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg 2640gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg 2700tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa 2760aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt 2820tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt 2880tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt 2940ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag 3000ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta 3060gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat 3120aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg 3180ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg 3240agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac 3300aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga 3360aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt 3420ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta 3480cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat 3540tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg 3600accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct 3660ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa 3720gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct 3780ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac 3840acaggaaaca gctatgacca tgattacgcc aagcttggta ccgagctcgg atccactagt 3900aacggccgcc agtgtgctgg aattcgccct t 393141694DNAEutreptiella cf_gymnastica CCMP1594 41cttgttggta ttagtaatgg gacacagcag tatattttcc cattgagaaa aacgctgaaa 60tactggttgg tgaaaacttg gtgagaacaa cggtccattt gaacacagct tcccaccgcc 120tttccccctt atctcatgtt gctggaccac actgcaagct gcatgagcga tagctgaacg 180agacttcacg ctgtcatccc ttcacttcat atgcgttgtg caagggaaag ggtgccatca 240ggtgtgactg tgcctccgtg ataaagtcga gggcacactc cgaattgggc agttctcgct 300accgtgacca gatgcgtgtc aaaactagat cccgaagaaa acgcccgcgc ggagagcctt 360gacacagttg tgttgaagaa aagtttgtgt ggcttcggag cgaaaaagac accgcaccat 420agctgtggca gtgcaagacc ccagatccgc tggtccctgc acttgttgaa gcctcaaaat 480gtcacctaaa cgggacgcat tgcctctgac aattgatggc accacgtacg acgtttccgc 540ttgggtaaac catcaccctg gaggggctca aatcattgaa aactaccgga accgagatgc 600taccgacgtg ttcatggtca tgcattcaca gcaggcgctc aacaagttga agcggatgcc 660tgttatggag ccctcttcac cacttactcc caag 69442648DNAEutreptiella sp. CCMP389 42cgtggcctga aattgcatct atgcaaatgg caactgccat gttgtgggac ttggcttgcc 60actacccact aaaaaggcca acgctgccat aatcaaatat aaagaccact gcctgggcat 120gaccactagt ggcgtatgca gcgccacatg ctccaacaca gcatgtcaag gcgctttgct 180gagcgtctac ttcatgatgc atttgcctcc atttaaaagt cattaaaaga catactcatg 240atgtaaccca acgcacgttg cactgcattt tgcgacctcc gcgtctacct ccattcaaaa 300tgtgtgaatc atgattgctc caatttggga ggagggggta ataaactcag cccatccact 360gcccttccct tgggacgtga cacgagtacc aacgcacttc tgcccgctgt ctttgctccg 420cgtagtcttg gaatgtcacc caagcgagag gccttgccca tcacgattga tggcacaacc 480tatgatgtgt ccgcatgggt gaaccatcac cccgggggcg cagacatcat ggagaattat 540cggaaccgag atgccacgga tgtgttcatg gtcatgcact cccacgatgc gttgaacaag 600ctgaagcgca tgcctgtgat ggagcccact tcgccacgaa gccccaag 64843717DNAEutreptiella sp. CCMP389 43ctctgcattg gattctgaaa ggggtattca tgttctgtta catgcccggc atactgacgg 60gcttggcctt cttcctcatc tcggagtgcc tgggcgggtt tgggattgcc attgtcgtgt 120tcttgaatca ctacccattg gagaaggtgg aggagtccgt gtgggacagc cacgggtttt 180gcgcggggca gatccacacg acgatgaaca tccaacgcgg ggtcatcgtt gactggttct 240ttggaggcct gaattatcag atcgaacacc atctgtggcc gacgctgccc cggcatcact 300tgaaagctgc ttcttttgag gtggagaaaa tttgccagaa gcacaaattg ccatacagag 360caccccccat gtccgatggt gttgctcaat tgcttggctt cttggggaag attgctaagc 420tggcagctgt cccagtgtaa ccctaaacgt accacggcgt tgtcaagaca gtcagctggg 480tttcggagtg gtagcagtgc gtgcagctgt gcagctgagg acgattgtgg ggtttgatca 540tgtctgtcag agttctttgt gcacgtagaa tgatgcacgg taccatcagg tcagcttggt 600tggctctgca tgaggctgac ggtcttaatt tggggtgtct caaagatact caaggacgaa 660gagtatgcac acgtgtttgg ccatttcmca gtgmmaaaaa aaaaaaaaaa aaaaaaa 717441164DNAEutreptiella cf_gymnastica CCMP1594misc_feature(596)..(596)n is a, c, g, or t 44gcccttcgtt tttccttatc tcggagtgtc tgggcggctt tgggatcgcg attgtggtgt 60ttttgaacca ctatccgctg gataaggttg aggaatccgt ttgggatggt cacggtttct 120gtgctgggca gatcctcaca accatgaaca tccaacgcgg actcatcact gactggttct 180ttggaggttt gaattaccag attgagcatc atctgtggcc caaccttcca agacaccatt 240tgaaagcagt ttcctttgag gttgagaaat tgtgccagaa gcacaacctg ccctacagag 300ctccgccgat gcatactggt gttgcacaat tgcttggata tttggggaag attgctcagt 360tggctgctgt cccagtataa ccctggatca ccttcatcga tcctattctg agtgttcagg 420gtgcgactgc atcccccgtt tttcttgcac agccgcgcat ttgcagggtg gtttctatta 480caattttttt tctgcccaaa acaacacctg atgtggcgag cgaggttcac tcttgctgca 540caccactcat tttgttctgg gttgagttat atgtgaatta atatgtaagc agtttncttg 600cacagcccgg ggcatttttt ntattcccaa gacagcgtga taacatttgg cgggcgaggt 660tcactcttgc tgcacaccat catgttttgg gtgcccagcc ccccccnncc ccnctctatt 720atgactgaat cgttgtagaa gcatggagtc caggtgtggt tttgcactgt aagcatgtcc 780gctttggtga actggttatg gtgactcagg tctcgtgcct aggtttacta aatgcgtgac 840atttgcagtc atcacatttc ttttttgatc acatgctgta acgttcacac tactgcctaa 900ggttaggttg tgtgtttggg ccatttagct gtcaaaaccc tccctctttc agaagtgttt 960gtcgaccaca aatattgcac caagttgtta catcattgtt tagttagttg cgttcagtgt 1020ccaatttttc gacggccaaa ttttcttgct gggcttattc gttgggtggt gatgtggcat 1080tgaaagatga tgcagtggtg cagatgagaa cagaaatggt ctgtgttgcg ttgatttgtt 1140caaaaaaaaa aaaaaaaaaa aaaa 116445435DNAEutreptiella cf_gymnastica CCMP1594 45gcccttcgtt tttccttatc tcggagtgtc tgggcggctt tgggatcgcg attgtggtgt 60ttttgaacca ctatccgctg gataaggttg aggaatccgt ttgggatggt cacggtttyt 120gtgctgggca gatcctcaca accatgaaca tccaacgcgg actcatcact gactggttct 180ttggaggttt gaattaccag attgagcatc atytgtggcc caaccttcca agacaccatt 240tgaaagcagt ttcctttgag gttgagaaat tgtgccagaa gcacaacctg ccctgcagag 300ctccgccgat gcatactggt gttgcacaat tgcttggata tttggggaag attgctcagt 360tggctgctgt cccagtataa ccctggatca ccttcatcga tccttttttg aaaaaaaaaa 420aaaaaaaaaa aaaaa 435461963DNAEutreptiella sp. CCMP389misc_feature(1934)..(1934)n is a, c, g, or t 46cgtggcctga aattgcatct atgcaaatgg caactgccat gttgtgggac ttggcttgcc 60actacccact aaaaaggcca acgctgccat aatcaaatat aaagaccact gcctgggcat 120gaccactagt ggcgtatgca gcgccacatg ctccaacaca gcatgtcaag gcgctttgct 180gagcgtctac ttcatgatgc atttgcctcc atttaaaagt cattaaaaga catactcatg 240atgtaaccca acgcacgttg cactgcattt tgcgacctcc gcgtctacct ccattcaaaa 300tgtgtgaatc

atgattgctc caatttggga ggagggggta ataaactcag cccatccact 360gcccttccct tgggacgtga cacgagtacc aacgcacttc tgcccgctgt ctttgctccg 420cgtagtcttg gaatgtcacc caagcgagag gccttgccca tcacgattga tggcacaacc 480tatgatgtgt ccgcatgggt gaaccatcac cccgggggcg cagacatcat ggagaattat 540cggaaccgag atgccacgga tgtgttcatg gtcatgcact cccacgatgc gttgaacaag 600ctgaagcgca tgcctgtgat ggagcccact tcgccacgaa gccccaagac tcccaacgac 660gaggttgctg aggacttccg caagcttcga aaggacatga ttgcaaaagg catgttcaac 720gcatcccctc tcttctacgt gtacaaaagt gcgaccacag tagccctggg cgccctggct 780attctgatgg tgatgcacct gcagtggtac tacatcccag ccattttgtt gggactttgc 840taccagcagc tggggtggtt ggcacacgat tactgccacc atcaggtgtt ctctaaccgg 900gcgtacaaca attttgctgg acttgtattc ggcaatgtga tgcaaggata ctccgggact 960tggtggaagg acaggcacaa cggccatcac gccgccacga acgtgcaagg gcacgatccc 1020gacatcgacg acctcccggt gttggcctgg tccccggagg acgtcaaaaa cgccggtccc 1080acgacgcgga agctcatcaa gtggcaacaa tactatttcc tccccaccat cgcaaccctc 1140cgattcatct ggtgcttcca gagcattctg gcggttatgg catacaagac agatgcaagg 1200aatatatatt accaacgcca gtacgcaaag gaggccgtgg ggctggctct gcattggatt 1260ctgaaagggg tattcatgtt ctgttacatg cccggcatac tgacgggctt ggccttcttc 1320ctcatctcgg agtgcctggg cgggtttggg attgccattg tcgtgttctt gaatcactac 1380ccattggaga aggtggagga gtccgtgtgg gacagccacg ggttttgcgc ggggcagatc 1440cacacgacga tgaacatcca acgcggggtc atcgttgact ggttctttgg aggcctgaac 1500taccaaatcg aacaccatct gtggccgacg ctgccccggc atcacttgaa agctgcttct 1560tttgaggtgg agaaaatttg ccagaagcac aaattgccat acagagcacc ccccatgtcc 1620gatggtgttg ctcaattgct tggcttcttg gggaagattg ctaagctggc agctgtccca 1680gtgtaaccct aaacgtacca cggcgttgtc aagacagtca gctgggtttc ggagtggtag 1740cagtgcgtgc agctgtgcag ctgaggacga ttgtggggtt tgatcatgtc tgtcagagtt 1800ctttgtgcac gtagaatgat gcacggtacc atcaggtcag cttggttggc tctgcatgag 1860gctgacggtc ttaatttggg gtgtctcaaa gatactcaag gacgaagagt atgcacacgt 1920gtttggccat ttcncagtgn naaaaaaaaa aaaaaaaaaa aaa 196347417PRTEutreptiella sp. CCMP389 47Met Ser Pro Lys Arg Glu Ala Leu Pro Ile Thr Ile Asp Gly Thr Thr1 5 10 15Tyr Asp Val Ser Ala Trp Val Asn His His Pro Gly Gly Ala Asp Ile 20 25 30Met Glu Asn Tyr Arg Asn Arg Asp Ala Thr Asp Val Phe Met Val Met 35 40 45His Ser His Asp Ala Leu Asn Lys Leu Lys Arg Met Pro Val Met Glu 50 55 60Pro Thr Ser Pro Arg Ser Pro Lys Thr Pro Asn Asp Glu Val Ala Glu65 70 75 80Asp Phe Arg Lys Leu Arg Lys Asp Met Ile Ala Lys Gly Met Phe Asn 85 90 95Ala Ser Pro Leu Phe Tyr Val Tyr Lys Ser Ala Thr Thr Val Ala Leu 100 105 110Gly Ala Leu Ala Ile Leu Met Val Met His Leu Gln Trp Tyr Tyr Ile 115 120 125Pro Ala Ile Leu Leu Gly Leu Cys Tyr Gln Gln Leu Gly Trp Leu Ala 130 135 140His Asp Tyr Cys His His Gln Val Phe Ser Asn Arg Ala Tyr Asn Asn145 150 155 160Phe Ala Gly Leu Val Phe Gly Asn Val Met Gln Gly Tyr Ser Gly Thr 165 170 175Trp Trp Lys Asp Arg His Asn Gly His His Ala Ala Thr Asn Val Gln 180 185 190Gly His Asp Pro Asp Ile Asp Asp Leu Pro Val Leu Ala Trp Ser Pro 195 200 205Glu Asp Val Lys Asn Ala Gly Pro Thr Thr Arg Lys Leu Ile Lys Trp 210 215 220Gln Gln Tyr Tyr Phe Leu Pro Thr Ile Ala Thr Leu Arg Phe Ile Trp225 230 235 240Cys Phe Gln Ser Ile Leu Ala Val Met Ala Tyr Lys Thr Asp Ala Arg 245 250 255Asn Ile Tyr Tyr Gln Arg Gln Tyr Ala Lys Glu Ala Val Gly Leu Ala 260 265 270Leu His Trp Ile Leu Lys Gly Val Phe Met Phe Cys Tyr Met Pro Gly 275 280 285Ile Leu Thr Gly Leu Ala Phe Phe Leu Ile Ser Glu Cys Leu Gly Gly 290 295 300Phe Gly Ile Ala Ile Val Val Phe Leu Asn His Tyr Pro Leu Glu Lys305 310 315 320Val Glu Glu Ser Val Trp Asp Ser His Gly Phe Cys Ala Gly Gln Ile 325 330 335His Thr Thr Met Asn Ile Gln Arg Gly Val Ile Val Asp Trp Phe Phe 340 345 350Gly Gly Leu Asn Tyr Gln Ile Glu His His Leu Trp Pro Thr Leu Pro 355 360 365Arg His His Leu Lys Ala Ala Ser Phe Glu Val Glu Lys Ile Cys Gln 370 375 380Lys His Lys Leu Pro Tyr Arg Ala Pro Pro Met Ser Asp Gly Val Ala385 390 395 400Gln Leu Leu Gly Phe Leu Gly Lys Ile Ala Lys Leu Ala Ala Val Pro 405 410 415Val482063DNAEutreptiella cf_gymnastica CCMP1594misc_feature(716)..(716)n is a, c, g, or t 48cttgttggta ttagtaatgg gacacagcag tatattttcc cattgagaaa aacgctgaaa 60tactggttgg tgaaaacttg gtgagaacaa cggtccattt gaacacagct tcccaccgcc 120tttccccctt atctcatgtt gctggaccac actgcaagct gcatgagcga tagctgaacg 180agacttcacg ctgtcatccc ttcacttcat atgcgttgtg caagggaaag ggtgccatca 240ggtgtgactg tgcctccgtg ataaagtcga gggcacactc cgaattgggc agttctcgct 300accgtgacca gatgcgtgtc aaaactagat cccgaagaaa acgcccgcgc ggagagcctt 360gacacagttg tgttgaagaa aagtttgtgt ggcttcggag cgaaaaagac accgcaccat 420agctgtggca gtgcaagacc ccagatccgc tggtccctgc acttgttgaa gcctcaaaat 480gtcacctaaa cgggacgcat tgcctctgac aattgatggc accacgtacg acgtttccgc 540ttgggtaaac catcaccctg gaggggctca aatcattgaa aactaccgga accgagatgc 600taccgacgtg ttcatggtca tgcattcaca gcaggcgctc aacaagttga agcggatgcc 660tgttatggag ccctcttcac cacttactcc caagagccca agtgacgaca tttccnagga 720tttccgcaag ctccgcaaca gtatggttga gaagggtatg ttcaacgcgt cccctctgtt 780ttatgtgtac aaatcactga ccactgtcgc ccttggcgcc gtgggtgttc tcatggttat 840gtacctgcag tggtactacg tttcagccat gtttttggga ctttgctacc aacagctggg 900ttgggtggcg catgactacg cgcatcacca ggttttcacg aaccgtgatt atggcaatct 960tggtgggctt ttctttggcn acgttctcca aggatattct ttgacttggt ggaaggacag 1020gcacaacggc catcacgccg ccacaaacgt gcaaggacat gaccccgaca ttgataatct 1080ccccgttttg gcttggtcgc cagaggacgt caagaatgcc ggacctggaa cccgcaatat 1140catcaagtac cagcagtatt atttcctccc taccatcgcc atccttcggt tcatctggtg 1200tttccaaagc attctggggg tgatgtcata caagacagac tccnagaatc tctattacaa 1260acggcagtac cggagagagg cagccggtct ggcgctgcac tggattctga agagcgtttt 1320cttgttctgt tacatgccaa gtttcctcac tggcctggcg tttttcctta tctcggagtg 1380tctgggcggc tttgggatcg cgattgtggt gtttttgaac cactatccgc tggataaggt 1440tgaggaatcc gtttgggatg gtcacggttt ctgtgctggg cagatcctca caaccatgaa 1500catccaacgc ggactcatca ctgactggtt ctttggaggt ttgaattacc agattgagca 1560tcatctgtgg cccaaccttc caagacacca tttgaaagca gtttcctttg aggttgagaa 1620attgtgccag aagcacaacc tgccctacag agctccgccg atgcatactg gtgttgcaca 1680attgcttgga tatttgggga agattgctca gttggctgct gtcccagtat aaccctggat 1740caccttcatc gatcctattc tgagtgttca gggtgcgact gcatcccccg tttttcttgc 1800acagccgcgc atttgcaggg tggtttctat tacaattttt tttctgccca aaacaacacc 1860tgatgtggcg agcgaggttc actcttgctg cacaccactc attttgttct gggttgagtt 1920atatgtgaat taatatgtaa gcagtttnct tgcacagccc ggggcatttt ttntattccc 1980aagacagcgt gataacattt ggcgggcgag gttcactctt gctgcacacc atcatgtttt 2040gggtgcccag cccccccccc cct 206349417PRTEutreptiella cf_gymnastica CCMP1594misc_feature(80)..(80)Xaa can be any naturally occurring amino acid 49Met Ser Pro Lys Arg Asp Ala Leu Pro Leu Thr Ile Asp Gly Thr Thr1 5 10 15Tyr Asp Val Ser Ala Trp Val Asn His His Pro Gly Gly Ala Gln Ile 20 25 30Ile Glu Asn Tyr Arg Asn Arg Asp Ala Thr Asp Val Phe Met Val Met 35 40 45His Ser Gln Gln Ala Leu Asn Lys Leu Lys Arg Met Pro Val Met Glu 50 55 60Pro Ser Ser Pro Leu Thr Pro Lys Ser Pro Ser Asp Asp Ile Ser Xaa65 70 75 80Asp Phe Arg Lys Leu Arg Asn Ser Met Val Glu Lys Gly Met Phe Asn 85 90 95Ala Ser Pro Leu Phe Tyr Val Tyr Lys Ser Leu Thr Thr Val Ala Leu 100 105 110Gly Ala Val Gly Val Leu Met Val Met Tyr Leu Gln Trp Tyr Tyr Val 115 120 125Ser Ala Met Phe Leu Gly Leu Cys Tyr Gln Gln Leu Gly Trp Val Ala 130 135 140His Asp Tyr Ala His His Gln Val Phe Thr Asn Arg Asp Tyr Gly Asn145 150 155 160Leu Gly Gly Leu Phe Phe Gly Xaa Val Leu Gln Gly Tyr Ser Leu Thr 165 170 175Trp Trp Lys Asp Arg His Asn Gly His His Ala Ala Thr Asn Val Gln 180 185 190Gly His Asp Pro Asp Ile Asp Asn Leu Pro Val Leu Ala Trp Ser Pro 195 200 205Glu Asp Val Lys Asn Ala Gly Pro Gly Thr Arg Asn Ile Ile Lys Tyr 210 215 220Gln Gln Tyr Tyr Phe Leu Pro Thr Ile Ala Ile Leu Arg Phe Ile Trp225 230 235 240Cys Phe Gln Ser Ile Leu Gly Val Met Ser Tyr Lys Thr Asp Ser Xaa 245 250 255Asn Leu Tyr Tyr Lys Arg Gln Tyr Arg Arg Glu Ala Ala Gly Leu Ala 260 265 270Leu His Trp Ile Leu Lys Ser Val Phe Leu Phe Cys Tyr Met Pro Ser 275 280 285Phe Leu Thr Gly Leu Ala Phe Phe Leu Ile Ser Glu Cys Leu Gly Gly 290 295 300Phe Gly Ile Ala Ile Val Val Phe Leu Asn His Tyr Pro Leu Asp Lys305 310 315 320Val Glu Glu Ser Val Trp Asp Gly His Gly Phe Cys Ala Gly Gln Ile 325 330 335Leu Thr Thr Met Asn Ile Gln Arg Gly Leu Ile Thr Asp Trp Phe Phe 340 345 350Gly Gly Leu Asn Tyr Gln Ile Glu His His Leu Trp Pro Asn Leu Pro 355 360 365Arg His His Leu Lys Ala Val Ser Phe Glu Val Glu Lys Leu Cys Gln 370 375 380Lys His Asn Leu Pro Tyr Arg Ala Pro Pro Met His Thr Gly Val Ala385 390 395 400Gln Leu Leu Gly Tyr Leu Gly Lys Ile Ala Gln Leu Ala Ala Val Pro 405 410 415Val5060DNAArtificial SequenceTOPO linker 50tagaaggcac agtcgaggac ttatcctagc ctctgaatac tttcaacaag ttacaccctt 605124DNAArtificial SequenceLinkAmp primer 1 51aggcacagtc gaggacttat ccta 245226DNAArtificial SequenceLinkAmp primer 2 52gcctctgaat actttcaaca agttac 2653601DNATetruetreptia pomquetensis CCMP1491 53gggctcatca cgcttaggct tgggtgcaac aggtgtgtca ggagaggaag gctccataac 60aggcattctc ttcaacttgg cgacggcttc ttgagagtgc atcaccatga agacatcggt 120cgcatcgcgg ttgcgatagt tctcgataat gtcagctcct ccagggtggt gattgaccca 180agcagacaca tcataagttg cgccatcaat tgtgattggc agagcttgcc gcttaggaga 240catcttagct gcagatgagt ttacacggtt ctagcattgg tcgaaagacg tttctacagc 300cccaaagaat gtttgcatgt atttcataca tttcctactg agatagttcc tgtgcaaacg 360tttttggata cattgtttct ggtgtgacag gacaattttg ctgactgtaa gtaccgtgcc 420ggtgtgcatg cgcttcagcg ggttgatgga gccttacaac tggaatgcaa ctgcaattgg 480agtgcaagtt aatccgcgga ttggacagat gtggcactta gggaggggat gatttcgatt 540tcatgccggg ataagccgaa acactccgga ccgagtcaag ccacacttca gcaatttgtg 600t 6015420DNAArtificial Sequenceprimer AUAP 54ggccacgcgt cgactagtac 20551028DNATetruetreptia pomquetensis CCMP1491misc_feature(793)..(794)n is a, c, g, or t 55gcccttcttt gccaccggtt tgggatgctt cttgatctcc gagctgctcg gaggatttgg 60cattgccatc gttgtgtttc tgaatcacta tcctttggac aaggttgagg agactgtctg 120ggatgagcac gggttcagcg ccagccagat ccacgagacg ttgaacatta agcccggcct 180tctcaccgat tgggtctttg gtggtctcaa ctaccagatt gagcaccact tgtggcccaa 240catgcccagg cacaacctca cggcagcttc cctggaggtg cagaagttgt gcgccaagca 300caacctgccc tacagggccc cagccatcat ccccggggtt cagaaattgg tcagcttctt 360aggcgagatt gcccagctgg ctgctgtccc tgaatgattg gtgactaagc aagcgtcggc 420atggcgtgcg tgtgtggggc gggggttccc gcactgtaac ccgcggtgta acgcgcggtg 480gccgttccac ccaatcaaat gacaccacct gcgccaacca tcgctccccc accaaaccaa 540cccggaccaa aactaataaa catgtggttt tttccatcca ctggggccgc ctactccgcg 600ccgtgtgccc atcgtgcggg ttgtaccccc tccccaccta tgtctaatgc gtgtgtgcgt 660gcgtgtgtgc ctccttaatt gtacctattt atatctttcc tcctggggtg gcttttttac 720acccccttat catactctgg tcagcgcagt gcctgtgttg ccgctgagga tgaagaatgc 780atggcagggc gcnntttttt ttgcaccagt cacacatcca ccccttcgat aatgcccatg 840ctacgcatgc aaccaggtgt tggccaccgg tgcctatgcg tagcggtgag cctcgcgagg 900tgcatttggt caccaatccc caaggggcac ggcccccagt ttggttgtac acttgcatac 960agggactcaa tgggattttt gaattttgat catttataag catgactgct gaaaaaaaaa 1020aaaaaaaa 1028562233DNATetruetreptia pomquetensis CCMP1491 56acacaaattg ctgaagtgtg gcttgactcg gtccggagtg tttcggctta tcccggcatg 60aaatcgaaat catcccctcc ctaagtgcca catctgtcca atccgcggat taacttgcac 120tccaattgca gttgcattcc agttgtaagg ctccatcaac ccgctgaagc gcatgcacac 180cggcacggta cttacagtca gcaaaattgt cctgtcacac cagaaacaat gtatccaaaa 240acgtttgcac aggaactatc tcagtaggaa atgtatgaaa tacatgcaaa cattctttgg 300ggctgtagaa acgtctttcg accaatgcta gaaccgtgta aactcatctg cagctaagat 360gtctcctaag cggcaagctc tgccaatcac aattgatggc gcaacttatg atgtgtctgc 420ttgggtcaat caccaccctg gaggagctga cattatcgag aactatcgca accgcgatgc 480gaccgatgtc ttcatggtga tgcactctca agaagccgtc gccaagttga agagaatgcc 540tgttatggag ccttcctctc ctgacacacc tgttgcaccc aagcctaagc gtgatgagcc 600ccaggaggat ttccgcaagt tgcgggagga attcatctcc aagggtatgt tcgagacgag 660tttcctttgg tatttttaca agacttcaac taccgtcggt ttgatggtcc tttccatctt 720gatgaccgtg tacacgaatt ggtatttcac cgctgctttg gttcttggcg tgtgctacca 780acagctaggc tggttgtccc acgactattg ccatcaccag gttttcacaa accgcaagat 840taacgacgct ttcggtctct ttttcggtaa cgtgatgcag ggatactcac agacttggtg 900gaaggatagg cacaatggtc accatgccgc caccaatgtg gtcggccatg acccagatat 960tgataacctc cccatcctgg cttggtctcc cgaagatgtc aagagggcta ctccttcgac 1020tcggaatctc atcaagtacc agcagtacta cttcattccc accattgcat cccttaggtt 1080catctggtgc ctccaatcca tcggcggcgt catgtcctac aagagcgagg agaggaacct 1140gtactacaag cgccagtaca ctaaggaggc gattggtctg gccctccatt gggtgctcaa 1200ggccactttc tattgcagtg ccatgcctag ctttgccacc ggtttgggat gcttcttgat 1260ctccgagctg ctcggaggat ttggcattgc catcgttgtg tttctgaatc actatccttt 1320ggacaaggtt gaggagactg tctgggatga gcacgggttc agcgccagcc agatccacga 1380gacgttgaac attaagcccg gccttctcac cgattgggtc tttggtggtc tcaactacca 1440gattgagcac cacttgtggc ccaacatgcc caggcacaac ctcacggcag cttccctgga 1500ggtgcagaag ttgtgcgcca agcacaacct gccctacagg gccccagcca tcatccccgg 1560ggttcagaaa ttggtcagct tcttaggcga gattgcccag ctggctgctg tccctgaatg 1620attggtgact aagcaagcgt cggcatggcg tgcgtgtgtg gggcgggggt tcccgcactg 1680taacccgcgg tgtaacgcgc ggtggccgtt ccacccaatc aaatgacacc acctgcgcca 1740accatcgctc ccccaccaaa ccaacccgga ccaaaactaa taaacatgtg gttttttcca 1800tccactgggg ccgcctactc cgcgccgtgt gcccatcgtg cgggttgtac cccctcccca 1860cctatgtcta atgcgtgtgt gcgtgcgtgt gtgcctcctt aattgtacct atttatatct 1920ttcctcctgg ggtggctttt ttacaccccc ttatcatact ctggtcagcg cagtgcctgt 1980gttgccgctg aggatgaaga atgcatggca gggcgctttt ttttgcacca gtcacacatc 2040caccccttcg ataatgccca tgctacgcat gcaaccaggt gttggccacc ggtgcctatg 2100cgtagcggtg agcctcgcga ggtgcatttg gtcaccaatc cccaaggggc acggccccca 2160gtttggttgt acacttgcat acagggactc aatgggattt ttgaattttg atcatttata 2220agcatgactg ctg 223357420PRTTetruetreptia pomquetensis CCMP1491 57Met Ser Pro Lys Arg Gln Ala Leu Pro Ile Thr Ile Asp Gly Ala Thr1 5 10 15Tyr Asp Val Ser Ala Trp Val Asn His His Pro Gly Gly Ala Asp Ile 20 25 30Ile Glu Asn Tyr Arg Asn Arg Asp Ala Thr Asp Ala Phe Met Val Met 35 40 45His Ser Gln Glu Ala Val Ala Lys Leu Lys Arg Met Pro Val Met Glu 50 55 60Pro Ser Ser Pro Asp Thr Pro Val Ala Pro Lys Pro Lys Arg Asp Glu65 70 75 80Pro Gln Glu Asp Phe Arg Lys Leu Arg Glu Glu Phe Ile Ser Lys Gly 85 90 95Met Phe Glu Thr Ser Phe Leu Trp Tyr Phe Tyr Lys Thr Ser Thr Thr 100 105 110Val Gly Leu Met Val Leu Ser Ile Leu Met Thr Val Tyr Thr Asn Trp 115 120 125Tyr Phe Thr Ala Ala Leu Val Leu Gly Val Cys Tyr Gln Gln Leu Gly 130 135 140Trp Leu Ser His Asp Tyr Cys His His Gln Val Phe Thr Asn Arg Lys145 150 155 160Ile Asn Asp Ala Phe Gly Leu Phe Phe Gly Asn Val Met Gln Gly Tyr 165 170 175Ser Gln Thr Trp Trp Lys Asp Arg His Asn Gly His His Ala Ala Thr 180 185 190Asn Val Val Gly His Asp Pro Asp Ile Asp Asn Leu Pro Ile Leu Ala 195 200 205Trp Ser Pro Glu Asp Val Lys Arg Ala Thr Pro Ser

Thr Arg Asn Leu 210 215 220Ile Lys Tyr Gln Gln Tyr Tyr Phe Ile Pro Thr Ile Ala Ser Leu Arg225 230 235 240Phe Ile Trp Cys Leu Gln Ser Ile Gly Gly Val Met Ser Tyr Lys Ser 245 250 255Glu Glu Arg Asn Leu Tyr Tyr Lys Arg Arg Tyr Thr Lys Glu Ala Ile 260 265 270Gly Leu Ala Leu Pro Trp Val Leu Lys Ala Thr Phe Tyr Cys Ser Ala 275 280 285Met Pro Ser Phe Ala Thr Gly Leu Gly Cys Phe Leu Ile Ser Glu Leu 290 295 300Leu Gly Gly Phe Gly Ile Ala Ile Val Val Phe Leu Asn His Tyr Pro305 310 315 320Leu Asp Lys Val Glu Glu Thr Val Trp Asp Glu His Gly Phe Ser Ala 325 330 335Ser Gln Ile His Glu Thr Leu Asn Ile Lys Pro Gly Leu Leu Thr Asp 340 345 350Trp Val Phe Gly Gly Leu Asn Tyr Gln Ile Glu His His Leu Trp Pro 355 360 365Asn Met Pro Arg His Asn Leu Thr Ala Ala Ser Leu Glu Val Gln Lys 370 375 380Leu Cys Ala Lys His Asn Leu Pro Tyr Arg Ala Pro Ala Ile Ile Pro385 390 395 400Gly Val Gln Lys Leu Val Ser Phe Leu Gly Glu Ile Ala Gln Leu Ala 405 410 415Ala Val Pro Glu 4205831DNAArtificial Sequenceprimer TpomNot-5 58gcggccgcac catgtctcct aagcggcaag c 315927DNAArtificial Sequenceprimer TpomNot-3 59gcggccgctc attcagggac agcagcc 276019DNAArtificial Sequenceprimer T7 60ggaaacagct atgaccatg 196122DNAArtificial Sequenceprimer M13-28Rev 61gtaatacgac tcactatagg gc 22621260DNATetruetreptia pomquetensis CCMP1491 62atgtctccta agcggcaagc tctgccaatc acaattgatg gcgcaactta tgatgtgtct 60gcttgggtca atcaccaccc tggaggagct gacattatcg agaactatcg caaccgcgat 120gcgaccgatg tcttcatggt gatgcactct caagaagccg tcgccaagtt gaagagaatg 180cctgttatgg agccttcctc tcctgacaca cctgttgcac ccaagcctaa gcgtgatgag 240ccccaggagg atttccgcaa gttgcgggag gaattcatct ccaagggtat gttcgagacg 300agtttccttt ggtattttta caagacttca actaccgtcg gtttgatggt cctttccatc 360ttgatgaccg tgtacacgaa ttggtatttc accgctgctt tggttcttgg cgtgtgctac 420caacagctag gctggttgtc ccacgactat tgccatcacc aggttttcac aaaccgcaag 480attaacgacg ctttcggtct ctttttcggt aacgtgatgc agggatactc acagacttgg 540tggaaggata ggcacaatgg tcaccatgcc gccaccaatg tggtcggcca tgacccagat 600attgataacc tccccatcct ggcttggtct cccgaagatg tcaagagggc tactccttcg 660actcggaatc tcatcaagta ccagcagtac tacttcattc ccaccattgc atcccttagg 720ttcatctggt gcctccaatc catcggcggc gtcatgtcct acaagagcga ggagaggaac 780ctgtactaca agcgccagta cactaaggag gcgattggtc tggccctcca ctgggtgctc 840aaggccactt tctattgcag tgccatgcct agctttgcca ccggtttggg atgcttcttg 900atctccgagc tgctcggagg atttggcatt gccatcgttg tgtttctgaa tcactatcct 960ttggacaagg ttgaggagac tgtctgggat gagcacgggt tcagcgccag ccagatccac 1020gagacgttga acattaagcc cggccttctc accgattggg tctttggtgg tctcaactac 1080cagattgagc accacttgtg gcccaacatg cccaggcaca acctcacggc agcttccctg 1140gaggtgcaga agttgtgcgc caagcacaac ctgccctaca gggccccagc catcatcccc 1200ggggttcaga aattggtcag cttcttaggc gagattgccc agctggctgc tgtccctgaa 1260634300DNAArtificial Sequenceplasmid pLF114-10 63taatacgact cactataggg cgaattgggc ccgacgtcgc atgctcccgg ccgccatggc 60ggccgcggga attcgattgg cggccgcacc atgtctccta agcggcaagc tctgccaatc 120acaattgatg gcgcaactta tgatgtgtct gcttgggtca atcaccaccc tggaggagct 180gacattatcg agaactatcg caaccgcgat gcgaccgatg tcttcatggt gatgcactct 240caagaagccg tcgccaagtt gaagagaatg cctgttatgg agccttcctc tcctgacaca 300cctgttgcac ccaagcctaa gcgtgatgag ccccaggagg atttccgcaa gttgcgggag 360gaattcatct ccaagggtat gttcgagacg agtttccttt ggtattttta caagacttca 420actaccgtcg gtttgatggt cctttccatc ttgatgaccg tgtacacgaa ttggtatttc 480accgctgctt tggttcttgg cgtgtgctac caacagctag gctggttgtc ccacgactat 540tgccatcacc aggttttcac aaaccgcaag attaacgacg ctttcggtct ctttttcggt 600aacgtgatgc agggatactc acagacttgg tggaaggata ggcacaatgg tcaccatgcc 660gccaccaatg tggtcggcca tgacccagat attgataacc tccccatcct ggcttggtct 720cccgaagatg tcaagagggc tactccttcg actcggaatc tcatcaagta ccagcagtac 780tacttcattc ccaccattgc atcccttagg ttcatctggt gcctccaatc catcggcggc 840gtcatgtcct acaagagcga ggagaggaac ctgtactaca agcgccagta cactaaggag 900gcgattggtc tggccctcca ctgggtgctc aaggccactt tctattgcag tgccatgcct 960agctttgcca ccggtttggg atgcttcttg atctccgagc tgctcggagg atttggcatt 1020gccatcgttg tgtttctgaa tcactatcct ttggacaagg ttgaggagac tgtctgggat 1080gagcacgggt tcagcgccag ccagatccac gagacgttga acattaagcc cggccttctc 1140accgattggg tctttggtgg tctcaactac cagattgagc accacttgtg gcccaacatg 1200cccaggcaca acctcacggc agcttccctg gaggtgcaga agttgtgcgc caagcacaac 1260ctgccctaca gggccccagc catcatcccc ggggttcaga aattggtcag cttcttaggc 1320gagattgccc agctggctgc tgtccctgaa tgagcggccg caatcactag tgaattcgcg 1380gccgcctgca ggtcgaccat atgggagagc tcccaacgcg ttggatgcat agcttgagta 1440ttctatagtg tcacctaaat agcttggcgt aatcatggtc atagctgttt cctgtgtgaa 1500attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct 1560ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc 1620agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 1680gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 1740ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 1800gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 1860aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 1920gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 1980ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 2040cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 2100cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 2160gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 2220cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 2280agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg 2340ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 2400ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 2460gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 2520cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 2580attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 2640accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 2700ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 2760gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc 2820agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 2880ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 2940ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 3000gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 3060ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 3120tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 3180tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 3240cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 3300tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 3360gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 3420tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 3480ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 3540attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc 3600cgcgcacatt tccccgaaaa gtgccacctg atgcggtgtg aaataccgca cagatgcgta 3660aggagaaaat accgcatcag gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa 3720atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata 3780aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac 3840tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc 3900cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa 3960atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg 4020cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg 4080tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcca 4140ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt 4200acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt 4260ttcccagtca cgacgttgta aaacgacggc cagtgaattg 4300647518DNAArtificial Sequenceprimer pY-75 64ggccgcaggg cggatccccc gggctgcagg aattcgatat caagcttatc gataccgtcg 60acctcgaggg ggggcccggt acccaattcg ccctatagtg agtcgtatta cgcgcgctca 120ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc 180cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc 240ccttcccaac agttgcgcag cctgaatggc gaatggcgcg acgcgccctg tagcggcgca 300ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta 360gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt 420caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac 480cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt 540tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga 600acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg 660gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata 720ttaacgttta caatttcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 780accgcatatc gacggtcgag gagaacttct agtatatcca catacctaat attattgcct 840tattaaaaat ggaatcccaa caattacatc aaaatccaca ttctcttcaa aatcaattgt 900cctgtacttc cttgttcatg tgtgttcaaa aacgttatat ttataggata attatactct 960atttctcaac aagtaattgg ttgtttggcc gagcggtcta aggcgcctga ttcaagaaat 1020atcttgaccg cagttaactg tgggaatact caggtatcgt aagatgcaag agttcgaatc 1080tcttagcaac cattattttt ttcctcaaca taacgagaac acacaggggc gctatcgcac 1140agaatcaaat tcgatgactg gaaatttttt gttaatttca gaggtcgcct gacgcatata 1200cctttttcaa ctgaaaaatt gggagaaaaa ggaaaggtga gaggccggaa ccggcttttc 1260atatagaata gagaagcgtt catgactaaa tgcttgcatc acaatacttg aagttgacaa 1320tattatttaa ggacctattg ttttttccaa taggtggtta gcaatcgtct tactttctaa 1380cttttcttac cttttacatt tcagcaatat atatatatat ttcaaggata taccattcta 1440atgtctgccc ctatgtctgc ccctaagaag atcgtcgttt tgccaggtga ccacgttggt 1500caagaaatca cagccgaagc cattaaggtt cttaaagcta tttctgatgt tcgttccaat 1560gtcaagttcg atttcgaaaa tcatttaatt ggtggtgctg ctatcgatgc tacaggtgtc 1620ccacttccag atgaggcgct ggaagcctcc aagaaggttg atgccgtttt gttaggtgct 1680gtggctggtc ctaaatgggg taccggtagt gttagacctg aacaaggttt actaaaaatc 1740cgtaaagaac ttcaattgta cgccaactta agaccatgta actttgcatc cgactctctt 1800ttagacttat ctccaatcaa gccacaattt gctaaaggta ctgacttcgt tgttgtcaga 1860gaattagtgg gaggtattta ctttggtaag agaaaggaag acgatggtga tggtgtcgct 1920tgggatagtg aacaatacac cgttccagaa gtgcaaagaa tcacaagaat ggccgctttc 1980atggccctac aacatgagcc accattgcct atttggtcct tggataaagc taatcttttg 2040gcctcttcaa gattatggag aaaaactgtg gaggaaacca tcaagaacga attccctaca 2100ttgaaggttc aacatcaatt gattgattct gccgccatga tcctagttaa gaacccaacc 2160cacctaaatg gtattataat caccagcaac atgtttggtg atatcatctc cgatgaagcc 2220tccgttatcc caggttcctt gggtttgttg ccatctgcgt ccttggcctc tttgccagac 2280aagaacaccg catttggttt gtacgaacca tgccacggtt ctgctccaga tttgccaaag 2340aataaggttg accctatcgc cactatcttg tctgctgcaa tgatgttgaa attgtcattg 2400aacttgcctg aagaaggtaa ggccattgaa gatgcagtta aaaaggtttt ggatgcaggt 2460atcagaactg gtgatttagg tggttccaac agtaccaccg aagtcggtga tgctgtcgcc 2520gaagaagtta agaaaatcct tgcttaaaaa gattctcttt ttttatgata tttgtacata 2580aactttataa atgaaattca taatagaaac gacacgaaat tacaaaatgg aatatgttca 2640tagggtagac gaaactatat acgcaatcta catacattta tcaagaagga gaaaaaggag 2700gatagtaaag gaatacaggt aagcaaattg atactaatgg ctcaacgtga taaggaaaaa 2760gaattgcact ttaacattaa tattgacaag gaggagggca ccacacaaaa agttaggtgt 2820aacagaaaat catgaaacta cgattcctaa tttgatattg gaggattttc tctaaaaaaa 2880aaaaaataca acaaataaaa aacactcaat gacctgacca tttgatggag tttaagtcaa 2940taccttcttg aagcatttcc cataatggtg aaagttccct caagaatttt actctgtcag 3000aaacggcctt acgacgtagt cgatatggtg cactctcagt acaatctgct ctgatgccgc 3060atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 3120gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 3180gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt 3240ataggttaat gtcatgataa taatggtttc ttagtatgat ccaatatcaa aggaaatgat 3300agcattgaag gatgagacta atccaattga ggagtggcag catatagaac agctaaaggg 3360tagtgctgaa ggaagcatac gataccccgc atggaatggg ataatatcac aggaggtact 3420agactacctt tcatcctaca taaatagacg catataagta cgcatttaag cataaacacg 3480cactatgccg ttcttctcat gtatatatat atacaggcaa cacgcagata taggtgcgac 3540gtgaacagtg agctgtatgt gcgcagctcg cgttgcattt tcggaagcgc tcgttttcgg 3600aaacgctttg aagttcctat tccgaagttc ctattctcta gaaagtatag gaacttcaga 3660gcgcttttga aaaccaaaag cgctctgaag acgcactttc aaaaaaccaa aaacgcaccg 3720gactgtaacg agctactaaa atattgcgaa taccgcttcc acaaacattg ctcaaaagta 3780tctctttgct atatatctct gtgctatatc cctatataac ctacccatcc acctttcgct 3840ccttgaactt gcatctaaac tcgacctcta cattttttat gtttatctct agtattactc 3900tttagacaaa aaaattgtag taagaactat tcatagagtg aatcgaaaac aatacgaaaa 3960tgtaaacatt tcctatacgt agtatataga gacaaaatag aagaaaccgt tcataatttt 4020ctgaccaatg aagaatcatc aacgctatca ctttctgttc acaaagtatg cgcaatccac 4080atcggtatag aatataatcg gggatgcctt tatcttgaaa aaatgcaccc gcagcttcgc 4140tagtaatcag taaacgcggg aagtggagtc aggctttttt tatggaagag aaaatagaca 4200ccaaagtagc cttcttctaa ccttaacgga cctacagtgc aaaaagttat caagagactg 4260cattatagag cgcacaaagg agaaaaaaag taatctaaga tgctttgtta gaaaaatagc 4320gctctcggga tgcatttttg tagaacaaaa aagaagtata gattctttgt tggtaaaata 4380gcgctctcgc gttgcatttc tgttctgtaa aaatgcagct cagattcttt gtttgaaaaa 4440ttagcgctct cgcgttgcat ttttgtttta caaaaatgaa gcacagattc ttcgttggta 4500aaatagcgct ttcgcgttgc atttctgttc tgtaaaaatg cagctcagat tctttgtttg 4560aaaaattagc gctctcgcgt tgcatttttg ttctacaaaa tgaagcacag atgcttcgtt 4620caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac 4680attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa 4740aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat 4800tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc 4860agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga 4920gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg 4980cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc 5040agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag 5100taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc 5160tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg 5220taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg 5280acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac 5340ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac 5400cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg 5460agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg 5520tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg 5580agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac 5640tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg 5700ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 5760tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 5820aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 5880tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt 5940agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 6000taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 6060caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 6120agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 6180aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 6240gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 6300tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 6360gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 6420ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct 6480ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg 6540aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt 6600aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta 6660atgtgagtta cctcactcat taggcacccc aggctttaca ctttatgctt ccggctccta 6720tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt 6780acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctc caccgcggga 6840tttcgaaact aagttcttgg tgttttaaaa ctaaaaaaaa gactaactat aaaagtagaa 6900tttaagaagt ttaagaaata gatttacaga attacaatca atacctaccg tctttatata 6960cttattagtc aagtagggga ataatttcag ggaactggtt tcaacctttt ttttcagctt 7020tttccaaatc agagagagca gaaggtaata gaaggtgtaa gaaaatgaga tagatacatg 7080cgtgggtcaa ttgccttgtg tcatcattta ctccaggcag gttgcatcac tccattgagg 7140ttgtgcccgt tttttgcctg tttgtgcccc tgttctctgt agttgcgcta agagaatgga 7200cctatgaact gatggttggt gaagaaaaca atattttggt gctgggattc tttttttttc 7260tggatgccag cttaaaaagc gggctccatt atatttagtg gatgccagga ataaactgtt 7320cacccagaca cctacgatgt tatatattct gtgtaacccg ccccctattt tgggcatgta 7380cgggttacag cagaattaaa aggctaattt tttgactaaa taaagttagg aaaatcacta 7440ctattaatta tttacgtatt ctttgaaatg gcagtattga taatgataaa ctcgaaatca 7500ctagtggatc cgcccagc 7518658792DNAArtificial Sequenceplasmid pY126 65ggccgcaggg cggatccccc gggctgcagg aattcgatat caagcttatc gataccgtcg 60acctcgaggg ggggcccggt acccaattcg ccctatagtg

agtcgtatta cgcgcgctca 120ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc 180cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc 240ccttcccaac agttgcgcag cctgaatggc gaatggcgcg acgcgccctg tagcggcgca 300ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta 360gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt 420caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac 480cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt 540tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga 600acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg 660gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata 720ttaacgttta caatttcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 780accgcatatc gacggtcgag gagaacttct agtatatcca catacctaat attattgcct 840tattaaaaat ggaatcccaa caattacatc aaaatccaca ttctcttcaa aatcaattgt 900cctgtacttc cttgttcatg tgtgttcaaa aacgttatat ttataggata attatactct 960atttctcaac aagtaattgg ttgtttggcc gagcggtcta aggcgcctga ttcaagaaat 1020atcttgaccg cagttaactg tgggaatact caggtatcgt aagatgcaag agttcgaatc 1080tcttagcaac cattattttt ttcctcaaca taacgagaac acacaggggc gctatcgcac 1140agaatcaaat tcgatgactg gaaatttttt gttaatttca gaggtcgcct gacgcatata 1200cctttttcaa ctgaaaaatt gggagaaaaa ggaaaggtga gaggccggaa ccggcttttc 1260atatagaata gagaagcgtt catgactaaa tgcttgcatc acaatacttg aagttgacaa 1320tattatttaa ggacctattg ttttttccaa taggtggtta gcaatcgtct tactttctaa 1380cttttcttac cttttacatt tcagcaatat atatatatat ttcaaggata taccattcta 1440atgtctgccc ctatgtctgc ccctaagaag atcgtcgttt tgccaggtga ccacgttggt 1500caagaaatca cagccgaagc cattaaggtt cttaaagcta tttctgatgt tcgttccaat 1560gtcaagttcg atttcgaaaa tcatttaatt ggtggtgctg ctatcgatgc tacaggtgtc 1620ccacttccag atgaggcgct ggaagcctcc aagaaggttg atgccgtttt gttaggtgct 1680gtggctggtc ctaaatgggg taccggtagt gttagacctg aacaaggttt actaaaaatc 1740cgtaaagaac ttcaattgta cgccaactta agaccatgta actttgcatc cgactctctt 1800ttagacttat ctccaatcaa gccacaattt gctaaaggta ctgacttcgt tgttgtcaga 1860gaattagtgg gaggtattta ctttggtaag agaaaggaag acgatggtga tggtgtcgct 1920tgggatagtg aacaatacac cgttccagaa gtgcaaagaa tcacaagaat ggccgctttc 1980atggccctac aacatgagcc accattgcct atttggtcct tggataaagc taatcttttg 2040gcctcttcaa gattatggag aaaaactgtg gaggaaacca tcaagaacga attccctaca 2100ttgaaggttc aacatcaatt gattgattct gccgccatga tcctagttaa gaacccaacc 2160cacctaaatg gtattataat caccagcaac atgtttggtg atatcatctc cgatgaagcc 2220tccgttatcc caggttcctt gggtttgttg ccatctgcgt ccttggcctc tttgccagac 2280aagaacaccg catttggttt gtacgaacca tgccacggtt ctgctccaga tttgccaaag 2340aataaggttg accctatcgc cactatcttg tctgctgcaa tgatgttgaa attgtcattg 2400aacttgcctg aagaaggtaa ggccattgaa gatgcagtta aaaaggtttt ggatgcaggt 2460atcagaactg gtgatttagg tggttccaac agtaccaccg aagtcggtga tgctgtcgcc 2520gaagaagtta agaaaatcct tgcttaaaaa gattctcttt ttttatgata tttgtacata 2580aactttataa atgaaattca taatagaaac gacacgaaat tacaaaatgg aatatgttca 2640tagggtagac gaaactatat acgcaatcta catacattta tcaagaagga gaaaaaggag 2700gatagtaaag gaatacaggt aagcaaattg atactaatgg ctcaacgtga taaggaaaaa 2760gaattgcact ttaacattaa tattgacaag gaggagggca ccacacaaaa agttaggtgt 2820aacagaaaat catgaaacta cgattcctaa tttgatattg gaggattttc tctaaaaaaa 2880aaaaaataca acaaataaaa aacactcaat gacctgacca tttgatggag tttaagtcaa 2940taccttcttg aagcatttcc cataatggtg aaagttccct caagaatttt actctgtcag 3000aaacggcctt acgacgtagt cgatatggtg cactctcagt acaatctgct ctgatgccgc 3060atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 3120gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 3180gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt 3240ataggttaat gtcatgataa taatggtttc ttagtatgat ccaatatcaa aggaaatgat 3300agcattgaag gatgagacta atccaattga ggagtggcag catatagaac agctaaaggg 3360tagtgctgaa ggaagcatac gataccccgc atggaatggg ataatatcac aggaggtact 3420agactacctt tcatcctaca taaatagacg catataagta cgcatttaag cataaacacg 3480cactatgccg ttcttctcat gtatatatat atacaggcaa cacgcagata taggtgcgac 3540gtgaacagtg agctgtatgt gcgcagctcg cgttgcattt tcggaagcgc tcgttttcgg 3600aaacgctttg aagttcctat tccgaagttc ctattctcta gaaagtatag gaacttcaga 3660gcgcttttga aaaccaaaag cgctctgaag acgcactttc aaaaaaccaa aaacgcaccg 3720gactgtaacg agctactaaa atattgcgaa taccgcttcc acaaacattg ctcaaaagta 3780tctctttgct atatatctct gtgctatatc cctatataac ctacccatcc acctttcgct 3840ccttgaactt gcatctaaac tcgacctcta cattttttat gtttatctct agtattactc 3900tttagacaaa aaaattgtag taagaactat tcatagagtg aatcgaaaac aatacgaaaa 3960tgtaaacatt tcctatacgt agtatataga gacaaaatag aagaaaccgt tcataatttt 4020ctgaccaatg aagaatcatc aacgctatca ctttctgttc acaaagtatg cgcaatccac 4080atcggtatag aatataatcg gggatgcctt tatcttgaaa aaatgcaccc gcagcttcgc 4140tagtaatcag taaacgcggg aagtggagtc aggctttttt tatggaagag aaaatagaca 4200ccaaagtagc cttcttctaa ccttaacgga cctacagtgc aaaaagttat caagagactg 4260cattatagag cgcacaaagg agaaaaaaag taatctaaga tgctttgtta gaaaaatagc 4320gctctcggga tgcatttttg tagaacaaaa aagaagtata gattctttgt tggtaaaata 4380gcgctctcgc gttgcatttc tgttctgtaa aaatgcagct cagattcttt gtttgaaaaa 4440ttagcgctct cgcgttgcat ttttgtttta caaaaatgaa gcacagattc ttcgttggta 4500aaatagcgct ttcgcgttgc atttctgttc tgtaaaaatg cagctcagat tctttgtttg 4560aaaaattagc gctctcgcgt tgcatttttg ttctacaaaa tgaagcacag atgcttcgtt 4620caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac 4680attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa 4740aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat 4800tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc 4860agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga 4920gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg 4980cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc 5040agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag 5100taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc 5160tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg 5220taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg 5280acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac 5340ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac 5400cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg 5460agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg 5520tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg 5580agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac 5640tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg 5700ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 5760tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 5820aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 5880tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt 5940agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 6000taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 6060caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 6120agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 6180aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 6240gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 6300tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 6360gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 6420ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct 6480ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg 6540aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt 6600aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta 6660atgtgagtta cctcactcat taggcacccc aggctttaca ctttatgctt ccggctccta 6720tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt 6780acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctc caccgcggga 6840tttcgaaact aagttcttgg tgttttaaaa ctaaaaaaaa gactaactat aaaagtagaa 6900tttaagaagt ttaagaaata gatttacaga attacaatca atacctaccg tctttatata 6960cttattagtc aagtagggga ataatttcag ggaactggtt tcaacctttt ttttcagctt 7020tttccaaatc agagagagca gaaggtaata gaaggtgtaa gaaaatgaga tagatacatg 7080cgtgggtcaa ttgccttgtg tcatcattta ctccaggcag gttgcatcac tccattgagg 7140ttgtgcccgt tttttgcctg tttgtgcccc tgttctctgt agttgcgcta agagaatgga 7200cctatgaact gatggttggt gaagaaaaca atattttggt gctgggattc tttttttttc 7260tggatgccag cttaaaaagc gggctccatt atatttagtg gatgccagga ataaactgtt 7320cacccagaca cctacgatgt tatatattct gtgtaacccg ccccctattt tgggcatgta 7380cgggttacag cagaattaaa aggctaattt tttgactaaa taaagttagg aaaatcacta 7440ctattaatta tttacgtatt ctttgaaatg gcagtattga taatgataaa ctcgaaatca 7500ctagtggatc cgcccagcgg ccgcaccatg tctcctaagc ggcaagctct gccaatcaca 7560attgatggcg caacttatga tgtgtctgct tgggtcaatc accaccctgg aggagctgac 7620attatcgaga actatcgcaa ccgcgatgcg accgatgtct tcatggtgat gcactctcaa 7680gaagccgtcg ccaagttgaa gagaatgcct gttatggagc cttcctctcc tgacacacct 7740gttgcaccca agcctaagcg tgatgagccc caggaggatt tccgcaagtt gcgggaggaa 7800ttcatctcca agggtatgtt cgagacgagt ttcctttggt atttttacaa gacttcaact 7860accgtcggtt tgatggtcct ttccatcttg atgaccgtgt acacgaattg gtatttcacc 7920gctgctttgg ttcttggcgt gtgctaccaa cagctaggct ggttgtccca cgactattgc 7980catcaccagg ttttcacaaa ccgcaagatt aacgacgctt tcggtctctt tttcggtaac 8040gtgatgcagg gatactcaca gacttggtgg aaggataggc acaatggtca ccatgccgcc 8100accaatgtgg tcggccatga cccagatatt gataacctcc ccatcctggc ttggtctccc 8160gaagatgtca agagggctac tccttcgact cggaatctca tcaagtacca gcagtactac 8220ttcattccca ccattgcatc ccttaggttc atctggtgcc tccaatccat cggcggcgtc 8280atgtcctaca agagcgagga gaggaacctg tactacaagc gccagtacac taaggaggcg 8340attggtctgg ccctccactg ggtgctcaag gccactttct attgcagtgc catgcctagc 8400tttgccaccg gtttgggatg cttcttgatc tccgagctgc tcggaggatt tggcattgcc 8460atcgttgtgt ttctgaatca ctatcctttg gacaaggttg aggagactgt ctgggatgag 8520cacgggttca gcgccagcca gatccacgag acgttgaaca ttaagcccgg ccttctcacc 8580gattgggtct ttggtggtct caactaccag attgagcacc acttgtggcc caacatgccc 8640aggcacaacc tcacggcagc ttccctggag gtgcagaagt tgtgcgccaa gcacaacctg 8700ccctacaggg ccccagccat catccccggg gttcagaaat tggtcagctt cttaggcgag 8760attgcccagc tggctgctgt ccctgaatga gc 8792664993DNAArtificial Sequenceplasmid pKR123r 66ctagacctgc aggatataat gagccgtaaa caaagatgat taagtagtaa ttaatacgta 60ctagtaaaag tggcaaaaga taacgagaaa gaaccaattt ctttgcattc ggccttagcg 120gaaggcatat ataagctttg attattttat ttagtgtaat gatttcgtac aaccaaagca 180tttatttagt actctcacac ttgtgtcgcg gccgcttggg gggctatgga agactttctt 240agttagttgt gtgaataagc aatgttggga gaatcgggac tacttatagg ataggaataa 300aacagaaaag tattaagtgc taatgaaata tttagactga taattaaaat cttcacgtat 360gtccacttga tataaaaacg tcaggaataa aggaagtaca gtagaattta aaggtactct 420ttttatatat acccgtgttc tctttttggc tagctagttg cataaaaaat aatctatatt 480tttatcatta ttttaaatat cttatgagat ggtaaatatt tatcataatt ttttttacta 540ttatttatta tttgtgtgtg taatacatat agaagttaat tacaaatttt atttactttt 600tcattatttt gatatgattc accattaatt tagtgttatt atttataata gttcatttta 660atctttttgt atatattatg cgtgcagtac ttttttccta catataacta ctattacatt 720ttatttatat aatattttta ttaatgaatt ttcgtgataa tatgtaatat tgttcattat 780tatttcagat tttttaaaaa tatttgtgtt attatttatg aaatatgtaa tttttttagt 840atttgatttt atgatgataa agtgttctaa attcaaaaga agggggaaag cgtaaacatt 900aaaaaacgtc atcaaacaaa aacaaaatct tgttaataaa gataaaactg tttgttttga 960tcactgttat ttcgtaatat aaaaacatta tttatattta tattgttgac aaccaaattt 1020gcctatcaaa tctaaccaat ataatgcatg cgtggcaggt aatgtactac catgaactta 1080agtcatgaca taataaaccg tgaatctgac caatgcatgt acctanctaa attgtatttg 1140tgacacgaag caaatgattc aattcacaat ggagatggga aacaaataat gaagaaccca 1200gaactaagaa agcttttctg aaaaataaaa taaaggcaat gtcaaaagta tactgcatca 1260tcagtccaga aagcacatga tattttttta tcagtatcaa tgcagctagt tttattttac 1320aatatcgata tagctagttt aaatatattg cagctagatt tataaatatt tgtgttatta 1380tttatcattt gtgtaatcct gtttttagta ttttagttta tatatgatga taatgtattc 1440caaatttaaa agaagggaaa taaatttaaa caagaaaaaa agtcatcaaa caaaaaacaa 1500atgaaagggt ggaaagatgt taccatgtaa tgtgaatgtt acagtatttc ttttattata 1560gagttaacaa attaactaat atgattttgt taataatgat aaaatatttt ttttattatt 1620atttcataat ataaaaatag tttacttaat ataaaaaaaa ttctatcgtt cacaacaaag 1680ttggccacct aatttaacca tgcatgtacc catggaccat attaggtaac catcaaacct 1740gatgaagaga taaagagatg aagacttaag tcataacaca aaaccataaa aaacaaaaat 1800acaatcaacc gtcaatctga ccaatgcatg aaaaagctgc aatagtgagt ggcgacacaa 1860agcacatgat tttcttacaa cggagataaa accaaaaaaa tatttcatga acaacctaga 1920acaaataaag cttttatata ataaatatat aaataaataa aggctatgga ataatatact 1980tcaatatatt tggattaaat aaattgttgg cggggttgat atatttatac acacctaaag 2040tcacttcaat ctcattttca cttaactttt attttttttt tctttttatt tatcataaag 2100agaatattga taatatactt tttaacatat ttttatgaca ttttttattg gtgaaaactt 2160attaaaaatc ataaattttg taagttagat ttatttaaag agttcctctt cttattttaa 2220attttttaat aaatttttaa ataactaaaa tttgtgttaa aaatgttaaa aaatgtgtta 2280ttaacccttc tcttcgagga cgtacgtcta gagtcgacct gcaggcatgc aagcttggcg 2340taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 2400atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 2460ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 2520taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 2580tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 2640aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 2700aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 2760ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 2820acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 2880ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 2940tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 3000tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 3060gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 3120agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 3180tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 3240agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 3300tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 3360acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 3420tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 3480agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 3540tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 3600acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 3660tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 3720ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 3780agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 3840tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 3900acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 3960agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 4020actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 4080tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 4140gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 4200ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 4260tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 4320aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 4380tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 4440tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 4500gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg 4560ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg 4620gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg 4680tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta 4740ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc 4800atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc 4860tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta 4920acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt 4980acccggggat cct 4993676267DNAArtificial Sequenceplasmid pKR1007 67ggccgcgaca caagtgtgag agtactaaat aaatgctttg gttgtacgaa atcattacac 60taaataaaat aatcaaagct tatatatgcc ttccgctaag gccgaatgca aagaaattgg 120ttctttctcg ttatcttttg ccacttttac tagtacgtat taattactac ttaatcatct 180ttgtttacgg ctcattatat cctgcaggtc tagaggatcc ccgggtaccg agctcgaatt 240cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc 300gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc 360gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg tattttctcc 420ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg 480atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg 540cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt 600gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc 660tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc 720ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc 780cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga 840gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt 900ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag 960tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag 1020aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta 1080ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg 1140agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca 1200gtgctgccat aaccatgagt

gataacactg cggccaactt acttctgaca acgatcggag 1260gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc 1320gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg 1380tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc 1440ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg 1500cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg 1560gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga 1620cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac 1680tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa 1740aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca 1800aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag 1860gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac 1920cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa 1980ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc 2040accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag 2100tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac 2160cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc 2220gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc 2280ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca 2340cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc 2400tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg 2460ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct 2520ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata 2580ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc 2640gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg 2700acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca 2760ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg 2820tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagcttgcat 2880gcctgcaggt cgactctaga cgtacgtcct cgaagagaag ggttaataac acatttttta 2940acatttttaa cacaaatttt agttatttaa aaatttatta aaaaatttaa aataagaaga 3000ggaactcttt aaataaatct aacttacaaa atttatgatt tttaataagt tttcaccaat 3060aaaaaatgtc ataaaaatat gttaaaaagt atattatcaa tattctcttt atgataaata 3120aaaagaaaaa aaaaataaaa gttaagtgaa aatgagattg aagtgacttt aggtgtgtat 3180aaatatatca accccgccaa caatttattt aatccaaata tattgaagta tattattcca 3240tagcctttat ttatttatat atttattata taaaagcttt atttgttcta ggttgttcat 3300gaaatatttt tttggtttta tctccgttgt aagaaaatca tgtgctttgt gtcgccactc 3360actattgcag ctttttcatg cattggtcag attgacggtt gattgtattt ttgtttttta 3420tggttttgtg ttatgactta agtcttcatc tctttatctc ttcatcaggt ttgatggtta 3480cctaatatgg tccatgggta catgcatggt taaattaggt ggccaacttt gttgtgaacg 3540atagaatttt ttttatatta agtaaactat ttttatatta tgaaataata ataaaaaaaa 3600tattttatca ttattaacaa aatcatatta gttaatttgt taactctata ataaaagaaa 3660tactgtaaca ttcacattac atggtaacat ctttccaccc tttcatttgt tttttgtttg 3720atgacttttt ttcttgttta aatttatttc ccttctttta aatttggaat acattatcat 3780catatataaa ctaaaatact aaaaacagga ttacacaaat gataaataat aacacaaata 3840tttataaatc tagctgcaat atatttaaac tagctatatc gatattgtaa aataaaacta 3900gctgcattga tactgataaa aaaatatcat gtgctttctg gactgatgat gcagtatact 3960tttgacattg cctttatttt atttttcaga aaagctttct tagttctggg ttcttcatta 4020tttgtttccc atctccattg tgaattgaat catttgcttc gtgtcacaaa tacaatttag 4080ntaggtacat gcattggtca gattcacggt ttattatgtc atgacttaag ttcatggtag 4140tacattacct gccacgcatg cattatattg gttagatttg ataggcaaat ttggttgtca 4200acaatataaa tataaataat gtttttatat tacgaaataa cagtgatcaa aacaaacagt 4260tttatcttta ttaacaagat tttgtttttg tttgatgacg ttttttaatg tttacgcttt 4320cccccttctt ttgaatttag aacactttat catcataaaa tcaaatacta aaaaaattac 4380atatttcata aataataaca caaatatttt taaaaaatct gaaataataa tgaacaatat 4440tacatattat cacgaaaatt cattaataaa aatattatat aaataaaatg taatagtagt 4500tatatgtagg aaaaaagtac tgcacgcata atatatacaa aaagattaaa atgaactatt 4560ataaataata acactaaatt aatggtgaat catatcaaaa taatgaaaaa gtaaataaaa 4620tttgtaatta acttctatat gtattacaca cacaaataat aaataatagt aaaaaaaatt 4680atgataaata tttaccatct cataagatat ttaaaataat gataaaaata tagattattt 4740tttatgcaac tagctagcca aaaagagaac acgggtatat ataaaaagag tacctttaaa 4800ttctactgta cttcctttat tcctgacgtt tttatatcaa gtggacatac gtgaagattt 4860taattatcag tctaaatatt tcattagcac ttaatacttt tctgttttat tcctatccta 4920taagtagtcc cgattctccc aacattgctt attcacacaa ctaactaaga aagtcttcca 4980tagcccccca agcggccgca ccatgtctcc taagcggcaa gctctgccaa tcacaattga 5040tggcgcaact tatgatgtgt ctgcttgggt caatcaccac cctggaggag ctgacattat 5100cgagaactat cgcaaccgcg atgcgaccga tgtcttcatg gtgatgcact ctcaagaagc 5160cgtcgccaag ttgaagagaa tgcctgttat ggagccttcc tctcctgaca cacctgttgc 5220acccaagcct aagcgtgatg agccccagga ggatttccgc aagttgcggg aggaattcat 5280ctccaagggt atgttcgaga cgagtttcct ttggtatttt tacaagactt caactaccgt 5340cggtttgatg gtcctttcca tcttgatgac cgtgtacacg aattggtatt tcaccgctgc 5400tttggttctt ggcgtgtgct accaacagct aggctggttg tcccacgact attgccatca 5460ccaggttttc acaaaccgca agattaacga cgctttcggt ctctttttcg gtaacgtgat 5520gcagggatac tcacagactt ggtggaagga taggcacaat ggtcaccatg ccgccaccaa 5580tgtggtcggc catgacccag atattgataa cctccccatc ctggcttggt ctcccgaaga 5640tgtcaagagg gctactcctt cgactcggaa tctcatcaag taccagcagt actacttcat 5700tcccaccatt gcatccctta ggttcatctg gtgcctccaa tccatcggcg gcgtcatgtc 5760ctacaagagc gaggagagga acctgtacta caagcgccag tacactaagg aggcgattgg 5820tctggccctc cactgggtgc tcaaggccac tttctattgc agtgccatgc ctagctttgc 5880caccggtttg ggatgcttct tgatctccga gctgctcgga ggatttggca ttgccatcgt 5940tgtgtttctg aatcactatc ctttggacaa ggttgaggag actgtctggg atgagcacgg 6000gttcagcgcc agccagatcc acgagacgtt gaacattaag cccggccttc tcaccgattg 6060ggtctttggt ggtctcaact accagattga gcaccacttg tggcccaaca tgcccaggca 6120caacctcacg gcagcttccc tggaggtgca gaagttgtgc gccaagcaca acctgcccta 6180cagggcccca gccatcatcc ccggggttca gaaattggtc agcttcttag gcgagattgc 6240ccagctggct gctgtccctg aatgagc 6267687887DNAArtificial Sequenceplasmid pKR607 68ggccgcagta tatcttaaat tctttaatac ggtgtactag gatattgaac tggttcttga 60tgatgaaaac ctgggccgag attgcagcta tttatagtca taggtcttgt taacatgcat 120ggacatttgg ccacggggtg gcatgcagtt tgacgggtgt tgaaataaac aaaaatgagg 180tggcggaaga gaatacgagt ttgaggttgg gttagaaaca acaaatgtga gggctcatga 240tgggttgagt tggtgaatgt tttgggctgc tcgattgaca cctttgtgag tacgtgttgt 300tgtgcatggc ttttggggtc cagttttttt ttcttgacgc ggcgatcctg atcagctagt 360ggataagtga tgtccactgt gtgtgattgc gtttttgttt gaattttatg aacttagaca 420ttgctatgca aaggatactc tcattgtgtt ttgtcttctt ttgttccttg gctttttctt 480atgatccaag agactagtca gtgttgtggc attcgagact accaagatta attatgatgg 540gggaaggata agtaactgat tagtacggac tgttaccaaa ttaattaata agcggcaaat 600gaagggcatg gatcaaaagc ttggatctcc tgcaggatct ggccggccgg atctcgtacg 660gatccgtcga cggcgcgccc gatcatccgg atatagttcc tcctttcagc aaaaaacccc 720tcaagacccg tttagaggcc ccaaggggtt atgctagtta ttgctcagcg gtggcagcag 780ccaactcagc ttcctttcgg gctttgttag cagccggatc gatccaagct gtacctcact 840attcctttgc cctcggacga gtgctggggc gtcggtttcc actatcggcg agtacttcta 900cacagccatc ggtccagacg gccgcgcttc tgcgggcgat ttgtgtacgc ccgacagtcc 960cggctccgga tcggacgatt gcgtcgcatc gaccctgcgc ccaagctgca tcatcgaaat 1020tgccgtcaac caagctctga tagagttggt caagaccaat gcggagcata tacgcccgga 1080gccgcggcga tcctgcaagc tccggatgcc tccgctcgaa gtagcgcgtc tgctgctcca 1140tacaagccaa ccacggcctc cagaagaaga tgttggcgac ctcgtattgg gaatccccga 1200acatcgcctc gctccagtca atgaccgctg ttatgcggcc attgtccgtc aggacattgt 1260tggagccgaa atccgcgtgc acgaggtgcc ggacttcggg gcagtcctcg gcccaaagca 1320tcagctcatc gagagcctgc gcgacggacg cactgacggt gtcgtccatc acagtttgcc 1380agtgatacac atggggatca gcaatcgcgc atatgaaatc acgccatgta gtgtattgac 1440cgattccttg cggtccgaat gggccgaacc cgctcgtctg gctaagatcg gccgcagcga 1500tcgcatccat agcctccgcg accggctgca gaacagcggg cagttcggtt tcaggcaggt 1560cttgcaacgt gacaccctgt gcacggcggg agatgcaata ggtcaggctc tcgctgaatt 1620ccccaatgtc aagcacttcc ggaatcggga gcgcggccga tgcaaagtgc cgataaacat 1680aacgatcttt gtagaaacca tcggcgcagc tatttacccg caggacatat ccacgccctc 1740ctacatcgaa gctgaaagca cgagattctt cgccctccga gagctgcatc aggtcggaga 1800cgctgtcgaa cttttcgatc agaaacttct cgacagacgt cgcggtgagt tcaggctttt 1860ccatgggtat atctccttct taaagttaaa caaaattatt tctagaggga aaccgttgtg 1920gtctccctat agtgagtcgt attaatttcg cgggatcgag atcgatccaa ttccaatccc 1980acaaaaatct gagcttaaca gcacagttgc tcctctcaga gcagaatcgg gtattcaaca 2040ccctcatatc aactactacg ttgtgtataa cggtccacat gccggtatat acgatgactg 2100gggttgtaca aaggcggcaa caaacggcgt tcccggagtt gcacacaaga aatttgccac 2160tattacagag gcaagagcag cagctgacgc gtacacaaca agtcagcaaa cagacaggtt 2220gaacttcatc cccaaaggag aagctcaact caagcccaag agctttgcta aggccctaac 2280aagcccacca aagcaaaaag cccactggct cacgctagga accaaaaggc ccagcagtga 2340tccagcccca aaagagatct cctttgcccc ggagattaca atggacgatt tcctctatct 2400ttacgatcta ggaaggaagt tcgaaggtga aggtgacgac actatgttca ccactgataa 2460tgagaaggtt agcctcttca atttcagaaa gaatgctgac ccacagatgg ttagagaggc 2520ctacgcagca ggtctcatca agacgatcta cccgagtaac aatctccagg agatcaaata 2580ccttcccaag aaggttaaag atgcagtcaa aagattcagg actaattgca tcaagaacac 2640agagaaagac atatttctca agatcagaag tactattcca gtatggacga ttcaaggctt 2700gcttcataaa ccaaggcaag taatagagat tggagtctct aaaaaggtag ttcctactga 2760atctaaggcc atgcatggag tctaagattc aaatcgagga tctaacagaa ctcgccgtga 2820agactggcga acagttcata cagagtcttt tacgactcaa tgacaagaag aaaatcttcg 2880tcaacatggt ggagcacgac actctggtct actccaaaaa tgtcaaagat acagtctcag 2940aagaccaaag ggctattgag acttttcaac aaaggataat ttcgggaaac ctcctcggat 3000tccattgccc agctatctgt cacttcatcg aaaggacagt agaaaaggaa ggtggctcct 3060acaaatgcca tcattgcgat aaaggaaagg ctatcattca agatgcctct gccgacagtg 3120gtcccaaaga tggaccccca cccacgagga gcatcgtgga aaaagaagac gttccaacca 3180cgtcttcaaa gcaagtggat tgatgtgaca tctccactga cgtaagggat gacgcacaat 3240cccactatcc ttcgcaagac ccttcctcta tataaggaag ttcatttcat ttggagagga 3300cacgctcgag ctcatttctc tattacttca gccataacaa aagaactctt ttctcttctt 3360attaaaccat gaaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa 3420agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca 3480gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct 3540acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc 3600ttgacattgg ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg 3660tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg 3720ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac 3780cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc 3840atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc 3900tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg 3960atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga 4020gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt 4080ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag 4140gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct 4200tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc 4260gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga 4320ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga 4380gggcaaagga atagtgaggt acctaaagaa ggagtgcgtc gaagcagatc gttcaaacat 4440ttggcaataa agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata 4500atttctgttg aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat 4560gagatgggtt tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa 4620aatatagcgc gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatcg 4680atgtcgaatc gatcaacctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc 4740gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 4800ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 4860acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 4920cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 4980caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 5040gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 5100tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc agttcggtgt 5160aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 5220ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 5280cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 5340tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc 5400tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 5460ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 5520aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 5580aagggatttt ggtcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc 5640tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 5700cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 5760ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 5820accatatgga catattgtcg ttagaacgcg gctacaatta atacataacc ttatgtatca 5880tacacatacg atttaggtga cactatagaa cggcgcgcca agcttgttga aacatccctg 5940aagtgtctca ttttatttta tttattcttt gctgataaaa aaataaaata aaagaagcta 6000agcacacggt caaccattgc tctactgcta aaagggttat gtgtagtgtt ttactgcata 6060aattatgcag caaacaagac aactcaaatt aaaaaatttc ctttgcttgt ttttttgttg 6120tctctgactt gactttcttg tggaagttgg ttgtataagg attgggacac cattgtcctt 6180cttaatttaa ttttattctt tgctgataaa aaaaaaaatt tcatatagtg ttaaataata 6240atttgttaaa taaccaaaaa gtcaaatatg tttactctcg tttaaataat tgagattcgt 6300ccagcaaggc taaacgattg tatagattta tgacaatatt tactttttta tagataaatg 6360ttatattata ataaatttat atacatatat tatatgttat ttattattat tttaaatcct 6420tcaatatttt atcaaaccaa ctcataattt tttttttatc tgtaagaagc aataaaatta 6480aatagaccca ctttaaggat gatccaacct ttatacagag taagagagtt caaatagtac 6540cctttcatat acatatcaac taaaatatta gaaatatcat ggatcaaacc ttataaagac 6600attaaataag tggataagta taatatataa atgggtagta tataatatat aaatggatac 6660aaacttctct ctttataatt gttatgtctc cttaacatcc taatataata cataagtggg 6720taatatataa tatataaatg gagacaaact tcttccatta taattgttat gtcttcttaa 6780cacttatgtc tcgttcacaa tgctaaggtt agaattgttt agaaagtctt atagtacaca 6840tttgtttttg tactatttga agcattccat aagccgtcac gattcagatg atttataata 6900ataagaggaa atttatcata gaacaataag gtgcatagat agagtgttaa tatatcataa 6960catcctttgt ttattcatag aagaagtgag atggagctca gttattatac tgttacatgg 7020tcggatacaa tattccatgc tctccatgag ctcttacacc tacatgcatt ttagttcata 7080cttgcggccg ctaaagctgc ttaccagcct tagcggattt cttggtggcc aggttgtcct 7140ggtaaaagaa gtgacagaac aggagaaaga cagatccgac gtaggcgtag ttgaaagccc 7200aggagaacag cttgcccttg tcagagttga agcagggaac gttgatgtag tcccagacca 7260ggagaaagcc accgacgaac tggcaaatct gcatggcagt gatcagaggc ttggccttga 7320acttgtagcc agcggcagtc agtccatagt aggtgtacat gatggtgtga atgaacgagt 7380taaagaacat gaagatccac acaccctcgt tgtgcagtcg aatgccgagg tagacgtccc 7440agggagctcc aaagtgatgg aaggcctgca gaaaggacac tcgcttgccc ttgaggacca 7500gccaagcggt gtcgaggtac tccacgtact tagaatagta gaaggccttg gcagtccagg 7560tgaacagctt ggagtcccag acaggagagg gacactgaaa gagaggctgg ggagtatcac 7620cggtctgtct tcgcagccag gctccagtac cgtagtccca gccgagagcg gtggcagtca 7680cgtagaagga cagggcagag aagagagcca ggaggacgtt gtaccagatc atggaggttc 7740ggtaggctcc tttcttctcg tccacgagac cagagtttcg caggagaggc ttcaggagca 7800ggtaggagaa ggtgccaatg aggatttcgg gatcggtgac ggcagcccag attcgctcgc 7860cagcgtcgtt ggccagagcc atggtgc 78876911473DNAArtificial Sequenceplasmid pKR1013 69ggagatccaa 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 gcaccatggc tctggccaac gacgctggcg 660agcgaatctg ggctgccgtc accgatcccg aaatcctcat tggcaccttc tcctacctgc 720tcctgaagcc tctcctgcga aactctggtc tcgtggacga gaagaaagga gcctaccgaa 780cctccatgat ctggtacaac gtcctcctgg ctctcttctc tgccctgtcc ttctacgtga 840ctgccaccgc tctcggctgg gactacggta ctggagcctg gctgcgaaga cagaccggtg 900atactcccca gcctctcttt cagtgtccct ctcctgtctg ggactccaag ctgttcacct 960ggactgccaa ggccttctac tattctaagt acgtggagta cctcgacacc gcttggctgg 1020tcctcaaggg caagcgagtg tcctttctgc aggccttcca tcactttgga gctccctggg 1080acgtctacct cggcattcga ctgcacaacg agggtgtgtg gatcttcatg ttctttaact 1140cgttcattca caccatcatg tacacctact atggactgac tgccgctggc tacaagttca 1200aggccaagcc tctgatcact gccatgcaga tttgccagtt cgtcggtggc tttctcctgg 1260tctgggacta catcaacgtt ccctgcttca actctgacaa gggcaagctg ttctcctggg 1320ctttcaacta cgcctacgtc ggatctgtct ttctcctgtt ctgtcacttc ttttaccagg 1380acaacctggc caccaagaaa tccgctaagg ctggtaagca gctttagcgg ccgcaagtat 1440gaactaaaat gcatgtaggt gtaagagctc atggagagca tggaatattg tatccgacca 1500tgtaacagta taataactga gctccatctc acttcttcta tgaataaaca aaggatgtta 1560tgatatatta acactctatc tatgcacctt attgttctat gataaatttc ctcttattat 1620tataaatcat ctgaatcgtg acggcttatg gaatgcttca aatagtacaa aaacaaatgt 1680gtactataag actttctaaa caattctaac cttagcattg tgaacgagac ataagtgtta 1740agaagacata acaattataa tggaagaagt ttgtctccat ttatatatta tatattaccc 1800acttatgtat tatattagga tgttaaggag acataacaat tataaagaga gaagtttgta 1860tccatttata tattatatac tacccattta tatattatac ttatccactt atttaatgtc 1920tttataaggt ttgatccatg atatttctaa tattttagtt gatatgtata tgaaagggta

1980ctatttgaac tctcttactc tgtataaagg ttggatcatc cttaaagtgg gtctatttaa 2040ttttattgct tcttacagat aaaaaaaaaa ttatgagttg gtttgataaa atattgaagg 2100atttaaaata ataataaata acatataata tatgtatata aatttattat aatataacat 2160ttatctataa aaaagtaaat attgtcataa atctatacaa tcgtttagcc ttgctggacg 2220aatctcaatt atttaaacga gagtaaacat atttgacttt ttggttattt aacaaattat 2280tatttaacac tatatgaaat tttttttttt atcagcaaag aataaaatta aattaagaag 2340gacaatggtg tcccaatcct tatacaacca acttccacaa gaaagtcaag tcagagacaa 2400caaaaaaaca agcaaaggaa attttttaat ttgagttgtc ttgtttgctg cataatttat 2460gcagtaaaac actacacata acccttttag cagtagagca atggttgacc gtgtgcttag 2520cttcttttat tttatttttt tatcagcaaa gaataaataa aataaaatga gacacttcag 2580ggatgtttca acaagcttgg cgcgccgttc tatagtgtca cctaaatcgt atgtgtatga 2640tacataaggt tatgtattaa ttgtagccgc gttctaacga caatatgtcc atatggtgca 2700ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac 2760ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga 2820ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac 2880gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgaccaaa atcccttaac 2940gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 3000atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 3060tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 3120gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga 3180actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 3240gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 3300agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 3360ccgaactgag atacctacag cgtgagcatt gagaaagcgc cacgcttccc gaagggagaa 3420aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 3480cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 3540gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 3600cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 3660cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 3720gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca 3780aaccgcctct ccccgcgcgt tggccgattc attaatgcag gttgatcgat tcgacatcga 3840tctagtaaca tagatgacac cgcgcgcgat aatttatcct agtttgcgcg ctatattttg 3900ttttctatcg cgtattaaat gtataattgc gggactctaa tcataaaaac ccatctcata 3960aataacgtca tgcattacat gttaattatt acatgcttaa cgtaattcaa cagaaattat 4020atgataatca tcgcaagacc ggcaacagga ttcaatctta agaaacttta ttgccaaatg 4080tttgaacgat ctgcttcgac gcactccttc tttaggtacc tcactattcc tttgccctcg 4140gacgagtgct ggggcgtcgg tttccactat cggcgagtac ttctacacag ccatcggtcc 4200agacggccgc gcttctgcgg gcgatttgtg tacgcccgac agtcccggct ccggatcgga 4260cgattgcgtc gcatcgaccc tgcgcccaag ctgcatcatc gaaattgccg tcaaccaagc 4320tctgatagag ttggtcaaga ccaatgcgga gcatatacgc ccggagccgc ggcgatcctg 4380caagctccgg atgcctccgc tcgaagtagc gcgtctgctg ctccatacaa gccaaccacg 4440gcctccagaa gaagatgttg gcgacctcgt attgggaatc cccgaacatc gcctcgctcc 4500agtcaatgac cgctgttatg cggccattgt ccgtcaggac attgttggag ccgaaatccg 4560cgtgcacgag gtgccggact tcggggcagt cctcggccca aagcatcagc tcatcgagag 4620cctgcgcgac ggacgcactg acggtgtcgt ccatcacagt ttgccagtga tacacatggg 4680gatcagcaat cgcgcatatg aaatcacgcc atgtagtgta ttgaccgatt ccttgcggtc 4740cgaatgggcc gaacccgctc gtctggctaa gatcggccgc agcgatcgca tccatggcct 4800ccgcgaccgg ctgcagaaca gcgggcagtt cggtttcagg caggtcttgc aacgtgacac 4860cctgtgcacg gcgggagatg caataggtca ggctctcgct gaattcccca atgtcaagca 4920cttccggaat cgggagcgcg gccgatgcaa agtgccgata aacataacga tctttgtaga 4980aaccatcggc gcagctattt acccgcagga catatccacg ccctcctaca tcgaagctga 5040aagcacgaga ttcttcgccc tccgagagct gcatcaggtc ggagacgctg tcgaactttt 5100cgatcagaaa cttctcgaca gacgtcgcgg tgagttcagg ctttttcatg gtttaataag 5160aagagaaaag agttcttttg ttatggctga agtaatagag aaatgagctc gagcgtgtcc 5220tctccaaatg aaatgaactt ccttatatag aggaagggtc ttgcgaagga tagtgggatt 5280gtgcgtcatc ccttacgtca gtggagatgt cacatcaatc cacttgcttt gaagacgtgg 5340ttggaacgtc ttctttttcc acgatgctcc tcgtgggtgg gggtccatct ttgggaccac 5400tgtcggcaga ggcatcttga atgatagcct ttcctttatc gcaatgatgg catttgtagg 5460agccaccttc cttttctact gtcctttcga tgaagtgaca gatagctggg caatggaatc 5520cgaggaggtt tcccgaaatt atcctttgtt gaaaagtctc aatagccctt tggtcttctg 5580agactgtatc tttgacattt ttggagtaga ccagagtgtc gtgctccacc atgttgacga 5640agattttctt cttgtcattg agtcgtaaaa gactctgtat gaactgttcg ccagtcttca 5700cggcgagttc tgttagatcc tcgatttgaa tcttagactc catgcatggc cttagattca 5760gtaggaacta cctttttaga gactccaatc tctattactt gccttggttt atgaagcaag 5820ccttgaatcg tccatactgg aatagtactt ctgatcttga gaaatatgtc tttctctgtg 5880ttcttgatgc aattagtcct gaatcttttg actgcatctt taaccttctt gggaaggtat 5940ttgatctcct ggagattgtt actcgggtag atcgtcttga tgagacctgc tgcgtaggcc 6000tctctaacca tctgtgggtc agcattcttt ctgaaattga agaggctaac cttctcatta 6060tcagtggtga acatagtgtc gtcaccttca ccttcgaact tccttcctag atcgtaaaga 6120tagaggaaat cgtccattgt aatctccggg gcaaaggaga tctcttttgg ggctggatca 6180ctgctgggcc ttttggttcc tagcgtgagc cagtgggctt tttgctttgg tgggcttgtt 6240agggccttag caaagctctt gggcttgagt tgagcttctc ctttggggat gaagttcaac 6300ctgtctgttt gctgacttgt tgtgtacgcg tcagctgctg ctcttgcctc tgtaatagtg 6360gcaaatttct tgtgtgcaac tccgggaacg ccgtttgttg ccgcctttgt acaaccccag 6420tcatcgtata taccggcatg tggaccgtta tacacaacgt agtagttgat atgagggtgt 6480tgaatacccg attctgctct gagaggagca actgtgctgt taagctcaga tttttgtggg 6540attggaattg gatcgatctc gatcccgcga aattaatacg actcactata gggagaccac 6600aacggtttcc ctctagaaat aattttgttt aactttaaga aggagatata cccatggaaa 6660agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct 6720ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag 6780ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg 6840tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat 6900tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc 6960tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggctatg gatgcgatcg 7020ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc 7080aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc 7140aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc 7200tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca 7260atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg 7320gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg 7380agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc 7440gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt 7500tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga 7560ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag 7620aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagt 7680gaggtacagc ttggatcgat ccggctgcta acaaagcccg aaaggaagct gagttggctg 7740ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg 7800gttttttgct gaaaggagga actatatccg gatgatcggg cgcgccgtcg acggatccgt 7860acgagatccg gccggccaga tcctgcagga tataatgagc cgtaaacaaa gatgattaag 7920tagtaattaa tacgtactag taaaagtggc aaaagataac gagaaagaac caatttcttt 7980gcattcggcc ttagcggaag gcatatataa gctttgatta ttttatttag tgtaatgatt 8040tcgtacaacc aaagcattta tttagtactc tcacacttgt gtcgcggccg ctcattcagg 8100gacagcagcc agctgggcaa tctcgcctaa gaagctgacc aatttctgaa ccccggggat 8160gatggctggg gccctgtagg gcaggttgtg cttggcgcac aacttctgca cctccaggga 8220agctgccgtg aggttgtgcc tgggcatgtt gggccacaag tggtgctcaa tctggtagtt 8280gagaccacca aagacccaat cggtgagaag gccgggctta atgttcaacg tctcgtggat 8340ctggctggcg ctgaacccgt gctcatccca gacagtctcc tcaaccttgt ccaaaggata 8400gtgattcaga aacacaacga tggcaatgcc aaatcctccg agcagctcgg agatcaagaa 8460gcatcccaaa ccggtggcaa agctaggcat ggcactgcaa tagaaagtgg ccttgagcac 8520ccagtggagg gccagaccaa tcgcctcctt agtgtactgg cgcttgtagt acaggttcct 8580ctcctcgctc ttgtaggaca tgacgccgcc gatggattgg aggcaccaga tgaacctaag 8640ggatgcaatg gtgggaatga agtagtactg ctggtacttg atgagattcc gagtcgaagg 8700agtagccctc ttgacatctt cgggagacca agccaggatg gggaggttat caatatctgg 8760gtcatggccg accacattgg tggcggcatg gtgaccattg tgcctatcct tccaccaagt 8820ctgtgagtat ccctgcatca cgttaccgaa aaagagaccg aaagcgtcgt taatcttgcg 8880gtttgtgaaa acctggtgat ggcaatagtc gtgggacaac cagcctagct gttggtagca 8940cacgccaaga accaaagcag cggtgaaata ccaattcgtg tacacggtca tcaagatgga 9000aaggaccatc aaaccgacgg tagttgaagt cttgtaaaaa taccaaagga aactcgtctc 9060gaacataccc ttggagatga attcctcccg caacttgcgg aaatcctcct ggggctcatc 9120acgcttaggc ttgggtgcaa caggtgtgtc aggagaggaa ggctccataa caggcattct 9180cttcaacttg gcgacggctt cttgagagtg catcaccatg aagacatcgg tcgcatcgcg 9240gttgcgatag ttctcgataa tgtcagctcc tccagggtgg tgattgaccc aagcagacac 9300atcataagtt gcgccatcaa ttgtgattgg cagagcttgc cgcttaggag acatggtgcg 9360gccgcttggg gggctatgga agactttctt agttagttgt gtgaataagc aatgttggga 9420gaatcgggac tacttatagg ataggaataa aacagaaaag tattaagtgc taatgaaata 9480tttagactga taattaaaat cttcacgtat gtccacttga tataaaaacg tcaggaataa 9540aggaagtaca gtagaattta aaggtactct ttttatatat acccgtgttc tctttttggc 9600tagctagttg cataaaaaat aatctatatt tttatcatta ttttaaatat cttatgagat 9660ggtaaatatt tatcataatt ttttttacta ttatttatta tttgtgtgtg taatacatat 9720agaagttaat tacaaatttt atttactttt tcattatttt gatatgattc accattaatt 9780tagtgttatt atttataata gttcatttta atctttttgt atatattatg cgtgcagtac 9840ttttttccta catataacta ctattacatt ttatttatat aatattttta ttaatgaatt 9900ttcgtgataa tatgtaatat tgttcattat tatttcagat tttttaaaaa tatttgtgtt 9960attatttatg aaatatgtaa tttttttagt atttgatttt atgatgataa agtgttctaa 10020attcaaaaga agggggaaag cgtaaacatt aaaaaacgtc atcaaacaaa aacaaaatct 10080tgttaataaa gataaaactg tttgttttga tcactgttat ttcgtaatat aaaaacatta 10140tttatattta tattgttgac aaccaaattt gcctatcaaa tctaaccaat ataatgcatg 10200cgtggcaggt aatgtactac catgaactta agtcatgaca taataaaccg tgaatctgac 10260caatgcatgt acctanctaa attgtatttg tgacacgaag caaatgattc aattcacaat 10320ggagatggga aacaaataat gaagaaccca gaactaagaa agcttttctg aaaaataaaa 10380taaaggcaat gtcaaaagta tactgcatca tcagtccaga aagcacatga tattttttta 10440tcagtatcaa tgcagctagt tttattttac aatatcgata tagctagttt aaatatattg 10500cagctagatt tataaatatt tgtgttatta tttatcattt gtgtaatcct gtttttagta 10560ttttagttta tatatgatga taatgtattc caaatttaaa agaagggaaa taaatttaaa 10620caagaaaaaa agtcatcaaa caaaaaacaa atgaaagggt ggaaagatgt taccatgtaa 10680tgtgaatgtt acagtatttc ttttattata gagttaacaa attaactaat atgattttgt 10740taataatgat aaaatatttt ttttattatt atttcataat ataaaaatag tttacttaat 10800ataaaaaaaa ttctatcgtt cacaacaaag ttggccacct aatttaacca tgcatgtacc 10860catggaccat attaggtaac catcaaacct gatgaagaga taaagagatg aagacttaag 10920tcataacaca aaaccataaa aaacaaaaat acaatcaacc gtcaatctga ccaatgcatg 10980aaaaagctgc aatagtgagt ggcgacacaa agcacatgat tttcttacaa cggagataaa 11040accaaaaaaa tatttcatga acaacctaga acaaataaag cttttatata ataaatatat 11100aaataaataa aggctatgga ataatatact tcaatatatt tggattaaat aaattgttgg 11160cggggttgat atatttatac acacctaaag tcacttcaat ctcattttca cttaactttt 11220attttttttt tctttttatt tatcataaag agaatattga taatatactt tttaacatat 11280ttttatgaca ttttttattg gtgaaaactt attaaaaatc ataaattttg taagttagat 11340ttatttaaag agttcctctt cttattttaa attttttaat aaatttttaa ataactaaaa 11400tttgtgttaa aaatgttaaa aaatgtgtta ttaacccttc tcttcgagga cgtacgtcta 11460gagtcgacct gca 1147370792DNAIsochrysis galbana 70atggccctcg caaacgacgc gggagagcgc atctgggcgg ctgtgaccga cccggaaatc 60ctcattggca ccttctcgta cttgctactc aaaccgctgc tccgcaattc cgggctggtg 120gatgagaaga agggcgcata caggacgtcc atgatctggt acaacgttct gctggcgctc 180ttctctgcgc tgagcttcta cgtgacggcg accgccctcg gctgggacta tggtacgggc 240gcgtggctgc gcaggcaaac cggcgacaca ccgcagccgc tcttccagtg cccgtccccg 300gtttgggact cgaagctctt cacatggacc gccaaggcat tctattactc caagtacgtg 360gagtacctcg acacggcctg gctggtgctc aagggcaaga gggtctcctt tctccaggcc 420ttccaccact ttggcgcgcc gtgggatgtg tacctcggca ttcggctgca caacgagggc 480gtatggatct tcatgttttt caactcgttc attcacacca tcatgtacac ctactacggc 540ctcaccgccg ccgggtataa gttcaaggcc aagccgctca tcaccgcgat gcagatctgc 600cagttcgtgg gcggcttcct gttggtctgg gactacatca acgtcccctg cttcaactcg 660gacaaaggga agttgttcag ctgggctttc aactatgcat acgtcggctc ggtcttcttg 720ctcttctgcc actttttcta ccaggacaac ttggcaacga agaaatcggc caaggcgggc 780aagcagctct ag 79271757DNAEuglena gracilismisc_feature(677)..(677)n is a, c, g, or t 71ttttttttcg aacacttaat ggaggtggtg aatgaaatag tctcaattgg gcaggaagtt 60ttacccaaag ttgattatgc ccaactctgg agtgatgcca gtcactgtga ggtgctttac 120ttgtccatcg catttgtcat cttgaagttc actcttggcc cccttggtcc aaaaggtcag 180tctcgtatga agtttgtttt caccaattac aaccttctca tgtccattta ttcgttggga 240tcattcctct caatggcata tgccatgtac accatcggtg ttatgtctga caactgcgag 300aaggcttttg acaacaacgt cttcaggatc accacgcagt tgttctattt gagcaagttc 360ctggagtata ttgactcctt ctatttgcca ctgatgggca agcctctgac ctggttgcaa 420ttcttccatc atttgggggc accgatggat atgtggctgt tctataatta ccgaaatgaa 480gctgtttgga tttttgtgct gttgaatggt ttcatccact ggatcatgta cggttattat 540tggaccagat tgatcaagct gaagttcccc atgccaaaat ccctgattac atcaatgcag 600atcattcaat tcaatgttgg tttctacatt gtctggaagt acaggaacat tccctgttat 660cgccaagatg ggatgangat gtttggctgg ttcttcaatt acttttatgt tggcacagtc 720ttgtgtttgt tcttgaattt ctatgtgcaa acgtata 75772774DNAEuglena gracilismisc_feature(34)..(34)n is a, c, g, or t 72tcaggatcac cacgcagttg ttctatttga gcangttcct ggagtatatt gactccttct 60atttgccant gatgggcaag cntctgacct ggttgcaatt cttccatcat tngggggcac 120cgatggatat gtggctgttc tataattacc gaaatgaagc tgtttggatt tttgtgctgt 180tgaatggttt catccactgg atcatgtacg gttattannn gaccagattg atcaagctga 240agttccccat gccaaaatcc ctgattacat caatgcagat cattcaattc aatgttggtt 300tctacattgt ctggaagtac aggaacattc cctgttatcg ccaagatggg atgaggatgt 360ttggctggtt cttcaattac ttttatgttg gcacagtctt gtgtttgttc ttgaatttct 420atgtgcaaac gtatatcgtc aggaagcaca agggagccaa aaagattcag tgatatttcc 480tcctctgcgg tggcctcttt tgacctcccc ttgacaccta taatgtggag gtgtcgggct 540ctctccgtct caccagcact tgactctgca ggtgctcact tttatttttt acccatcttt 600gcttgttgac cattcacctc tcccacttcc acatagtcca ttctaactgt tgcagactgc 660ggtccatttt ttccagagct cccaatgacc atacgcgaca ccttgtnnnc ncccagccca 720ttgtgcacaa ttcatagtgg catcgttttg ccttgatacg tgtgcatcca gcgg 774731201DNAEuglena gracilismisc_feature(1134)..(1136)n is a, c, g, or t 73gacatggcaa ctatgatttt attttgactg atagtgacct gttcgttgca acaaattgat 60gagcaatgct tttttataat gccaactttg tacaaaaaag ttggattttt tttcgaacac 120ttaatggagg tggtgaatga aatagtctca attgggcagg aagttttacc caaagttgat 180tatgcccaac tctggagtga tgccagtcac tgtgaggtgc tttacttgtc catcgcattt 240gtcatcttga agttcactct tggccccctt ggtccaaaag gtcagtctcg tatgaagttt 300gttttcacca attacaacct tctcatgtcc atttattcgt tgggatcatt cctctcaatg 360gcatatgcca tgtacaccat cggtgttatg tctgacaact gcgagaaggc ttttgacaac 420aacgtcttca ggatcaccac gcagttgttc tatttgagca agttcctgga gtatattgac 480tccttctatt tgccactgat gggcaagcct ctgacctggt tgcaattctt ccatcatttg 540ggggcaccga tggatatgtg gctgttctat aattaccgaa atgaagctgt ttggattttt 600gtgctgttga atggtttcat ccactggatc atgtacggtt attattggac cagattgatc 660aagctgaagt tccccatgcc aaaatccctg attacatcaa tgcagatcat tcaattcaat 720gttggtttct acattgtctg gaagtacagg aacattccct gttatcgcca agatgggatg 780aggatgtttg gctggttctt caattacttt tatgttggca cagtcttgtg tttgttcttg 840aatttctatg tgcaaacgta tatcgtcagg aagcacaagg gagccaaaaa gattcagtga 900tatttcctcc tctgcggtgg cctcttttga cctccccttg acacctataa tgtggaggtg 960tcgggctctc tccgtctcac cagcacttga ctctgcaggt gctcactttt attttttacc 1020catctttgct tgttgaccat tcacctctcc cacttccaca tagtccattc taactgttgc 1080agactgcggt ccattttttc cagagctccc aatgaccata cgcgacacct tgtnnncncc 1140cagcccattg tgcacaattc atagtggcat cgttttgcct tgatacgtgt gcatccagcg 1200g 120174777DNAEuglena gracilis 74atggaggtgg 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 tcagtga 77775258PRTEuglena gracilis 75Met Glu Val Val Asn Glu Ile Val Ser Ile Gly Gln Glu Val Leu Pro1 5 10 15Lys Val Asp Tyr Ala Gln Leu Trp Ser Asp Ala Ser His Cys Glu Val 20 25 30Leu Tyr Leu Ser Ile Ala Phe Val Ile Leu Lys Phe Thr Leu Gly Pro 35 40 45Leu Gly Pro Lys Gly Gln Ser Arg Met Lys Phe Val Phe Thr Asn Tyr 50 55 60Asn Leu Leu Met Ser Ile Tyr Ser Leu Gly Ser Phe Leu Ser Met Ala65 70 75 80Tyr Ala Met Tyr Thr Ile Gly Val Met Ser Asp Asn Cys Glu Lys Ala 85 90 95Phe Asp Asn Asn Val Phe Arg Ile Thr Thr Gln Leu Phe Tyr Leu Ser 100 105 110Lys Phe Leu Glu Tyr Ile Asp Ser Phe Tyr Leu Pro Leu Met Gly Lys 115 120 125Pro Leu Thr Trp

Leu Gln Phe Phe His His Leu Gly Ala Pro Met Asp 130 135 140Met Trp Leu Phe Tyr Asn Tyr Arg Asn Glu Ala Val Trp Ile Phe Val145 150 155 160Leu Leu Asn Gly Phe Ile His Trp Ile Met Tyr Gly Tyr Tyr Trp Thr 165 170 175Arg Leu Ile Lys Leu Lys Phe Pro Met Pro Lys Ser Leu Ile Thr Ser 180 185 190Met Gln Ile Ile Gln Phe Asn Val Gly Phe Tyr Ile Val Trp Lys Tyr 195 200 205Arg Asn Ile Pro Cys Tyr Arg Gln Asp Gly Met Arg Met Phe Gly Trp 210 215 220Phe Phe Asn Tyr Phe Tyr Val Gly Thr Val Leu Cys Leu Phe Leu Asn225 230 235 240Phe Tyr Val Gln Thr Tyr Ile Val Arg Lys His Lys Gly Ala Lys Lys 245 250 255Ile Gln76263PRTIsochrysis galbana 76Met Ala Leu Ala Asn Asp Ala Gly Glu Arg Ile Trp Ala Ala Val Thr1 5 10 15Asp Pro Glu Ile Leu Ile Gly Thr Phe Ser Tyr Leu Leu Leu Lys Pro 20 25 30Leu Leu Arg Asn Ser Gly Leu Val Asp Glu Lys Lys Gly Ala Tyr Arg 35 40 45Thr Ser Met Ile Trp Tyr Asn Val Leu Leu Ala Leu Phe Ser Ala Leu 50 55 60Ser Phe Tyr Val Thr Ala Thr Ala Leu Gly Trp Asp Tyr Gly Thr Gly65 70 75 80Ala Trp Leu Arg Arg Gln Thr Gly Asp Thr Pro Gln Pro Leu Phe Gln 85 90 95Cys Pro Ser Pro Val Trp Asp Ser Lys Leu Phe Thr Trp Thr Ala Lys 100 105 110Ala Phe Tyr Tyr Ser Lys Tyr Val Glu Tyr Leu Asp Thr Ala Trp Leu 115 120 125Val Leu Lys Gly Lys Arg Val Ser Phe Leu Gln Ala Phe His His Phe 130 135 140Gly Ala Pro Trp Asp Val Tyr Leu Gly Ile Arg Leu His Asn Glu Gly145 150 155 160Val Trp Ile Phe Met Phe Phe Asn Ser Phe Ile His Thr Ile Met Tyr 165 170 175Thr Tyr Tyr Gly Leu Thr Ala Ala Gly Tyr Lys Phe Lys Ala Lys Pro 180 185 190Leu Ile Thr Ala Met Gln Ile Cys Gln Phe Val Gly Gly Phe Leu Leu 195 200 205Val Trp Asp Tyr Ile Asn Val Pro Cys Phe Asn Ser Asp Lys Gly Lys 210 215 220Leu Phe Ser Trp Ala Phe Asn Tyr Ala Tyr Val Gly Ser Val Phe Leu225 230 235 240Leu Phe Cys His Phe Phe Tyr Gln Asp Asn Leu Ala Thr Lys Lys Ser 245 250 255Ala Lys Ala Gly Lys Gln Leu 2607730DNAArtificial Sequenceprimer sense oligonucleotide oEugEL1-1 77agcggccgca ccatggaggt ggtgaatgaa 307830DNAArtificial Sequenceprimer sense oligonucleotide oEugEL1-2 78tgcggccgct cactgaatct ttttggctcc 30794311DNAArtificial Sequenceplasmid pKR906 79agcggccgca 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 4311807085DNAArtificial Sequenceplasmid pKR72 (ATCC Accession No. PTA-6019) 80gtacggatcc 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 7085817873DNAArtificial Sequenceplasmid pKR912 81ggccgcaagt 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 aggttgatcg 2400attcgacatc gatctagtaa catagatgac accgcgcgcg ataatttatc ctagtttgcg 2460cgctatattt tgttttctat cgcgtattaa atgtataatt gcgggactct aatcataaaa 2520acccatctca taaataacgt catgcattac atgttaatta ttacatgctt aacgtaattc 2580aacagaaatt atatgataat catcgcaaga ccggcaacag gattcaatct taagaaactt 2640tattgccaaa tgtttgaacg atctgcttcg acgcactcct tctttaggta cctcactatt 2700cctttgccct cggacgagtg ctggggcgtc ggtttccact atcggcgagt acttctacac 2760agccatcggt ccagacggcc gcgcttctgc gggcgatttg tgtacgcccg acagtcccgg 2820ctccggatcg gacgattgcg tcgcatcgac cctgcgccca agctgcatca tcgaaattgc 2880cgtcaaccaa gctctgatag agttggtcaa gaccaatgcg gagcatatac gcccggagcc 2940gcggcgatcc tgcaagctcc ggatgcctcc gctcgaagta gcgcgtctgc tgctccatac 3000aagccaacca cggcctccag aagaagatgt tggcgacctc gtattgggaa tccccgaaca 3060tcgcctcgct ccagtcaatg accgctgtta tgcggccatt gtccgtcagg acattgttgg 3120agccgaaatc cgcgtgcacg aggtgccgga cttcggggca gtcctcggcc caaagcatca 3180gctcatcgag agcctgcgcg acggacgcac tgacggtgtc gtccatcaca gtttgccagt 3240gatacacatg gggatcagca atcgcgcata tgaaatcacg ccatgtagtg tattgaccga 3300ttccttgcgg tccgaatggg ccgaacccgc tcgtctggct aagatcggcc gcagcgatcg 3360catccatggc ctccgcgacc ggctgcagaa cagcgggcag ttcggtttca ggcaggtctt 3420gcaacgtgac accctgtgca cggcgggaga tgcaataggt caggctctcg ctgaattccc 3480caatgtcaag cacttccgga atcgggagcg cggccgatgc aaagtgccga taaacataac 3540gatctttgta gaaaccatcg gcgcagctat ttacccgcag gacatatcca cgccctccta 3600catcgaagct gaaagcacga gattcttcgc cctccgagag ctgcatcagg tcggagacgc 3660tgtcgaactt ttcgatcaga aacttctcga cagacgtcgc ggtgagttca ggctttttca 3720tggtttaata agaagagaaa agagttcttt tgttatggct gaagtaatag agaaatgagc 3780tcgagcgtgt cctctccaaa tgaaatgaac ttccttatat agaggaaggg tcttgcgaag 3840gatagtggga ttgtgcgtca tcccttacgt cagtggagat gtcacatcaa tccacttgct 3900ttgaagacgt ggttggaacg tcttcttttt ccacgatgct cctcgtgggt gggggtccat 3960ctttgggacc actgtcggca gaggcatctt gaatgatagc ctttccttta tcgcaatgat 4020ggcatttgta ggagccacct tccttttcta ctgtcctttc gatgaagtga cagatagctg 4080ggcaatggaa tccgaggagg tttcccgaaa ttatcctttg ttgaaaagtc tcaatagccc 4140tttggtcttc tgagactgta tctttgacat ttttggagta gaccagagtg tcgtgctcca 4200ccatgttgac gaagattttc ttcttgtcat tgagtcgtaa aagactctgt atgaactgtt 4260cgccagtctt cacggcgagt tctgttagat cctcgatttg aatcttagac tccatgcatg 4320gccttagatt cagtaggaac taccttttta gagactccaa tctctattac ttgccttggt 4380ttatgaagca agccttgaat cgtccatact ggaatagtac ttctgatctt gagaaatatg 4440tctttctctg tgttcttgat gcaattagtc ctgaatcttt tgactgcatc tttaaccttc 4500ttgggaaggt atttgatctc ctggagattg ttactcgggt agatcgtctt gatgagacct 4560gctgcgtagg cctctctaac catctgtggg tcagcattct ttctgaaatt gaagaggcta 4620accttctcat tatcagtggt gaacatagtg tcgtcacctt caccttcgaa cttccttcct 4680agatcgtaaa gatagaggaa atcgtccatt gtaatctccg gggcaaagga gatctctttt 4740ggggctggat cactgctggg ccttttggtt cctagcgtga gccagtgggc tttttgcttt 4800ggtgggcttg ttagggcctt agcaaagctc ttgggcttga gttgagcttc tcctttgggg 4860atgaagttca acctgtctgt ttgctgactt gttgtgtacg cgtcagctgc tgctcttgcc 4920tctgtaatag tggcaaattt cttgtgtgca actccgggaa cgccgtttgt tgccgccttt 4980gtacaacccc agtcatcgta tataccggca tgtggaccgt tatacacaac gtagtagttg 5040atatgagggt gttgaatacc cgattctgct ctgagaggag caactgtgct gttaagctca 5100gatttttgtg ggattggaat tggatcgatc tcgatcccgc gaaattaata cgactcacta 5160tagggagacc acaacggttt ccctctagaa ataattttgt ttaactttaa gaaggagata 5220tacccatgga aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt 5280tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct 5340tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca 5400aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg 5460acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca 5520cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcta 5580tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc 5640aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg 5700tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg 5760atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt 5820tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg 5880aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt 5940tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat 6000cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg 6060ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat 6120ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg 6180atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg 6240caaaggaata gtgaggtaca gcttggatcg atccggctgc taacaaagcc cgaaaggaag 6300ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac 6360gggtcttgag gggttttttg ctgaaaggag gaactatatc cggatgatcg ggcgcgccgt 6420cgacggatcc gtacgagatc cggccggcca gatcctgcag gagatccaag cttttgatcc 6480atgcccttca tttgccgctt attaattaat ttggtaacag tccgtactaa tcagttactt 6540atccttcccc catcataatt aatcttggta gtctcgaatg ccacaacact gactagtctc 6600ttggatcata agaaaaagcc aaggaacaaa agaagacaaa acacaatgag agtatccttt 6660gcatagcaat gtctaagttc ataaaattca aacaaaaacg caatcacaca cagtggacat 6720cacttatcca ctagctgatc aggatcgccg cgtcaagaaa aaaaaactgg accccaaaag 6780ccatgcacaa caacacgtac tcacaaaggt gtcaatcgag cagcccaaaa cattcaccaa 6840ctcaacccat catgagccct cacatttgtt gtttctaacc caacctcaaa ctcgtattct 6900cttccgccac ctcatttttg tttatttcaa cacccgtcaa actgcatgcc accccgtggc 6960caaatgtcca tgcatgttaa caagacctat gactataaat agctgcaatc tcggcccagg 7020ttttcatcat caagaaccag ttcaatatcc tagtacaccg tattaaagaa tttaagatat 7080actgcggccg caccatggag gtggtgaatg aaatagtctc aattgggcag gaagttttac 7140ccaaagttga ttatgcccaa ctctggagtg atgccagtca ctgtgaggtg ctttacttgt 7200ccatcgcatt tgtcatcttg aagttcactc ttggccccct tggtccaaaa ggtcagtctc 7260gtatgaagtt tgttttcacc aattacaacc ttctcatgtc catttattcg ttgggatcat 7320tcctctcaat ggcatatgcc atgtacacca tcggtgttat gtctgacaac tgcgagaagg 7380cttttgacaa caacgtcttc aggatcacca cgcagttgtt ctatttgagc aagttcctgg 7440agtatattga ctccttctat ttgccactga tgggcaagcc tctgacctgg ttgcaattct 7500tccatcattt gggggcaccg atggatatgt ggctgttcta taattaccga aatgaagctg 7560tttggatttt tgtgctgttg aatggtttca tccactggat catgtacggt tattattgga 7620ccagattgat caagctgaag ttccccatgc caaaatccct gattacatca atgcagatca 7680ttcaattcaa tgttggtttc tacattgtct ggaagtacag gaacattccc tgttatcgcc 7740aagatgggat gaggatgttt ggctggttct tcaattactt ttatgttggc acagtcttgt 7800gtttgttctt gaatttctat gtgcaaacgt atatcgtcag gaagcacaag ggagccaaaa 7860agattcagtg agc 78738211459DNAArtificial Sequenceplasmid pKR1014 82ggagatccaa 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 atcgattcga catcgatcta gtaacataga 3840tgacaccgcg cgcgataatt tatcctagtt tgcgcgctat attttgtttt ctatcgcgta 3900ttaaatgtat aattgcggga ctctaatcat aaaaacccat ctcataaata acgtcatgca 3960ttacatgtta attattacat gcttaacgta attcaacaga aattatatga taatcatcgc 4020aagaccggca acaggattca atcttaagaa actttattgc caaatgtttg aacgatctgc 4080ttcgacgcac tccttcttta ggtacctcac tattcctttg ccctcggacg agtgctgggg 4140cgtcggtttc cactatcggc gagtacttct acacagccat cggtccagac ggccgcgctt 4200ctgcgggcga tttgtgtacg cccgacagtc ccggctccgg atcggacgat tgcgtcgcat 4260cgaccctgcg cccaagctgc atcatcgaaa ttgccgtcaa ccaagctctg atagagttgg 4320tcaagaccaa tgcggagcat atacgcccgg agccgcggcg atcctgcaag ctccggatgc 4380ctccgctcga agtagcgcgt ctgctgctcc atacaagcca accacggcct ccagaagaag 4440atgttggcga cctcgtattg ggaatccccg aacatcgcct cgctccagtc aatgaccgct 4500gttatgcggc cattgtccgt caggacattg ttggagccga aatccgcgtg cacgaggtgc 4560cggacttcgg ggcagtcctc ggcccaaagc atcagctcat cgagagcctg cgcgacggac 4620gcactgacgg tgtcgtccat cacagtttgc cagtgataca catggggatc agcaatcgcg 4680catatgaaat cacgccatgt agtgtattga ccgattcctt gcggtccgaa tgggccgaac 4740ccgctcgtct ggctaagatc ggccgcagcg atcgcatcca tggcctccgc gaccggctgc 4800agaacagcgg gcagttcggt ttcaggcagg tcttgcaacg tgacaccctg tgcacggcgg 4860gagatgcaat aggtcaggct ctcgctgaat tccccaatgt caagcacttc cggaatcggg 4920agcgcggccg atgcaaagtg ccgataaaca taacgatctt tgtagaaacc atcggcgcag 4980ctatttaccc gcaggacata tccacgccct cctacatcga agctgaaagc acgagattct 5040tcgccctccg agagctgcat caggtcggag acgctgtcga acttttcgat cagaaacttc 5100tcgacagacg tcgcggtgag ttcaggcttt ttcatggttt aataagaaga gaaaagagtt 5160cttttgttat ggctgaagta atagagaaat gagctcgagc gtgtcctctc caaatgaaat 5220gaacttcctt atatagagga agggtcttgc gaaggatagt gggattgtgc gtcatccctt 5280acgtcagtgg agatgtcaca tcaatccact tgctttgaag acgtggttgg aacgtcttct 5340ttttccacga tgctcctcgt gggtgggggt ccatctttgg gaccactgtc ggcagaggca 5400tcttgaatga tagcctttcc tttatcgcaa tgatggcatt tgtaggagcc accttccttt 5460tctactgtcc tttcgatgaa gtgacagata gctgggcaat ggaatccgag gaggtttccc 5520gaaattatcc tttgttgaaa agtctcaata gccctttggt cttctgagac tgtatctttg 5580acatttttgg agtagaccag agtgtcgtgc tccaccatgt tgacgaagat tttcttcttg 5640tcattgagtc gtaaaagact ctgtatgaac tgttcgccag tcttcacggc gagttctgtt 5700agatcctcga tttgaatctt agactccatg catggcctta gattcagtag gaactacctt 5760tttagagact ccaatctcta ttacttgcct tggtttatga agcaagcctt gaatcgtcca 5820tactggaata gtacttctga tcttgagaaa tatgtctttc tctgtgttct tgatgcaatt 5880agtcctgaat cttttgactg catctttaac cttcttggga aggtatttga tctcctggag 5940attgttactc gggtagatcg tcttgatgag acctgctgcg taggcctctc taaccatctg 6000tgggtcagca ttctttctga aattgaagag gctaaccttc tcattatcag tggtgaacat 6060agtgtcgtca ccttcacctt cgaacttcct tcctagatcg taaagataga ggaaatcgtc 6120cattgtaatc tccggggcaa aggagatctc ttttggggct ggatcactgc tgggcctttt 6180ggttcctagc gtgagccagt gggctttttg ctttggtggg cttgttaggg ccttagcaaa 6240gctcttgggc ttgagttgag cttctccttt ggggatgaag ttcaacctgt ctgtttgctg 6300acttgttgtg tacgcgtcag ctgctgctct tgcctctgta atagtggcaa atttcttgtg 6360tgcaactccg ggaacgccgt ttgttgccgc ctttgtacaa ccccagtcat cgtatatacc 6420ggcatgtgga ccgttataca caacgtagta gttgatatga gggtgttgaa tacccgattc 6480tgctctgaga ggagcaactg tgctgttaag ctcagatttt tgtgggattg gaattggatc 6540gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct 6600agaaataatt ttgtttaact ttaagaagga gatataccca tggaaaagcc tgaactcacc 6660gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag 6720ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc 6780ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt 6840gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg 6900acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa 6960ctgcccgctg ttctgcagcc ggtcgcggag gctatggatg cgatcgctgc ggccgatctt 7020agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg 7080cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac 7140gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac 7200tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac 7260aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac 7320gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc 7380tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc 7440cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct 7500tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca 7560caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat 7620agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagtgagg tacagcttgg 7680atcgatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag 7740caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa 7800ggaggaacta tatccggatg atcgggcgcg ccgtcgacgg atccgtacga gatccggccg 7860gccagatcct gcaggatata atgagccgta aacaaagatg

attaagtagt aattaatacg 7920tactagtaaa agtggcaaaa gataacgaga aagaaccaat ttctttgcat tcggccttag 7980cggaaggcat atataagctt tgattatttt atttagtgta atgatttcgt acaaccaaag 8040catttattta gtactctcac acttgtgtcg cggccgctca ttcagggaca gcagccagct 8100gggcaatctc gcctaagaag ctgaccaatt tctgaacccc ggggatgatg gctggggccc 8160tgtagggcag gttgtgcttg gcgcacaact tctgcacctc cagggaagct gccgtgaggt 8220tgtgcctggg catgttgggc cacaagtggt gctcaatctg gtagttgaga ccaccaaaga 8280cccaatcggt gagaaggccg ggcttaatgt tcaacgtctc gtggatctgg ctggcgctga 8340acccgtgctc atcccagaca gtctcctcaa ccttgtccaa aggatagtga ttcagaaaca 8400caacgatggc aatgccaaat cctccgagca gctcggagat caagaagcat cccaaaccgg 8460tggcaaagct aggcatggca ctgcaataga aagtggcctt gagcacccag tggagggcca 8520gaccaatcgc ctccttagtg tactggcgct tgtagtacag gttcctctcc tcgctcttgt 8580aggacatgac gccgccgatg gattggaggc accagatgaa cctaagggat gcaatggtgg 8640gaatgaagta gtactgctgg tacttgatga gattccgagt cgaaggagta gccctcttga 8700catcttcggg agaccaagcc aggatgggga ggttatcaat atctgggtca tggccgacca 8760cattggtggc ggcatggtga ccattgtgcc tatccttcca ccaagtctgt gagtatccct 8820gcatcacgtt accgaaaaag agaccgaaag cgtcgttaat cttgcggttt gtgaaaacct 8880ggtgatggca atagtcgtgg gacaaccagc ctagctgttg gtagcacacg ccaagaacca 8940aagcagcggt gaaataccaa ttcgtgtaca cggtcatcaa gatggaaagg accatcaaac 9000cgacggtagt tgaagtcttg taaaaatacc aaaggaaact cgtctcgaac atacccttgg 9060agatgaattc ctcccgcaac ttgcggaaat cctcctgggg ctcatcacgc ttaggcttgg 9120gtgcaacagg tgtgtcagga gaggaaggct ccataacagg cattctcttc aacttggcga 9180cggcttcttg agagtgcatc accatgaaga catcggtcgc atcgcggttg cgatagttct 9240cgataatgtc agctcctcca gggtggtgat tgacccaagc agacacatca taagttgcgc 9300catcaattgt gattggcaga gcttgccgct taggagacat ggtgcggccg cttggggggc 9360tatggaagac tttcttagtt agttgtgtga ataagcaatg ttgggagaat cgggactact 9420tataggatag gaataaaaca gaaaagtatt aagtgctaat gaaatattta gactgataat 9480taaaatcttc acgtatgtcc acttgatata aaaacgtcag gaataaagga agtacagtag 9540aatttaaagg tactcttttt atatataccc gtgttctctt tttggctagc tagttgcata 9600aaaaataatc tatattttta tcattatttt aaatatctta tgagatggta aatatttatc 9660ataatttttt ttactattat ttattatttg tgtgtgtaat acatatagaa gttaattaca 9720aattttattt actttttcat tattttgata tgattcacca ttaatttagt gttattattt 9780ataatagttc attttaatct ttttgtatat attatgcgtg cagtactttt ttcctacata 9840taactactat tacattttat ttatataata tttttattaa tgaattttcg tgataatatg 9900taatattgtt cattattatt tcagattttt taaaaatatt tgtgttatta tttatgaaat 9960atgtaatttt tttagtattt gattttatga tgataaagtg ttctaaattc aaaagaaggg 10020ggaaagcgta aacattaaaa aacgtcatca aacaaaaaca aaatcttgtt aataaagata 10080aaactgtttg ttttgatcac tgttatttcg taatataaaa acattattta tatttatatt 10140gttgacaacc aaatttgcct atcaaatcta accaatataa tgcatgcgtg gcaggtaatg 10200tactaccatg aacttaagtc atgacataat aaaccgtgaa tctgaccaat gcatgtacct 10260anctaaattg tatttgtgac acgaagcaaa tgattcaatt cacaatggag atgggaaaca 10320aataatgaag aacccagaac taagaaagct tttctgaaaa ataaaataaa ggcaatgtca 10380aaagtatact gcatcatcag tccagaaagc acatgatatt tttttatcag tatcaatgca 10440gctagtttta ttttacaata tcgatatagc tagtttaaat atattgcagc tagatttata 10500aatatttgtg ttattattta tcatttgtgt aatcctgttt ttagtatttt agtttatata 10560tgatgataat gtattccaaa tttaaaagaa gggaaataaa tttaaacaag aaaaaaagtc 10620atcaaacaaa aaacaaatga aagggtggaa agatgttacc atgtaatgtg aatgttacag 10680tatttctttt attatagagt taacaaatta actaatatga ttttgttaat aatgataaaa 10740tatttttttt attattattt cataatataa aaatagttta cttaatataa aaaaaattct 10800atcgttcaca acaaagttgg ccacctaatt taaccatgca tgtacccatg gaccatatta 10860ggtaaccatc aaacctgatg aagagataaa gagatgaaga cttaagtcat aacacaaaac 10920cataaaaaac aaaaatacaa tcaaccgtca atctgaccaa tgcatgaaaa agctgcaata 10980gtgagtggcg acacaaagca catgattttc ttacaacgga gataaaacca aaaaaatatt 11040tcatgaacaa cctagaacaa ataaagcttt tatataataa atatataaat aaataaaggc 11100tatggaataa tatacttcaa tatatttgga ttaaataaat tgttggcggg gttgatatat 11160ttatacacac ctaaagtcac ttcaatctca ttttcactta acttttattt tttttttctt 11220tttatttatc ataaagagaa tattgataat atacttttta acatattttt atgacatttt 11280ttattggtga aaacttatta aaaatcataa attttgtaag ttagatttat ttaaagagtt 11340cctcttctta ttttaaattt tttaataaat ttttaaataa ctaaaatttg tgttaaaaat 11400gttaaaaaat gtgttattaa cccttctctt cgaggacgta cgtctagagt cgacctgca 11459836021DNAArtificial Sequenceplasmid pKR271 83ggccgcgaat tcaatcacta gtgaattcgc ggccgcatga gccgtaaagg ttcaatacaa 60cgagtgcttg ttttcttagg gacaagcatt gtacttatgt atgattctgt gtaaccatga 120gtcttccacg ttgtactaat gtgaagggca aaaataaaac acagaacaag ttcgtttttc 180tcaaataatg tgaaggtaga aaatggaacc atgcctcctc tcttgcatgt gatttaaaat 240attagcagat ggtaccgtac gtgggcggat cccccgggct gcaggaattc actggccgtc 300gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca 360catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa 420cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg 480tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag 540ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc 600ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt 660tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag 720gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg 780cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga 840caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat 900ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca 960gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc 1020gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca 1080atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg 1140caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca 1200gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata 1260accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag 1320ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg 1380gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca 1440acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta 1500atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct 1560ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca 1620gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag 1680gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat 1740tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt 1800taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 1860cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1920gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1980gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 2040agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 2100aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 2160agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 2220cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 2280accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 2340aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 2400ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2460cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2520gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2580tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2640agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc 2700aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc 2760gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca 2820ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa 2880caatttcaca caggaaacag ctatgaccat gattacgcca agcttgcatg cctgcaggtc 2940gactcgacgt acgatcccac atgcaagttt ttatttcaat cccttttcct ttgaataact 3000gaccaagaac aacaagaaaa aaaaaaaaaa agaaaaggat cattttgaaa ggatattttt 3060cgctcctatt caaatactgt atttttacca aaaaaactgt atttttccta cactctcaag 3120ctttgttttt cgcttcgact ctcatgattt ccttcatatg ccaatcactc tatttataaa 3180tggcataagg tagtgtgaac aattgcaaag cttgtcatca aaagcttgca atgtacaaat 3240taatgttttt catgcctttc aaaattatct gcacccccta gctattaatc taacatctaa 3300gtaaggctag tgaatttttt cgaatagtca tgcagtgcat taatttcccc gtgactattt 3360tggctttgac tccaacactg gccccgtaca tccgtccctc attacatgaa aagaaatatt 3420gtttatattc ttaattaaaa atattgtccc ttctaaattt tcatatagtt aattattata 3480ttactttttt ctctattcta ttagttctat tttcaaatta ttatttatgc atatgtaaag 3540tacattatat ttttgctata tacttaaata tttctaaatt attaaaaaaa gactgatatg 3600aaaaatttat tctttttaaa gctatatcat tttatatata ctttttcttt tcttttcttt 3660cattttctat tcaatttaat aagaaataaa ttttgtaaat ttttatttat caatttataa 3720aaatatttta ctttatatgt tttttcacat ttttgttaaa caaatcatat cattatgatt 3780gaaagagagg aaattgacag tgagtaataa gtgatgagaa aaaaatgtgt tatttcctaa 3840aaaaaaccta aacaaacatg tatctactct ctatttcatc tatctctcat ttcatttttc 3900tctttatctc tttctttatt tttttatcat atcatttcac attaattatt tttactctct 3960ttattttttc tctctatccc tctcttattt ccactcatat atacactcca aaattggggc 4020atgcctttat cactactcta tctcctccac taaatcattt aaatgaaact gaaaagcatt 4080ggcaagtctc ctcccctcct caagtgattt ccaactcagc attggcatct aattgattca 4140gtatatctat tgcatgtgta aaagtctttc cacaatacat aactattaat taatcttaaa 4200taaataaagg ataaaatatt tttttttctt cataaaatta aaatatgtta ttttttgttt 4260agatgtatat tcgaataaat ctaaatatat gataatgatt ttttatattg attaaacata 4320taatcaatat taaatatgat atttttttat ataggttgta cacataattt tataaggata 4380aaaaatatga taaaaataaa ttttaaatat ttttatattt acgagaaaaa aaaatatttt 4440agccataaat aaatgaccag catattttac aaccttagta attcataaat tcctatatgt 4500atatttgaaa ttaaaaacag ataatcgtta agggaaggaa tcctacgtca tctcttgcca 4560tttgtttttc atgcaaacag aaagggacga aaaaccacct caccatgaat cactcttcac 4620accattttta ctagcaaaca agtctcaaca actgaagcca gctctctttc cgtttctttt 4680tacaacactt tctttgaaat agtagtattt ttttttcaca tgatttatta acgtgccaaa 4740agatgcttat tgaatagagt gcacatttgt aatgtactac taattagaac atgaaaaagc 4800attgttctaa cacgataatc ctgtgaaggc gttaactcca aagatccaat ttcactatat 4860aaattgtgac gaaagcaaaa tgaattcaca tagctgagag agaaaggaaa ggttaactaa 4920gaagcaatac ttcagcggcc gcatgactga ggataagacg aaggtcgagt tcccgacgct 4980cacggagctc aagcactcga tcccgaacgc gtgctttgag tcgaacctcg gcctctcgct 5040ctactacacg gcccgcgcga tcttcaacgc gtcggcctcg gcggcgctgc tctacgcggc 5100gcgctcgacg ccgttcattg ccgataacgt tctgctccac gcgctcgttt gcgccaccta 5160catctacgtg cagggcgtca tcttctgggg cttcttcacg gtcggccacg actgcggcca 5220ctcggccttc tcgcgctacc acagcgtcaa ctttatcatc ggctgcatca tgcactctgc 5280gattttgacg ccgttcgaga gctggcgcgt gacgcaccgc caccaccaca agaacacggg 5340caacattgat aaggacgaga tcttttaccc gcaccggtcg gtcaaggacc tccaggacgt 5400gcgccaatgg gtctacacgc tcggcggtgc gtggtttgtc tacttgaagg tcgggtatgc 5460cccgcgcacg atgagccact ttgacccgtg ggacccgctc ctccttcgcc gcgcgtcggc 5520cgtcatcgtg tcgctcggcg tctgggccgc cttcttcgcc gcgtacgcgt acctcacata 5580ctcgctcggc tttgccgtca tgggcctcta ctactatgcg ccgctctttg tctttgcttc 5640gttcctcgtc attacgacct tcttgcacca caacgacgaa gcgacgccgt ggtacggcga 5700ctcggagtgg acgtacgtca agggcaacct ctcgagcgtc gaccgctcgt acggcgcgtt 5760cgtggacaac ctgagccacc acattggcac gcaccaggtc caccacttgt tcccgatcat 5820tccgcactac aagctcaacg aagccaccaa gcactttgcg gccgcgtacc cgcacctcgt 5880gcgcaggaac gacgagccca tcatcacggc cttcttcaag accgcgcacc tctttgtcaa 5940ctacggcgct gtgcccgaga cggcgcagat cttcacgctc aaagagtcgg ccgcggccgc 6000caaggccaag tcggactaag c 6021846524DNAArtificial Sequenceplasmid pKR226 84gtacgtctag aggatccgtc gacggcgcgc ccgatcatcc ggatatagtt cctcctttca 60gcaaaaaacc cctcaagacc cgtttagagg ccccaagggg ttatgctagt tattgctcag 120cggtggcagc agccaactca gcttcctttc gggctttgtt agcagccgga tcgatccaag 180ctgtacctca ctattccttt gccctcggac gagtgctggg gcgtcggttt ccactatcgg 240cgagtacttc tacacagcca tcggtccaga cggccgcgct tctgcgggcg atttgtgtac 300gcccgacagt cccggctccg gatcggacga ttgcgtcgca tcgaccctgc gcccaagctg 360catcatcgaa attgccgtca accaagctct gatagagttg gtcaagacca atgcggagca 420tatacgcccg gagccgcggc gatcctgcaa gctccggatg cctccgctcg aagtagcgcg 480tctgctgctc catacaagcc aaccacggcc tccagaagaa gatgttggcg acctcgtatt 540gggaatcccc gaacatcgcc tcgctccagt caatgaccgc tgttatgcgg ccattgtccg 600tcaggacatt gttggagccg aaatccgcgt gcacgaggtg ccggacttcg gggcagtcct 660cggcccaaag catcagctca tcgagagcct gcgcgacgga cgcactgacg gtgtcgtcca 720tcacagtttg ccagtgatac acatggggat cagcaatcgc gcatatgaaa tcacgccatg 780tagtgtattg accgattcct tgcggtccga atgggccgaa cccgctcgtc tggctaagat 840cggccgcagc gatcgcatcc atagcctccg cgaccggctg cagaacagcg ggcagttcgg 900tttcaggcag gtcttgcaac gtgacaccct gtgcacggcg ggagatgcaa taggtcaggc 960tctcgctgaa ttccccaatg tcaagcactt ccggaatcgg gagcgcggcc gatgcaaagt 1020gccgataaac ataacgatct ttgtagaaac catcggcgca gctatttacc cgcaggacat 1080atccacgccc tcctacatcg aagctgaaag cacgagattc ttcgccctcc gagagctgca 1140tcaggtcgga gacgctgtcg aacttttcga tcagaaactt ctcgacagac gtcgcggtga 1200gttcaggctt ttccatgggt atatctcctt cttaaagtta aacaaaatta tttctagagg 1260gaaaccgttg tggtctccct atagtgagtc gtattaattt cgcgggatcg agatctgatc 1320aacctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct 1380tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 1440gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 1500atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 1560ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 1620cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 1680tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 1740gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 1800aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 1860tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 1920aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 1980aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc 2040ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 2100ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 2160atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 2220atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt 2280gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa 2340gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg 2400ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca tatggacata 2460ttgtcgttag aacgcggcta caattaatac ataaccttat gtatcataca catacgattt 2520aggtgacact atagaacggc gcgccaagct gggtctagaa ctagaaacgt gatgccactt 2580gttattgaag tcgattacag catctattct gttttactat ttataacttt gccatttctg 2640acttttgaaa actatctctg gatttcggta tcgctttgtg aagatcgagc aaaagagacg 2700ttttgtggac gcaatggtcc aaatccgttc tacatgaaca aattggtcac aatttccact 2760aaaagtaaat aaatggcaag ttaaaaaagg aatatgcatt ttactgattg cctaggtgag 2820ctccaagaga agttgaatct acacgtctac caaccgctaa aaaaagaaaa acattgatat 2880gtaacctgat tccattagct tttgacttct tcaacagatt ctctacttag atttctaaca 2940gaaatattat tactagcaca tcattttcag tctcactaca gcaaaaaatc caacggcaca 3000atacagacaa caggagatat cagactacag agatagatag atgctactgc atgtagtaag 3060ttaaataaaa ggaaaataaa atgtcttgct accaaaacta ctacagacta tgatgctcac 3120cacaggccaa atcctgcaac taggacagca ttatcttata tatattgtac aaaacaagca 3180tcaaggaaca tttggtctag gcaatcagta cctcgttcta ccatcaccct cagttatcac 3240atccttgaag gatccattac tgggaatcat cggcaacaca tgctcctgat ggggcacaat 3300gacatcaaga aggtaggggc caggggtgtc caacattctc tgaattgccg ctctaagctc 3360ttccttcttc gtcactcgcg ctgccggtat cccacaagca tcagcaaact tgagcatgtt 3420tgggaatatc tcgctctcgc tagacggatc tccaagatag gtgtgagctc tattggactt 3480gtagaaccta tcctccaact gaaccaccat acccaaatgc tgattgttca acaacaatat 3540cttaactggg agattctcca ctcttatagt ggccaactcc tgaacattca tgatgaaact 3600accatcccca tcaatgtcaa ccacaacagc cccagggtta gcaacagcag caccaatagc 3660cgcaggcaat ccaaaaccca tggctccaag accccctgag gtcaaccact gcctcggtct 3720cttgtacttg taaaactgcg cagcccacat ttgatgctgc ccaaccccag tactaacaat 3780agcatctcca ttagtcaact catcaagaac ctcgatagca tgctgcggag aaatcgcgtc 3840ctggaatgtc ttgtaaccca atggaaactt gtgtttctgc acattaatct cttctctcca 3900acctccaaga tcaaacttac cctccactcc tttctcctcc aaaatcatat taattccctt 3960caaggccaac ttcaaatccg cgcaaaccga cacgtgcgcc tgcttgttct tcccaatctc 4020ggcagaatca atatcaatgt gaacaatctt agccctacta gcaaaagcct caagcttccc 4080agtaacacgg tcatcaaacc ttaccccaaa ggcaagcaac aaatcactat tgtcaacagc 4140atagttagca taaacagtac catgcatacc cagcatctga agggaatatt catcaccaat 4200aggaaaagtt ccaagaccca ttaaagtgct agcaacggga ataccagtga gttcaacaaa 4260gcgcctcaat tcagcactgg aattcaaact gccaccgccg acgtagagaa cgggcttttg 4320ggcctccatg atgagtctga caatgtgttc caattgggcc tcggcggggg gcctgggcag 4380cctggcgagg taaccgggga ggttaacggg ctcgtcccaa ttaggcacgg cgagttgctg 4440ctgaacgtct ttgggaatgt cgatgaggac cggaccgggg cggccggagg tggcgacgaa 4500gaaagcctcg gcgacgacgc gggggatgtc gtcgacgtcg aggatgaggt agttgtgctt 4560cgtgatggat ctgctcacct ccacgatcgg ggtttcttgg aaggcgtcgg tgccgatcat 4620ccggcgggcg acctggccgg tgatggcgac gactgggacg ctgtccatta aagcgtcggc 4680gaggccgctc acgaggttgg tggcgccggg gccggaggtg gcaatgcaga cgccggggag 4740gccggaggaa cgcgcgtagc cttcggcggc gaagacgccg ccctgctcgt ggcgcgggag 4800cacgttgcgg atggcggcgg agcgcgtgag cgcctggtgg atctccatcg acgcaccgcc 4860ggggtacgcg aacaccgtcg tcacgccctg cctctccagc gcctccacaa ggatgtccgc 4920gcccttgcga ggttcgccgg aggcgaaccg tgacacgaag ggctccgtgg tcggcgcttc 4980cttggtgaag ggcgccgccg tggggggttt ggagatggaa catttgattt tgagagcgtg 5040gttgggtttg gtgagggttt gatgagagag agggagggtg gatctagtaa tgcgtttggg 5100gaaggtgggg tgtgaagagg aagaagagaa tcgggtggtt ctggaagcgg tggccgccat 5160tgtgttgtgt ggcatggtta tacttcaaaa actgcacaac aagcctagag ttagtaccta 5220aacagtaaat ttacaacaga gagcaaagac acatgcaaaa atttcagcca taaaaaaagt 5280tataatagaa tttaaagcaa aagtttcatt ttttaaacat atatacaaac aaactggatt 5340tgaaggaagg gattaattcc

cctgctcaaa gtttgaattc ctattgtgac ctatactcga 5400ataaaattga agcctaagga atgtatgaga aacaagaaaa caaaacaaaa ctacagacaa 5460acaagtacaa ttacaaaatt cgctaaaatt ctgtaatcac caaaccccat ctcagtcagc 5520acaaggccca aggtttattt tgaaataaaa aaaaagtgat tttatttctc ataagctaaa 5580agaaagaaag gcaattatga aatgatttcg actagatctg aaagtccaac gcgtattccg 5640cagatattaa agaaagagta gagtttcaca tggatcctag atggacccag ttgaggaaaa 5700agcaaggcaa agcaaaccag aagtgcaaga tccgaaattg aaccacggaa tctaggattt 5760ggtagaggga gaagaaaagt accttgagag gtagaagaga agagaagagc agagagatat 5820atgaacgagt gtgtcttggt ctcaactctg aagcgatacg agtttagagg ggagcattga 5880gttccaattt atagggaaac cgggtggcag gggtgagtta atgacggaaa agcccctaag 5940taacgagatt ggattgtggg ttagattcaa ccgtttgcat ccgcggctta gattggggaa 6000gtcagagtga atctcaaccg ttgactgagt tgaaaattga atgtagcaac caattgagcc 6060aaccccagcc tttgcccttt gattttgatt tgtttgttgc atacttttta tttgtcttct 6120ggttctgact ctctttctct cgtttcaatg ccaggttgcc tactcccaca ccactcacaa 6180gaagattcta ctgttagtat taaatatttt ttaatgtatt aaatgatgaa tgcttttgta 6240aacagaacaa gactatgtct aataagtgtc ttgcaacatt ttttaagaaa ttaaaaaaaa 6300tatatttatt atcaaaatca aatgtatgaa aaatcatgaa taatataatt ttatacattt 6360ttttaaaaaa tcttttaatt tcttaattaa tatcttaaaa ataatgatta atatttaacc 6420caaaataatt agtatgattg gtaaggaaga tatccatgtt atgtttggat gtgagtttga 6480tctagagcaa agcttactag agtcgacctg caggtcgact cgac 6524859892DNAArtificial Sequenceplasmid pKR886r 85ggtcgactcg acgtacgtct agaggatccg tcgacggcgc gcccgatcat ccggatatag 60ttcctccttt cagcaaaaaa cccctcaaga cccgtttaga ggccccaagg ggttatgcta 120gttattgctc agcggtggca gcagccaact cagcttcctt tcgggctttg ttagcagccg 180gatcgatcca agctgtacct cactattcct ttgccctcgg acgagtgctg gggcgtcggt 240ttccactatc ggcgagtact tctacacagc catcggtcca gacggccgcg cttctgcggg 300cgatttgtgt acgcccgaca gtcccggctc cggatcggac gattgcgtcg catcgaccct 360gcgcccaagc tgcatcatcg aaattgccgt caaccaagct ctgatagagt tggtcaagac 420caatgcggag catatacgcc cggagccgcg gcgatcctgc aagctccgga tgcctccgct 480cgaagtagcg cgtctgctgc tccatacaag ccaaccacgg cctccagaag aagatgttgg 540cgacctcgta ttgggaatcc ccgaacatcg cctcgctcca gtcaatgacc gctgttatgc 600ggccattgtc cgtcaggaca ttgttggagc cgaaatccgc gtgcacgagg tgccggactt 660cggggcagtc ctcggcccaa agcatcagct catcgagagc ctgcgcgacg gacgcactga 720cggtgtcgtc catcacagtt tgccagtgat acacatgggg atcagcaatc gcgcatatga 780aatcacgcca tgtagtgtat tgaccgattc cttgcggtcc gaatgggccg aacccgctcg 840tctggctaag atcggccgca gcgatcgcat ccatagcctc cgcgaccggc tgcagaacag 900cgggcagttc ggtttcaggc aggtcttgca acgtgacacc ctgtgcacgg cgggagatgc 960aataggtcag gctctcgctg aattccccaa tgtcaagcac ttccggaatc gggagcgcgg 1020ccgatgcaaa gtgccgataa acataacgat ctttgtagaa accatcggcg cagctattta 1080cccgcaggac atatccacgc cctcctacat cgaagctgaa agcacgagat tcttcgccct 1140ccgagagctg catcaggtcg gagacgctgt cgaacttttc gatcagaaac ttctcgacag 1200acgtcgcggt gagttcaggc ttttccatgg gtatatctcc ttcttaaagt taaacaaaat 1260tatttctaga gggaaaccgt tgtggtctcc ctatagtgag tcgtattaat ttcgcgggat 1320cgagatctga tcaacctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 1380attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 1440cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 1500gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 1560ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 1620agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 1680tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 1740ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag 1800gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 1860ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 1920gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 1980aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg 2040aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 2100ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 2160gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 2220gggattttgg tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc 2280gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca 2340gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt 2400ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac 2460catatggaca tattgtcgtt agaacgcggc tacaattaat acataacctt atgtatcata 2520cacatacgat ttaggtgaca ctatagaacg gcgcgccaag ctgggtctag aactagaaac 2580gtgatgccac ttgttattga agtcgattac agcatctatt ctgttttact atttataact 2640ttgccatttc tgacttttga aaactatctc tggatttcgg tatcgctttg tgaagatcga 2700gcaaaagaga cgttttgtgg acgcaatggt ccaaatccgt tctacatgaa caaattggtc 2760acaatttcca ctaaaagtaa ataaatggca agttaaaaaa ggaatatgca ttttactgat 2820tgcctaggtg agctccaaga gaagttgaat ctacacgtct accaaccgct aaaaaaagaa 2880aaacattgat atgtaacctg attccattag cttttgactt cttcaacaga ttctctactt 2940agatttctaa cagaaatatt attactagca catcattttc agtctcacta cagcaaaaaa 3000tccaacggca caatacagac aacaggagat atcagactac agagatagat agatgctact 3060gcatgtagta agttaaataa aaggaaaata aaatgtcttg ctaccaaaac tactacagac 3120tatgatgctc accacaggcc aaatcctgca actaggacag cattatctta tatatattgt 3180acaaaacaag catcaaggaa catttggtct aggcaatcag tacctcgttc taccatcacc 3240ctcagttatc acatccttga aggatccatt actgggaatc atcggcaaca catgctcctg 3300atggggcaca atgacatcaa gaaggtaggg gccaggggtg tccaacattc tctgaattgc 3360cgctctaagc tcttccttct tcgtcactcg cgctgccggt atcccacaag catcagcaaa 3420cttgagcatg tttgggaata tctcgctctc gctagacgga tctccaagat aggtgtgagc 3480tctattggac ttgtagaacc tatcctccaa ctgaaccacc atacccaaat gctgattgtt 3540caacaacaat atcttaactg ggagattctc cactcttata gtggccaact cctgaacatt 3600catgatgaaa ctaccatccc catcaatgtc aaccacaaca gccccagggt tagcaacagc 3660agcaccaata gccgcaggca atccaaaacc catggctcca agaccccctg aggtcaacca 3720ctgcctcggt ctcttgtact tgtaaaactg cgcagcccac atttgatgct gcccaacccc 3780agtactaaca atagcatctc cattagtcaa ctcatcaaga acctcgatag catgctgcgg 3840agaaatcgcg tcctggaatg tcttgtaacc caatggaaac ttgtgtttct gcacattaat 3900ctcttctctc caacctccaa gatcaaactt accctccact cctttctcct ccaaaatcat 3960attaattccc ttcaaggcca acttcaaatc cgcgcaaacc gacacgtgcg cctgcttgtt 4020cttcccaatc tcggcagaat caatatcaat gtgaacaatc ttagccctac tagcaaaagc 4080ctcaagcttc ccagtaacac ggtcatcaaa ccttacccca aaggcaagca acaaatcact 4140attgtcaaca gcatagttag cataaacagt accatgcata cccagcatct gaagggaata 4200ttcatcacca ataggaaaag ttccaagacc cattaaagtg ctagcaacgg gaataccagt 4260gagttcaaca aagcgcctca attcagcact ggaattcaaa ctgccaccgc cgacgtagag 4320aacgggcttt tgggcctcca tgatgagtct gacaatgtgt tccaattggg cctcggcggg 4380gggcctgggc agcctggcga ggtaaccggg gaggttaacg ggctcgtccc aattaggcac 4440ggcgagttgc tgctgaacgt ctttgggaat gtcgatgagg accggaccgg ggcggccgga 4500ggtggcgacg aagaaagcct cggcgacgac gcgggggatg tcgtcgacgt cgaggatgag 4560gtagttgtgc ttcgtgatgg atctgctcac ctccacgatc ggggtttctt ggaaggcgtc 4620ggtgccgatc atccggcggg cgacctggcc ggtgatggcg acgactggga cgctgtccat 4680taaagcgtcg gcgaggccgc tcacgaggtt ggtggcgccg gggccggagg tggcaatgca 4740gacgccgggg aggccggagg aacgcgcgta gccttcggcg gcgaagacgc cgccctgctc 4800gtggcgcggg agcacgttgc ggatggcggc ggagcgcgtg agcgcctggt ggatctccat 4860cgacgcaccg ccggggtacg cgaacaccgt cgtcacgccc tgcctctcca gcgcctccac 4920aaggatgtcc gcgcccttgc gaggttcgcc ggaggcgaac cgtgacacga agggctccgt 4980ggtcggcgct tccttggtga agggcgccgc cgtggggggt ttggagatgg aacatttgat 5040tttgagagcg tggttgggtt tggtgagggt ttgatgagag agagggaggg tggatctagt 5100aatgcgtttg gggaaggtgg ggtgtgaaga ggaagaagag aatcgggtgg ttctggaagc 5160ggtggccgcc attgtgttgt gtggcatggt tatacttcaa aaactgcaca acaagcctag 5220agttagtacc taaacagtaa atttacaaca gagagcaaag acacatgcaa aaatttcagc 5280cataaaaaaa gttataatag aatttaaagc aaaagtttca ttttttaaac atatatacaa 5340acaaactgga tttgaaggaa gggattaatt cccctgctca aagtttgaat tcctattgtg 5400acctatactc gaataaaatt gaagcctaag gaatgtatga gaaacaagaa aacaaaacaa 5460aactacagac aaacaagtac aattacaaaa ttcgctaaaa ttctgtaatc accaaacccc 5520atctcagtca gcacaaggcc caaggtttat tttgaaataa aaaaaaagtg attttatttc 5580tcataagcta aaagaaagaa aggcaattat gaaatgattt cgactagatc tgaaagtcca 5640acgcgtattc cgcagatatt aaagaaagag tagagtttca catggatcct agatggaccc 5700agttgaggaa aaagcaaggc aaagcaaacc agaagtgcaa gatccgaaat tgaaccacgg 5760aatctaggat ttggtagagg gagaagaaaa gtaccttgag aggtagaaga gaagagaaga 5820gcagagagat atatgaacga gtgtgtcttg gtctcaactc tgaagcgata cgagtttaga 5880ggggagcatt gagttccaat ttatagggaa accgggtggc aggggtgagt taatgacgga 5940aaagccccta agtaacgaga ttggattgtg ggttagattc aaccgtttgc atccgcggct 6000tagattgggg aagtcagagt gaatctcaac cgttgactga gttgaaaatt gaatgtagca 6060accaattgag ccaaccccag cctttgccct ttgattttga tttgtttgtt gcatactttt 6120tatttgtctt ctggttctga ctctctttct ctcgtttcaa tgccaggttg cctactccca 6180caccactcac aagaagattc tactgttagt attaaatatt ttttaatgta ttaaatgatg 6240aatgcttttg taaacagaac aagactatgt ctaataagtg tcttgcaaca ttttttaaga 6300aattaaaaaa aatatattta ttatcaaaat caaatgtatg aaaaatcatg aataatataa 6360ttttatacat ttttttaaaa aatcttttaa tttcttaatt aatatcttaa aaataatgat 6420taatatttaa cccaaaataa ttagtatgat tggtaaggaa gatatccatg ttatgtttgg 6480atgtgagttt gatctagagc aaagcttact agagtcgacc tgcagcccgg gggatccgcc 6540cacgtacggt accatctgct aatattttaa atcacatgca agagaggagg catggttcca 6600ttttctacct tcacattatt tgagaaaaac gaacttgttc tgtgttttat ttttgccctt 6660cacattagta caacgtggaa gactcatggt tacacagaat catacataag tacaatgctt 6720gtccctaaga aaacaagcac tcgttgtatt gaacctttac ggctcatgcg gccgcgaatt 6780cactagtgat tgaattcgcg gccgcttagt ccgacttggc cttggcggcc gcggccgact 6840ctttgagcgt gaagatctgc gccgtctcgg gcacagcgcc gtagttgaca aagaggtgcg 6900cggtcttgaa gaaggccgtg atgatgggct cgtcgttcct gcgcacgagg tgcgggtacg 6960cggccgcaaa gtgcttggtg gcttcgttga gcttgtagtg cggaatgatc gggaacaagt 7020ggtggacctg gtgcgtgcca atgtggtggc tcaggttgtc cacgaacgcg ccgtacgagc 7080ggtcgacgct cgagaggttg cccttgacgt acgtccactc cgagtcgccg taccacggcg 7140tcgcttcgtc gttgtggtgc aagaaggtcg taatgacgag gaacgaagca aagacaaaga 7200gcggcgcata gtagtagagg cccatgacgg caaagccgag cgagtatgtg aggtacgcgt 7260acgcggcgaa gaaggcggcc cagacgccga gcgacacgat gacggccgac gcgcggcgaa 7320ggaggagcgg gtcccacggg tcaaagtggc tcatcgtgcg cggggcatac ccgaccttca 7380agtagacaaa ccacgcaccg ccgagcgtgt agacccattg gcgcacgtcc tggaggtcct 7440tgaccgaccg gtgcgggtaa aagatctcgt ccttatcaat gttgcccgtg ttcttgtggt 7500ggtggcggtg cgtcacgcgc cagctctcga acggcgtcaa aatcgcagag tgcatgatgc 7560agccgatgat aaagttgacg ctgtggtagc gcgagaaggc cgagtggccg cagtcgtggc 7620cgaccgtgaa gaagccccag aagatgacgc cctgcacgta gatgtaggtg gcgcaaacga 7680gcgcgtggag cagaacgtta tcggcaatga acggcgtcga gcgcgccgcg tagagcagcg 7740ccgccgaggc cgacgcgttg aagatcgcgc gggccgtgta gtagagcgag aggccgaggt 7800tcgactcaaa gcacgcgttc gggatcgagt gcttgagctc cgtgagcgtc gggaactcga 7860ccttcgtctt atcctcagtc atgcggccgc tgaagtattg cttcttagtt aacctttcct 7920ttctctctca gctatgtgaa ttcattttgc tttcgtcaca atttatatag tgaaattgga 7980tctttggagt taacgccttc acaggattat cgtgttagaa caatgctttt tcatgttcta 8040attagtagta cattacaaat gtgcactcta ttcaataagc atcttttggc acgttaataa 8100atcatgtgaa aaaaaaatac tactatttca aagaaagtgt tgtaaaaaga aacggaaaga 8160gagctggctt cagttgttga gacttgtttg ctagtaaaaa tggtgtgaag agtgattcat 8220ggtgaggtgg tttttcgtcc ctttctgttt gcatgaaaaa caaatggcaa gagatgacgt 8280aggattcctt cccttaacga ttatctgttt ttaatttcaa atatacatat aggaatttat 8340gaattactaa ggttgtaaaa tatgctggtc atttatttat ggctaaaata tttttttttc 8400tcgtaaatat aaaaatattt aaaatttatt tttatcatat tttttatcct tataaaatta 8460tgtgtacaac ctatataaaa aaatatcata tttaatattg attatatgtt taatcaatat 8520aaaaaatcat tatcatatat ttagatttat tcgaatatac atctaaacaa aaaataacat 8580attttaattt tatgaagaaa aaaaaatatt ttatccttta tttatttaag attaattaat 8640agttatgtat tgtggaaaga cttttacaca tgcaatagat atactgaatc aattagatgc 8700caatgctgag ttggaaatca cttgaggagg ggaggagact tgccaatgct tttcagtttc 8760atttaaatga tttagtggag gagatagagt agtgataaag gcatgcccca attttggagt 8820gtatatatga gtggaaataa gagagggata gagagaaaaa ataaagagag taaaaataat 8880taatgtgaaa tgatatgata aaaaaataaa gaaagagata aagagaaaaa tgaaatgaga 8940gatagatgaa atagagagta gatacatgtt tgtttaggtt ttttttagga aataacacat 9000ttttttctca tcacttatta ctcactgtca atttcctctc tttcaatcat aatgatatga 9060tttgtttaac aaaaatgtga aaaaacatat aaagtaaaat atttttataa attgataaat 9120aaaaatttac aaaatttatt tcttattaaa ttgaatagaa aatgaaagaa aagaaaagaa 9180aaagtatata taaaatgata tagctttaaa aagaataaat ttttcatatc agtctttttt 9240taataattta gaaatattta agtatatagc aaaaatataa tgtactttac atatgcataa 9300ataataattt gaaaatagaa ctaatagaat agagaaaaaa gtaatataat aattaactat 9360atgaaaattt agaagggaca atatttttaa ttaagaatat aaacaatatt tcttttcatg 9420taatgaggga cggatgtacg gggccagtgt tggagtcaaa gccaaaatag tcacggggaa 9480attaatgcac tgcatgacta ttcgaaaaaa ttcactagcc ttacttagat gttagattaa 9540tagctagggg gtgcagataa ttttgaaagg catgaaaaac attaatttgt acattgcaag 9600cttttgatga caagctttgc aattgttcac actaccttat gccatttata aatagagtga 9660ttggcatatg aaggaaatca tgagagtcga agcgaaaaac aaagcttgag agtgtaggaa 9720aaatacagtt tttttggtaa aaatacagta tttgaatagg agcgaaaaat atcctttcaa 9780aatgatcctt ttcttttttt ttttttttct tgttgttctt ggtcagttat tcaaaggaaa 9840agggattgaa ataaaaactt gcatgtggga tcgtacgtcg agtcgacctg ca 98928632DNAArtificial Sequenceprimer oCon1 86aatctagacc tgcaggatcc atgcccttca tt 328733DNAArtificial Sequenceprimer oCon2 87tttctagacc tgcaggttga aacatccctg aag 33884480DNAArtificial Sequenceplasmid pKR179 88ctagacctgc aggatccatg cccttcattt gccgcttatt aattaatttg gtaacagtcc 60gtactaatca gttacttatc cttcccccat cataattaat cttggtagtc tcgaatgcca 120caacactgac tagtctcttg gatcataaga aaaagccaag gaacaaaaga agacaaaaca 180caatgagagt atcctttgca tagcaatgtc taagttcata aaattcaaac aaaaacgcaa 240tcacacacag tggacatcac ttatccacta gctgatcagg atcgccgcgt caagaaaaaa 300aaactggacc ccaaaagcca tgcacaacaa cacgtactca caaaggtgtc aatcgagcag 360cccaaaacat tcaccaactc aacccatcat gagccctcac atttgttgtt tctaacccaa 420cctcaaactc gtattctctt ccgccacctc atttttgttt atttcaacac ccgtcaaact 480gcatgccacc ccgtggccaa atgtccatgc atgttaacaa gacctatgac tataaatagc 540tgcaatctcg gcccaggttt tcatcatcaa gaaccagttc aatatcctag tacaccgtat 600taaagaattt aagatatact gcggccgcaa gtatgaacta aaatgcatgt aggtgtaaga 660gctcatggag agcatggaat attgtatccg accatgtaac agtataataa ctgagctcca 720tctcacttct tctatgaata aacaaaggat gttatgatat attaacactc tatctatgca 780ccttattgtt ctatgataaa tttcctctta ttattataaa tcatctgaat cgtgacggct 840tatggaatgc ttcaaatagt acaaaaacaa atgtgtacta taagactttc taaacaattc 900taaccttagc attgtgaacg agacataagt gttaagaaga cataacaatt ataatggaag 960aagtttgtct ccatttatat attatatatt acccacttat gtattatatt aggatgttaa 1020ggagacataa caattataaa gagagaagtt tgtatccatt tatatattat atactaccca 1080tttatatatt atacttatcc acttatttaa tgtctttata aggtttgatc catgatattt 1140ctaatatttt agttgatatg tatatgaaag ggtactattt gaactctctt actctgtata 1200aaggttggat catccttaaa gtgggtctat ttaattttat tgcttcttac agataaaaaa 1260aaaattatga gttggtttga taaaatattg aaggatttaa aataataata aataacatat 1320aatatatgta tataaattta ttataatata acatttatct ataaaaaagt aaatattgtc 1380ataaatctat acaatcgttt agccttgctg gacgaatctc aattatttaa acgagagtaa 1440acatatttga ctttttggtt atttaacaaa ttattattta acactatatg aaattttttt 1500ttttatcagc aaagaataaa attaaattaa gaaggacaat ggtgtcccaa tccttataca 1560accaacttcc acaagaaagt caagtcagag acaacaaaaa aacaagcaaa ggaaattttt 1620taatttgagt tgtcttgttt gctgcataat ttatgcagta aaacactaca cataaccctt 1680ttagcagtag agcaatggtt gaccgtgtgc ttagcttctt ttattttatt tttttatcag 1740caaagaataa ataaaataaa atgagacact tcagggatgt ttcaacctgc aggtctagag 1800gatccccggg taccgagctc gaattcactg gccgtcgttt tacaacgtcg tgactgggaa 1860aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt 1920aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa 1980tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg 2040tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca 2100acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct 2160gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg 2220agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt 2280tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt 2340ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa 2400taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt 2460tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat 2520gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag 2580atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg 2640ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata 2700cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat 2760ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc 2820aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg 2880ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac 2940gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact 3000ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa 3060gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct 3120ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc 3180tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga 3240cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac 3300tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag 3360atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg 3420tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc 3480tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag 3540ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc 3600cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac 3660ctcgctctgc taatcctgtt accagtggct gctgccagtg

gcgataagtc gtgtcttacc 3720gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt 3780tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt 3840gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc 3900ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt 3960tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca 4020ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt 4080tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt 4140attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag 4200tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg 4260ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc 4320aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt 4380ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat 4440gaccatgatt acgccaagct tgcatgcctg caggtcgact 4480895754DNAArtificial Sequenceplasmid pKR1002 89ggccgcaagt 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 caacctgcag gtctagagga tccccgggta ccgagctcga 1200attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta 1260atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg 1320atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc 1380tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct 1440ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac 1500gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca 1560tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac 1620gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt 1680ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 1740atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta 1800tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 1860tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac 1920gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg 1980aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 2040gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 2100ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat 2160gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg 2220gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 2280atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 2340ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt 2400cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct 2460cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc 2520gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca 2580cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct 2640cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt 2700taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga 2760ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 2820aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 2880caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 2940taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag 3000gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 3060cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 3120taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 3180agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc 3240ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 3300gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 3360acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 3420acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 3480tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg 3540ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag 3600agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 3660acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 3720tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 3780ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg 3840catgcctgca ggtcgactct agacctgcag gatccatgcc cttcatttgc cgcttattaa 3900ttaatttggt aacagtccgt actaatcagt tacttatcct tcccccatca taattaatct 3960tggtagtctc gaatgccaca acactgacta gtctcttgga tcataagaaa aagccaagga 4020acaaaagaag acaaaacaca atgagagtat cctttgcata gcaatgtcta agttcataaa 4080attcaaacaa aaacgcaatc acacacagtg gacatcactt atccactagc tgatcaggat 4140cgccgcgtca agaaaaaaaa actggacccc aaaagccatg cacaacaaca cgtactcaca 4200aaggtgtcaa tcgagcagcc caaaacattc accaactcaa cccatcatga gccctcacat 4260ttgttgtttc taacccaacc tcaaactcgt attctcttcc gccacctcat ttttgtttat 4320ttcaacaccc gtcaaactgc atgccacccc gtggccaaat gtccatgcat gttaacaaga 4380cctatgacta taaatagctg caatctcggc ccaggttttc atcatcaaga accagttcaa 4440tatcctagta caccgtatta aagaatttaa gatatactgc ggccgcacca tgtctcctaa 4500gcggcaagct ctgccaatca caattgatgg cgcaacttat gatgtgtctg cttgggtcaa 4560tcaccaccct ggaggagctg acattatcga gaactatcgc aaccgcgatg cgaccgatgt 4620cttcatggtg atgcactctc aagaagccgt cgccaagttg aagagaatgc ctgttatgga 4680gccttcctct cctgacacac ctgttgcacc caagcctaag cgtgatgagc cccaggagga 4740tttccgcaag ttgcgggagg aattcatctc caagggtatg ttcgagacga gtttcctttg 4800gtatttttac aagacttcaa ctaccgtcgg tttgatggtc ctttccatct tgatgaccgt 4860gtacacgaat tggtatttca ccgctgcttt ggttcttggc gtgtgctacc aacagctagg 4920ctggttgtcc cacgactatt gccatcacca ggttttcaca aaccgcaaga ttaacgacgc 4980tttcggtctc tttttcggta acgtgatgca gggatactca cagacttggt ggaaggatag 5040gcacaatggt caccatgccg ccaccaatgt ggtcggccat gacccagata ttgataacct 5100ccccatcctg gcttggtctc ccgaagatgt caagagggct actccttcga ctcggaatct 5160catcaagtac cagcagtact acttcattcc caccattgca tcccttaggt tcatctggtg 5220cctccaatcc atcggcggcg tcatgtccta caagagcgag gagaggaacc tgtactacaa 5280gcgccagtac actaaggagg cgattggtct ggccctccac tgggtgctca aggccacttt 5340ctattgcagt gccatgccta gctttgccac cggtttggga tgcttcttga tctccgagct 5400gctcggagga tttggcattg ccatcgttgt gtttctgaat cactatcctt tggacaaggt 5460tgaggagact gtctgggatg agcacgggtt cagcgccagc cagatccacg agacgttgaa 5520cattaagccc ggccttctca ccgattgggt ctttggtggt ctcaactacc agattgagca 5580ccacttgtgg cccaacatgc ccaggcacaa cctcacggca gcttccctgg aggtgcagaa 5640gttgtgcgcc aagcacaacc tgccctacag ggccccagcc atcatccccg gggttcagaa 5700attggtcagc ttcttaggcg agattgccca gctggctgct gtccctgaat gagc 57549012947DNAArtificial Sequenceplasmid pKR1005 90ggtcgactcg acgtacgtct agaggatccg tcgacggcgc gcccgatcat ccggatatag 60ttcctccttt cagcaaaaaa cccctcaaga cccgtttaga ggccccaagg ggttatgcta 120gttattgctc agcggtggca gcagccaact cagcttcctt tcgggctttg ttagcagccg 180gatcgatcca agctgtacct cactattcct ttgccctcgg acgagtgctg gggcgtcggt 240ttccactatc ggcgagtact tctacacagc catcggtcca gacggccgcg cttctgcggg 300cgatttgtgt acgcccgaca gtcccggctc cggatcggac gattgcgtcg catcgaccct 360gcgcccaagc tgcatcatcg aaattgccgt caaccaagct ctgatagagt tggtcaagac 420caatgcggag catatacgcc cggagccgcg gcgatcctgc aagctccgga tgcctccgct 480cgaagtagcg cgtctgctgc tccatacaag ccaaccacgg cctccagaag aagatgttgg 540cgacctcgta ttgggaatcc ccgaacatcg cctcgctcca gtcaatgacc gctgttatgc 600ggccattgtc cgtcaggaca ttgttggagc cgaaatccgc gtgcacgagg tgccggactt 660cggggcagtc ctcggcccaa agcatcagct catcgagagc ctgcgcgacg gacgcactga 720cggtgtcgtc catcacagtt tgccagtgat acacatgggg atcagcaatc gcgcatatga 780aatcacgcca tgtagtgtat tgaccgattc cttgcggtcc gaatgggccg aacccgctcg 840tctggctaag atcggccgca gcgatcgcat ccatagcctc cgcgaccggc tgcagaacag 900cgggcagttc ggtttcaggc aggtcttgca acgtgacacc ctgtgcacgg cgggagatgc 960aataggtcag gctctcgctg aattccccaa tgtcaagcac ttccggaatc gggagcgcgg 1020ccgatgcaaa gtgccgataa acataacgat ctttgtagaa accatcggcg cagctattta 1080cccgcaggac atatccacgc cctcctacat cgaagctgaa agcacgagat tcttcgccct 1140ccgagagctg catcaggtcg gagacgctgt cgaacttttc gatcagaaac ttctcgacag 1200acgtcgcggt gagttcaggc ttttccatgg gtatatctcc ttcttaaagt taaacaaaat 1260tatttctaga gggaaaccgt tgtggtctcc ctatagtgag tcgtattaat ttcgcgggat 1320cgagatctga tcaacctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 1380attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 1440cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 1500gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 1560ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 1620agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 1680tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 1740ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag 1800gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 1860ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 1920gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 1980aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg 2040aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 2100ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 2160gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 2220gggattttgg tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc 2280gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca 2340gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt 2400ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac 2460catatggaca tattgtcgtt agaacgcggc tacaattaat acataacctt atgtatcata 2520cacatacgat ttaggtgaca ctatagaacg gcgcgccaag ctgggtctag aactagaaac 2580gtgatgccac ttgttattga agtcgattac agcatctatt ctgttttact atttataact 2640ttgccatttc tgacttttga aaactatctc tggatttcgg tatcgctttg tgaagatcga 2700gcaaaagaga cgttttgtgg acgcaatggt ccaaatccgt tctacatgaa caaattggtc 2760acaatttcca ctaaaagtaa ataaatggca agttaaaaaa ggaatatgca ttttactgat 2820tgcctaggtg agctccaaga gaagttgaat ctacacgtct accaaccgct aaaaaaagaa 2880aaacattgaa tatgtaacct gattccatta gcttttgact tcttcaacag attctctact 2940tagatttcta acagaaatat tattactagc acatcatttt cagtctcact acagcaaaaa 3000atccaacggc acaatacaga caacaggaga tatcagacta cagagataga tagatgctac 3060tgcatgtagt aagttaaata aaaggaaaat aaaatgtctt gctaccaaaa ctactacaga 3120ctatgatgct caccacaggc caaatcctgc aactaggaca gcattatctt atatatattg 3180tacaaaacaa gcatcaagga acatttggtc taggcaatca gtacctcgtt ctaccatcac 3240cctcagttat cacatccttg aaggatccat tactgggaat catcggcaac acatgctcct 3300gatggggcac aatgacatca agaaggtagg ggccaggggt gtccaacatt ctctgaattg 3360ccgctctaag ctcttccttc ttcgtcactc gcgctgccgg tatcccacaa gcatcagcaa 3420acttgagcat gtttgggaat atctcgctct cgctagacgg atctccaaga taggtgtgag 3480ctctattgga cttgtagaac ctatcctcca actgaaccac catacccaaa tgctgattgt 3540tcaacaacaa tatcttaact gggagattct ccactcttat agtggccaac tcctgaacat 3600tcatgatgaa actaccatcc ccatcaatgt caaccacaac agccccaggg ttagcaacag 3660cagcaccaat agccgcaggc aatccaaaac ccatggctcc aagaccccct gaggtcaacc 3720actgcctcgg tctcttgtac ttgtaaaact gcgcagccca catttgatgc tgcccaaccc 3780cagtactaac aatagcatct ccattagtca actcatcaag aacctcgata gcatgctgcg 3840gagaaatcgc gtcctggaat gtcttgtaac ccaatggaaa cttgtgtttc tgcacattaa 3900tctcttctct ccaacctcca agatcaaact taccctccac tcctttctcc tccaaaatca 3960tattaattcc cttcaaggcc aacttcaaat ccgcgcaaac cgacacgtgc gcctgcttgt 4020tcttcccaat ctcggcagaa tcaatatcaa tgtgaacaat cttagcccta ctagcaaaag 4080cctcaagctt cccagtaaca cggtcatcaa accttacccc aaaggcaagc aacaaatcac 4140tattgtcaac agcatagtta gcataaacag taccatgcat acccagcatc tgaagggaat 4200attcatcacc aataggaaaa gttccaagac ccattaaagt gctagcaacg ggaataccag 4260tgagttcaac aaagcgcctc aattcagcac tggaattcaa actgccaccg ccgacgtaga 4320gaacgggctt ttgggcctcc atgatgagtc tgacaatgtg ttccaattgg gcctcggcgg 4380ggggcctggg cagcctggcg aggtaaccgg ggaggttaac gggctcgtcc caattaggca 4440cggcgagttg ctgctgaacg tctttgggaa tgtcgatgag gaccggaccg gggcggccgg 4500aggtggcgac gaagaaagcc tcggcgacga cgcgggggat gtcgtcgacg tcgaggatga 4560ggtagttgtg cttcgtgatg gatctgctca cctccacgat cggggtttct tggaaggcgt 4620cggtgccgat catccggcgg gcgacctggc cggtgatggc gacgactggg acgctgtcca 4680ttaaagcgtc ggcgaggccg ctcacgaggt tggtggcgcc ggggccggag gtggcaatgc 4740agacgccggg gaggccggag gaacgcgcgt agccttcggc ggcgaagacg ccgccctgct 4800cgtggcgcgg gagcacgttg cggatggcgg cggagcgcgt gagcgcctgg tggatctcca 4860tcgacgcacc gccggggtac gcgaacaccg tcgtcacgcc ctgcctctcc agcgcctcca 4920caaggatgtc cgcgcccttg cgaggttcgc cggaggcgaa ccgtgacacg aagggctccg 4980tggtcggcgc ttccttggtg aagggcgccg ccgtgggggg tttggagatg gaacatttga 5040ttttgagagc gtggttgggt ttggtgaggg tttgatgaga gagagggagg gtggatctag 5100taatgcgttt ggggaaggtg gggtgtgaag aggaagaaga gaatcgggtg gttctggaag 5160cggtggccgc cattgtgttg tgtggcatgg ttatacttca aaaactgcac aacaagccta 5220gagttagtac ctaaacagta aatttacaac agagagcaaa gacacatgca aaaatttcag 5280ccataaaaaa agttataata gaatttaaag caaaagtttc attttttaaa catatataca 5340aacaaactgg atttgaagga agggattaat tcccctgctc aaagtttgaa ttcctattgt 5400gacctatact cgaataaaat tgaagcctaa ggaatgtatg agaaacaaga aaacaaaaca 5460aaactacaga caaacaagta caattacaaa attcgctaaa attctgtaat caccaaaccc 5520catctcagtc agcacaaggc ccaaggttta ttttgaaata aaaaaaaagt gattttattt 5580ctcataagct aaaagaaaga aaggcaatta tgaaatgatt tcgactagat ctgaaagtca 5640aacgcgtatt ccgcagatat taaagaaaga gtagagtttc acatggatcc tagatggacc 5700cagttgagga aaaagcaagg caaagcaaac cagaagtgca agatccgaaa ttgaaccacg 5760gaatctagga tttggtagag ggagaagaaa agtaccttga gaggtagaag agaagagaag 5820agcagagaga tatatgaacg agtgtgtctt ggtctcaact ctgaagcgat acgagtttag 5880aggggagcat tgagttccaa tttataggga aaccgggtgg caggggtgag ttaatgacgg 5940aaaagcccct aagtaacgag attggattgt gggttagatt caaccgtttg catccgcggc 6000ttagattggg gaagtcagag tgaatctcaa ccgttgactg agttgaaaat tgaatgtagc 6060aaccaattga gccaacccca gcctttgccc tttgattttg atttgtttgt tgcatacttt 6120ttatttgtct tctggttctg actctctttc tctcgtttca atgccaggtt gcctactccc 6180acaccactca caagaagatt ctactgttag tattaaatat tttttaatgt attaaatgat 6240gaatgctttt gtaaacagaa caagactatg tctaataagt gtcttgcaac attttttaag 6300aaattaaaaa aaatatattt attatcaaaa tcaaatgtat gaaaaatcat gaataatata 6360attttataca tttttttaaa aaatctttta atttcttaat taatatctta aaaataatga 6420ttaatattta acccaaaata attagtatga ttggtaagga agatatccat gttatgtttg 6480gatgtgagtt tgatctagag caaagcttac tagagtcgac ctgcagcccg ggggatccgc 6540ccacgtacgg taccatctgc taatatttta aatcacatgc aagagaggag gcatggttcc 6600attttctacc ttcacattat ttgagaaaaa cgaacttgtt ctgtgtttta tttttgccct 6660tcacattagt acaacgtgga agactcatgg ttacacagaa tcatacataa gtacaatgct 6720tgtccctaag aaaacaagca ctcgttgtat tgaaccttta cggctcatgc ggccgcgaat 6780tcactagtga ttgaattcgc ggccgcttag tccgacttgg ccttggcggc cgcggccgac 6840tctttgagcg tgaagatctg cgccgtctcg ggcacagcgc cgtagttgac aaagaggtgc 6900gcggtcttga agaaggccgt gatgatgggc tcgtcgttcc tgcgcacgag gtgcgggtac 6960gcggccgcaa agtgcttggt ggcttcgttg agcttgtagt gcggaatgat cgggaacaag 7020tggtggacct ggtgcgtgcc aatgtggtgg ctcaggttgt ccacgaacgc gccgtacgag 7080cggtcgacgc tcgagaggtt gcccttgacg tacgtccact ccgagtcgcc gtaccacggc 7140gtcgcttcgt cgttgtggtg caagaaggtc gtaatgacga ggaacgaagc aaagacaaag 7200agcggcgcat agtagtagag gcccatgacg gcaaagccga gcgagtatgt gaggtacgcg 7260tacgcggcga agaaggcggc ccagacgccg agcgacacga tgacggccga cgcgcggcga 7320aggaggagcg ggtcccacgg gtcaaagtgg ctcatcgtgc gcggggcata cccgaccttc 7380aagtagacaa accacgcacc gccgagcgtg tagacccatt ggcgcacgtc ctggaggtcc 7440ttgaccgacc ggtgcgggta aaagatctcg tccttatcaa tgttgcccgt gttcttgtgg 7500tggtggcggt gcgtcacgcg ccagctctcg aacggcgtca aaatcgcaga gtgcatgatg 7560cagccgatga taaagttgac gctgtggtag cgcgagaagg ccgagtggcc gcagtcgtgg 7620ccgaccgtga agaagcccca gaagatgacg ccctgcacgt agatgtaggt ggcgcaaacg 7680agcgcgtgga gcagaacgtt atcggcaatg aacggcgtcg agcgcgccgc gtagagcagc 7740gccgccgagg ccgacgcgtt gaagatcgcg cgggccgtgt agtagagcga gaggccgagg 7800ttcgactcaa agcacgcgtt cgggatcgag tgcttgagct ccgtgagcgt cgggaactcg 7860accttcgtct tatcctcagt catgcggccg ctgaagtatt gcttcttagt taacctttcc 7920tttctctctc agctatgtga attcattttg ctttcgtcac aatttatata gtgaaattgg 7980atctttggag ttaacgcctt cacaggatta tcgtgttaga acaatgcttt ttcatgttct 8040aattagtagt acattacaaa tgtgcactct attcaataag catcttttgg cacgttaata 8100aatcatgtga aaaaaaaata ctactatttc aaagaaagtg ttgtaaaaag aaacggaaag 8160agagctggct tcagttgttg agacttgttt gctagtaaaa atggtgtgaa gagtgattca 8220tggtgaggtg gtttttcgtc cctttctgtt tgcatgaaaa acaaatggca agagatgacg 8280taggattcct tcccttaacg attatctgtt tttaatttca aatatacata taggaattta 8340tgaattacta aggttgtaaa atatgctggt catttattta tggctaaaat attttttttt 8400ctcgtaaata taaaaatatt

taaaatttat ttttatcata ttttttatcc ttataaaatt 8460atgtgtacaa cctatataaa aaaatatcat atttaatatt gattatatgt ttaatcaata 8520taaaaaatca ttatcatata tttagattta ttcgaatata catctaaaca aaaaataaca 8580tattttaatt ttatgaagaa aaaaaaatat tttatccttt atttatttaa gattaattaa 8640tagttatgta ttgtggaaag acttttacac atgcaataga tatactgaat caattagatg 8700ccaatgctga gttggaaatc acttgaggag gggaggagac ttgccaatgc ttttcagttt 8760catttaaatg atttagtgga ggagatagag tagtgataaa ggcatgcccc aattttggag 8820tgtatatatg agtggaaata agagagggat agagagaaaa aataaagaga gtaaaaataa 8880ttaatgtgaa atgatatgat aaaaaaataa agaaagagat aaagagaaaa atgaaatgag 8940agatagatga aatagagagt agatacatgt ttgtttaggt tttttttagg aaataacaca 9000tttttttctc atcacttatt actcactgtc aatttcctct ctttcaatca taatgatatg 9060atttgtttaa caaaaatgtg aaaaaacata taaagtaaaa tatttttata aattgataaa 9120taaaaattta caaaatttat ttcttattaa attgaataga aaatgaaaga aaagaaaaga 9180aaaagtatat ataaaatgat atagctttaa aaagaataaa tttttcatat cagtcttttt 9240ttaataattt agaaatattt aagtatatag caaaaatata atgtacttta catatgcata 9300aataataatt tgaaaataga actaatagaa tagagaaaaa agtaatataa taattaacta 9360tatgaaaatt tagaagggac aatattttta attaagaata taaacaatat ttcttttcat 9420gtaatgaggg acggatgtac ggggccagtg ttggagtcaa agccaaaata gtcacgggga 9480aattaatgca ctgcatgact attcgaaaaa attcactagc cttacttaga tgttagatta 9540atagctaggg ggtgcagata attttgaaag gcatgaaaaa cattaatttg tacattgcaa 9600gcttttgatg acaagctttg caattgttca cactacctta tgccatttat aaatagagtg 9660attggcatat gaaggaaatc atgagagtcg aagcgaaaaa caaagcttga gagtgtagga 9720aaaatacagt ttttttggta aaaatacagt atttgaatag gagcgaaaaa tatcctttca 9780aaatgatcct tttctttttt tttttttttc ttgttgttct tggtcagtta ttcaaaggaa 9840aagggattga aataaaaact tgcatgtggg atcgtacgtc gagtcgacct gcaggatcca 9900tgcccttcat ttgccgctta ttaattaatt tggtaacagt ccgtactaat cagttactta 9960tccttccccc atcataatta atcttggtag tctcgaatgc cacaacactg actagtctct 10020tggatcataa gaaaaagcca aggaacaaaa gaagacaaaa cacaatgaga gtatcctttg 10080catagcaatg tctaagttca taaaattcaa acaaaaacgc aatcacacac agtggacatc 10140acttatccac tagctgatca ggatcgccgc gtcaagaaaa aaaaactgga ccccaaaagc 10200catgcacaac aacacgtact cacaaaggtg tcaatcgagc agcccaaaac attcaccaac 10260tcaacccatc atgagccctc acatttgttg tttctaaccc aacctcaaac tcgtattctc 10320ttccgccacc tcatttttgt ttatttcaac acccgtcaaa ctgcatgcca ccccgtggcc 10380aaatgtccat gcatgttaac aagacctatg actataaata gctgcaatct cggcccaggt 10440tttcatcatc aagaaccagt tcaatatcct agtacaccgt attaaagaat ttaagatata 10500ctgcggccgc accatgtctc ctaagcggca agctctgcca atcacaattg atggcgcaac 10560ttatgatgtg tctgcttggg tcaatcacca ccctggagga gctgacatta tcgagaacta 10620tcgcaaccgc gatgcgaccg atgtcttcat ggtgatgcac tctcaagaag ccgtcgccaa 10680gttgaagaga atgcctgtta tggagccttc ctctcctgac acacctgttg cacccaagcc 10740taagcgtgat gagccccagg aggatttccg caagttgcgg gaggaattca tctccaaggg 10800tatgttcgag acgagtttcc tttggtattt ttacaagact tcaactaccg tcggtttgat 10860ggtcctttcc atcttgatga ccgtgtacac gaattggtat ttcaccgctg ctttggttct 10920tggcgtgtgc taccaacagc taggctggtt gtcccacgac tattgccatc accaggtttt 10980cacaaaccgc aagattaacg acgctttcgg tctctttttc ggtaacgtga tgcagggata 11040ctcacagact tggtggaagg ataggcacaa tggtcaccat gccgccacca atgtggtcgg 11100ccatgaccca gatattgata acctccccat cctggcttgg tctcccgaag atgtcaagag 11160ggctactcct tcgactcgga atctcatcaa gtaccagcag tactacttca ttcccaccat 11220tgcatccctt aggttcatct ggtgcctcca atccatcggc ggcgtcatgt cctacaagag 11280cgaggagagg aacctgtact acaagcgcca gtacactaag gaggcgattg gtctggccct 11340ccactgggtg ctcaaggcca ctttctattg cagtgccatg cctagctttg ccaccggttt 11400gggatgcttc ttgatctccg agctgctcgg aggatttggc attgccatcg ttgtgtttct 11460gaatcactat cctttggaca aggttgagga gactgtctgg gatgagcacg ggttcagcgc 11520cagccagatc cacgagacgt tgaacattaa gcccggcctt ctcaccgatt gggtctttgg 11580tggtctcaac taccagattg agcaccactt gtggcccaac atgcccaggc acaacctcac 11640ggcagcttcc ctggaggtgc agaagttgtg cgccaagcac aacctgccct acagggcccc 11700agccatcatc cccggggttc agaaattggt cagcttctta ggcgagattg cccagctggc 11760tgctgtccct gaatgagcgg ccgcaagtat gaactaaaat gcatgtaggt gtaagagctc 11820atggagagca tggaatattg tatccgacca tgtaacagta taataactga gctccatctc 11880acttcttcta tgaataaaca aaggatgtta tgatatatta acactctatc tatgcacctt 11940attgttctat gataaatttc ctcttattat tataaatcat ctgaatcgtg acggcttatg 12000gaatgcttca aatagtacaa aaacaaatgt gtactataag actttctaaa caattctaac 12060cttagcattg tgaacgagac ataagtgtta agaagacata acaattataa tggaagaagt 12120ttgtctccat ttatatatta tatattaccc acttatgtat tatattagga tgttaaggag 12180acataacaat tataaagaga gaagtttgta tccatttata tattatatac tacccattta 12240tatattatac ttatccactt atttaatgtc tttataaggt ttgatccatg atatttctaa 12300tattttagtt gatatgtata tgaaagggta ctatttgaac tctcttactc tgtataaagg 12360ttggatcatc cttaaagtgg gtctatttaa ttttattgct tcttacagat aaaaaaaaaa 12420ttatgagttg gtttgataaa atattgaagg atttaaaata ataataaata acatataata 12480tatgtatata aatttattat aatataacat ttatctataa aaaagtaaat attgtcataa 12540atctatacaa tcgtttagcc ttgctggacg aatctcaatt atttaaacga gagtaaacat 12600atttgacttt ttggttattt aacaaattat tatttaacac tatatgaaat tttttttttt 12660atcagcaaag aataaaatta aattaagaag gacaatggtg tcccaatcct tatacaacca 12720acttccacaa gaaagtcaag tcagagacaa caaaaaaaca agcaaaggaa attttttaat 12780ttgagttgtc ttgtttgctg cataatttat gcagtaaaac actacacata acccttttag 12840cagtagagca atggttgacc gtgtgcttag cttcttttat tttatttttt tatcagcaaa 12900gaataaataa aataaaatga gacacttcag ggatgtttca acctgca 129479118DNAArtificial Sequenceuniversal primer M13F 91tgtaaaacga cggccagt 18921254DNAEutreptiella sp. CCMP389 92atgtcaccca agcgagaggc cttgcccatc acgattgatg gcacaaccta tgatgtgtcc 60gcatgggtga accatcaccc cgggggcgca gacatcatgg agaattatcg gaaccgagat 120gccacggatg tgttcatggt catgcactcc cacgatgcgt tgaacaagct gaagcgcatg 180cctgtgatgg agcccacttc gccacgaagc cccaagactc ccaacgacga ggttgctgag 240gacttccgca agcttcgaaa ggacatgatt gcaaaaggca tgttcaacgc atcccctctc 300ttctacgtgt acaaaagtgc gaccacagta gccctgggcg ccctggctat tctgatggtg 360atgcacctgc agtggtacta catcccagcc attttgttgg gactttgcta ccagcagctg 420gggtggttgg cacacgatta ctgccaccat caggtgttct ctaaccgggc gtacaacaat 480tttgctggac ttgtattcgg caatgtgatg caaggatact ccgggacttg gtggaaggac 540aggcacaacg gccatcacgc cgccacgaac gtgcaagggc acgatcccga catcgacgac 600ctcccggtgt tggcctggtc cccggaggac gtcaaaaacg ccggtcccac gacgcggaag 660ctcatcaagt ggcaacaata ctatttcctc cccaccatcg caaccctccg attcatctgg 720tgcttccaga gcattctggc ggttatggca tacaagacag atgcaaggaa tatatattac 780caacgccagt acgcaaagga ggccgtgggg ctggctctgc attggattct gaaaggggta 840ttcatgttct gttacatgcc cggcatactg acgggcttgg ccttcttcct catctcggag 900tgcctgggcg ggtttgggat tgccattgtc gtgttcttga atcactaccc attggagaag 960gtggaggagt ccgtgtggga cagccacggg ttttgcgcgg ggcagatcca cacgacgatg 1020aacatccaac gcggggtcat cgttgactgg ttctttggag gcctgaacta ccaaatcgaa 1080caccatctgt ggccgacgct gccccggcat cacttgaaag ctgcttcttt tgaggtggag 1140aaaatttgcc agaagcacaa attgccatac agagcacccc ccatgtccga tggtgttgct 1200caattgcttg gcttcttggg gaagattgct aagctggcag ctgtcccagt gtaa 1254931254DNAEutreptiella cf_gymnastica CCMP1594misc_feature(238)..(238)n is a, c, g, or t 93atgtcaccta aacgggacgc attgcctctg acaattgatg gcaccacgta cgacgtttcc 60gcttgggtaa accatcaccc tggaggggct caaatcattg aaaactaccg gaaccgagat 120gctaccgacg tgttcatggt catgcattca cagcaggcgc tcaacaagtt gaagcggatg 180cctgttatgg agccctcttc accacttact cccaagagcc caagtgacga catttccnag 240gatttccgca agctccgcaa cagtatggtt gagaagggta tgttcaacgc gtcccctctg 300ttttatgtgt acaaatcact gaccactgtc gcccttggcg ccgtgggtgt tctcatggtt 360atgtacctgc agtggtacta cgtttcagcc atgtttttgg gactttgcta ccaacagctg 420ggttgggtgg cgcatgacta cgcgcatcac caggttttca cgaaccgtga ttatggcaat 480cttggtgggc ttttctttgg cnacgttctc caaggatatt ctttgacttg gtggaaggac 540aggcacaacg gccatcacgc cgccacaaac gtgcaaggac atgaccccga cattgataat 600ctccccgttt tggcttggtc gccagaggac gtcaagaatg ccggacctgg aacccgcaat 660atcatcaagt accagcagta ttatttcctc cctaccatcg ccatccttcg gttcatctgg 720tgtttccaaa gcattctggg ggtgatgtca tacaagacag actccnagaa tctctattac 780aaacggcagt accggagaga ggcagccggt ctggcgctgc actggattct gaagagcgtt 840ttcttgttct gttacatgcc aagtttcctc actggcctgg cgtttttcct tatctcggag 900tgtctgggcg gctttgggat cgcgattgtg gtgtttttga accactatcc gctggataag 960gttgaggaat ccgtttggga tggtcacggt ttctgtgctg ggcagatcct cacaaccatg 1020aacatccaac gcggactcat cactgactgg ttctttggag gtttgaatta ccagattgag 1080catcatctgt ggcccaacct tccaagacac catttgaaag cagtttcctt tgaggttgag 1140aaattgtgcc agaagcacaa cctgccctac agagctccgc cgatgcatac tggtgttgca 1200caattgcttg gatatttggg gaagattgct cagttggctg ctgtcccagt ataa 1254947222DNAArtificial SequenceYarrowia lipolytica expression vector pFBAIn-MOD-1 94catggatcca ggcctgttaa cggccattac ggcctgcagg atccgaaaaa acctcccaca 60cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc 120agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt 180ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgcgg 240ccgcaagtgt ggatggggaa gtgagtgccc ggttctgtgt gcacaattgg caatccaaga 300tggatggatt caacacaggg atatagcgag ctacgtggtg gtgcgaggat atagcaacgg 360atatttatgt ttgacacttg agaatgtacg atacaagcac tgtccaagta caatactaaa 420catactgtac atactcatac tcgtacccgg gcaacggttt cacttgagtg cagtggctag 480tgctcttact cgtacagtgt gcaatactgc gtatcatagt ctttgatgta tatcgtattc 540attcatgtta gttgcgtacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 600gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 660tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 720cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 780gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 840aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 900gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 960aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 1020gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 1080ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 1140cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 1200ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 1260actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 1320tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca 1380gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 1440ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 1500cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 1560ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 1620tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 1680agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 1740gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 1800ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 1860gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 1920cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 1980acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 2040cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 2100cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 2160ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 2220tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 2280atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 2340tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 2400actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 2460aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 2520ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 2580ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 2640cgaaaagtgc cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt 2700acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc 2760ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct 2820ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat 2880ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc 2940acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc 3000tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg 3060atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttccattcgc 3120cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc 3180agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc 3240agtcacgacg ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat 3300tgggtaccgg gccccccctc gaggtcgatg gtgtcgataa gcttgatatc gaattcatgt 3360cacacaaacc gatcttcgcc tcaaggaaac ctaattctac atccgagaga ctgccgagat 3420ccagtctaca ctgattaatt ttcgggccaa taatttaaaa aaatcgtgtt atataatatt 3480atatgtatta tatatataca tcatgatgat actgacagtc atgtcccatt gctaaataga 3540cagactccat ctgccgcctc caactgatgt tctcaatatt taaggggtca tctcgcattg 3600tttaataata aacagactcc atctaccgcc tccaaatgat gttctcaaaa tatattgtat 3660gaacttattt ttattactta gtattattag acaacttact tgctttatga aaaacacttc 3720ctatttagga aacaatttat aatggcagtt cgttcattta acaatttatg tagaataaat 3780gttataaatg cgtatgggaa atcttaaata tggatagcat aaatgatatc tgcattgcct 3840aattcgaaat caacagcaac gaaaaaaatc ccttgtacaa cataaatagt catcgagaaa 3900tatcaactat caaagaacag ctattcacac gttactattg agattattat tggacgagaa 3960tcacacactc aactgtcttt ctctcttcta gaaatacagg tacaagtatg tactattctc 4020attgttcata cttctagtca tttcatccca catattcctt ggatttctct ccaatgaatg 4080acattctatc ttgcaaattc aacaattata ataagatata ccaaagtagc ggtatagtgg 4140caatcaaaaa gcttctctgg tgtgcttctc gtatttattt ttattctaat gatccattaa 4200aggtatatat ttatttcttg ttatataatc cttttgttta ttacatgggc tggatacata 4260aaggtatttt gatttaattt tttgcttaaa ttcaatcccc cctcgttcag tgtcaactgt 4320aatggtagga aattaccata cttttgaaga agcaaaaaaa atgaaagaaa aaaaaaatcg 4380tatttccagg ttagacgttc cgcagaatct agaatgcggt atgcggtaca ttgttcttcg 4440aacgtaaaag ttgcgctccc tgagatattg tacatttttg cttttacaag tacaagtaca 4500tcgtacaact atgtactact gttgatgcat ccacaacagt ttgttttgtt tttttttgtt 4560tttttttttt ctaatgattc attaccgcta tgtataccta cttgtacttg tagtaagccg 4620ggttattggc gttcaattaa tcatagactt atgaatctgc acggtgtgcg ctgcgagtta 4680cttttagctt atgcatgcta cttgggtgta atattgggat ctgttcggaa atcaacggat 4740gctcaatcga tttcgacagt aattaattaa gtcatacaca agtcagcttt cttcgagcct 4800catataagta taagtagttc aacgtattag cactgtaccc agcatctccg tatcgagaaa 4860cacaacaaca tgccccattg gacagatcat gcggatacac aggttgtgca gtatcataca 4920tactcgatca gacaggtcgt ctgaccatca tacaagctga acaagcgctc catacttgca 4980cgctctctat atacacagtt aaattacata tccatagtct aacctctaac agttaatctt 5040ctggtaagcc tcccagccag ccttctggta tcgcttggcc tcctcaatag gatctcggtt 5100ctggccgtac agacctcggc cgacaattat gatatccgtt ccggtagaca tgacatcctc 5160aacagttcgg tactgctgtc cgagagcgtc tcccttgtcg tcaagaccca ccccgggggt 5220cagaataagc cagtcctcag agtcgccctt aggtcggttc tgggcaatga agccaaccac 5280aaactcgggg tcggatcggg caagctcaat ggtctgcttg gagtactcgc cagtggccag 5340agagcccttg caagacagct cggccagcat gagcagacct ctggccagct tctcgttggg 5400agaggggact aggaactcct tgtactggga gttctcgtag tcagagacgt cctccttctt 5460ctgttcagag acagtttcct cggcaccagc tcgcaggcca gcaatgattc cggttccggg 5520tacaccgtgg gcgttggtga tatcggacca ctcggcgatt cggtgacacc ggtactggtg 5580cttgacagtg ttgccaatat ctgcgaactt tctgtcctcg aacaggaaga aaccgtgctt 5640aagagcaagt tccttgaggg ggagcacagt gccggcgtag gtgaagtcgt caatgatgtc 5700gatatgggtt ttgatcatgc acacataagg tccgacctta tcggcaagct caatgagctc 5760cttggtggtg gtaacatcca gagaagcaca caggttggtt ttcttggctg ccacgagctt 5820gagcactcga gcggcaaagg cggacttgtg gacgttagct cgagcttcgt aggagggcat 5880tttggtggtg aagaggagac tgaaataaat ttagtctgca gaacttttta tcggaacctt 5940atctggggca gtgaagtata tgttatggta atagttacga gttagttgaa cttatagata 6000gactggacta tacggctatc ggtccaaatt agaaagaacg tcaatggctc tctgggcgtc 6060gcctttgccg acaaaaatgt gatcatgatg aaagccagca atgacgttgc agctgatatt 6120gttgtcggcc aaccgcgccg aaaacgcagc tgtcagaccc acagcctcca acgaagaatg 6180tatcgtcaaa gtgatccaag cacactcata gttggagtcg tactccaaag gcggcaatga 6240cgagtcagac agatactcgt cgaaaacagt gtacgcagat ctactataga ggaacattta 6300aattgccccg gagaagacgg ccaggccgcc tagatgacaa attcaacaac tcacagctga 6360ctttctgcca ttgccactag gggggggcct ttttatatgg ccaagccaag ctctccacgt 6420cggttgggct gcacccaaca ataaatgggt agggttgcac caacaaaggg atgggatggg 6480gggtagaaga tacgaggata acggggctca atggcacaaa taagaacgaa tactgccatt 6540aagactcgtg atccagcgac tgacaccatt gcatcatcta agggcctcaa aactacctcg 6600gaactgctgc gctgatctgg acaccacaga ggttccgagc actttaggtt gcaccaaatg 6660tcccaccagg tgcaggcaga aaacgctgga acagcgtgta cagtttgtct taacaaaaag 6720tgagggcgct gaggtcgagc agggtggtgt gacttgttat agcctttaga gctgcgaaag 6780cgcgtatgga tttggctcat caggccagat tgagggtctg tggacacatg tcatgttagt 6840gtacttcaat cgccccctgg atatagcccc gacaataggc cgtggcctca tttttttgcc 6900ttccgcacat ttccattgct cggtacccac accttgcttc tcctgcactt gccaacctta 6960atactggttt acattgacca acatcttaca agcggggggc ttgtctaggg tatatataaa 7020cagtggctct cccaatcggt tgccagtctc ttttttcctt tctttcccca cagattcgaa 7080atctaaacta cacatcacag aattccgagc cgtgagtatc cacgacaaga tcagtgtcga 7140gacgacgcgt tttgtgtaat gacacaatcc gaaagtcgct agcaacacac actctctaca 7200caaactaacc cagctctggt ac 7222958256DNAArtificial SequenceYarrowia lipolytica expression vector pFBAIn-389D8 95ggccgcaagt 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 gtcgaaaaca gtgtacgcag atctactata gaggaacatt 6060taaattgccc cggagaagac ggccaggccg cctagatgac aaattcaaca actcacagct 6120gactttctgc cattgccact aggggggggc ctttttatat ggccaagcca agctctccac 6180gtcggttggg ctgcacccaa caataaatgg gtagggttgc accaacaaag ggatgggatg 6240gggggtagaa gatacgagga taacggggct caatggcaca aataagaacg aatactgcca 6300ttaagactcg tgatccagcg actgacacca ttgcatcatc taagggcctc aaaactacct 6360cggaactgct gcgctgatct ggacaccaca gaggttccga gcactttagg ttgcaccaaa 6420tgtcccacca ggtgcaggca gaaaacgctg gaacagcgtg tacagtttgt cttaacaaaa 6480agtgagggcg ctgaggtcga gcagggtggt gtgacttgtt atagccttta gagctgcgaa 6540agcgcgtatg gatttggctc atcaggccag attgagggtc tgtggacaca tgtcatgtta 6600gtgtacttca atcgccccct ggatatagcc ccgacaatag gccgtggcct catttttttg 6660ccttccgcac atttccattg ctcggtaccc acaccttgct tctcctgcac ttgccaacct 6720taatactggt ttacattgac caacatctta caagcggggg gcttgtctag ggtatatata 6780aacagtggct ctcccaatcg gttgccagtc tcttttttcc tttctttccc cacagattcg 6840aaatctaaac tacacatcac agaattccga gccgtgagta tccacgacaa gatcagtgtc 6900gagacgacgc gttttgtgta atgacacaat ccgaaagtcg ctagcaacac acactctcta 6960cacaaactaa cccagctctg gtaccatggc acccaagcga gaggccttgc ccatcacgat 7020tgatggcaca acctatgatg tgtccgcatg ggtgaaccat caccccgggg gcgcagacat 7080catggagaat tatcggaacc gagatgccac ggatgtgttc atggtcatgc actcccacga 7140tgcgttgaac aagctgaagc gcatgcctgt gatggagccc acttcgccac gaagccccaa 7200gactcccaac gacgaggttg ctgaggactt ccgcaagctt cgaaaggaca tgattgcaaa 7260aggcatgttc aacgcatccc ctctcttcta cgtgtacaaa agtgcgacca cagtagccct 7320gggcgccctg gctattctga tggtgatgca cctgcagtgg tactacatcc cagccatttt 7380gttgggactt tgctaccagc agctggggtg gttggcacac gattactgcc accatcaggt 7440gttctctaac cgggcgtaca acaattttgc tggacttgta ttcggcaatg tgatgcaagg 7500atactccggg acttggtgga aggacaggca caacggccat cacgccgcca cgaacgtgca 7560agggcacgat cccgacatcg acgacctccc ggtgttggcc tggtccccgg aggacgtcaa 7620aaacgccggt cccacgacgc ggaagctcat caagtggcaa caatactatt tcctccccac 7680catcgcaacc ctccgattca tctggtgctt ccagagcatt ctggcggtta tggcatacaa 7740gacagatgca aggaatatat attaccaacg ccagtacgca aaggaggccg tggggctggc 7800tctgcattgg attctgaaag gggtattcat gttctgttac atgcccggca tactgacggg 7860cttggccttc ttcctcatct cggagtgcct gggcgggttt gggattgcca ttgtcgtgtt 7920cttgaatcac tacccattgg agaaggtgga ggagtccgtg tgggacagcc acgggttttg 7980cgcggggcag atccacacga cgatgaacat ccaacgcggg gtcatcgttg actggttctt 8040tggaggcctg aactaccaaa tcgaacacca tctgtggccg acgctgcccc ggcatcactt 8100gaaagctgct tcttttgagg tggagaaaat ttgccagaag cacaaattgc catacagagc 8160accccccatg tccgatggtg ttgctcaatt gcttggcttc ttggggaaga ttgctaagct 8220ggcagctgtc ccagtgtaac cctaaacgta ccacgc 8256968262DNAArtificial SequenceYarrowia lipolytica expression vector pFBAIn-1594D8 96ggccgcaagt 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 gtcgaaaaca gtgtacgcag atctactata gaggaacatt 6060taaattgccc cggagaagac ggccaggccg cctagatgac aaattcaaca actcacagct 6120gactttctgc cattgccact aggggggggc ctttttatat ggccaagcca agctctccac 6180gtcggttggg ctgcacccaa caataaatgg gtagggttgc accaacaaag ggatgggatg 6240gggggtagaa gatacgagga taacggggct caatggcaca aataagaacg aatactgcca 6300ttaagactcg tgatccagcg actgacacca ttgcatcatc taagggcctc aaaactacct 6360cggaactgct gcgctgatct ggacaccaca gaggttccga gcactttagg ttgcaccaaa 6420tgtcccacca ggtgcaggca gaaaacgctg gaacagcgtg tacagtttgt cttaacaaaa 6480agtgagggcg ctgaggtcga gcagggtggt gtgacttgtt atagccttta gagctgcgaa 6540agcgcgtatg gatttggctc atcaggccag attgagggtc tgtggacaca tgtcatgtta 6600gtgtacttca atcgccccct ggatatagcc ccgacaatag gccgtggcct catttttttg 6660ccttccgcac atttccattg ctcggtaccc acaccttgct tctcctgcac ttgccaacct 6720taatactggt ttacattgac caacatctta caagcggggg gcttgtctag ggtatatata 6780aacagtggct ctcccaatcg gttgccagtc tcttttttcc tttctttccc cacagattcg 6840aaatctaaac tacacatcac agaattccga gccgtgagta tccacgacaa gatcagtgtc 6900gagacgacgc gttttgtgta atgacacaat ccgaaagtcg ctagcaacac acactctcta 6960cacaaactaa cccagctctg gtaccatggc acctaaacgg gacgcattgc ctctgacaat 7020tgatggcacc acgtacgacg tttccgcttg ggtaaaccat caccctggag gggctcaaat

7080cattgaaaac taccggaacc gagatgctac cgacgtgttc atggtcatgc attcacagca 7140ggcgctcaac aagttgaagc ggatgcctgt tatggagccc tcttcaccac ttactcccaa 7200gagcccaagt gacgacattt ccnaggattt ccgcaagctc cgcaacagta tggttgagaa 7260gggtatgttc aacgcgtccc ctctgtttta tgtgtacaaa tcactgacca ctgtcgccct 7320tggcgccgtg ggtgttctca tggttatgta cctgcagtgg tactacgttt cagccatgtt 7380tttgggactt tgctaccaac agctgggttg ggtggcgcat gactacgcgc atcaccaggt 7440tttcacgaac cgtgattatg gcaatcttgg tgggcttttc tttggcnacg ttctccaagg 7500atattctttg acttggtgga aggacaggca caacggccat cacgccgcca caaacgtgca 7560aggacatgac cccgacattg ataatctccc cgttttggct tggtcgccag aggacgtcaa 7620gaatgccgga cctggaaccc gcaatatcat caagtaccag cagtattatt tcctccctac 7680catcgccatc cttcggttca tctggtgttt ccaaagcatt ctgggggtga tgtcatacaa 7740gacagactcc nagaatctct attacaaacg gcagtaccgg agagaggcag ccggtctggc 7800gctgcactgg attctgaaga gcgttttctt gttctgttac atgccaagtt tcctcactgg 7860cctggcgttt ttccttatct cggagtgtct gggcggcttt gggatcgcga ttgtggtgtt 7920tttgaaccac tatccgctgg ataaggttga ggaatccgtt tgggatggtc acggtttctg 7980tgctgggcag atcctcacaa ccatgaacat ccaacgcgga ctcatcactg actggttctt 8040tggaggtttg aattaccaga ttgagcatca tctgtggccc aaccttccaa gacaccattt 8100gaaagcagtt tcctttgagg ttgagaaatt gtgccagaag cacaacctgc cctacagagc 8160tccgccgatg catactggtg ttgcacaatt gcttggatat ttggggaaga ttgctcagtt 8220ggctgctgtc ccagtataac cctggatcac cttcatcgat gc 8262978363DNAArtificial SequenceYarrowia lipolytica expression vector pFBAIn-1491D8 97ggccgcaagt 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 gtcgaaaaca gtgtacgcag atctactata gaggaacatt 6060taaattgccc cggagaagac ggccaggccg cctagatgac aaattcaaca actcacagct 6120gactttctgc cattgccact aggggggggc ctttttatat ggccaagcca agctctccac 6180gtcggttggg ctgcacccaa caataaatgg gtagggttgc accaacaaag ggatgggatg 6240gggggtagaa gatacgagga taacggggct caatggcaca aataagaacg aatactgcca 6300ttaagactcg tgatccagcg actgacacca ttgcatcatc taagggcctc aaaactacct 6360cggaactgct gcgctgatct ggacaccaca gaggttccga gcactttagg ttgcaccaaa 6420tgtcccacca ggtgcaggca gaaaacgctg gaacagcgtg tacagtttgt cttaacaaaa 6480agtgagggcg ctgaggtcga gcagggtggt gtgacttgtt atagccttta gagctgcgaa 6540agcgcgtatg gatttggctc atcaggccag attgagggtc tgtggacaca tgtcatgtta 6600gtgtacttca atcgccccct ggatatagcc ccgacaatag gccgtggcct catttttttg 6660ccttccgcac atttccattg ctcggtaccc acaccttgct tctcctgcac ttgccaacct 6720taatactggt ttacattgac caacatctta caagcggggg gcttgtctag ggtatatata 6780aacagtggct ctcccaatcg gttgccagtc tcttttttcc tttctttccc cacagattcg 6840aaatctaaac tacacatcac agaattccga gccgtgagta tccacgacaa gatcagtgtc 6900gagacgacgc gttttgtgta atgacacaat ccgaaagtcg ctagcaacac acactctcta 6960cacaaactaa cccagctctg gtaccatggc tcctaagcgg caagctctgc caatcacaat 7020tgatggcgca acttatgatg tgtctgcttg ggtcaatcac caccctggag gagctgacat 7080tatcgagaac tatcgcaacc gcgatgcgac cgatgtcttc atggtgatgc actctcaaga 7140agccgtcgcc aagttgaaga gaatgcctgt tatggagcct tcctctcctg acacacctgt 7200tgcacccaag cctaagcgtg atgagcccca ggaggatttc cgcaagttgc gggaggaatt 7260catctccaag ggtatgttcg agacgagttt cctttggtat ttttacaaga cttcaactac 7320cgtcggtttg atggtccttt ccatcttgat gaccgtgtac acgaattggt atttcaccgc 7380tgctttggtt cttggcgtgt gctaccaaca gctaggctgg ttgtcccacg actattgcca 7440tcaccaggtt ttcacaaacc gcaagattaa cgacgctttc ggtctctttt tcggtaacgt 7500gatgcaggga tactcacaga cttggtggaa ggataggcac aatggtcacc atgccgccac 7560caatgtggtc ggccatgacc cagatattga taacctcccc atcctggctt ggtctcccga 7620agatgtcaag agggctactc cttcgactcg gaatctcatc aagtaccagc agtactactt 7680cattcccacc attgcatccc ttaggttcat ctggtgcctc caatccatcg gcggcgtcat 7740gtcctacaag agcgaggaga ggaacctgta ctacaagcgc cagtacacta aggaggcgat 7800tggtctggcc ctccattggg tgctcaaggc cactttctat tgcagtgcca tgcctagctt 7860tgccaccggt ttgggatgct tcttgatctc cgagctgctc ggaggatttg gcattgccat 7920cgttgtgttt ctgaatcact atcctttgga caaggttgag gagactgtct gggatgagca 7980cgggttcagc gccagccaga tccacgagac gttgaacatt aagcccggcc ttctcaccga 8040ttgggtcttt ggtggtctca actaccagat tgagcaccac ttgtggccca acatgcccag 8100gcacaacctc acggcagctt ccctggaggt gcagaagttg tgcgccaagc acaacctgcc 8160ctacagggcc ccagccatca tccccggggt tcagaaattg gtcagcttct taggcgagat 8220tgcccagctg gctgctgtcc ctgaatgatt ggtgactaag caagcgtcgg catggcgtgc 8280gtgtgtgggg cgggggttcc cgcactgtaa cccgcggtgt aacgcgcggt ggccgttcca 8340cccaatcaaa tgacaccacc tgc 836398419PRTEuglena gracilis 98Met Lys Ser Lys Arg Gln Ala Leu Ser Pro Leu Gln Leu Met Glu Gln1 5 10 15Thr Tyr Asp Val Val Asn Phe His Pro Gly Gly Ala Glu Ile Ile Glu 20 25 30Asn Tyr Gln Gly Arg Asp Ala Thr Asp Ala Phe Met Val Met His Phe 35 40 45Gln Glu Ala Phe Asp Lys Leu Lys Arg Met Pro Lys Ile Asn Pro Ser 50 55 60Phe Glu Leu Pro Pro Gln Ala Ala Val Asn Glu Ala Gln Glu Asp Phe65 70 75 80Arg Lys Leu Arg Glu Glu Leu Ile Ala Thr Gly Met Phe Asp Ala Ser 85 90 95Pro Leu Trp Tyr Ser Tyr Lys Ile Ser Thr Thr Leu Gly Leu Gly Val 100 105 110Leu Gly Tyr Phe Leu Met Val Gln Tyr Gln Met Tyr Phe Ile Gly Ala 115 120 125Val Leu Leu Gly Met His Tyr Gln Gln Met Gly Trp Leu Ser His Asp 130 135 140Ile Cys His His Gln Thr Phe Lys Asn Arg Asn Trp Asn Asn Leu Val145 150 155 160Gly Leu Val Phe Gly Asn Gly Leu Gln Gly Phe Ser Val Thr Cys Trp 165 170 175Lys Asp Arg His Asn Ala His His Ser Ala Thr Asn Val Gln Gly His 180 185 190Asp Pro Asp Ile Asp Asn Leu Pro Pro Leu Ala Trp Ser Glu Asp Asp 195 200 205Val Thr Arg Ala Ser Pro Ile Ser Arg Lys Leu Ile Gln Phe Gln Gln 210 215 220Tyr Tyr Phe Leu Val Ile Cys Ile Leu Leu Arg Phe Ile Trp Cys Phe225 230 235 240Gln Cys Val Leu Thr Val Arg Ser Leu Lys Asp Arg Asp Asn Gln Phe 245 250 255Tyr Arg Ser Gln Tyr Lys Lys Glu Ala Ile Gly Leu Ala Leu His Trp 260 265 270Thr Leu Lys Ala Leu Phe His Leu Phe Phe Met Pro Ser Ile Leu Thr 275 280 285Ser Leu Leu Val Phe Phe Val Ser Glu Leu Val Gly Gly Phe Gly Ile 290 295 300Ala Ile Val Val Phe Met Asn His Tyr Pro Leu Glu Lys Ile Gly Asp305 310 315 320Pro Val Trp Asp Gly His Gly Phe Ser Val Gly Gln Ile His Glu Thr 325 330 335Met Asn Ile Arg Arg Gly Ile Ile Thr Asp Trp Phe Phe Gly Gly Leu 340 345 350Asn Tyr Gln Ile Glu His His Leu Trp Pro Thr Leu Pro Arg His Asn 355 360 365Leu Thr Ala Val Ser Tyr Gln Val Glu Gln Leu Cys Gln Lys His Asn 370 375 380Leu Pro Tyr Arg Asn Pro Leu Pro His Glu Gly Leu Val Ile Leu Leu385 390 395 400Arg Tyr Leu Ala Val Phe Ala Arg Met Ala Glu Lys Gln Pro Ala Gly 405 410 415Lys Ala Leu9932DNAArtificial Sequenceprimer 389D8-F 99cggatcccat ggcacccaag cgagaggcct tg 3210037DNAArtificial Sequenceprimer 389D8-R 100aagatcgcgg ccgcgtggta cgtttagggt tacactg 3710135DNAArtificial Sequenceprimer 1491D8-F 101ggaactgcac catggctcct aagcggcaag ctctg 3510237DNAArtificial Sequenceprimer 1491D8-R 102gcttagcgcg gccgccgcag gtggtgtcat ttgattg 3710333DNAArtificial Sequenceprimer 1594D8-F 103cggatcccat ggcacctaaa cgggacgcat tgc 3310435DNAArtificial Sequenceprimer 1594D8-R 104aagatcgcgg ccgcggatcg atgaaggtga tccag 3510523DNAArtificial Sequence5' PCR primer 105aagcagtggt atcaacgcag agt 2310614655DNAArtificial Sequenceplasmid pZKLeuN-29E3 106cgattgttgt ctactaacta tcgtacgata acttcgtata gcatacatta tacgaagtta 60tcgcgtcgac gagtatctgt ctgactcgtc attgccgcct ttggagtacg actccaacta 120tgagtgtgct tggatcactt tgacgataca ttcttcgttg gaggctgtgg gtctgacagc 180tgcgttttcg gcgcggttgg ccgacaacaa tatcagctgc aacgtcattg ctggctttca 240tcatgatcac atttttgtcg gcaaaggcga cgcccagaga gccattgacg ttctttctaa 300tttggaccga tagccgtata gtccagtcta tctataagtt caactaactc gtaactatta 360ccataacata tacttcactg ccccagataa ggttccgata aaaagttctg cagactaaat 420ttatttcagt ctcctcttca ccaccaaaat gccctcctac gaagctcgag ctaacgtcca 480caagtccgcc tttgccgctc gagtgctcaa gctcgtggca gccaagaaaa ccaacctgtg 540tgcttctctg gatgttacca ccaccaagga gctcattgag cttgccgata aggtcggacc 600ttatgtgtgc atgatcaaaa cccatatcga catcattgac gacttcacct acgccggcac 660tgtgctcccc ctcaaggaac ttgctcttaa gcacggtttc ttcctgttcg aggacagaaa 720gttcgcagat attggcaaca ctgtcaagca ccagtaccgg tgtcaccgaa tcgccgagtg 780gtccgatatc accaacgccc acggtgtacc cggaaccgga atcattgctg gcctgcgagc 840tggtgccgag gaaactgtct ctgaacagaa gaaggaggac gtctctgact acgagaactc 900ccagtacaag gagttcctag tcccctctcc caacgagaag ctggccagag gtctgctcat 960gctggccgag ctgtcttgca agggctctct ggccactggc gagtactcca agcagaccat 1020tgagcttgcc cgatccgacc ccgagtttgt ggttggcttc attgcccaga accgacctaa 1080gggcgactct gaggactggc ttattctgac ccccggggtg ggtcttgacg acaagggaga 1140cgctctcgga cagcagtacc gaactgttga ggatgtcatg tctaccggaa cggatatcat 1200aattgtcggc cgaggtctgt acggccagaa ccgagatcct attgaggagg ccaagcgata 1260ccagaaggct ggctgggagg cttaccagaa gattaactgt tagaggttag actatggata 1320tgtaatttaa ctgtgtatat agagagcgtg caagtatgga gcgcttgttc agcttgtatg 1380atggtcagac gacctgtctg atcgagtatg tatgatactg cacaacctgt gtatccgcat 1440gatctgtcca atggggcatg ttgttgtgtt tctcgatacg gagatgctgg gtacagtgct 1500aatacgttga actacttata cttatatgag gctcgaagaa agctgacttg tgtatgactt 1560attctcaact acatccccag tcacaatacc accactgcac taccactaca ccaaaaccat 1620gatcaaacca cccatggact tcctggaggc agaagaactt gttatggaaa agctcaagag 1680agagatcata acttcgtata gcatacatta tacgaagtta tcctgcaggt aaaggaattc 1740tggagtttct gagagaaaaa ggcaagatac gtatgtaaca aagcgacgca tggtacaata 1800ataccggagg catgtatcat agagagttag tggttcgatg atggcactgg tgcctggtat 1860gactttatac ggctgactac atatttgtcc tcagacatac aattacagtc aagcacttac 1920ccttggacat ctgtaggtac cccccggcca agacgatctc agcgtgtcgt atgtcggatt 1980ggcgtagctc cctcgctcgt caattggctc ccatctactt tcttctgctt ggctacaccc 2040agcatgtctg ctatggctcg ttttcgtgcc

ttatctatcc tcccagtatt accaactcta 2100aatgacatga tgtgattggg tctacacttt catatcagag ataaggagta gcacagttgc 2160ataaaaagcc caactctaat cagcttcttc ctttcttgta attagtacaa aggtgattag 2220cgaaatctgg aagcttagtt ggccctaaaa aaatcaaaaa aagcaaaaaa cgaaaaacga 2280aaaaccacag ttttgagaac agggaggtaa cgaaggatcg tatatatata tatatatata 2340tatacccacg gatcccgaga ccggcctttg attcttccct acaaccaacc attctcacca 2400ccctaattca caaccatgga gtctggaccc atgcctgctg gcattccctt ccctgagtac 2460tatgacttct ttatggactg gaagactccc ctggccatcg ctgccaccta cactgctgcc 2520gtcggtctct tcaaccccaa ggttggcaag gtctcccgag tggttgccaa gtcggctaac 2580gcaaagcctg ccgagcgaac ccagtccgga gctgccatga ctgccttcgt ctttgtgcac 2640aacctcattc tgtgtgtcta ctctggcatc accttctact acatgtttcc tgctatggtc 2700aagaacttcc gaacccacac actgcacgaa gcctactgcg acacggatca gtccctctgg 2760aacaacgcac ttggctactg gggttacctc ttctacctgt ccaagttcta cgaggtcatt 2820gacaccatca tcatcatcct gaagggacga cggtcctcgc tgcttcagac ctaccaccat 2880gctggagcca tgattaccat gtggtctggc atcaactacc aagccactcc catttggatc 2940tttgtggtct tcaactcctt cattcacacc atcatgtact gttactatgc cttcacctct 3000atcggattcc atcctcctgg caaaaagtac ctgacttcga tgcagattac tcagtttctg 3060gtcggtatca ccattgccgt gtcctacctc ttcgttcctg gctgcatccg aacacccggt 3120gctcagatgg ctgtctggat caacgtcggc tacctgtttc ccttgaccta tctgttcgtg 3180gactttgcca agcgaaccta ctccaagcga tctgccattg ccgctcagaa aaaggctcag 3240taagcggccg cattgatgat tggaaacaca cacatgggtt atatctaggt gagagttagt 3300tggacagtta tatattaaat cagctatgcc aacggtaact tcattcatgt caacgaggaa 3360ccagtgactg caagtaatat agaatttgac caccttgcca ttctcttgca ctcctttact 3420atatctcatt tatttcttat atacaaatca cttcttcttc ccagcatcga gctcggaaac 3480ctcatgagca ataacatcgt ggatctcgtc aatagagggc tttttggact ccttgctgtt 3540ggccaccttg tccttgctgt ctggctcatt ctgtttcaac gccttttaat taacggagta 3600ggtctcggtg tcggaagcga cgccagatcc gtcatcctcc tttcgctctc caaagtagat 3660acctccgacg agctctcgga caatgatgaa gtcggtgccc tcaacgtttc ggatggggga 3720gagatcggcg agcttgggcg acagcagctg gcagggtcgc aggttggcgt acaggttcag 3780gtcctttcgc agcttgagga gaccctgctc gggtcgcacg tcggttcgtc cgtcgggagt 3840ggtccatacg gtgttggcag cgcctccgac agcaccgagc ataatagagt cagcctttcg 3900gcagatgtcg agagtagcgt cggtgatggg ctcgccctcc ttctcaatgg cagctcctcc 3960aatgagtcgg tcctcaaaca caaactcggt gccggaggcc tcagcaacag acttgagcac 4020cttgacggcc tcggcaatca cctcggggcc acagaagtcg ccgccgagaa gaacaatctt 4080cttggagtca gtcttggtct tcttagtttc gggttccatt gtggatgtgt gtggttgtat 4140gtgtgatgtg gtgtgtggag tgaaaatctg tggctggcaa acgctcttgt atatatacgc 4200acttttgccc gtgctatgtg gaagactaaa cctccgaaga ttgtgactca ggtagtgcgg 4260tatcggctag ggacccaaac cttgtcgatg ccgatagcat gcgacgtcgg gcccaattcg 4320ccctatagtg agtcgtatta caattcactg gccgtcgttt tacaacgtcg tgactgggaa 4380aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt 4440aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa 4500tggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 4560ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 4620ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat 4680ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg 4740ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata 4800gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt 4860tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat 4920ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc ctgatgcggt attttctcct 4980tacgcatctg tgcggtattt cacaccgcat caggtggcac ttttcgggga aatgtgcgcg 5040gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat 5100aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc 5160gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa 5220cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac 5280tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga 5340tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag 5400agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca 5460cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca 5520tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa 5580ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc 5640tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa 5700cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag 5760actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct 5820ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac 5880tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa 5940ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt 6000aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat 6060ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg 6120agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc 6180ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 6240tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag 6300cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact 6360ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 6420gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 6480ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 6540aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 6600cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 6660ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 6720gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct 6780ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc 6840ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc 6900gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac 6960cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcgcgcccac tgagctcgtc 7020taacggactt gatatacaac caattaaaac aaatgaaaag aaatacagtt ctttgtatca 7080tttgtaacaa ttaccctgta caaactaagg tattgaaatc ccacaatatt cccaaagtcc 7140acccctttcc aaattgtcat gcctacaact catataccaa gcactaacct accaaacacc 7200actaaaaccc cacaaaatat atcttaccga atatacagta acaagctacc accacactcg 7260ttgggtgcag tcgccagctt aaagatatct atccacatca gccacaactc ccttccttta 7320ataaaccgac tacacccttg gctattgagg ttatgagtga atatactgta gacaagacac 7380tttcaagaag actgtttcca aaacgtacca ctgtcctcca ctacaaacac acccaatctg 7440cttcttctag tcaaggttgc tacaccggta aattataaat catcatttca ttagcagggc 7500agggcccttt ttatagagtc ttatacacta gcggaccctg ccggtagacc aacccgcagg 7560cgcgtcagtt tgctccttcc atcaatgcgt cgtagaaacg acttactcct tcttgagcag 7620ctccttgacc ttgttggcaa caagtctccg acctcggagg tggaggaaga gcctccgata 7680tcggcggtag tgataccagc ctcgacggac tccttgacgg cagcctcaac agcgtcaccg 7740gcgggcttca tgttaagaga gaacttgagc atcatggcgg cagacagaat ggtggcgtac 7800gcaactaaca tgaatgaata cgatatacat caaagactat gatacgcagt attgcacact 7860gtacgagtaa gagcactagc cactgcactc aagtgaaacc gttgcccggg tacgagtatg 7920agtatgtaca gtatgtttag tattgtactt ggacagtgct tgtatcgtac attctcaagt 7980gtcaaacata aatatccgtt gctatatcct cgcaccacca cgtagctcgc tatatccctg 8040tgttgaatcc atccatcttg gattgccaat tgtgcacaca gaaccgggca ctcacttccc 8100catccacact tgcggccgcg cctacttaag caacgggctt gataacagcg gggggggtgc 8160ccacgttgtt gcggttgcgg aagaacagaa cacccttacc agcaccctcg gcaccagcgc 8220tgggctcaac ccactggcac atacgcgcac tgcggtacat ggcgcggatg aagccacgag 8280gaccatcctg gacatcagcc cggtagtgct tgcccatgat gggcttaatg gcctcggtgg 8340cctcgtccgc gttgtagaag gggatgctgc tgacgtagtg gtggaggaca tgagtctcga 8400tgatgccgtg gagaaggtgg cggccgatga agcccatctc acggtcaatg gtagcagcgg 8460caccacggac gaagttccac tcgtcgttgg tgtagtgggg aagggtaggg tcggtgtgct 8520ggaggaaggt gatggcaacg agccagtggt taacccagag gtagggaaca aagtaccaga 8580tggccatgtt gtagaaaccg aacttctgaa cgaggaagta cagagcagtg gccatcagac 8640cgataccaat atcgctgagg acgatgagct tagcgtcact gttctcgtac agagggctgc 8700ggggatcgaa gtggttaaca ccaccgccga ggccgttatg cttgcccttg ccgcgaccct 8760cacgctggcg ctcgtggtag ttgtggccgg taacattggt gatgaggtag ttgggccagc 8820cnacgannnn ctcagtaaga tgagcgagct cgtgggtcat ctttccgaga cgagtagcct 8880gctgctcgcg ggttcgggga acgaagacca tgtcacgctc catgttgcca gtggccttgt 8940ggtgctttcg gtgggagatt tgccagctga agtaggggac aaggagggaa gagtgaagaa 9000cccagccagt aatgtcgttg atgatgcgag aatcggagaa agcaccgtga ccgcactcat 9060gggcaataac ccagagacca gtaccgaaaa gaccctgaag aacggtgtac acggcccaca 9120gaccagcgcg ggcgggggtg gaggggatat attcgggggt cacaaagttg taccagatgc 9180tgaaagtggt agtcaggagg acaatgtcgc ggaggatata accgtatccc ttgagagcgg 9240agcgcttgaa gcagtgctta gggatggcat tgtagatgtc cttgatggta aagtcgggaa 9300cctcgaactg gttgccgtag gtgtcgagca tgacaccata ctcggacttg ggcttggcga 9360tatcaacctc ggacatggac gagagcgatg tggaagaggc cgagtggcgg ggagagtctg 9420aaggagagac ggcggcagac tcagaatccg tcacagtagt tgaggtgacg gtgcgtctaa 9480gcgcagggtt ctgcttgggc agagccgaag tggacgccat ggttgatgtg tgtttaattc 9540aagaatgaat atagagaaga gaagaagaaa aaagattcaa ttgagccggc gatgcagacc 9600cttatataaa tgttgccttg gacagacgga gcaagcccgc ccaaacctac gttcggtata 9660atatgttaag ctttttaaca caaaggtttg gcttggggta acctgatgtg gtgcaaaaga 9720ccgggcgttg gcgagccatt gcgcgggcga atggggccgt gactcgtctc aaattcgagg 9780gcgtgcctca attcgtgccc ccgtggcttt ttcccgccgt ttccgccccg tttgcaccac 9840tgcagccgct tctttggttc ggacaccttg ctgcgagcta ggtgccttgt gctacttaaa 9900aagtggcctc ccaacaccaa catgacatga gtgcgtgggc caagacacgt tggcggggtc 9960gcagtcggct caatggcccg gaaaaaacgc tgctggagct ggttcggacg cagtccgccg 10020cggcgtatgg atatccgcaa ggttccatag cgccattgcc ctccgtcggc gtctatcccg 10080caacctctaa atagagcggg aatataaccc aagcttcttt tttttccttt aacacgcaca 10140cccccaacta tcatgttgct gctgctgttt gactctactc tgtggagggg tgctcccacc 10200caacccaacc tacaggtgga tccggcgctg tgattggctg ataagtctcc tatccggact 10260aattctgacc aatgggacat gcgcgcagga cccaaatgcc gcaattacgt aaccccaacg 10320aaatgcctac ccctctttgg agcccagcgg ccccaaatcc ccccaagcag cccggttcta 10380ccggcttcca tctccaagca caagcagccc ggttctaccg gcttccatct ccaagcaccc 10440ctttctccac accccacaaa aagacccgtg caggacatcc tactgcgtcg acatcattta 10500aattccttca cttcaagttc attcttcatc tgcttctgtt ttactttgac aggcaaatga 10560agacatggta cgacttgatg gaggccaaga acgccatttc accccgagac accgaagtgc 10620ctgaaatcct ggctgccccc attgataaca tcggaaacta cggtattccg gaaagtgtat 10680atagaacctt tccccagctt gtgtctgtgg atatggatgg tgtaatcccc tttgagtact 10740cgtcttggct tctctccgag cagtatgagg ctctctaatc tagcgcattt aatatctcaa 10800tgtatttata tatttatctt ctcatgcggc cgctcactga atctttttgg ctcccttgtg 10860cttcctgacg atatacgttt gcacatagaa attcaagaac aaacacaaga ctgtgccaac 10920ataaaagtaa ttgaagaacc agccaaacat cctcatccca tcttggcgat aacagggaat 10980gttcctgtac ttccagacaa tgtagaaacc aacattgaat tgaatgatct gcattgatgt 11040aatcagggat tttggcatgg ggaacttcag cttgatcaat ctggtccaat aataaccgta 11100catgatccag tggatgaaac cattcaacag cacaaaaatc caaacagctt catttcggta 11160attatagaac agccacatat ccatcggtgc ccccaaatga tggaagaatt gcaaccaggt 11220cagaggcttg cccatcagtg gcaaatagaa ggagtcaata tactccagga acttgctcaa 11280atagaacaac tgcgtggtga tcctgaagac gttgttgtca aaagccttct cgcagttgtc 11340agacataaca ccgatggtgt acatggcata tgccattgag aggaatgatc ccaacgaata 11400aatggacatg agaaggttgt aattggtgaa aacaaacttc atacgagact gaccttttgg 11460accaaggggg ccaagagtga acttcaagat gacaaatgcg atggacaagt aaagcacctc 11520acagtgactg gcatcactcc agagttgggc ataatcaact ggttgggtaa aacttcctgc 11580ccaattgaga ctatttcatt caccacctcc atggccattg ctgtagatat gtcttgtgtg 11640taagggggtt ggggtggttg tttgtgttct tgacttttgt gttagcaagg gaagacgggc 11700aaaaaagtga gtgtggttgg gagggagaga cgagccttat atataatgct tgtttgtgtt 11760tgtgcaagtg gacgccgaaa cgggcaggag ccaaactaaa caaggcagac aatgcgagct 11820taattggatt gcctgatggg caggggttag ggctcgatca atgggggtgc gaagtgacaa 11880aattgggaat taggttcgca agcaaggctg acaagacttt ggcccaaaca tttgtacgcg 11940gtggacaaca ggagccaccc atcgtctgtc acgggctagc cggtcgtgcg tcctgtcagg 12000ctccacctag gctccatgcc actccataca atcccactag tgtaccgcta ggccgctttt 12060agctcccatc taagaccccc ccaaaacctc cactgtacag tgcactgtac tgtgtggcga 12120tcaagggcaa gggaaaaaag gcgcaaacat gcacgcatgg aatgacgtag gtaaggcgtt 12180actagactga aaagtggcac atttcggcgt gccaaagggt cctaggtgcg tttcgcgagc 12240tgggcgccag gccaagccgc tccaaaacgc ctctccgact ccctccagcg gcctccatat 12300ccccatccct ctccacagca atgttgttaa gccttgcaaa cgaaaaaata gaaaggctaa 12360taagcttcca atattgtggt gtacgctgca taacgcaaca atgagcgcca aacaacacac 12420acacacagca cacagcagca ttaaccacga tgaacagcat gacattacag gtgggtgtgt 12480aatcagggcc ctgattgctg gtggtgggag cccccatcat gggcagatct gcgtacactg 12540tttaaacagt gtacgcagat ctactataga ggaacattta aattgccccg gagaagacgg 12600ccaggccgcc tagatgacaa attcaacaac tcacagctga ctttctgcca ttgccactag 12660gggggggcct ttttatatgg ccaagccaag ctctccacgt cggttgggct gcacccaaca 12720ataaatgggt agggttgcac caacaaaggg atgggatggg gggtagaaga tacgaggata 12780acggggctca atggcacaaa taagaacgaa tactgccatt aagactcgtg atccagcgac 12840tgacaccatt gcatcatcta agggcctcaa aactacctcg gaactgctgc gctgatctgg 12900acaccacaga ggttccgagc actttaggtt gcaccaaatg tcccaccagg tgcaggcaga 12960aaacgctgga acagcgtgta cagtttgtct taacaaaaag tgagggcgct gaggtcgagc 13020agggtggtgt gacttgttat agcctttaga gctgcgaaag cgcgtatgga tttggctcat 13080caggccagat tgagggtctg tggacacatg tcatgttagt gtacttcaat cgccccctgg 13140atatagcccc gacaataggc cgtggcctca tttttttgcc ttccgcacat ttccattgct 13200cgatacccac accttgcttc tcctgcactt gccaacctta atactggttt acattgacca 13260acatcttaca agcggggggc ttgtctaggg tatatataaa cagtggctct cccaatcggt 13320tgccagtctc ttttttcctt tctttcccca cagattcgaa atctaaacta cacatcacag 13380aattccgagc cgtgagtatc cacgacaaga tcagtgtcga gacgacgcgt tttgtgtaat 13440gacacaatcc gaaagtcgct agcaacacac actctctaca caaactaacc cagctctggt 13500accatggagg tcgtgaacga aatcgtctcc attggccagg aggttcttcc caaggtcgac 13560tatgctcagc tctggtctga tgcctcgcac tgcgaggtgc tgtacctctc catcgccttc 13620gtcatcctga agttcaccct tggtcctctc ggacccaagg gtcagtctcg aatgaagttt 13680gtgttcacca actacaacct gctcatgtcc atctactcgc tgggctcctt cctctctatg 13740gcctacgcca tgtacaccat tggtgtcatg tccgacaact gcgagaaggc tttcgacaac 13800aatgtcttcc gaatcaccac tcagctgttc tacctcagca agttcctcga gtacattgac 13860tccttctatc tgcccctcat gggcaagcct ctgacctggt tgcagttctt tcaccatctc 13920ggagctccta tggacatgtg gctgttctac aactaccgaa acgaagccgt ttggatcttt 13980gtgctgctca acggcttcat tcactggatc atgtacggct actattggac ccgactgatc 14040aagctcaagt tccctatgcc caagtccctg attacttcta tgcagatcat tcagttcaac 14100gttggcttct acatcgtctg gaagtaccgg aacattccct gctaccgaca agatggaatg 14160agaatgtttg gctggttttt caactacttc tacgttggta ctgtcctgtg tctgttcctc 14220aacttctacg tgcagaccta catcgtccga aagcacaagg gagccaaaaa gattcagtga 14280gcggccgcat gtacatacaa gattatttat agaaatgaat cgcgatcgaa caaagagtac 14340gagtgtacga gtaggggatg atgataaaag tggaagaagt tccgcatctt tggatttatc 14400aacgtgtagg acgatacttc ctgtaaaaat gcaatgtctt taccataggt tctgctgtag 14460atgttattaa ctaccattaa catgtctact tgtacagttg cagaccagtt ggagtataga 14520atggtacact taccaaaaag tgttgatggt tgtaactacg atatataaaa ctgttgacgg 14580gatccccgct gatatgccta aggaacaatc aaagaggaag atattaattc agaatgctag 14640tatacagtta gggat 14655107777DNAArtificial Sequencesynthetic delta-9 elongase (initially from Euglena gracilis) codon-optimized for Yarrowia lipolytica; U.S. Patent Application No. 60/739989, filed 11/23/2005 107atggaggtcg tgaacgaaat cgtctccatt ggccaggagg ttcttcccaa ggtcgactat 60gctcagctct ggtctgatgc ctcgcactgc gaggtgctgt acctctccat cgccttcgtc 120atcctgaagt tcacccttgg tcctctcgga cccaagggtc agtctcgaat gaagtttgtg 180ttcaccaact acaacctgct catgtccatc tactcgctgg gctccttcct ctctatggcc 240tacgccatgt acaccattgg tgtcatgtcc gacaactgcg agaaggcttt cgacaacaat 300gtcttccgaa tcaccactca gctgttctac ctcagcaagt tcctcgagta cattgactcc 360ttctatctgc ccctcatggg caagcctctg acctggttgc agttctttca ccatctcgga 420gctcctatgg acatgtggct gttctacaac taccgaaacg aagccgtttg gatctttgtg 480ctgctcaacg gcttcattca ctggatcatg tacggctact attggacccg actgatcaag 540ctcaagttcc ctatgcccaa gtccctgatt acttctatgc agatcattca gttcaacgtt 600ggcttctaca tcgtctggaa gtaccggaac attccctgct accgacaaga tggaatgaga 660atgtttggct ggtttttcaa ctacttctac gttggtactg tcctgtgtct gttcctcaac 720ttctacgtgc agacctacat cgtccgaaag cacaagggag ccaaaaagat tcagtga 77710834DNAEscherichia coli 108ataacttcgt ataatgtatg ctatacgaag ttat 34109828DNAArtificial Sequencesynthetic C16/18 elongase (initally from M. alpina) codon-optimized for Yarrowia lipolytica; see also U.S. Patent Application No. 11/253882, filed October 19, 2005 109atggagtctg gacccatgcc tgctggcatt cccttccctg agtactatga cttctttatg 60gactggaaga ctcccctggc catcgctgcc acctacactg ctgccgtcgg tctcttcaac 120cccaaggttg gcaaggtctc ccgagtggtt gccaagtcgg ctaacgcaaa gcctgccgag 180cgaacccagt ccggagctgc catgactgcc ttcgtctttg tgcacaacct cattctgtgt 240gtctactctg gcatcacctt ctactacatg tttcctgcta tggtcaagaa cttccgaacc 300cacacactgc acgaagccta ctgcgacacg gatcagtccc tctggaacaa cgcacttggc 360tactggggtt acctcttcta cctgtccaag ttctacgagg tcattgacac catcatcatc 420atcctgaagg gacgacggtc ctcgctgctt cagacctacc accatgctgg agccatgatt 480accatgtggt ctggcatcaa ctaccaagcc actcccattt ggatctttgt ggtcttcaac 540tccttcattc acaccatcat gtactgttac tatgccttca cctctatcgg attccatcct 600cctggcaaaa agtacctgac ttcgatgcag attactcagt ttctggtcgg tatcaccatt 660gccgtgtcct acctcttcgt tcctggctgc atccgaacac ccggtgctca gatggctgtc 720tggatcaacg tcggctacct gtttcccttg acctatctgt tcgtggactt tgccaagcga 780acctactcca agcgatctgc cattgccgct cagaaaaagg ctcagtaa 828110792DNAArtificial SequenceIgD9e synthetic delta-9 elongase (initally from Isochrysis galbana) codon-optimized for Yarrowia lipolytica 110atggctctgg ccaacgacgc tggcgagcga atctgggctg ccgtcaccga tcccgaaatc 60ctcattggca ccttctccta cctgctcctg aagcctctcc tgcgaaactc tggtctcgtg 120gacgagaaga aaggagccta ccgaacctcc atgatctggt acaacgtcct cctggctctc 180ttctctgccc tgtccttcta cgtgactgcc accgctctcg gctgggacta cggtactgga

240gcctggctgc gaagacagac cggtgatact ccccagcctc tctttcagtg tccctctcct 300gtctgggact ccaagctgtt cacctggact gccaaggcct tctactattc taagtacgtg 360gagtacctcg acaccgcttg gctggtcctc aagggcaagc gagtgtcctt tctgcaggcc 420ttccatcact ttggagctcc ctgggacgtc tacctcggca ttcgactgca caacgagggt 480gtgtggatct tcatgttctt taactcgttc attcacacca tcatgtacac ctactatgga 540ctgactgccg ctggctacaa gttcaaggcc aagcctctga tcactgccat gcagatttgc 600cagttcgtcg gtggctttct cctggtctgg gactacatca acgttccctg cttcaactct 660gacaagggca agctgttctc ctgggctttc aactacgcct acgtcggatc tgtctttctc 720ctgttctgtc acttctttta ccaggacaac ctggccacca agaaatccgc taaggctggt 780aagcagcttt ag 7921118DNAArtificial SequenceGenomeWalker adaptor-2 111accagccc 8112416PRTEuglena gracilis 112Met Lys Ser Lys Arg Gln Ala Leu Pro Leu Thr Ile Asp Gly Thr Thr1 5 10 15Tyr Asp Val Ser Ala Trp Val Asn Phe His Pro Gly Gly Ala Glu Ile 20 25 30Ile Glu Asn Tyr Gln Gly Arg Asp Ala Thr Asp Ala Phe Met Val Met 35 40 45His Ser Gln Glu Ala Phe Asp Lys Leu Lys Arg Met Pro Lys Ile Asn 50 55 60Pro Ser Ser Glu Leu Pro Pro Gln Ala Ala Val Asn Glu Ala Gln Glu65 70 75 80Asp Phe Arg Lys Leu Arg Glu Glu Leu Ile Ala Thr Gly Met Phe Asp 85 90 95Ala Ser Pro Leu Trp Tyr Ser Tyr Lys Ile Ser Thr Thr Leu Gly Leu 100 105 110Gly Val Leu Gly Tyr Phe Leu Met Val Gln Tyr Gln Met Tyr Phe Ile 115 120 125Gly Ala Val Leu Leu Gly Met His Tyr Gln Gln Met Gly Trp Leu Ser 130 135 140His Asp Ile Cys His His Gln Thr Phe Lys Asn Arg Asn Trp Asn Asn145 150 155 160Leu Val Gly Leu Val Phe Gly Asn Gly Leu Gln Gly Phe Ser Val Thr 165 170 175Trp Trp Lys Asp Arg His Asn Ala His His Ser Ala Thr Asn Val Gln 180 185 190Gly His Asp Pro Asp Ile Asp Asn Leu Pro Leu Leu Ala Trp Ser Glu 195 200 205Asp Asp Val Thr Arg Ala Ser Pro Ile Ser Arg Lys Leu Ile Gln Phe 210 215 220Gln Gln Tyr Tyr Phe Leu Val Ile Cys Ile Leu Leu Arg Phe Ile Trp225 230 235 240Cys Phe Gln Ser Val Leu Thr Val Arg Ser Leu Lys Asp Arg Asp Asn 245 250 255Gln Phe Tyr Arg Ser Gln Tyr Lys Lys Glu Ala Ile Gly Leu Ala Leu 260 265 270His Trp Thr Leu Lys Thr Leu Phe His Leu Phe Phe Met Pro Ser Ile 275 280 285Leu Thr Ser Leu Leu Val Phe Phe Val Ser Glu Leu Val Gly Gly Phe 290 295 300Gly Ile Ala Ile Val Val Phe Met Asn His Tyr Pro Leu Glu Lys Ile305 310 315 320Gly Asp Ser Val Trp Asp Gly His Gly Phe Ser Val Gly Gln Ile His 325 330 335Glu Thr Met Asn Ile Arg Arg Gly Ile Ile Thr Asp Trp Phe Phe Gly 340 345 350Gly Leu Asn Tyr Gln Ile Glu His His Leu Trp Pro Thr Leu Pro Arg 355 360 365His Asn Leu Thr Ala Val Ser Tyr Gln Val Glu Gln Leu Cys Gln Lys 370 375 380His Asn Leu Pro Tyr Arg Asn Pro Leu Pro His Glu Gly Leu Val Ile385 390 395 400Leu Leu Arg Tyr Leu Ala Val Phe Ala Arg Met Ala Glu Lys Gln Pro 405 410 4151133983DNAArtificial SequencepKR132 113ctagagtcga cctgcaggca tgcaagcttg gcgtaatcat ggtcatagct gtttcctgtg 60tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa 120gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct 180ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga 240ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc 300gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa 360tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 420aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 480aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 540ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 600tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc 660agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 720gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 780tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 840acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc 900tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 960caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 1020aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 1080aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt 1140ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac 1200agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc 1260atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc 1320cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata 1380aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc 1440cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc 1500aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca 1560ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa 1620gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca 1680ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt 1740tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt 1800tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg 1860ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga 1920tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc 1980agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg 2040acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag 2100ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg 2160gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg 2220acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat 2280gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg 2340gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc 2400tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa 2460ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc attcaggctg 2520cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa 2580gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt 2640tgtaaaacga cggccagtga attcgagctc ggtacccggg gatcctctag acctgcaggc 2700caactgcgtt tggggctcca gattaaacga cgccgtttcg ttcctttcgc ttcacggctt 2760aacgatgtcg tttctgtctg tgcccaaaaa ataaaggcat ttgttatttg caccagatat 2820ttactaagtg caccctagtt tgacaagtag gcgataatta caaatagatg cggtgcaaat 2880aataaatttt gaaggaaata attacaaaag aacagaactt atatttactt tattttaaaa 2940aactaaaatg aaagaacaaa aaaagtaaaa aatacaaaaa atgtgcttta accactttca 3000ttatttgtta cagaaagtat gattctactc aaattgatct gttgtatctg gtgctgcctt 3060gtcacactgg cgatttcaat cccctaaaga tatggtgcaa actgcgaagt gatcaatatc 3120tgctcggtta atttagatta attaataata ttcaacgtga tgtaccaaaa aaagacaatt 3180ttttgctcca ttgacaaatt aaacctcatc aaggtaattt ccaaacctat aagcaaaaaa 3240atttcacatt aattggcccg caatcctatt agtcttatta tactagagta ggaaaaaaaa 3300caattacaca acttgtctta ttattctcta tgctaatgaa tatttttccc ttttgttaga 3360aatcagtgtt tcctaattta ttgagtatta attccactca ccgcatatat ttaccgttga 3420ataagaaaat tttacacata attcttttta agataaataa tttttttata ctagatctta 3480tatgattacg tgaagccaag tgggttatac taatgatata taatgtttga tagtaatcag 3540tttataaacc aaatgcatgg aaatgttacg tggaagcacg taaattaaca agcattgaag 3600caaatgcagc caccgcacca aaaccacccc acttcacttc cacgtaccat attccatgca 3660actacaacac cctaaaactt caataaatgc ccccaccttc acttcacttc acccatcaat 3720agcaagcggc cgcgaagtta aaagcaatgt tgtcacttgt cgtactaaca catgatgtga 3780tagtttatgc tagctagcta taacataagc tgtctctgag tgtgttgtat attaataaag 3840atcatcactg gtgaatggtg atcgtgtacg taccctactt agtaggcaat ggaagcactt 3900agagtgtgct ttgtgcatgg ccttgcctct gttttgagac ttttgtaatg ttttcgagtt 3960taaatctttg cctttgcgta cgt 39831144771DNAArtificial SequencepKR953 114ggccgcgaag ttaaaagcaa tgttgtcact tgtcgtacta acacatgatg tgatagttta 60tgctagctag ctataacata agctgtctct gagtgtgttg tatattaata aagatcatca 120ctggtgaatg gtgatcgtgt acgtacccta cttagtaggc aatggaagca cttagagtgt 180gctttgtgca tggccttgcc tctgttttga gacttttgta atgttttcga gtttaaatct 240ttgcctttgc gtacgtctag agtcgacctg caggcatgca agcttggcgt aatcatggtc 300atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg 360aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt 420gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg 480ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 540ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 600acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 660aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 720tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 780aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 840gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 900acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 960accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 1020ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 1080gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 1140gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 1200ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 1260gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 1320cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 1380cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 1440gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 1500tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 1560gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 1620agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 1680tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 1740agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 1800gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 1860catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 1920ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 1980atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 2040tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 2100cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 2160cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 2220atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 2280aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 2340ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 2400aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga 2460aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct 2520cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac 2580agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt 2640tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca 2700ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgcca 2760ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt 2820acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt 2880ttcccagtca cgacgttgta aaacgacggc cagtgaattc gagctcggta cccggggatc 2940ctctagacct gcaggccaac tgcgtttggg gctccagatt aaacgacgcc gtttcgttcc 3000tttcgcttca cggcttaacg atgtcgtttc tgtctgtgcc caaaaaataa aggcatttgt 3060tatttgcacc agatatttac taagtgcacc ctagtttgac aagtaggcga taattacaaa 3120tagatgcggt gcaaataata aattttgaag gaaataatta caaaagaaca gaacttatat 3180ttactttatt ttaaaaaact aaaatgaaag aacaaaaaaa gtaaaaaata caaaaaatgt 3240gctttaacca ctttcattat ttgttacaga aagtatgatt ctactcaaat tgatctgttg 3300tatctggtgc tgccttgtca cactggcgat ttcaatcccc taaagatatg gtgcaaactg 3360cgaagtgatc aatatctgct cggttaattt agattaatta ataatattca acgtgatgta 3420ccaaaaaaag acaatttttt gctccattga caaattaaac ctcatcaagg taatttccaa 3480acctataagc aaaaaaattt cacattaatt ggcccgcaat cctattagtc ttattatact 3540agagtaggaa aaaaaacaat tacacaactt gtcttattat tctctatgct aatgaatatt 3600tttccctttt gttagaaatc agtgtttcct aatttattga gtattaattc cactcaccgc 3660atatatttac cgttgaataa gaaaatttta cacataattc tttttaagat aaataatttt 3720tttatactag atcttatatg attacgtgaa gccaagtggg ttatactaat gatatataat 3780gtttgatagt aatcagttta taaaccaaat gcatggaaat gttacgtgga agcacgtaaa 3840ttaacaagca ttgaagcaaa tgcagccacc gcaccaaaac caccccactt cacttccacg 3900taccatattc catgcaacta caacacccta aaacttcaat aaatgccccc accttcactt 3960cacttcaccc atcaatagca agcggccgca ccatggaggt ggtgaatgaa atagtctcaa 4020ttgggcagga agttttaccc aaagttgatt atgcccaact ctggagtgat gccagtcact 4080gtgaggtgct ttacttgtcc atcgcatttg tcatcttgaa gttcactctt ggcccccttg 4140gtccaaaagg tcagtctcgt atgaagtttg ttttcaccaa ttacaacctt ctcatgtcca 4200tttattcgtt gggatcattc ctctcaatgg catatgccat gtacaccatc ggtgttatgt 4260ctgacaactg cgagaaggct tttgacaaca acgtcttcag gatcaccacg cagttgttct 4320atttgagcaa gttcctggag tatattgact ccttctattt gccactgatg ggcaagcctc 4380tgacctggtt gcaattcttc catcatttgg gggcaccgat ggatatgtgg ctgttctata 4440attaccgaaa tgaagctgtt tggatttttg tgctgttgaa tggtttcatc cactggatca 4500tgtacggtta ttattggacc agattgatca agctgaagtt ccccatgcca aaatccctga 4560ttacatcaat gcagatcatt caattcaatg ttggtttcta cattgtctgg aagtacagga 4620acattccctg ttatcgccaa gatgggatga ggatgtttgg ctggttcttc aattactttt 4680atgttggcac agtcttgtgt ttgttcttga atttctatgt gcaaacgtat atcgtcagga 4740agcacaaggg agccaaaaag attcagtgag c 47711155492DNAArtificial SequencepKR287 115ggccgcattt 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 300tggttggaga ggaggaccaa ccgatgggac aacattggga gaaagagatt caatggagat 360ttggatagga gaacaacatt ctttttcact tcaatacaag atgagtgcaa cactaaggat 420atgtatgaga ctttcagaag ctacgacaac atagatgagt gaggtggtga ttcctagcaa 480gaaagacatt agaggaagcc aaaatcgaac aaggaagaca tcaagggcaa gagacaggac 540catccatctc aggaaaagga gctttgggat agtccgagaa gttgtacaag aaattttttg 600gagggtgagt gatgcattgc tggtgacttt aactcaatca aaattgagaa agaaagaaaa 660gggagggggc tcacatgtga atagaaggga aacgggagaa ttttacagtt ttgatctaat 720gggcatccca gctagtggta acatattcac catgtttaac cttcacgtac gtctagagga 780tccccgggta ccgagctcga attcactggc cgtcgtttta caacgtcgtg actgggaaaa 840ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa 900tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg 960gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg 1020cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac 1080acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt 1140gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag 1200acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc 1260ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt 1320ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata 1380atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt 1440tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc 1500tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat 1560ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct 1620atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca 1680ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg 1740catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa 1800cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg 1860ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga 1920cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg 1980cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt 2040tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg 2100agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc 2160ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca 2220gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc 2280atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat 2340cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc 2400agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg 2460ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct 2520accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct 2580tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct 2640cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg 2700gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc 2760gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga 2820gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc

cggtaagcgg 2880cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta 2940tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg 3000ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg 3060ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat 3120taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc 3180agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc 3240gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa 3300cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc 3360ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga 3420ccatgattac gccaagcttg catgcctgca ggctagccta agtacgtact caaaatgcca 3480acaaataaaa aaaaagttgc tttaataatg ccaaaacaaa ttaataaaac acttacaaca 3540ccggattttt tttaattaaa atgtgccatt taggataaat agttaatatt tttaataatt 3600atttaaaaag ccgtatctac taaaatgatt tttatttggt tgaaaatatt aatatgttta 3660aatcaacaca atctatcaaa attaaactaa aaaaaaaata agtgtacgtg gttaacatta 3720gtacagtaat ataagaggaa aatgagaaat taagaaattg aaagcgagtc taatttttaa 3780attatgaacc tgcatatata aaaggaaaga aagaatccag gaagaaaaga aatgaaacca 3840tgcatggtcc cctcgtcatc acgagtttct gccatttgca atagaaacac tgaaacacct 3900ttctctttgt cacttaattg agatgccgaa gccacctcac accatgaact tcatgaggtg 3960tagcacccaa ggcttccata gccatgcata ctgaagaatg tctcaagctc agcaccctac 4020ttctgtgacg tgtccctcat tcaccttcct ctcttcccta taaataacca cgcctcaggt 4080tctccgcttc acaactcaaa cattctctcc attggtcctt aaacactcat cagtcatcac 4140cgcggccgca tgggaacgga ccaaggaaaa accttcacct gggaagagct ggcggcccat 4200aacaccaagg acgacctact cttggccatc cgcggcaggg tgtacgatgt cacaaagttc 4260ttgagccgcc atcctggtgg agtggacact ctcctgctcg gagctggccg agatgttact 4320ccggtctttg agatgtatca cgcgtttggg gctgcagatg ccattatgaa gaagtactat 4380gtcggtacac tggtctcgaa tgagctgccc atcttcccgg agccaacggt gttccacaaa 4440accatcaaga cgagagtcga gggctacttt acggatcgga acattgatcc caagaataga 4500ccagagatct ggggacgata cgctcttatc tttggatcct tgatcgcttc ctactacgcg 4560cagctctttg tgcctttcgt tgtcgaacgc acatggcttc aggtggtgtt tgcaatcatc 4620atgggatttg cgtgcgcaca agtcggactc aaccctcttc atgatgcgtc tcacttttca 4680gtgacccaca accccactgt ctggaagatt ctgggagcca cgcacgactt tttcaacgga 4740gcatcgtacc tggtgtggat gtaccaacat atgctcggcc atcaccccta caccaacatt 4800gctggagcag atcccgacgt gtcgacgtct gagcccgatg ttcgtcgtat caagcccaac 4860caaaagtggt ttgtcaacca catcaaccag cacatgtttg ttcctttcct gtacggactg 4920ctggcgttca aggtgcgcat tcaggacatc aacattttgt actttgtcaa gaccaatgac 4980gctattcgtg tcaatcccat ctcgacatgg cacactgtga tgttctgggg cggcaaggct 5040ttctttgtct ggtatcgcct gattgttccc ctgcagtatc tgcccctggg caaggtgctg 5100ctcttgttca cggtcgcgga catggtgtcg tcttactggc tggcgctgac cttccaggcg 5160aaccacgttg ttgaggaagt tcagtggccg ttgcctgacg agaacgggat catccaaaag 5220gactgggcag ctatgcaggt cgagactacg caggattacg cacacgattc gcacctctgg 5280accagcatca ctggcagctt gaactaccag gctgtgcacc atctgttccc caacgtgtcg 5340cagcaccatt atcccgatat tctggccatc atcaagaaca cctgcagcga gtacaaggtt 5400ccataccttg tcaaggatac gttttggcaa gcatttgctt cacatttgga gcacttgcgt 5460gttcttggac tccgtcccaa ggaagagtag gc 54921161338DNAMortierella alpina 116atgggaacgg 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 13381172577DNAArtificial SequencepKR277 117agcttggatc tcctgcagga tctggccggc cggatctcgt acggatccgt cgacggcgcg 60cccgatcatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag 120gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt 180cgggctttgt tagcagccgg atcgatccaa gctgtacctc actattcctt tgccctcgga 240cgagtgctgg ggcgtcggtt tccactatcg gcgagtactt ctacacagcc atcggtccag 300acggccgcgc ttctgcgggc gatttgtgta cgcccgacag tcccggctcc ggatcggacg 360attgcgtcgc atcgaccctg cgcccaagct gcatcatcga aattgccgtc aaccaagctc 420tgatagagtt ggtcaagacc aatgcggagc atatacgccc ggagccgcgg cgatcctgca 480agctccggat gcctccgctc gaagtagcgc gtctgctgct ccatacaagc caaccacggc 540ctccagaaga agatgttggc gacctcgtat tgggaatccc cgaacatcgc ctcgctccag 600tcaatgaccg ctgttatgcg gccattgtcc gtcaggacat tgttggagcc gaaatccgcg 660tgcacgaggt gccggacttc ggggcagtcc tcggcccaaa gcatcagctc atcgagagcc 720tgcgcgacgg acgcactgac ggtgtcgtcc atcacagttt gccagtgata cacatgggga 780tcagcaatcg cgcatatgaa atcacgccat gtagtgtatt gaccgattcc ttgcggtccg 840aatgggccga acccgctcgt ctggctaaga tcggccgcag cgatcgcatc catagcctcc 900gcgaccggct gcagaacagc gggcagttcg gtttcaggca ggtcttgcaa cgtgacaccc 960tgtgcacggc gggagatgca ataggtcagg ctctcgctga attccccaat gtcaagcact 1020tccggaatcg ggagcgcggc cgatgcaaag tgccgataaa cataacgatc tttgtagaaa 1080ccatcggcgc agctatttac ccgcaggaca tatccacgcc ctcctacatc gaagctgaaa 1140gcacgagatt cttcgccctc cgagagctgc atcaggtcgg agacgctgtc gaacttttcg 1200atcagaaact tctcgacaga cgtcgcggtg agttcaggct tttccatggg tatatctcct 1260tcttaaagtt aaacaaaatt atttctagag ggaaaccgtt gtggtctccc tatagtgagt 1320cgtattaatt tcgcgggatc gagatctgat caacctgcat taatgaatcg gccaacgcgc 1380ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg 1440ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 1500cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 1560gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 1620tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 1680ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 1740atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag 1800gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 1860tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 1920cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 1980cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt 2040tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 2100cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 2160cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 2220gaacgaaaac tcacgttaag ggattttggt catgacatta acctataaaa ataggcgtat 2280cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca 2340gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca 2400gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca 2460gattgtactg agagtgcacc atatggacat attgtcgtta gaacgcggct acaattaata 2520cataacctta tgtatcatac acatacgatt taggtgacac tatagaacgg cgcgcca 25771185364DNAArtificial SequencepKR952 118ggctagccta agtacgtact caaaatgcca acaaataaaa aaaaagttgc tttaataatg 60ccaaaacaaa ttaataaaac acttacaaca ccggattttt tttaattaaa atgtgccatt 120taggataaat agttaatatt tttaataatt atttaaaaag ccgtatctac taaaatgatt 180tttatttggt tgaaaatatt aatatgttta aatcaacaca atctatcaaa attaaactaa 240aaaaaaaata agtgtacgtg gttaacatta gtacagtaat ataagaggaa aatgagaaat 300taagaaattg aaagcgagtc taatttttaa attatgaacc tgcatatata aaaggaaaga 360aagaatccag gaagaaaaga aatgaaacca tgcatggtcc cctcgtcatc acgagtttct 420gccatttgca atagaaacac tgaaacacct ttctctttgt cacttaattg agatgccgaa 480gccacctcac accatgaact tcatgaggtg tagcacccaa ggcttccata gccatgcata 540ctgaagaatg tctcaagctc agcaccctac ttctgtgacg tgtccctcat tcaccttcct 600ctcttcccta taaataacca cgcctcaggt tctccgcttc acaactcaaa cattctctcc 660attggtcctt aaacactcat cagtcatcac cgcggccgca tgggaacgga ccaaggaaaa 720accttcacct gggaagagct ggcggcccat aacaccaagg acgacctact cttggccatc 780cgcggcaggg tgtacgatgt cacaaagttc ttgagccgcc atcctggtgg agtggacact 840ctcctgctcg gagctggccg agatgttact ccggtctttg agatgtatca cgcgtttggg 900gctgcagatg ccattatgaa gaagtactat gtcggtacac tggtctcgaa tgagctgccc 960atcttcccgg agccaacggt gttccacaaa accatcaaga cgagagtcga gggctacttt 1020acggatcgga acattgatcc caagaataga ccagagatct ggggacgata cgctcttatc 1080tttggatcct tgatcgcttc ctactacgcg cagctctttg tgcctttcgt tgtcgaacgc 1140acatggcttc aggtggtgtt tgcaatcatc atgggatttg cgtgcgcaca agtcggactc 1200aaccctcttc atgatgcgtc tcacttttca gtgacccaca accccactgt ctggaagatt 1260ctgggagcca cgcacgactt tttcaacgga gcatcgtacc tggtgtggat gtaccaacat 1320atgctcggcc atcaccccta caccaacatt gctggagcag atcccgacgt gtcgacgtct 1380gagcccgatg ttcgtcgtat caagcccaac caaaagtggt ttgtcaacca catcaaccag 1440cacatgtttg ttcctttcct gtacggactg ctggcgttca aggtgcgcat tcaggacatc 1500aacattttgt actttgtcaa gaccaatgac gctattcgtg tcaatcccat ctcgacatgg 1560cacactgtga tgttctgggg cggcaaggct ttctttgtct ggtatcgcct gattgttccc 1620ctgcagtatc tgcccctggg caaggtgctg ctcttgttca cggtcgcgga catggtgtcg 1680tcttactggc tggcgctgac cttccaggcg aaccacgttg ttgaggaagt tcagtggccg 1740ttgcctgacg agaacgggat catccaaaag gactgggcag ctatgcaggt cgagactacg 1800caggattacg cacacgattc gcacctctgg accagcatca ctggcagctt gaactaccag 1860gctgtgcacc atctgttccc caacgtgtcg cagcaccatt atcccgatat tctggccatc 1920atcaagaaca cctgcagcga gtacaaggtt ccataccttg tcaaggatac gttttggcaa 1980gcatttgctt cacatttgga gcacttgcgt gttcttggac tccgtcccaa ggaagagtag 2040gcggccgcat ttcgcaccaa atcaatgaaa gtaataatga aaagtctgaa taagaatact 2100taggcttaga tgcctttgtt acttgtgtaa aataacttga gtcatgtacc tttggcggaa 2160acagaataaa taaaaggtga aattccaatg ctctatgtat aagttagtaa tacttaatgt 2220gttctacggt tgtttcaata tcatcaaact ctaattgaaa ctttagaacc acaaatctca 2280atcttttctt aatgaaatga aaaatcttaa ttgtaccatg tttatgttaa acaccttaca 2340attggttgga gaggaggacc aaccgatggg acaacattgg gagaaagaga ttcaatggag 2400atttggatag gagaacaaca ttctttttca cttcaataca agatgagtgc aacactaagg 2460atatgtatga gactttcaga agctacgaca acatagatga gtgaggtggt gattcctagc 2520aagaaagaca ttagaggaag ccaaaatcga acaaggaaga catcaagggc aagagacagg 2580accatccatc tcaggaaaag gagctttggg atagtccgag aagttgtaca agaaattttt 2640tggagggtga gtgatgcatt gctggtgact ttaactcaat caaaattgag aaagaaagaa 2700aagggagggg gctcacatgt gaatagaagg gaaacgggag aattttacag ttttgatcta 2760atgggcatcc cagctagtgg taacatattc accatgttta accttcacgt acggatccgt 2820cgacggcgcg cccgatcatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac 2880ccgtttagag gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc 2940agcttccttt cgggctttgt tagcagccgg atcgatccaa gctgtacctc actattcctt 3000tgccctcgga cgagtgctgg ggcgtcggtt tccactatcg gcgagtactt ctacacagcc 3060atcggtccag acggccgcgc ttctgcgggc gatttgtgta cgcccgacag tcccggctcc 3120ggatcggacg attgcgtcgc atcgaccctg cgcccaagct gcatcatcga aattgccgtc 3180aaccaagctc tgatagagtt ggtcaagacc aatgcggagc atatacgccc ggagccgcgg 3240cgatcctgca agctccggat gcctccgctc gaagtagcgc gtctgctgct ccatacaagc 3300caaccacggc ctccagaaga agatgttggc gacctcgtat tgggaatccc cgaacatcgc 3360ctcgctccag tcaatgaccg ctgttatgcg gccattgtcc gtcaggacat tgttggagcc 3420gaaatccgcg tgcacgaggt gccggacttc ggggcagtcc tcggcccaaa gcatcagctc 3480atcgagagcc tgcgcgacgg acgcactgac ggtgtcgtcc atcacagttt gccagtgata 3540cacatgggga tcagcaatcg cgcatatgaa atcacgccat gtagtgtatt gaccgattcc 3600ttgcggtccg aatgggccga acccgctcgt ctggctaaga tcggccgcag cgatcgcatc 3660catagcctcc gcgaccggct gcagaacagc gggcagttcg gtttcaggca ggtcttgcaa 3720cgtgacaccc tgtgcacggc gggagatgca ataggtcagg ctctcgctga attccccaat 3780gtcaagcact tccggaatcg ggagcgcggc cgatgcaaag tgccgataaa cataacgatc 3840tttgtagaaa ccatcggcgc agctatttac ccgcaggaca tatccacgcc ctcctacatc 3900gaagctgaaa gcacgagatt cttcgccctc cgagagctgc atcaggtcgg agacgctgtc 3960gaacttttcg atcagaaact tctcgacaga cgtcgcggtg agttcaggct tttccatggg 4020tatatctcct tcttaaagtt aaacaaaatt atttctagag ggaaaccgtt gtggtctccc 4080tatagtgagt cgtattaatt tcgcgggatc gagatctgat caacctgcat taatgaatcg 4140gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg 4200actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 4260tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 4320aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 4380ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 4440aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 4500cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct 4560cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 4620aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 4680cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 4740ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 4800ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 4860gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 4920agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 4980acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgacatta acctataaaa 5040ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct 5100gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac 5160aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg 5220catcagagca gattgtactg agagtgcacc atatggacat attgtcgtta gaacgcggct 5280acaattaata cataacctta tgtatcatac acatacgatt taggtgacac tatagaacgg 5340cgcgccaagc ttggatctcc tgca 53641195252DNAArtificial SequencepKR457 119gtacgtgggc ggatcccccg ggctgcagga attcactggc cgtcgtttta caacgtcgtg 60actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca 120gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 180atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc 240gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac 300acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca 360gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga 420aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa 480taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt 540gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa 600tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta 660ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag 720taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca 780gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta 840aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc 900gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc 960ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca 1020ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc 1080acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca 1140taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac 1200tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg 1260cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg 1320ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg 1380gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac 1440gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc 1500aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct 1560aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc 1620actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc 1680gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 1740atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa 1800atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc 1860ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 1920gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 1980cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 2040tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 2100cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct 2160ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 2220gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc 2280tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg 2340ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc 2400gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg 2460cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca 2520gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact 2580ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa 2640acagctatga ccatgattac gccaagcttg catgcctgca ggtcgactcg acgtacgtcc 2700tcgaagagaa gggttaataa cacatttttt aacattttta acacaaattt tagttattta 2760aaaatttatt aaaaaattta aaataagaag aggaactctt taaataaatc taacttacaa 2820aatttatgat ttttaataag ttttcaccaa taaaaaatgt cataaaaata tgttaaaaag 2880tatattatca atattctctt

tatgataaat aaaaagaaaa aaaaaataaa agttaagtga 2940aaatgagatt gaagtgactt taggtgtgta taaatatatc aaccccgcca acaatttatt 3000taatccaaat atattgaagt atattattcc atagccttta tttatttata tatttattat 3060ataaaagctt tatttgttct aggttgttca tgaaatattt ttttggtttt atctccgttg 3120taagaaaatc atgtgctttg tgtcgccact cactattgca gctttttcat gcattggtca 3180gattgacggt tgattgtatt tttgtttttt atggttttgt gttatgactt aagtcttcat 3240ctctttatct cttcatcagg tttgatggtt acctaatatg gtccatgggt acatgcatgg 3300ttaaattagg tggccaactt tgttgtgaac gatagaattt tttttatatt aagtaaacta 3360tttttatatt atgaaataat aataaaaaaa atattttatc attattaaca aaatcatatt 3420agttaatttg ttaactctat aataaaagaa atactgtaac attcacatta catggtaaca 3480tctttccacc ctttcatttg ttttttgttt gatgactttt tttcttgttt aaatttattt 3540cccttctttt aaatttggaa tacattatca tcatatataa actaaaatac taaaaacagg 3600attacacaaa tgataaataa taacacaaat atttataaat ctagctgcaa tatatttaaa 3660ctagctatat cgatattgta aaataaaact agctgcattg atactgataa aaaaatatca 3720tgtgctttct ggactgatga tgcagtatac ttttgacatt gcctttattt tatttttcag 3780aaaagctttc ttagttctgg gttcttcatt atttgtttcc catctccatt gtgaattgaa 3840tcatttgctt cgtgtcacaa atacaattta gntaggtaca tgcattggtc agattcacgg 3900tttattatgt catgacttaa gttcatggta gtacattacc tgccacgcat gcattatatt 3960ggttagattt gataggcaaa tttggttgtc aacaatataa atataaataa tgtttttata 4020ttacgaaata acagtgatca aaacaaacag ttttatcttt attaacaaga ttttgttttt 4080gtttgatgac gttttttaat gtttacgctt tcccccttct tttgaattta gaacacttta 4140tcatcataaa atcaaatact aaaaaaatta catatttcat aaataataac acaaatattt 4200ttaaaaaatc tgaaataata atgaacaata ttacatatta tcacgaaaat tcattaataa 4260aaatattata taaataaaat gtaatagtag ttatatgtag gaaaaaagta ctgcacgcat 4320aatatataca aaaagattaa aatgaactat tataaataat aacactaaat taatggtgaa 4380tcatatcaaa ataatgaaaa agtaaataaa atttgtaatt aacttctata tgtattacac 4440acacaaataa taaataatag taaaaaaaat tatgataaat atttaccatc tcataagata 4500tttaaaataa tgataaaaat atagattatt ttttatgcaa ctagctagcc aaaaagagaa 4560cacgggtata tataaaaaga gtacctttaa attctactgt acttccttta ttcctgacgt 4620ttttatatca agtggacata cgtgaagatt ttaattatca gtctaaatat ttcattagca 4680cttaatactt ttctgtttta ttcctatcct ataagtagtc ccgattctcc caacattgct 4740tattcacaca actaactaag aaagtcttcc atagcccccc aagcggccgc gacacaagtg 4800tgagagtact aaataaatgc tttggttgta cgaaatcatt acactaaata aaataatcaa 4860agcttatata tgccttccgc taaggccgaa tgcaaagaaa ttggttcttt ctcgttatct 4920tttgccactt ttactagtac gtattaatta ctacttaatc atctttgttt acggctcatt 4980atatccggtc tagaggatcc aaggccgcga agttaaaagc aatgttgtca cttgtcgtac 5040taacacatga tgtgatagtt tatgctagct agctataaca taagctgtct ctgagtgtgt 5100tgtatattaa taaagatcat cactggtgaa tggtgatcgt gtacgtaccc tacttagtag 5160gcaatggaag cacttagagt gtgctttgtg catggccttg cctctgtttt gagacttttg 5220taatgttttc gagtttaaat ctttgccttt gc 52521202635DNAArtificial Sequencemodified Kti-NotI-Kti3'Salb3'cassette 120ggtaccgggc cccccctcga ggtcgcccgg gggatccgcc ctaagcttcg tacgtcctcg 60aagagaaggg ttaataacac attttttaac atttttaaca caaattttag ttatttaaaa 120atttattaaa aaatttaaaa taagaagagg aactctttaa ataaatctaa cttacaaaat 180ttatgatttt taataagttt tcaccaataa aaaatgtcat aaaaatatgt taaaaagtat 240attatcaata ttctctttat gataaataaa aagaaaaaaa aaataaaagt taagtgaaaa 300tgagattgaa gtgactttag gtgtgtataa atatatcaac cccgccaaca atttatttaa 360tccaaatata ttgaagtata ttattccata gcctttattt atttatatat ttattatata 420aaagctttat ttgttctagg ttgttcatga aatatttttt tggttttatc tccgttgtaa 480gaaaatcatg tgctttgtgt cgccactcac tattgcagct ttttcatgca ttggtcagat 540tgacggttga ttgtattttt gttttttatg gttttgtgtt atgacttaag tcttcatctc 600tttatctctt catcaggttt gatggttacc taatatggtc catgggtaca tgcatggtta 660aattaggtgg ccaactttgt tgtgaacgat agaatttttt ttatattaag taaactattt 720ttatattatg aaataataat aaaaaaaata ttttatcatt attaacaaaa tcatattagt 780taatttgtta actctataat aaaagaaata ctgtaacatt cacattacat ggtaacatct 840ttccaccctt tcatttgttt tttgtttgat gacttttttt cttgtttaaa tttatttccc 900ttcttttaaa tttggaatac attatcatca tatataaact aaaatactaa aaacaggatt 960acacaaatga taaataataa cacaaatatt tataaatcta gctgcaatat atttaaacta 1020gctatatcga tattgtaaaa taaaactagc tgcattgata ctgataaaaa aatatcatgt 1080gctttctgga ctgatgatgc agtatacttt tgacattgcc tttattttat ttttcagaaa 1140agctttctta gttctgggtt cttcattatt tgtttcccat ctccattgtg aattgaatca 1200tttgcttcgt gtcacaaata caatttagnt aggtacatgc attggtcaga ttcacggttt 1260attatgtcat gacttaagtt catggtagta cattacctgc cacgcatgca ttatattggt 1320tagatttgat aggcaaattt ggttgtcaac aatataaata taaataatgt ttttatatta 1380cgaaataaca gtgatcaaaa caaacagttt tatctttatt aacaagattt tgtttttgtt 1440tgatgacgtt ttttaatgtt tacgctttcc cccttctttt gaatttagaa cactttatca 1500tcataaaatc aaatactaaa aaaattacat atttcataaa taataacaca aatattttta 1560aaaaatctga aataataatg aacaatatta catattatca cgaaaattca ttaataaaaa 1620tattatataa ataaaatgta atagtagtta tatgtaggaa aaaagtactg cacgcataat 1680atatacaaaa agattaaaat gaactattat aaataataac actaaattaa tggtgaatca 1740tatcaaaata atgaaaaagt aaataaaatt tgtaattaac ttctatatgt attacacaca 1800caaataataa ataatagtaa aaaaaattat gataaatatt taccatctca taagatattt 1860aaaataatga taaaaatata gattattttt tatgcaacta gctagccaaa aagagaacac 1920gggtatatat aaaaagagta cctttaaatt ctactgtact tcctttattc ctgacgtttt 1980tatatcaagt ggacatacgt gaagatttta attatcagtc taaatatttc attagcactt 2040aatacttttc tgttttattc ctatcctata agtagtcccg attctcccaa cattgcttat 2100tcacacaact aactaagaaa gtcttccata gccccccaag cggccgcgac acaagtgtga 2160gagtactaaa taaatgcttt ggttgtacga aatcattaca ctaaataaaa taatcaaagc 2220ttatatatgc cttccgctaa ggccgaatgc aaagaaattg gttctttctc gttatctttt 2280gccactttta ctagtacgta ttaattacta cttaatcatc tttgtttacg gctcattata 2340tccggtctag aggatccaag gccgcgaagt taaaagcaat gttgtcactt gtcgtactaa 2400cacatgatgt gatagtttat gctagctagc tataacataa gctgtctctg agtgtgttgt 2460atattaataa agatcatcac tggtgaatgg tgatcgtgta cgtaccctac ttagtaggca 2520atggaagcac ttagagtgtg ctttgtgcat ggccttgcct ctgttttgag acttttgtaa 2580tgttttcgag tttaaatctt tgcctttgcg tacgtctaga ggatccccgg gtacc 26351211269DNAPavlova lutheri 121atgggcaagg gtggagacgg cggcgcgcag gcggtgagcg ggaccgacgc gtctctcgct 60gaggtgagct ccgtcgatag caagagcgtg cacgtcgtgc tctacggcaa gcgcgtggat 120gtcacaaagt tccagaaggc acacccgggc gggagcaagg tgttccgcat cttccaggag 180cgcgacgcga cggagcagtt cgagtcttac cactcgccca aggccatcaa gatgatggag 240ggcatgctca agaagtcgga ggatgcgccc gcttccgtgc ccctgccctc gcggtccacc 300atgggcacgg agttcaagga gatgattgag cgccacaaga gggctggtct ctacgaccct 360tgcccgttgg acgagctgtt caagctcacc atcgtccttg cgcccatctt cgtgggcgcc 420tatctcgtgc ggagcggcgt ctcgcccctc gcgggcgcgc tctccatggg ctttggcttc 480tacctcgacg gctggcttgc tcacgactac ctgcatcacg cagtcttcaa gggctcggtc 540aacacgctcg tcaaggcgaa caacgccatg ggatacgccc tcggcttcct ccagggctac 600gacgtggcct ggtggcgcgc gcgccataac acgcaccacg tgtgcaccaa cgaggatggt 660tcggacccgg acatcaagac ggcgcccctg ctcatctacg tgcgagagaa cccgtccatt 720gccaagcggc tcaacttctt ccagcgctgg cagcagtact actatgtgcc gaccatggcc 780atcctcgacc tctactggcg cctggagtcc atcgcgtacg tggctgtgcg cctgcctaag 840atgtggatgc aggccgccgc tcttgccgct cactacgcgc tcctgtgctg ggtcttcgca 900gcgcatctca acctcatccc tctcatgatg gttgcacgcg gcttcgcgac gggcatcgtt 960gtctttgcaa cccactatgg tgaggacatc ctcgaccgcg agcacgtcga gggcatgacg 1020ctcgtcgagc agaccgccaa gacctcccgt aacatcacgg gcggctggct agtgaacgtg 1080ctcacgggct tcatctccct gcagaccgag catcacctct tccccatgat gcccaccggc 1140aacctaatga ctatccagcc cgaggtacgc gacttcttca agaagcatgg cctcgagtac 1200cgcgagggca acctcttcca gtgcgtgcac cagaacatca aggctctcgc cttcgagcac 1260ctcctccac 126912230DNAArtificialprimer PvDES5'Not-1 122gcggccgcac catgggcaag ggtggagacg 3012327DNAArtificialprimer PvDES3'Not-1 123gcggccgctc agtggaggag gtgctcg 271249276DNAArtificial SequencepKR970 124gtacggatcc 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 tcgagatctg atcaacctgc 1320attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt 1380cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 1440caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 1500caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 1560ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 1620cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 1680ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 1740tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 1800gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 1860ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 1920ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 1980gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 2040aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 2100tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 2160ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgacat 2220taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg 2280gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg 2340ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc 2400ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatggac atattgtcgt 2460tagaacgcgg ctacaattaa tacataacct tatgtatcat acacatacga tttaggtgac 2520actatagaac ggcgcgccaa gcttggatct cctgcaggct agcctaagta cgtactcaaa 2580atgccaacaa ataaaaaaaa agttgcttta ataatgccaa aacaaattaa taaaacactt 2640acaacaccgg atttttttta attaaaatgt gccatttagg ataaatagtt aatattttta 2700ataattattt aaaaagccgt atctactaaa atgattttta tttggttgaa aatattaata 2760tgtttaaatc aacacaatct atcaaaatta aactaaaaaa aaaataagtg tacgtggtta 2820acattagtac agtaatataa gaggaaaatg agaaattaag aaattgaaag cgagtctaat 2880ttttaaatta tgaacctgca tatataaaag gaaagaaaga atccaggaag aaaagaaatg 2940aaaccatgca tggtcccctc gtcatcacga gtttctgcca tttgcaatag aaacactgaa 3000acacctttct ctttgtcact taattgagat gccgaagcca cctcacacca tgaacttcat 3060gaggtgtagc acccaaggct tccatagcca tgcatactga agaatgtctc aagctcagca 3120ccctacttct gtgacgtgtc cctcattcac cttcctctct tccctataaa taaccacgcc 3180tcaggttctc cgcttcacaa ctcaaacatt ctctccattg gtccttaaac actcatcagt 3240catcaccgcg gccgcatggg aacggaccaa ggaaaaacct tcacctggga agagctggcg 3300gcccataaca ccaaggacga cctactcttg gccatccgcg gcagggtgta cgatgtcaca 3360aagttcttga gccgccatcc tggtggagtg gacactctcc tgctcggagc tggccgagat 3420gttactccgg tctttgagat gtatcacgcg tttggggctg cagatgccat tatgaagaag 3480tactatgtcg gtacactggt ctcgaatgag ctgcccatct tcccggagcc aacggtgttc 3540cacaaaacca tcaagacgag agtcgagggc tactttacgg atcggaacat tgatcccaag 3600aatagaccag agatctgggg acgatacgct cttatctttg gatccttgat cgcttcctac 3660tacgcgcagc tctttgtgcc tttcgttgtc gaacgcacat ggcttcaggt ggtgtttgca 3720atcatcatgg gatttgcgtg cgcacaagtc ggactcaacc ctcttcatga tgcgtctcac 3780ttttcagtga cccacaaccc cactgtctgg aagattctgg gagccacgca cgactttttc 3840aacggagcat cgtacctggt gtggatgtac caacatatgc tcggccatca cccctacacc 3900aacattgctg gagcagatcc cgacgtgtcg acgtctgagc ccgatgttcg tcgtatcaag 3960cccaaccaaa agtggtttgt caaccacatc aaccagcaca tgtttgttcc tttcctgtac 4020ggactgctgg cgttcaaggt gcgcattcag gacatcaaca ttttgtactt tgtcaagacc 4080aatgacgcta ttcgtgtcaa tcccatctcg acatggcaca ctgtgatgtt ctggggcggc 4140aaggctttct ttgtctggta tcgcctgatt gttcccctgc agtatctgcc cctgggcaag 4200gtgctgctct tgttcacggt cgcggacatg gtgtcgtctt actggctggc gctgaccttc 4260caggcgaacc acgttgttga ggaagttcag tggccgttgc ctgacgagaa cgggatcatc 4320caaaaggact gggcagctat gcaggtcgag actacgcagg attacgcaca cgattcgcac 4380ctctggacca gcatcactgg cagcttgaac taccaggctg tgcaccatct gttccccaac 4440gtgtcgcagc accattatcc cgatattctg gccatcatca agaacacctg cagcgagtac 4500aaggttccat accttgtcaa ggatacgttt tggcaagcat ttgcttcaca tttggagcac 4560ttgcgtgttc ttggactccg tcccaaggaa gagtaggcgg ccgcatttcg caccaaatca 4620atgaaagtaa taatgaaaag tctgaataag aatacttagg cttagatgcc tttgttactt 4680gtgtaaaata acttgagtca tgtacctttg gcggaaacag aataaataaa aggtgaaatt 4740ccaatgctct atgtataagt tagtaatact taatgtgttc tacggttgtt tcaatatcat 4800caaactctaa ttgaaacttt agaaccacaa atctcaatct tttcttaatg aaatgaaaaa 4860tcttaattgt accatgttta tgttaaacac cttacaattg gttggagagg aggaccaacc 4920gatgggacaa cattgggaga aagagattca atggagattt ggataggaga acaacattct 4980ttttcacttc aatacaagat gagtgcaaca ctaaggatat gtatgagact ttcagaagct 5040acgacaacat agatgagtga ggtggtgatt cctagcaaga aagacattag aggaagccaa 5100aatcgaacaa ggaagacatc aagggcaaga gacaggacca tccatctcag gaaaaggagc 5160tttgggatag tccgagaagt tgtacaagaa attttttgga gggtgagtga tgcattgctg 5220gtgactttaa ctcaatcaaa attgagaaag aaagaaaagg gagggggctc acatgtgaat 5280agaagggaaa cgggagaatt ttacagtttt gatctaatgg gcatcccagc tagtggtaac 5340atattcacca tgtttaacct tcacgtaccg ggccccccct cgaggtcgcc cgggggatcc 5400gccctaagct tcgtacgtcc tcgaagagaa gggttaataa cacatttttt aacattttta 5460acacaaattt tagttattta aaaatttatt aaaaaattta aaataagaag aggaactctt 5520taaataaatc taacttacaa aatttatgat ttttaataag ttttcaccaa taaaaaatgt 5580cataaaaata tgttaaaaag tatattatca atattctctt tatgataaat aaaaagaaaa 5640aaaaaataaa agttaagtga aaatgagatt gaagtgactt taggtgtgta taaatatatc 5700aaccccgcca acaatttatt taatccaaat atattgaagt atattattcc atagccttta 5760tttatttata tatttattat ataaaagctt tatttgttct aggttgttca tgaaatattt 5820ttttggtttt atctccgttg taagaaaatc atgtgctttg tgtcgccact cactattgca 5880gctttttcat gcattggtca gattgacggt tgattgtatt tttgtttttt atggttttgt 5940gttatgactt aagtcttcat ctctttatct cttcatcagg tttgatggtt acctaatatg 6000gtccatgggt acatgcatgg ttaaattagg tggccaactt tgttgtgaac gatagaattt 6060tttttatatt aagtaaacta tttttatatt atgaaataat aataaaaaaa atattttatc 6120attattaaca aaatcatatt agttaatttg ttaactctat aataaaagaa atactgtaac 6180attcacatta catggtaaca tctttccacc ctttcatttg ttttttgttt gatgactttt 6240tttcttgttt aaatttattt cccttctttt aaatttggaa tacattatca tcatatataa 6300actaaaatac taaaaacagg attacacaaa tgataaataa taacacaaat atttataaat 6360ctagctgcaa tatatttaaa ctagctatat cgatattgta aaataaaact agctgcattg 6420atactgataa aaaaatatca tgtgctttct ggactgatga tgcagtatac ttttgacatt 6480gcctttattt tatttttcag aaaagctttc ttagttctgg gttcttcatt atttgtttcc 6540catctccatt gtgaattgaa tcatttgctt cgtgtcacaa atacaattta gntaggtaca 6600tgcattggtc agattcacgg tttattatgt catgacttaa gttcatggta gtacattacc 6660tgccacgcat gcattatatt ggttagattt gataggcaaa tttggttgtc aacaatataa 6720atataaataa tgtttttata ttacgaaata acagtgatca aaacaaacag ttttatcttt 6780attaacaaga ttttgttttt gtttgatgac gttttttaat gtttacgctt tcccccttct 6840tttgaattta gaacacttta tcatcataaa atcaaatact aaaaaaatta catatttcat 6900aaataataac acaaatattt ttaaaaaatc tgaaataata atgaacaata ttacatatta 6960tcacgaaaat tcattaataa aaatattata taaataaaat gtaatagtag ttatatgtag 7020gaaaaaagta ctgcacgcat aatatataca aaaagattaa aatgaactat tataaataat 7080aacactaaat taatggtgaa tcatatcaaa ataatgaaaa agtaaataaa atttgtaatt 7140aacttctata tgtattacac acacaaataa taaataatag taaaaaaaat tatgataaat 7200atttaccatc tcataagata tttaaaataa tgataaaaat atagattatt ttttatgcaa 7260ctagctagcc aaaaagagaa cacgggtata tataaaaaga gtacctttaa attctactgt 7320acttccttta ttcctgacgt ttttatatca agtggacata cgtgaagatt ttaattatca 7380gtctaaatat ttcattagca cttaatactt ttctgtttta ttcctatcct ataagtagtc 7440ccgattctcc caacattgct tattcacaca actaactaag aaagtcttcc atagcccccc 7500aagcggccgc accatgggca agggtggaga cggcggcgcg caggcggtga gcgggaccga 7560cgcgtctctc gctgaggtga gctccgtcga tagcaagagc gtgcacgtcg tgctctacgg 7620caagcgcgtg gatgtcacaa agttccagaa ggcacacccg ggcgggagca aggtgttccg 7680catcttccag gagcgcgacg cgacggagca gttcgagtct taccactcgc ccaaggccat 7740caagatgatg gagggcatgc tcaagaagtc ggaggatgcg cccgcttccg tgcccctgcc 7800ctcgcggtcc accatgggca cggagttcaa ggagatgatt gagcgccaca agagggctgg 7860tctctacgac ccttgcccgt tggacgagct gttcaagctc accatcgtcc ttgcgcccat 7920cttcgtgggc gcctatctcg tgcggagcgg cgtctcgccc ctcgcgggcg cgctctccat 7980gggctttggc ttctacctcg acggctggct tgctcacgac tacctgcatc acgcagtctt 8040caagggctcg gtcaacacgc tcgtcaaggc gaacaacgcc atgggatacg ccctcggctt 8100cctccagggc tacgacgtgg cctggtggcg cgcgcgccat aacacgcacc acgtgtgcac 8160caacgaggat ggttcggacc cggacatcaa gacggcgccc ctgctcatct acgtgcgaga 8220gaacccgtcc attgccaagc ggctcaactt cttccagcgc tggcagcagt actactatgt 8280gccgaccatg gccatcctcg acctctactg gcgcctggag tccatcgcgt acgtggctgt 8340gcgcctgcct aagatgtgga tgcaggccgc cgctcttgcc gctcactacg

cgctcctgtg 8400ctgggtcttc gcagcgcatc tcaacctcat ccctctcatg atggttgcac gcggcttcgc 8460gacgggcatc gttgtctttg caacccacta tggtgaggac atcctcgacc gcgagcacgt 8520cgagggcatg acgctcgtcg agcagaccgc caagacctcc cgtaacatca cgggcggctg 8580gctagtgaac gtgctcacgg gcttcatctc cctgcagacc gagcatcacc tcttccccat 8640gatgcccacc ggcaacctaa tgactatcca gcccgaggta cgcgacttct tcaagaagca 8700tggcctcgag taccgcgagg gcaacctctt ccagtgcgtg caccagaaca tcaaggctct 8760cgccttcgag cacctcctcc actgagcggc cgcgacacaa gtgtgagagt actaaataaa 8820tgctttggtt gtacgaaatc attacactaa ataaaataat caaagcttat atatgccttc 8880cgctaaggcc gaatgcaaag aaattggttc tttctcgtta tcttttgcca cttttactag 8940tacgtattaa ttactactta atcatctttg tttacggctc attatatccg gtctagagga 9000tccaaggccg cgaagttaaa agcaatgttg tcacttgtcg tactaacaca tgatgtgata 9060gtttatgcta gctagctata acataagctg tctctgagtg tgttgtatat taataaagat 9120catcactggt gaatggtgat cgtgtacgta ccctacttag taggcaatgg aagcacttag 9180agtgtgcttt gtgcatggcc ttgcctctgt tttgagactt ttgtaatgtt ttcgagttta 9240aatctttgcc tttgcgtacg tctagaggat ccccgg 927612511366DNAArtificial SequencepKR973 125ggagatccaa gcttggcgcg ccgttctata gtgtcaccta aatcgtatgt gtatgataca 60taaggttatg tattaattgt agccgcgttc taacgacaat atgtccatat ggtgcactct 120cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc 180tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt 240ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa 300gggcctcgtg atacgcctat ttttataggt taatgtcatg accaaaatcc cttaacgtga 360gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 420tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 480ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 540gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc 600tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 660cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 720gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 780actgagatac ctacagcgtg agcattgaga aagcgccacg cttcccgaag ggagaaaggc 840ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 900gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 960atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt 1020tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc 1080tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg 1140aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc 1200gcctctcccc gcgcgttggc cgattcatta atgcaggttg atcagatctc gatcccgcga 1260aattaatacg actcactata gggagaccac aacggtttcc ctctagaaat aattttgttt 1320aactttaaga aggagatata cccatggaaa agcctgaact caccgcgacg tctgtcgaga 1380agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag 1440aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct 1500gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc 1560cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc 1620gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc 1680agccggtcgc ggaggctatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt 1740tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg 1800cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt 1860ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc 1920acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag 1980cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct 2040tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc 2100atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc 2160aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat 2220gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa 2280gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc 2340ccagcactcg tccgagggca aaggaatagt gaggtacagc ttggatcgat ccggctgcta 2400acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac 2460cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg 2520gatgatcggg cgcgccgtcg acggatccgt acccggggat cctctagacg tacgcaaagg 2580caaagattta aactcgaaaa cattacaaaa gtctcaaaac agaggcaagg ccatgcacaa 2640agcacactct aagtgcttcc attgcctact aagtagggta cgtacacgat caccattcac 2700cagtgatgat ctttattaat atacaacaca ctcagagaca gcttatgtta tagctagcta 2760gcataaacta tcacatcatg tgttagtacg acaagtgaca acattgcttt taacttcgcg 2820gccttggatc ctctagaccg gatataatga gccgtaaaca aagatgatta agtagtaatt 2880aatacgtact agtaaaagtg gcaaaagata acgagaaaga accaatttct ttgcattcgg 2940ccttagcgga aggcatatat aagctttgat tattttattt agtgtaatga tttcgtacaa 3000ccaaagcatt tatttagtac tctcacactt gtgtcgcggc cgctcagtgg aggaggtgct 3060cgaaggcgag agccttgatg ttctggtgca cgcactggaa gaggttgccc tcgcggtact 3120cgaggccatg cttcttgaag aagtcgcgta cctcgggctg gatagtcatt aggttgccgg 3180tgggcatcat ggggaagagg tgatgctcgg tctgcaggga gatgaagccc gtgagcacgt 3240tcactagcca gccgcccgtg atgttacggg aggtcttggc ggtctgctcg acgagcgtca 3300tgccctcgac gtgctcgcgg tcgaggatgt cctcaccata gtgggttgca aagacaacga 3360tgcccgtcgc gaagccgcgt gcaaccatca tgagagggat gaggttgaga tgcgctgcga 3420agacccagca caggagcgcg tagtgagcgg caagagcggc ggcctgcatc cacatcttag 3480gcaggcgcac agccacgtac gcgatggact ccaggcgcca gtagaggtcg aggatggcca 3540tggtcggcac atagtagtac tgctgccagc gctggaagaa gttgagccgc ttggcaatgg 3600acgggttctc tcgcacgtag atgagcaggg gcgccgtctt gatgtccggg tccgaaccat 3660cctcgttggt gcacacgtgg tgcgtgttat ggcgcgcgcg ccaccaggcc acgtcgtagc 3720cctggaggaa gccgagggcg tatcccatgg cgttgttcgc cttgacgagc gtgttgaccg 3780agcccttgaa gactgcgtga tgcaggtagt cgtgagcaag ccagccgtcg aggtagaagc 3840caaagcccat ggagagcgcg cccgcgaggg gcgagacgcc gctccgcacg agataggcgc 3900ccacgaagat gggcgcaagg acgatggtga gcttgaacag ctcgtccaac gggcaagggt 3960cgtagagacc agccctcttg tggcgctcaa tcatctcctt gaactccgtg cccatggtgg 4020accgcgaggg caggggcacg gaagcgggcg catcctccga cttcttgagc atgccctcca 4080tcatcttgat ggccttgggc gagtggtaag actcgaactg ctccgtcgcg tcgcgctcct 4140ggaagatgcg gaacaccttg ctcccgcccg ggtgtgcctt ctggaacttt gtgacatcca 4200cgcgcttgcc gtagagcacg acgtgcacgc tcttgctatc gacggagctc acctcagcga 4260gagacgcgtc ggtcccgctc accgcctgcg cgccgccgtc tccacccttg cccatggtgc 4320ggccgcttgg ggggctatgg aagactttct tagttagttg tgtgaataag caatgttggg 4380agaatcggga ctacttatag gataggaata aaacagaaaa gtattaagtg ctaatgaaat 4440atttagactg ataattaaaa tcttcacgta tgtccacttg atataaaaac gtcaggaata 4500aaggaagtac agtagaattt aaaggtactc tttttatata tacccgtgtt ctctttttgg 4560ctagctagtt gcataaaaaa taatctatat ttttatcatt attttaaata tcttatgaga 4620tggtaaatat ttatcataat tttttttact attatttatt atttgtgtgt gtaatacata 4680tagaagttaa ttacaaattt tatttacttt ttcattattt tgatatgatt caccattaat 4740ttagtgttat tatttataat agttcatttt aatctttttg tatatattat gcgtgcagta 4800cttttttcct acatataact actattacat tttatttata taatattttt attaatgaat 4860tttcgtgata atatgtaata ttgttcatta ttatttcaga ttttttaaaa atatttgtgt 4920tattatttat gaaatatgta atttttttag tatttgattt tatgatgata aagtgttcta 4980aattcaaaag aagggggaaa gcgtaaacat taaaaaacgt catcaaacaa aaacaaaatc 5040ttgttaataa agataaaact gtttgttttg atcactgtta tttcgtaata taaaaacatt 5100atttatattt atattgttga caaccaaatt tgcctatcaa atctaaccaa tataatgcat 5160gcgtggcagg taatgtacta ccatgaactt aagtcatgac ataataaacc gtgaatctga 5220ccaatgcatg tacctancta aattgtattt gtgacacgaa gcaaatgatt caattcacaa 5280tggagatggg aaacaaataa tgaagaaccc agaactaaga aagcttttct gaaaaataaa 5340ataaaggcaa tgtcaaaagt atactgcatc atcagtccag aaagcacatg atattttttt 5400atcagtatca atgcagctag ttttatttta caatatcgat atagctagtt taaatatatt 5460gcagctagat ttataaatat ttgtgttatt atttatcatt tgtgtaatcc tgtttttagt 5520attttagttt atatatgatg ataatgtatt ccaaatttaa aagaagggaa ataaatttaa 5580acaagaaaaa aagtcatcaa acaaaaaaca aatgaaaggg tggaaagatg ttaccatgta 5640atgtgaatgt tacagtattt cttttattat agagttaaca aattaactaa tatgattttg 5700ttaataatga taaaatattt tttttattat tatttcataa tataaaaata gtttacttaa 5760tataaaaaaa attctatcgt tcacaacaaa gttggccacc taatttaacc atgcatgtac 5820ccatggacca tattaggtaa ccatcaaacc tgatgaagag ataaagagat gaagacttaa 5880gtcataacac aaaaccataa aaaacaaaaa tacaatcaac cgtcaatctg accaatgcat 5940gaaaaagctg caatagtgag tggcgacaca aagcacatga ttttcttaca acggagataa 6000aaccaaaaaa atatttcatg aacaacctag aacaaataaa gcttttatat aataaatata 6060taaataaata aaggctatgg aataatatac ttcaatatat ttggattaaa taaattgttg 6120gcggggttga tatatttata cacacctaaa gtcacttcaa tctcattttc acttaacttt 6180tatttttttt ttctttttat ttatcataaa gagaatattg ataatatact ttttaacata 6240tttttatgac attttttatt ggtgaaaact tattaaaaat cataaatttt gtaagttaga 6300tttatttaaa gagttcctct tcttatttta aattttttaa taaattttta aataactaaa 6360atttgtgtta aaaatgttaa aaaatgtgtt attaaccctt ctcttcgagg acgtacgaag 6420cttagggcgg atcccccggg cgacctcgag ggggggcccg gtacgtgaag gttaaacatg 6480gtgaatatgt taccactagc tgggatgccc attagatcaa aactgtaaaa ttctcccgtt 6540tcccttctat tcacatgtga gccccctccc ttttctttct ttctcaattt tgattgagtt 6600aaagtcacca gcaatgcatc actcaccctc caaaaaattt cttgtacaac ttctcggact 6660atcccaaagc tccttttcct gagatggatg gtcctgtctc ttgcccttga tgtcttcctt 6720gttcgatttt ggcttcctct aatgtctttc ttgctaggaa tcaccacctc actcatctat 6780gttgtcgtag cttctgaaag tctcatacat atccttagtg ttgcactcat cttgtattga 6840agtgaaaaag aatgttgttc tcctatccaa atctccattg aatctctttc tcccaatgtt 6900gtcccatcgg ttggtcctcc tctccaacca attgtaaggt gtttaacata aacatggtac 6960aattaagatt tttcatttca ttaagaaaag attgagattt gtggttctaa agtttcaatt 7020agagtttgat gatattgaaa caaccgtaga acacattaag tattactaac ttatacatag 7080agcattggaa tttcaccttt tatttattct gtttccgcca aaggtacatg actcaagtta 7140ttttacacaa gtaacaaagg catctaagcc taagtattct tattcagact tttcattatt 7200actttcattg atttggtgcg aaatgcggcc gcctactctt ccttgggacg gagtccaaga 7260acacgcaagt gctccaaatg tgaagcaaat gcttgccaaa acgtatcctt gacaaggtat 7320ggaaccttgt actcgctgca ggtgttcttg atgatggcca gaatatcggg ataatggtgc 7380tgcgacacgt tggggaacag atggtgcaca gcctggtagt tcaagctgcc agtgatgctg 7440gtccagaggt gcgaatcgtg tgcgtaatcc tgcgtagtct cgacctgcat agctgcccag 7500tccttttgga tgatcccgtt ctcgtcaggc aacggccact gaacttcctc aacaacgtgg 7560ttcgcctgga aggtcagcgc cagccagtaa gacgacacca tgtccgcgac cgtgaacaag 7620agcagcacct tgcccagggg cagatactgc aggggaacaa tcaggcgata ccagacaaag 7680aaagccttgc cgccccagaa catcacagtg tgccatgtcg agatgggatt gacacgaata 7740gcgtcattgg tcttgacaaa gtacaaaatg ttgatgtcct gaatgcgcac cttgaacgcc 7800agcagtccgt acaggaaagg aacaaacatg tgctggttga tgtggttgac aaaccacttt 7860tggttgggct tgatacgacg aacatcgggc tcagacgtcg acacgtcggg atctgctcca 7920gcaatgttgg tgtaggggtg atggccgagc atatgttggt acatccacac caggtacgat 7980gctccgttga aaaagtcgtg cgtggctccc agaatcttcc agacagtggg gttgtgggtc 8040actgaaaagt gagacgcatc atgaagaggg ttgagtccga cttgtgcgca cgcaaatccc 8100atgatgattg caaacaccac ctgaagccat gtgcgttcga caacgaaagg cacaaagagc 8160tgcgcgtagt aggaagcgat caaggatcca aagataagag cgtatcgtcc ccagatctct 8220ggtctattct tgggatcaat gttccgatcc gtaaagtagc cctcgactct cgtcttgatg 8280gttttgtgga acaccgttgg ctccgggaag atgggcagct cattcgagac cagtgtaccg 8340acatagtact tcttcataat ggcatctgca gccccaaacg cgtgatacat ctcaaagacc 8400ggagtaacat ctcggccagc tccgagcagg agagtgtcca ctccaccagg atggcggctc 8460aagaactttg tgacatcgta caccctgccg cggatggcca agagtaggtc gtccttggtg 8520ttatgggccg ccagctcttc ccaggtgaag gtttttcctt ggtccgttcc catgcggccg 8580cggtgatgac tgatgagtgt ttaaggacca atggagagaa tgtttgagtt gtgaagcgga 8640gaacctgagg cgtggttatt tatagggaag agaggaaggt gaatgaggga cacgtcacag 8700aagtagggtg ctgagcttga gacattcttc agtatgcatg gctatggaag ccttgggtgc 8760tacacctcat gaagttcatg gtgtgaggtg gcttcggcat ctcaattaag tgacaaagag 8820aaaggtgttt cagtgtttct attgcaaatg gcagaaactc gtgatgacga ggggaccatg 8880catggtttca tttcttttct tcctggattc tttctttcct tttatatatg caggttcata 8940atttaaaaat tagactcgct ttcaatttct taatttctca ttttcctctt atattactgt 9000actaatgtta accacgtaca cttatttttt ttttagttta attttgatag attgtgttga 9060tttaaacata ttaatatttt caaccaaata aaaatcattt tagtagatac ggctttttaa 9120ataattatta aaaatattaa ctatttatcc taaatggcac attttaatta aaaaaaatcc 9180ggtgttgtaa gtgttttatt aatttgtttt ggcattatta aagcaacttt ttttttattt 9240gttggcattt tgagtacgta cttaggctag cctgcaggcc aactgcgttt ggggctccag 9300attaaacgac gccgtttcgt tcctttcgct tcacggctta acgatgtcgt ttctgtctgt 9360gcccaaaaaa taaaggcatt tgttatttgc accagatatt tactaagtgc accctagttt 9420gacaagtagg cgataattac aaatagatgc ggtgcaaata ataaattttg aaggaaataa 9480ttacaaaaga acagaactta tatttacttt attttaaaaa actaaaatga aagaacaaaa 9540aaagtaaaaa atacaaaaaa tgtgctttaa ccactttcat tatttgttac agaaagtatg 9600attctactca aattgatctg ttgtatctgg tgctgccttg tcacactggc gatttcaatc 9660ccctaaagat atggtgcaaa ctgcgaagtg atcaatatct gctcggttaa tttagattaa 9720ttaataatat tcaacgtgat gtaccaaaaa aagacaattt tttgctccat tgacaaatta 9780aacctcatca aggtaatttc caaacctata agcaaaaaaa tttcacatta attggcccgc 9840aatcctatta gtcttattat actagagtag gaaaaaaaac aattacacaa cttgtcttat 9900tattctctat gctaatgaat atttttccct tttgttagaa atcagtgttt cctaatttat 9960tgagtattaa ttccactcac cgcatatatt taccgttgaa taagaaaatt ttacacataa 10020ttctttttaa gataaataat ttttttatac tagatcttat atgattacgt gaagccaagt 10080gggttatact aatgatatat aatgtttgat agtaatcagt ttataaacca aatgcatgga 10140aatgttacgt ggaagcacgt aaattaacaa gcattgaagc aaatgcagcc accgcaccaa 10200aaccacccca cttcacttcc acgtaccata ttccatgcaa ctacaacacc ctaaaacttc 10260aataaatgcc cccaccttca cttcacttca cccatcaata gcaagcggcc gcaccatgga 10320ggtggtgaat gaaatagtct caattgggca ggaagtttta cccaaagttg attatgccca 10380actctggagt gatgccagtc actgtgaggt gctttacttg tccatcgcat ttgtcatctt 10440gaagttcact cttggccccc ttggtccaaa aggtcagtct cgtatgaagt ttgttttcac 10500caattacaac cttctcatgt ccatttattc gttgggatca ttcctctcaa tggcatatgc 10560catgtacacc atcggtgtta tgtctgacaa ctgcgagaag gcttttgaca acaacgtctt 10620caggatcacc acgcagttgt tctatttgag caagttcctg gagtatattg actccttcta 10680tttgccactg atgggcaagc ctctgacctg gttgcaattc ttccatcatt tgggggcacc 10740gatggatatg tggctgttct ataattaccg aaatgaagct gtttggattt ttgtgctgtt 10800gaatggtttc atccactgga tcatgtacgg ttattattgg accagattga tcaagctgaa 10860gttccccatg ccaaaatccc tgattacatc aatgcagatc attcaattca atgttggttt 10920ctacattgtc tggaagtaca ggaacattcc ctgttatcgc caagatggga tgaggatgtt 10980tggctggttc ttcaattact tttatgttgg cacagtcttg tgtttgttct tgaatttcta 11040tgtgcaaacg tatatcgtca ggaagcacaa gggagccaaa aagattcagt gagcggccgc 11100gaagttaaaa gcaatgttgt cacttgtcgt actaacacat gatgtgatag tttatgctag 11160ctagctataa cataagctgt ctctgagtgt gttgtatatt aataaagatc atcactggtg 11220aatggtgatc gtgtacgtac cctacttagt aggcaatgga agcacttaga gtgtgctttg 11280tgcatggcct tgcctctgtt ttgagacttt tgtaatgttt tcgagtttaa atctttgcct 11340ttgcgtacgt ctagagtcga cctgca 113661265671DNAArtificial SequencepKS129 126ggccgcaagt 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 ggatccgtcg acggcgcgcc cgatcatccg 1200gatatagttc ctcctttcag caaaaaaccc ctcaagaccc gtttagaggc cccaaggggt 1260tatgctagtt attgctcagc ggtggcagca gccaactcag cttcctttcg ggctttgtta 1320gcagccggat cgatccaagc tgtacctcac tattcctttg ccctcggacg agtgctgggg 1380cgtcggtttc cactatcggc gagtacttct acacagccat cggtccagac ggccgcgctt 1440ctgcgggcga tttgtgtacg cccgacagtc ccggctccgg atcggacgat tgcgtcgcat 1500cgaccctgcg cccaagctgc atcatcgaaa ttgccgtcaa ccaagctctg atagagttgg 1560tcaagaccaa tgcggagcat atacgcccgg agccgcggcg atcctgcaag ctccggatgc 1620ctccgctcga agtagcgcgt ctgctgctcc atacaagcca accacggcct ccagaagaag 1680atgttggcga cctcgtattg ggaatccccg aacatcgcct cgctccagtc aatgaccgct 1740gttatgcggc cattgtccgt caggacattg ttggagccga aatccgcgtg cacgaggtgc 1800cggacttcgg ggcagtcctc ggcccaaagc atcagctcat cgagagcctg cgcgacggac 1860gcactgacgg tgtcgtccat cacagtttgc cagtgataca catggggatc agcaatcgcg 1920catatgaaat cacgccatgt agtgtattga ccgattcctt gcggtccgaa tgggccgaac 1980ccgctcgtct ggctaagatc ggccgcagcg atcgcatcca tagcctccgc gaccggctgc 2040agaacagcgg gcagttcggt ttcaggcagg tcttgcaacg tgacaccctg tgcacggcgg 2100gagatgcaat aggtcaggct ctcgctgaat tccccaatgt caagcacttc cggaatcggg 2160agcgcggccg atgcaaagtg ccgataaaca taacgatctt tgtagaaacc atcggcgcag 2220ctatttaccc gcaggacata tccacgccct cctacatcga agctgaaagc acgagattct 2280tcgccctccg agagctgcat caggtcggag acgctgtcga acttttcgat cagaaacttc 2340tcgacagacg tcgcggtgag ttcaggcttt tccatgggta tatctccttc ttaaagttaa 2400acaaaattat ttctagaggg aaaccgttgt ggtctcccta tagtgagtcg tattaatttc 2460gcgggatcga gatctgatca acctgcatta atgaatcggc caacgcgcgg ggagaggcgg 2520tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 2580gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 2640ggataacgca ggaaagaaca

tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 2700ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 2760acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 2820tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 2880ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc 2940ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 3000ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 3060actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 3120gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 3180tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 3240caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 3300atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 3360acgttaaggg attttggtca tgacattaac ctataaaaat aggcgtatca cgaggccctt 3420tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac 3480ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc 3540gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag 3600agtgcaccat atggacatat tgtcgttaga acgcggctac aattaataca taaccttatg 3660tatcatacac atacgattta ggtgacacta tagaacggcg cgccaagctt ttgatccatg 3720cccttcattt gccgcttatt aattaatttg gtaacagtcc gtactaatca gttacttatc 3780cttcccccat cataattaat cttggtagtc tcgaatgcca caacactgac tagtctcttg 3840gatcataaga aaaagccaag gaacaaaaga agacaaaaca caatgagagt atcctttgca 3900tagcaatgtc taagttcata aaattcaaac aaaaacgcaa tcacacacag tggacatcac 3960ttatccacta gctgatcagg atcgccgcgt caagaaaaaa aaactggacc ccaaaagcca 4020tgcacaacaa cacgtactca caaaggtgtc aatcgagcag cccaaaacat tcaccaactc 4080aacccatcat gagccctcac atttgttgtt tctaacccaa cctcaaactc gtattctctt 4140ccgccacctc atttttgttt atttcaacac ccgtcaaact gcatgccacc ccgtggccaa 4200atgtccatgc atgttaacaa gacctatgac tataaatagc tgcaatctcg gcccaggttt 4260tcatcatcaa gaaccagttc aatatcctag tacaccgtat taaagaattt aagatatact 4320gcggccgcat gggaacggac caaggaaaaa ccttcacctg ggaagagctg gcggcccata 4380acaccaagga cgacctactc ttggccatcc gcggcagggt gtacgatgtc acaaagttct 4440tgagccgcca tcctggtgga gtggacactc tcctgctcgg agctggccga gatgttactc 4500cggtctttga gatgtatcac gcgtttgggg ctgcagatgc cattatgaag aagtactatg 4560tcggtacact ggtctcgaat gagctgccca tcttcccgga gccaacggtg ttccacaaaa 4620ccatcaagac gagagtcgag ggctacttta cggatcggaa cattgatccc aagaatagac 4680cagagatctg gggacgatac gctcttatct ttggatcctt gatcgcttcc tactacgcgc 4740agctctttgt gcctttcgtt gtcgaacgca catggcttca ggtggtgttt gcaatcatca 4800tgggatttgc gtgcgcacaa gtcggactca accctcttca tgatgcgtct cacttttcag 4860tgacccacaa ccccactgtc tggaagattc tgggagccac gcacgacttt ttcaacggag 4920catcgtacct ggtgtggatg taccaacata tgctcggcca tcacccctac accaacattg 4980ctggagcaga tcccgacgtg tcgacgtctg agcccgatgt tcgtcgtatc aagcccaacc 5040aaaagtggtt tgtcaaccac atcaaccagc acatgtttgt tcctttcctg tacggactgc 5100tggcgttcaa ggtgcgcatt caggacatca acattttgta ctttgtcaag accaatgacg 5160ctattcgtgt caatcccatc tcgacatggc acactgtgat gttctggggc ggcaaggctt 5220tctttgtctg gtatcgcctg attgttcccc tgcagtatct gcccctgggc aaggtgctgc 5280tcttgttcac ggtcgcggac atggtgtcgt cttactggct ggcgctgacc ttccaggcga 5340accacgttgt tgaggaagtt cagtggccgt tgcctgacga gaacgggatc atccaaaagg 5400actgggcagc tatgcaggtc gagactacgc aggattacgc acacgattcg cacctctgga 5460ccagcatcac tggcagcttg aactaccagg ctgtgcacca tctgttcccc aacgtgtcgc 5520agcaccatta tcccgatatt ctggccatca tcaagaacac ctgcagcgag tacaaggttc 5580cataccttgt caaggatacg ttttggcaag catttgcttc acatttggag cacttgcgtg 5640ttcttggact ccgtcccaag gaagagtagg c 56711276601DNAArtificial SequencepKR606 127ggccgcatgg gaacggacca aggaaaaacc ttcacctggg aagagctggc ggcccataac 60accaaggacg acctactctt ggccatccgc ggcagggtgt acgatgtcac aaagttcttg 120agccgccatc ctggtggagt ggacactctc ctgctcggag ctggccgaga tgttactccg 180gtctttgaga tgtatcacgc gtttggggct gcagatgcca ttatgaagaa gtactatgtc 240ggtacactgg tctcgaatga gctgcccatc ttcccggagc caacggtgtt ccacaaaacc 300atcaagacga gagtcgaggg ctactttacg gatcggaaca ttgatcccaa gaatagacca 360gagatctggg gacgatacgc tcttatcttt ggatccttga tcgcttccta ctacgcgcag 420ctctttgtgc ctttcgttgt cgaacgcaca tggcttcagg tggtgtttgc aatcatcatg 480ggatttgcgt gcgcacaagt cggactcaac cctcttcatg atgcgtctca cttttcagtg 540acccacaacc ccactgtctg gaagattctg ggagccacgc acgacttttt caacggagca 600tcgtacctgg tgtggatgta ccaacatatg ctcggccatc acccctacac caacattgct 660ggagcagatc ccgacgtgtc gacgtctgag cccgatgttc gtcgtatcaa gcccaaccaa 720aagtggtttg tcaaccacat caaccagcac atgtttgttc ctttcctgta cggactgctg 780gcgttcaagg tgcgcattca ggacatcaac attttgtact ttgtcaagac caatgacgct 840attcgtgtca atcccatctc gacatggcac actgtgatgt tctggggcgg caaggctttc 900tttgtctggt atcgcctgat tgttcccctg cagtatctgc ccctgggcaa ggtgctgctc 960ttgttcacgg tcgcggacat ggtgtcgtct tactggctgg cgctgacctt ccaggcgaac 1020cacgttgttg aggaagttca gtggccgttg cctgacgaga acgggatcat ccaaaaggac 1080tgggcagcta tgcaggtcga gactacgcag gattacgcac acgattcgca cctctggacc 1140agcatcactg gcagcttgaa ctaccaggct gtgcaccatc tgttccccaa cgtgtcgcag 1200caccattatc ccgatattct ggccatcatc aagaacacct gcagcgagta caaggttcca 1260taccttgtca aggatacgtt ttggcaagca tttgcttcac atttggagca cttgcgtgtt 1320cttggactcc gtcccaagga agagtaggcg gccgcgacac aagtgtgaga gtactaaata 1380aatgctttgg ttgtacgaaa tcattacact aaataaaata atcaaagctt atatatgcct 1440tccgctaagg ccgaatgcaa agaaattggt tctttctcgt tatcttttgc cacttttact 1500agtacgtatt aattactact taatcatctt tgtttacggc tcattatatc cggtctagag 1560gatccaaggc cgcgaagtta aaagcaatgt tgtcacttgt cgtactaaca catgatgtga 1620tagtttatgc tagctagcta taacataagc tgtctctgag tgtgttgtat attaataaag 1680atcatcactg gtgaatggtg atcgtgtacg taccctactt agtaggcaat ggaagcactt 1740agagtgtgct ttgtgcatgg ccttgcctct gttttgagac ttttgtaatg ttttcgagtt 1800taaatctttg cctttgcgta cgtgggcgga tcccccgggc tgcaggaatt cactggccgt 1860cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc 1920acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca 1980acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg tattttctcc ttacgcatct 2040gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata 2100gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct 2160cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt 2220ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata 2280ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt 2340gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag 2400acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca 2460tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc 2520agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat 2580cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc 2640aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg 2700gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc 2760agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat 2820aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga 2880gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc 2940ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc 3000aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt 3060aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc 3120tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc 3180agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca 3240ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca 3300ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt 3360ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta 3420acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 3480agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 3540ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 3600cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa 3660gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 3720cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 3780gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 3840caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag 3900aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 3960tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 4020gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc 4080ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt 4140atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg 4200cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg 4260caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc 4320cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc 4380accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata 4440acaatttcac acaggaaaca gctatgacca tgattacgcc aagcttgcat gcctgcaggt 4500cgactcgacg tacgtcctcg aagagaaggg ttaataacac attttttaac atttttaaca 4560caaattttag ttatttaaaa atttattaaa aaatttaaaa taagaagagg aactctttaa 4620ataaatctaa cttacaaaat ttatgatttt taataagttt tcaccaataa aaaatgtcat 4680aaaaatatgt taaaaagtat attatcaata ttctctttat gataaataaa aagaaaaaaa 4740aaataaaagt taagtgaaaa tgagattgaa gtgactttag gtgtgtataa atatatcaac 4800cccgccaaca atttatttaa tccaaatata ttgaagtata ttattccata gcctttattt 4860atttatatat ttattatata aaagctttat ttgttctagg ttgttcatga aatatttttt 4920tggttttatc tccgttgtaa gaaaatcatg tgctttgtgt cgccactcac tattgcagct 4980ttttcatgca ttggtcagat tgacggttga ttgtattttt gttttttatg gttttgtgtt 5040atgacttaag tcttcatctc tttatctctt catcaggttt gatggttacc taatatggtc 5100catgggtaca tgcatggtta aattaggtgg ccaactttgt tgtgaacgat agaatttttt 5160ttatattaag taaactattt ttatattatg aaataataat aaaaaaaata ttttatcatt 5220attaacaaaa tcatattagt taatttgtta actctataat aaaagaaata ctgtaacatt 5280cacattacat ggtaacatct ttccaccctt tcatttgttt tttgtttgat gacttttttt 5340cttgtttaaa tttatttccc ttcttttaaa tttggaatac attatcatca tatataaact 5400aaaatactaa aaacaggatt acacaaatga taaataataa cacaaatatt tataaatcta 5460gctgcaatat atttaaacta gctatatcga tattgtaaaa taaaactagc tgcattgata 5520ctgataaaaa aatatcatgt gctttctgga ctgatgatgc agtatacttt tgacattgcc 5580tttattttat ttttcagaaa agctttctta gttctgggtt cttcattatt tgtttcccat 5640ctccattgtg aattgaatca tttgcttcgt gtcacaaata caatttagnt aggtacatgc 5700attggtcaga ttcacggttt attatgtcat gacttaagtt catggtagta cattacctgc 5760cacgcatgca ttatattggt tagatttgat aggcaaattt ggttgtcaac aatataaata 5820taaataatgt ttttatatta cgaaataaca gtgatcaaaa caaacagttt tatctttatt 5880aacaagattt tgtttttgtt tgatgacgtt ttttaatgtt tacgctttcc cccttctttt 5940gaatttagaa cactttatca tcataaaatc aaatactaaa aaaattacat atttcataaa 6000taataacaca aatattttta aaaaatctga aataataatg aacaatatta catattatca 6060cgaaaattca ttaataaaaa tattatataa ataaaatgta atagtagtta tatgtaggaa 6120aaaagtactg cacgcataat atatacaaaa agattaaaat gaactattat aaataataac 6180actaaattaa tggtgaatca tatcaaaata atgaaaaagt aaataaaatt tgtaattaac 6240ttctatatgt attacacaca caaataataa ataatagtaa aaaaaattat gataaatatt 6300taccatctca taagatattt aaaataatga taaaaatata gattattttt tatgcaacta 6360gctagccaaa aagagaacac gggtatatat aaaaagagta cctttaaatt ctactgtact 6420tcctttattc ctgacgtttt tatatcaagt ggacatacgt gaagatttta attatcagtc 6480taaatatttc attagcactt aatacttttc tgttttattc ctatcctata agtagtcccg 6540attctcccaa cattgcttat tcacacaact aactaagaaa gtcttccata gccccccaag 6600c 66011286494DNAArtificial SequencepKR804 128ccggccggat ctcgtacgga tccgtcgacg gcgcgcccga tcatccggat atagttcctc 60ctttcagcaa aaaacccctc aagacccgtt tagaggcccc aaggggttat gctagttatt 120gctcagcggt ggcagcagcc aactcagctt cctttcgggc tttgttagca gccggatcga 180tccaagctgt acctcactat tcctttgccc tcggacgagt gctggggcgt cggtttccac 240tatcggcgag tacttctaca cagccatcgg tccagacggc cgcgcttctg cgggcgattt 300gtgtacgccc gacagtcccg gctccggatc ggacgattgc gtcgcatcga ccctgcgccc 360aagctgcatc atcgaaattg ccgtcaacca agctctgata gagttggtca agaccaatgc 420ggagcatata cgcccggagc cgcggcgatc ctgcaagctc cggatgcctc cgctcgaagt 480agcgcgtctg ctgctccata caagccaacc acggcctcca gaagaagatg ttggcgacct 540cgtattggga atccccgaac atcgcctcgc tccagtcaat gaccgctgtt atgcggccat 600tgtccgtcag gacattgttg gagccgaaat ccgcgtgcac gaggtgccgg acttcggggc 660agtcctcggc ccaaagcatc agctcatcga gagcctgcgc gacggacgca ctgacggtgt 720cgtccatcac agtttgccag tgatacacat ggggatcagc aatcgcgcat atgaaatcac 780gccatgtagt gtattgaccg attccttgcg gtccgaatgg gccgaacccg ctcgtctggc 840taagatcggc cgcagcgatc gcatccatag cctccgcgac cggctgcaga acagcgggca 900gttcggtttc aggcaggtct tgcaacgtga caccctgtgc acggcgggag atgcaatagg 960tcaggctctc gctgaattcc ccaatgtcaa gcacttccgg aatcgggagc gcggccgatg 1020caaagtgccg ataaacataa cgatctttgt agaaaccatc ggcgcagcta tttacccgca 1080ggacatatcc acgccctcct acatcgaagc tgaaagcacg agattcttcg ccctccgaga 1140gctgcatcag gtcggagacg ctgtcgaact tttcgatcag aaacttctcg acagacgtcg 1200cggtgagttc aggcttttcc atgggtatat ctccttctta aagttaaaca aaattatttc 1260tagagggaaa ccgttgtggt ctccctatag tgagtcgtat taatttcgcg ggatcgagat 1320ctgatcaacc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 1380cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 1440gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 1500aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 1560gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 1620aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 1680gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 1740ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 1800cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 1860ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 1920actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 1980tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca 2040gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 2100ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 2160cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 2220ttggtcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt 2280ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt 2340ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg 2400tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg 2460gacatattgt cgttagaacg cggctacaat taatacataa ccttatgtat catacacata 2520cgatttaggt gacactatag aacggcgcgc caagcttgga tctcctgcag gatctggccg 2580ggtacgtcct cgaagagaag ggttaataac acatttttta acatttttaa cacaaatttt 2640agttatttaa aaatttatta aaaaatttaa aataagaaga ggaactcttt aaataaatct 2700aacttacaaa atttatgatt tttaataagt tttcaccaat aaaaaatgtc ataaaaatat 2760gttaaaaagt atattatcaa tattctcttt atgataaata aaaagaaaaa aaaaataaaa 2820gttaagtgaa aatgagattg aagtgacttt aggtgtgtat aaatatatca accccgccaa 2880caatttattt aatccaaata tattgaagta tattattcca tagcctttat ttatttatat 2940atttattata taaaagcttt atttgttcta ggttgttcat gaaatatttt tttggtttta 3000tctccgttgt aagaaaatca tgtgctttgt gtcgccactc actattgcag ctttttcatg 3060cattggtcag attgacggtt gattgtattt ttgtttttta tggttttgtg ttatgactta 3120agtcttcatc tctttatctc ttcatcaggt ttgatggtta cctaatatgg tccatgggta 3180catgcatggt taaattaggt ggccaacttt gttgtgaacg atagaatttt ttttatatta 3240agtaaactat ttttatatta tgaaataata ataaaaaaaa tattttatca ttattaacaa 3300aatcatatta gttaatttgt taactctata ataaaagaaa tactgtaaca ttcacattac 3360atggtaacat ctttccaccc tttcatttgt tttttgtttg atgacttttt ttcttgttta 3420aatttatttc ccttctttta aatttggaat acattatcat catatataaa ctaaaatact 3480aaaaacagga ttacacaaat gataaataat aacacaaata tttataaatc tagctgcaat 3540atatttaaac tagctatatc gatattgtaa aataaaacta gctgcattga tactgataaa 3600aaaatatcat gtgctttctg gactgatgat gcagtatact tttgacattg cctttatttt 3660atttttcaga aaagctttct tagttctggg ttcttcatta tttgtttccc atctccattg 3720tgaattgaat catttgcttc gtgtcacaaa tacaatttag ntaggtacat gcattggtca 3780gattcacggt ttattatgtc atgacttaag ttcatggtag tacattacct gccacgcatg 3840cattatattg gttagatttg ataggcaaat ttggttgtca acaatataaa tataaataat 3900gtttttatat tacgaaataa cagtgatcaa aacaaacagt tttatcttta ttaacaagat 3960tttgtttttg tttgatgacg ttttttaatg tttacgcttt cccccttctt ttgaatttag 4020aacactttat catcataaaa tcaaatacta aaaaaattac atatttcata aataataaca 4080caaatatttt taaaaaatct gaaataataa tgaacaatat tacatattat cacgaaaatt 4140cattaataaa aatattatat aaataaaatg taatagtagt tatatgtagg aaaaaagtac 4200tgcacgcata atatatacaa aaagattaaa atgaactatt ataaataata acactaaatt 4260aatggtgaat catatcaaaa taatgaaaaa gtaaataaaa tttgtaatta acttctatat 4320gtattacaca cacaaataat aaataatagt aaaaaaaatt atgataaata tttaccatct 4380cataagatat ttaaaataat gataaaaata tagattattt tttatgcaac tagctagcca 4440aaaagagaac acgggtatat ataaaaagag tacctttaaa ttctactgta cttcctttat 4500tcctgacgtt tttatatcaa gtggacatac gtgaagattt taattatcag tctaaatatt 4560tcattagcac ttaatacttt tctgttttat tcctatccta taagtagtcc cgattctccc 4620aacattgctt attcacacaa ctaactaaga aagtcttcca tagcccccca agcggccgca 4680tgggaacgga ccaaggaaaa accttcacct gggaagagct ggcggcccat aacaccaagg 4740acgacctact cttggccatc cgcggcaggg tgtacgatgt cacaaagttc ttgagccgcc 4800atcctggtgg agtggacact ctcctgctcg gagctggccg agatgttact ccggtctttg 4860agatgtatca cgcgtttggg gctgcagatg ccattatgaa gaagtactat gtcggtacac 4920tggtctcgaa tgagctgccc atcttcccgg agccaacggt gttccacaaa accatcaaga 4980cgagagtcga gggctacttt acggatcgga acattgatcc caagaataga ccagagatct 5040ggggacgata cgctcttatc tttggatcct tgatcgcttc ctactacgcg cagctctttg 5100tgcctttcgt tgtcgaacgc acatggcttc aggtggtgtt tgcaatcatc atgggatttg 5160cgtgcgcaca agtcggactc aaccctcttc atgatgcgtc tcacttttca gtgacccaca 5220accccactgt ctggaagatt ctgggagcca cgcacgactt tttcaacgga gcatcgtacc 5280tggtgtggat

gtaccaacat atgctcggcc atcaccccta caccaacatt gctggagcag 5340atcccgacgt gtcgacgtct gagcccgatg ttcgtcgtat caagcccaac caaaagtggt 5400ttgtcaacca catcaaccag cacatgtttg ttcctttcct gtacggactg ctggcgttca 5460aggtgcgcat tcaggacatc aacattttgt actttgtcaa gaccaatgac gctattcgtg 5520tcaatcccat ctcgacatgg cacactgtga tgttctgggg cggcaaggct ttctttgtct 5580ggtatcgcct gattgttccc ctgcagtatc tgcccctggg caaggtgctg ctcttgttca 5640cggtcgcgga catggtgtcg tcttactggc tggcgctgac cttccaggcg aaccacgttg 5700ttgaggaagt tcagtggccg ttgcctgacg agaacgggat catccaaaag gactgggcag 5760ctatgcaggt cgagactacg caggattacg cacacgattc gcacctctgg accagcatca 5820ctggcagctt gaactaccag gctgtgcacc atctgttccc caacgtgtcg cagcaccatt 5880atcccgatat tctggccatc atcaagaaca cctgcagcga gtacaaggtt ccataccttg 5940tcaaggatac gttttggcaa gcatttgctt cacatttgga gcacttgcgt gttcttggac 6000tccgtcccaa ggaagagtag gcggccgcga cacaagtgtg agagtactaa ataaatgctt 6060tggttgtacg aaatcattac actaaataaa ataatcaaag cttatatatg ccttccgcta 6120aggccgaatg caaagaaatt ggttctttct cgttatcttt tgccactttt actagtacgt 6180attaattact acttaatcat ctttgtttac ggctcattat atccggtcta gaggatccaa 6240ggccgcgaag ttaaaagcaa tgttgtcact tgtcgtacta acacatgatg tgatagttta 6300tgctagctag ctataacata agctgtctct gagtgtgttg tatattaata aagatcatca 6360ctggtgaatg gtgatcgtgt acgtacccta cttagtaggc aatggaagca cttagagtgt 6420gctttgtgca tggccttgcc tctgttttga gacttttgta atgttttcga gtttaaatct 6480ttgcctttgc gtac 64941298584DNAArtificial SequencepKR1084 129ggagatccaa gcttggcgcg ccgttctata gtgtcaccta aatcgtatgt gtatgataca 60taaggttatg tattaattgt agccgcgttc taacgacaat atgtccatat ggtgcactct 120cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc 180tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt 240ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa 300gggcctcgtg atacgcctat ttttataggt taatgtcatg accaaaatcc cttaacgtga 360gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 420tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 480ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 540gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc 600tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 660cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 720gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 780actgagatac ctacagcgtg agcattgaga aagcgccacg cttcccgaag ggagaaaggc 840ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 900gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 960atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt 1020tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc 1080tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg 1140aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc 1200gcctctcccc gcgcgttggc cgattcatta atgcaggttg atcagatctc gatcccgcga 1260aattaatacg actcactata gggagaccac aacggtttcc ctctagaaat aattttgttt 1320aactttaaga aggagatata cccatggaaa agcctgaact caccgcgacg tctgtcgaga 1380agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag 1440aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct 1500gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc 1560cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc 1620gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc 1680agccggtcgc ggaggctatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt 1740tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg 1800cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt 1860ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc 1920acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag 1980cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct 2040tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc 2100atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc 2160aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat 2220gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa 2280gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc 2340ccagcactcg tccgagggca aaggaatagt gaggtacagc ttggatcgat ccggctgcta 2400acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac 2460cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg 2520gatgatcggg cgcgccgtcg acggatccgt acgagatccg gccgggtacg caaaggcaaa 2580gatttaaact cgaaaacatt acaaaagtct caaaacagag gcaaggccat gcacaaagca 2640cactctaagt gcttccattg cctactaagt agggtacgta cacgatcacc attcaccagt 2700gatgatcttt attaatatac aacacactca gagacagctt atgttatagc tagctagcat 2760aaactatcac atcatgtgtt agtacgacaa gtgacaacat tgcttttaac ttcgcggcct 2820tggatcctct agaccggata taatgagccg taaacaaaga tgattaagta gtaattaata 2880cgtactagta aaagtggcaa aagataacga gaaagaacca atttctttgc attcggcctt 2940agcggaaggc atatataagc tttgattatt ttatttagtg taatgatttc gtacaaccaa 3000agcatttatt tagtactctc acacttgtgt cgcggccgcc tactcttcct tgggacggag 3060tccaagaaca cgcaagtgct ccaaatgtga agcaaatgct tgccaaaacg tatccttgac 3120aaggtatgga accttgtact cgctgcaggt gttcttgatg atggccagaa tatcgggata 3180atggtgctgc gacacgttgg ggaacagatg gtgcacagcc tggtagttca agctgccagt 3240gatgctggtc cagaggtgcg aatcgtgtgc gtaatcctgc gtagtctcga cctgcatagc 3300tgcccagtcc ttttggatga tcccgttctc gtcaggcaac ggccactgaa cttcctcaac 3360aacgtggttc gcctggaagg tcagcgccag ccagtaagac gacaccatgt ccgcgaccgt 3420gaacaagagc agcaccttgc ccaggggcag atactgcagg ggaacaatca ggcgatacca 3480gacaaagaaa gccttgccgc cccagaacat cacagtgtgc catgtcgaga tgggattgac 3540acgaatagcg tcattggtct tgacaaagta caaaatgttg atgtcctgaa tgcgcacctt 3600gaacgccagc agtccgtaca ggaaaggaac aaacatgtgc tggttgatgt ggttgacaaa 3660ccacttttgg ttgggcttga tacgacgaac atcgggctca gacgtcgaca cgtcgggatc 3720tgctccagca atgttggtgt aggggtgatg gccgagcata tgttggtaca tccacaccag 3780gtacgatgct ccgttgaaaa agtcgtgcgt ggctcccaga atcttccaga cagtggggtt 3840gtgggtcact gaaaagtgag acgcatcatg aagagggttg agtccgactt gtgcgcacgc 3900aaatcccatg atgattgcaa acaccacctg aagccatgtg cgttcgacaa cgaaaggcac 3960aaagagctgc gcgtagtagg aagcgatcaa ggatccaaag ataagagcgt atcgtcccca 4020gatctctggt ctattcttgg gatcaatgtt ccgatccgta aagtagccct cgactctcgt 4080cttgatggtt ttgtggaaca ccgttggctc cgggaagatg ggcagctcat tcgagaccag 4140tgtaccgaca tagtacttct tcataatggc atctgcagcc ccaaacgcgt gatacatctc 4200aaagaccgga gtaacatctc ggccagctcc gagcaggaga gtgtccactc caccaggatg 4260gcggctcaag aactttgtga catcgtacac cctgccgcgg atggccaaga gtaggtcgtc 4320cttggtgtta tgggccgcca gctcttccca ggtgaaggtt tttccttggt ccgttcccat 4380gcggccgctt ggggggctat ggaagacttt cttagttagt tgtgtgaata agcaatgttg 4440ggagaatcgg gactacttat aggataggaa taaaacagaa aagtattaag tgctaatgaa 4500atatttagac tgataattaa aatcttcacg tatgtccact tgatataaaa acgtcaggaa 4560taaaggaagt acagtagaat ttaaaggtac tctttttata tatacccgtg ttctcttttt 4620ggctagctag ttgcataaaa aataatctat atttttatca ttattttaaa tatcttatga 4680gatggtaaat atttatcata atttttttta ctattattta ttatttgtgt gtgtaataca 4740tatagaagtt aattacaaat tttatttact ttttcattat tttgatatga ttcaccatta 4800atttagtgtt attatttata atagttcatt ttaatctttt tgtatatatt atgcgtgcag 4860tacttttttc ctacatataa ctactattac attttattta tataatattt ttattaatga 4920attttcgtga taatatgtaa tattgttcat tattatttca gattttttaa aaatatttgt 4980gttattattt atgaaatatg taattttttt agtatttgat tttatgatga taaagtgttc 5040taaattcaaa agaaggggga aagcgtaaac attaaaaaac gtcatcaaac aaaaacaaaa 5100tcttgttaat aaagataaaa ctgtttgttt tgatcactgt tatttcgtaa tataaaaaca 5160ttatttatat ttatattgtt gacaaccaaa tttgcctatc aaatctaacc aatataatgc 5220atgcgtggca ggtaatgtac taccatgaac ttaagtcatg acataataaa ccgtgaatct 5280gaccaatgca tgtacctanc taaattgtat ttgtgacacg aagcaaatga ttcaattcac 5340aatggagatg ggaaacaaat aatgaagaac ccagaactaa gaaagctttt ctgaaaaata 5400aaataaaggc aatgtcaaaa gtatactgca tcatcagtcc agaaagcaca tgatattttt 5460ttatcagtat caatgcagct agttttattt tacaatatcg atatagctag tttaaatata 5520ttgcagctag atttataaat atttgtgtta ttatttatca tttgtgtaat cctgttttta 5580gtattttagt ttatatatga tgataatgta ttccaaattt aaaagaaggg aaataaattt 5640aaacaagaaa aaaagtcatc aaacaaaaaa caaatgaaag ggtggaaaga tgttaccatg 5700taatgtgaat gttacagtat ttcttttatt atagagttaa caaattaact aatatgattt 5760tgttaataat gataaaatat tttttttatt attatttcat aatataaaaa tagtttactt 5820aatataaaaa aaattctatc gttcacaaca aagttggcca cctaatttaa ccatgcatgt 5880acccatggac catattaggt aaccatcaaa cctgatgaag agataaagag atgaagactt 5940aagtcataac acaaaaccat aaaaaacaaa aatacaatca accgtcaatc tgaccaatgc 6000atgaaaaagc tgcaatagtg agtggcgaca caaagcacat gattttctta caacggagat 6060aaaaccaaaa aaatatttca tgaacaacct agaacaaata aagcttttat ataataaata 6120tataaataaa taaaggctat ggaataatat acttcaatat atttggatta aataaattgt 6180tggcggggtt gatatattta tacacaccta aagtcacttc aatctcattt tcacttaact 6240tttatttttt ttttcttttt atttatcata aagagaatat tgataatata ctttttaaca 6300tatttttatg acatttttta ttggtgaaaa cttattaaaa atcataaatt ttgtaagtta 6360gatttattta aagagttcct cttcttattt taaatttttt aataaatttt taaataacta 6420aaatttgtgt taaaaatgtt aaaaaatgtg ttattaaccc ttctcttcga ggacgtaccc 6480ggccagatcc tgcaggccaa ctgcgtttgg ggctccagat taaacgacgc cgtttcgttc 6540ctttcgcttc acggcttaac gatgtcgttt ctgtctgtgc ccaaaaaata aaggcatttg 6600ttatttgcac cagatattta ctaagtgcac cctagtttga caagtaggcg ataattacaa 6660atagatgcgg tgcaaataat aaattttgaa ggaaataatt acaaaagaac agaacttata 6720tttactttat tttaaaaaac taaaatgaaa gaacaaaaaa agtaaaaaat acaaaaaatg 6780tgctttaacc actttcatta tttgttacag aaagtatgat tctactcaaa ttgatctgtt 6840gtatctggtg ctgccttgtc acactggcga tttcaatccc ctaaagatat ggtgcaaact 6900gcgaagtgat caatatctgc tcggttaatt tagattaatt aataatattc aacgtgatgt 6960accaaaaaaa gacaattttt tgctccattg acaaattaaa cctcatcaag gtaatttcca 7020aacctataag caaaaaaatt tcacattaat tggcccgcaa tcctattagt cttattatac 7080tagagtagga aaaaaaacaa ttacacaact tgtcttatta ttctctatgc taatgaatat 7140ttttcccttt tgttagaaat cagtgtttcc taatttattg agtattaatt ccactcaccg 7200catatattta ccgttgaata agaaaatttt acacataatt ctttttaaga taaataattt 7260ttttatacta gatcttatat gattacgtga agccaagtgg gttatactaa tgatatataa 7320tgtttgatag taatcagttt ataaaccaaa tgcatggaaa tgttacgtgg aagcacgtaa 7380attaacaagc attgaagcaa atgcagccac cgcaccaaaa ccaccccact tcacttccac 7440gtaccatatt ccatgcaact acaacaccct aaaacttcaa taaatgcccc caccttcact 7500tcacttcacc catcaatagc aagcggccgc accatggagg tggtgaatga aatagtctca 7560attgggcagg aagttttacc caaagttgat tatgcccaac tctggagtga tgccagtcac 7620tgtgaggtgc tttacttgtc catcgcattt gtcatcttga agttcactct tggccccctt 7680ggtccaaaag gtcagtctcg tatgaagttt gttttcacca attacaacct tctcatgtcc 7740atttattcgt tgggatcatt cctctcaatg gcatatgcca tgtacaccat cggtgttatg 7800tctgacaact gcgagaaggc ttttgacaac aacgtcttca ggatcaccac gcagttgttc 7860tatttgagca agttcctgga gtatattgac tccttctatt tgccactgat gggcaagcct 7920ctgacctggt tgcaattctt ccatcatttg ggggcaccga tggatatgtg gctgttctat 7980aattaccgaa atgaagctgt ttggattttt gtgctgttga atggtttcat ccactggatc 8040atgtacggtt attattggac cagattgatc aagctgaagt tccccatgcc aaaatccctg 8100attacatcaa tgcagatcat tcaattcaat gttggtttct acattgtctg gaagtacagg 8160aacattccct gttatcgcca agatgggatg aggatgtttg gctggttctt caattacttt 8220tatgttggca cagtcttgtg tttgttcttg aatttctatg tgcaaacgta tatcgtcagg 8280aagcacaagg gagccaaaaa gattcagtga gcggccgcga agttaaaagc aatgttgtca 8340cttgtcgtac taacacatga tgtgatagtt tatgctagct agctataaca taagctgtct 8400ctgagtgtgt tgtatattaa taaagatcat cactggtgaa tggtgatcgt gtacgtaccc 8460tacttagtag gcaatggaag cacttagagt gtgctttgtg catggccttg cctctgtttt 8520gagacttttg taatgttttc gagtttaaat ctttgccttt gcgtacgtct agagtcgacc 8580tgca 85841305095DNAArtificial SeqeuncepKR908 130catggttggg tctatttgcc aactggttca cgggtggatt gaactatcag atcgagcacc 60acttgttccc ttcgatgcct cgccacaact tttcaaagat ccagcctgct gtcgagaccc 120tgtgcaaaaa gtacaatgtc cgataccaca ccaccggtat gatcgaggga actgcagagg 180tctttagccg tctgaacgag gtctccaagg ctacctccaa gatgggtaag gcgcagtaag 240cggccgcatt tcgcaccaaa tcaatgaaag taataatgaa aagtctgaat aagaatactt 300aggcttagat gcctttgtta cttgtgtaaa ataacttgag tcatgtacct ttggcggaaa 360cagaataaat aaaaggtgaa attccaatgc tctatgtata agttagtaat acttaatgtg 420ttctacggtt gtttcaatat catcaaactc taattgaaac tttagaacca caaatctcaa 480tcttttctta atgaaatgaa aaatcttaat tgtaccatgt ttatgttaaa caccttacaa 540ttggttggag aggaggacca accgatggga caacattggg agaaagagat tcaatggaga 600tttggatagg agaacaacat tctttttcac ttcaatacaa gatgagtgca acactaagga 660tatgtatgag actttcagaa gctacgacaa catagatgag tgaggtggtg attcctagca 720agaaagacat tagaggaagc caaaatcgaa caaggaagac atcaagggca agagacagga 780ccatccatct caggaaaagg agctttggga tagtccgaga agttgtacaa gaaatttttt 840ggagggtgag tgatgcattg ctggtgactt taactcaatc aaaattgaga aagaaagaaa 900agggaggggg ctcacatgtg aatagaaggg aaacgggaga attttacagt tttgatctaa 960tgggcatccc agctagtggt aacatattca ccatgtttaa ccttcacgta cgtctagagg 1020atcccccggg ctgcaggaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc 1080ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata 1140gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc 1200gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca 1260ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac 1320ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga 1380ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac 1440gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt 1500agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct 1560aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat 1620attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg 1680cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg 1740aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc 1800ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat 1860gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact 1920attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca 1980tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact 2040tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg 2100atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg 2160agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg 2220aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg 2280caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag 2340ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc 2400gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga 2460tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat 2520atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc 2580tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 2640accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 2700gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 2760caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc 2820tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 2880ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 2940tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 3000gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 3060tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca 3120gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 3180gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 3240ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 3300ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta 3360ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag 3420tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga 3480ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg 3540caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg 3600ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc 3660atgattacgc caagcttgca tgcctgcagg ctagcctaag tacgtactca aaatgccaac 3720aaataaaaaa aaagttgctt taataatgcc aaaacaaatt aataaaacac ttacaacacc 3780ggattttttt taattaaaat gtgccattta ggataaatag ttaatatttt taataattat 3840ttaaaaagcc gtatctacta aaatgatttt tatttggttg aaaatattaa tatgtttaaa 3900tcaacacaat ctatcaaaat taaactaaaa aaaaaataag tgtacgtggt taacattagt 3960acagtaatat aagaggaaaa tgagaaatta agaaattgaa agcgagtcta atttttaaat 4020tatgaacctg catatataaa aggaaagaaa gaatccagga agaaaagaaa tgaaaccatg 4080catggtcccc tcgtcatcac gagtttctgc catttgcaat agaaacactg aaacaccttt 4140ctctttgtca cttaattgag atgccgaagc cacctcacac catgaacttc atgaggtgta 4200gcacccaagg cttccatagc catgcatact gaagaatgtc tcaagctcag caccctactt 4260ctgtgacgtg tccctcattc accttcctct cttccctata aataaccacg cctcaggttc 4320tccgcttcac aactcaaaca ttctctccat tggtccttaa acactcatca gtcatcaccg 4380cggccgcaaa ccatggccca cagcaagcac ggcctgaagg aggagatgac catgaagtac 4440cacatggagg gctgcgtgaa cggccacaag ttcgtgatca ccggcgaggg catcggctac 4500cccttcaagg gcaagcagac catcaacctg tgcgtgatcg agggcggccc cctgcccttc 4560agcgaggaca tcctgagcgc cggcttcaag tacggcgacc ggatcttcac cgagtacccc 4620caggacatcg tggactactt caagaacagc tgccccgccg gctacacctg gggccggagc 4680ttcctgttcg aggacggcgc cgtgtgcatc tgtaacgtgg acatcaccgt gagcgtgaag 4740gagaactgca tctaccacaa gagcatcttc aacggcgtga acttccccgc cgacggcccc 4800gtgatgaaga agatgaccac caactgggag gccagctgcg agaagatcat gcccgtgcct 4860aagcagggca tcctgaaggg cgacgtgagc atgtacctgc tgctgaagga cggcggccgg 4920taccggtgcc agttcgacac cgtgtacaag gccaagagcg tgcccagcaa gatgcccgag 4980tggcacttca tccagcacaa gctgctgcgg gaggaccgga gcgacgccaa gaaccagaag 5040tggcagctga ccgagcacgc catcgccttc cccagcgccc tggcctgaag

cggcc 50951314716DNAArtificial SeqeuncepKR1118 131ctagaggatc ccccgggctg caggaattca ctggccgtcg ttttacaacg tcgtgactgg 60gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg 120cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc 180gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata 240tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg 300ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa 360gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc 420gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg 480gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 540tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt 600caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc 660ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa 720gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt 780aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt 840ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc 900atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg 960gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg 1020gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac 1080atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca 1140aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta 1200actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat 1260aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa 1320tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag 1380ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat 1440agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt 1500tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg 1560aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 1620gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 1680atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 1740gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 1800gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 1860tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 1920accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 1980ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 2040cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 2100agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 2160ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 2220tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 2280ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 2340cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 2400gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt 2460tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag 2520cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg 2580cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc 2640tatgaccatg attacgccaa gcttgcatgc ctgcaggcta gcctaagtac gtactcaaaa 2700tgccaacaaa taaaaaaaaa gttgctttaa taatgccaaa acaaattaat aaaacactta 2760caacaccgga ttttttttaa ttaaaatgtg ccatttagga taaatagtta atatttttaa 2820taattattta aaaagccgta tctactaaaa tgatttttat ttggttgaaa atattaatat 2880gtttaaatca acacaatcta tcaaaattaa actaaaaaaa aaataagtgt acgtggttaa 2940cattagtaca gtaatataag aggaaaatga gaaattaaga aattgaaagc gagtctaatt 3000tttaaattat gaacctgcat atataaaagg aaagaaagaa tccaggaaga aaagaaatga 3060aaccatgcat ggtcccctcg tcatcacgag tttctgccat ttgcaataga aacactgaaa 3120cacctttctc tttgtcactt aattgagatg ccgaagccac ctcacaccat gaacttcatg 3180aggtgtagca cccaaggctt ccatagccat gcatactgaa gaatgtctca agctcagcac 3240cctacttctg tgacgtgtcc ctcattcacc ttcctctctt ccctataaat aaccacgcct 3300caggttctcc gcttcacaac tcaaacattc tctccattgg tccttaaaca ctcatcagtc 3360atcaccgcgg ccgcaaacca tggcacctaa acgggacgca ttgcctctga caattgatgg 3420caccacgtac gacgtttccg cttgggtaaa ccatcaccct ggaggggctc aaatcattga 3480aaactaccgg aaccgagatg ctaccgacgt gttcatggtc atgcattcac agcaggcgct 3540caacaagttg aagcggatgc ctgttatgga gccctcttca ccacttactc ccaagagccc 3600aagtgacgac atttccnagg atttccgcaa gctccgcaac agtatggttg agaagggtat 3660gttcaacgcg tcccctctgt tttatgtgta caaatcactg accactgtcg cccttggcgc 3720cgtgggtgtt ctcatggtta tgtacctgca gtggtactac gtttcagcca tgtttttggg 3780actttgctac caacagctgg gttgggtggc gcatgactac gcgcatcacc aggttttcac 3840gaaccgtgat tatggcaatc ttggtgggct tttctttggc nacgttctcc aaggatattc 3900tttgacttgg tggaaggaca ggcacaacgg ccatcacgcc gccacaaacg tgcaaggaca 3960tgaccccgac attgataatc tccccgtttt ggcttggtcg ccagaggacg tcaagaatgc 4020cggacctgga acccgcaata tcatcaagta ccagcagtat tatttcctcc ctaccatcgc 4080catccttcgg ttcatctggt gtttccaaag cattctgggg gtgatgtcat acaagacaga 4140ctccnagaat ctctattaca aacggcagta ccggagagag gcagccggtc tggcgctgca 4200ctggattctg aagagcgttt tcttgttctg ttacatgcca agtttcctca ctggcctggc 4260gtttttcctt atctcggagt gtctgggcgg ctttgggatc gcgattgtgg tgtttttgaa 4320ccactatccg ctggataagg ttgaggaatc cgtttgggat ggtcacggtt tctgtgctgg 4380gcagatcctc acaaccatga acatccaacg cggactcatc actgactggt tctttggagg 4440tttgaattac cagattgagc atcatctgtg gcccaacctt ccaagacacc atttgaaagc 4500agtttccttt gaggttgaga aattgtgcca gaagcacaac ctgccctaca gagctccgcc 4560gatgcatact ggtgttgcac aattgcttgg atatttgggg aagattgctc agttggctgc 4620tgtcccagta taaccctgga tcaccttcat cgatgcggcc gccaccgcgg cccgagattc 4680cggcctcttc ggccgccaag cgacccgggt ggacgt 47161326281DNAArtificial SequencepKR1120 132ggccgcgaca caagtgtgag agtactaaat aaatgctttg gttgtacgaa atcattacac 60taaataaaat aatcaaagct tatatatgcc ttccgctaag gccgaatgca aagaaattgg 120ttctttctcg ttatcttttg ccacttttac tagtacgtat taattactac ttaatcatct 180ttgtttacgg ctcattatat cctgcaggtc tagaggatcc ccgggtaccg agctcgaatt 240cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc 300gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc 360gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg tattttctcc 420ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg 480atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg 540cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt 600gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc 660tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc 720ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc 780cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga 840gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt 900ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag 960tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag 1020aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta 1080ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg 1140agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca 1200gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag 1260gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc 1320gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg 1380tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc 1440ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg 1500cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg 1560gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga 1620cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac 1680tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa 1740aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca 1800aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag 1860gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac 1920cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa 1980ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc 2040accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag 2100tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac 2160cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc 2220gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc 2280ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca 2340cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc 2400tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg 2460ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct 2520ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata 2580ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc 2640gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg 2700acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca 2760ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg 2820tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagcttgcat 2880gcctgcaggt cgactctaga cgtacgtcct cgaagagaag ggttaataac acatttttta 2940acatttttaa cacaaatttt agttatttaa aaatttatta aaaaatttaa aataagaaga 3000ggaactcttt aaataaatct aacttacaaa atttatgatt tttaataagt tttcaccaat 3060aaaaaatgtc ataaaaatat gttaaaaagt atattatcaa tattctcttt atgataaata 3120aaaagaaaaa aaaaataaaa gttaagtgaa aatgagattg aagtgacttt aggtgtgtat 3180aaatatatca accccgccaa caatttattt aatccaaata tattgaagta tattattcca 3240tagcctttat ttatttatat atttattata taaaagcttt atttgttcta ggttgttcat 3300gaaatatttt tttggtttta tctccgttgt aagaaaatca tgtgctttgt gtcgccactc 3360actattgcag ctttttcatg cattggtcag attgacggtt gattgtattt ttgtttttta 3420tggttttgtg ttatgactta agtcttcatc tctttatctc ttcatcaggt ttgatggtta 3480cctaatatgg tccatgggta catgcatggt taaattaggt ggccaacttt gttgtgaacg 3540atagaatttt ttttatatta agtaaactat ttttatatta tgaaataata ataaaaaaaa 3600tattttatca ttattaacaa aatcatatta gttaatttgt taactctata ataaaagaaa 3660tactgtaaca ttcacattac atggtaacat ctttccaccc tttcatttgt tttttgtttg 3720atgacttttt ttcttgttta aatttatttc ccttctttta aatttggaat acattatcat 3780catatataaa ctaaaatact aaaaacagga ttacacaaat gataaataat aacacaaata 3840tttataaatc tagctgcaat atatttaaac tagctatatc gatattgtaa aataaaacta 3900gctgcattga tactgataaa aaaatatcat gtgctttctg gactgatgat gcagtatact 3960tttgacattg cctttatttt atttttcaga aaagctttct tagttctggg ttcttcatta 4020tttgtttccc atctccattg tgaattgaat catttgcttc gtgtcacaaa tacaatttag 4080ntaggtacat gcattggtca gattcacggt ttattatgtc atgacttaag ttcatggtag 4140tacattacct gccacgcatg cattatattg gttagatttg ataggcaaat ttggttgtca 4200acaatataaa tataaataat gtttttatat tacgaaataa cagtgatcaa aacaaacagt 4260tttatcttta ttaacaagat tttgtttttg tttgatgacg ttttttaatg tttacgcttt 4320cccccttctt ttgaatttag aacactttat catcataaaa tcaaatacta aaaaaattac 4380atatttcata aataataaca caaatatttt taaaaaatct gaaataataa tgaacaatat 4440tacatattat cacgaaaatt cattaataaa aatattatat aaataaaatg taatagtagt 4500tatatgtagg aaaaaagtac tgcacgcata atatatacaa aaagattaaa atgaactatt 4560ataaataata acactaaatt aatggtgaat catatcaaaa taatgaaaaa gtaaataaaa 4620tttgtaatta acttctatat gtattacaca cacaaataat aaataatagt aaaaaaaatt 4680atgataaata tttaccatct cataagatat ttaaaataat gataaaaata tagattattt 4740tttatgcaac tagctagcca aaaagagaac acgggtatat ataaaaagag tacctttaaa 4800ttctactgta cttcctttat tcctgacgtt tttatatcaa gtggacatac gtgaagattt 4860taattatcag tctaaatatt tcattagcac ttaatacttt tctgttttat tcctatccta 4920taagtagtcc cgattctccc aacattgctt attcacacaa ctaactaaga aagtcttcca 4980tagcccccca agcggccgca aaccatggca cctaaacggg acgcattgcc tctgacaatt 5040gatggcacca cgtacgacgt ttccgcttgg gtaaaccatc accctggagg ggctcaaatc 5100attgaaaact accggaaccg agatgctacc gacgtgttca tggtcatgca ttcacagcag 5160gcgctcaaca agttgaagcg gatgcctgtt atggagccct cttcaccact tactcccaag 5220agcccaagtg acgacatttc cnaggatttc cgcaagctcc gcaacagtat ggttgagaag 5280ggtatgttca acgcgtcccc tctgttttat gtgtacaaat cactgaccac tgtcgccctt 5340ggcgccgtgg gtgttctcat ggttatgtac ctgcagtggt actacgtttc agccatgttt 5400ttgggacttt gctaccaaca gctgggttgg gtggcgcatg actacgcgca tcaccaggtt 5460ttcacgaacc gtgattatgg caatcttggt gggcttttct ttggcnacgt tctccaagga 5520tattctttga cttggtggaa ggacaggcac aacggccatc acgccgccac aaacgtgcaa 5580ggacatgacc ccgacattga taatctcccc gttttggctt ggtcgccaga ggacgtcaag 5640aatgccggac ctggaacccg caatatcatc aagtaccagc agtattattt cctccctacc 5700atcgccatcc ttcggttcat ctggtgtttc caaagcattc tgggggtgat gtcatacaag 5760acagactccn agaatctcta ttacaaacgg cagtaccgga gagaggcagc cggtctggcg 5820ctgcactgga ttctgaagag cgttttcttg ttctgttaca tgccaagttt cctcactggc 5880ctggcgtttt tccttatctc ggagtgtctg ggcggctttg ggatcgcgat tgtggtgttt 5940ttgaaccact atccgctgga taaggttgag gaatccgttt gggatggtca cggtttctgt 6000gctgggcaga tcctcacaac catgaacatc caacgcggac tcatcactga ctggttcttt 6060ggaggtttga attaccagat tgagcatcat ctgtggccca accttccaag acaccatttg 6120aaagcagttt cctttgaggt tgagaaattg tgccagaagc acaacctgcc ctacagagct 6180ccgccgatgc atactggtgt tgcacaattg cttggatatt tggggaagat tgctcagttg 6240gctgctgtcc cagtataacc ctggatcacc ttcatcgatg c 628113311473DNAArtificial SequencepKR1123 133ggagatccaa 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 atcgattcga catcgatcta gtaacataga 3840tgacaccgcg cgcgataatt tatcctagtt tgcgcgctat attttgtttt

ctatcgcgta 3900ttaaatgtat aattgcggga ctctaatcat aaaaacccat ctcataaata acgtcatgca 3960ttacatgtta attattacat gcttaacgta attcaacaga aattatatga taatcatcgc 4020aagaccggca acaggattca atcttaagaa actttattgc caaatgtttg aacgatctgc 4080ttcgacgcac tccttcttta ggtacctcac tattcctttg ccctcggacg agtgctgggg 4140cgtcggtttc cactatcggc gagtacttct acacagccat cggtccagac ggccgcgctt 4200ctgcgggcga tttgtgtacg cccgacagtc ccggctccgg atcggacgat tgcgtcgcat 4260cgaccctgcg cccaagctgc atcatcgaaa ttgccgtcaa ccaagctctg atagagttgg 4320tcaagaccaa tgcggagcat atacgcccgg agccgcggcg atcctgcaag ctccggatgc 4380ctccgctcga agtagcgcgt ctgctgctcc atacaagcca accacggcct ccagaagaag 4440atgttggcga cctcgtattg ggaatccccg aacatcgcct cgctccagtc aatgaccgct 4500gttatgcggc cattgtccgt caggacattg ttggagccga aatccgcgtg cacgaggtgc 4560cggacttcgg ggcagtcctc ggcccaaagc atcagctcat cgagagcctg cgcgacggac 4620gcactgacgg tgtcgtccat cacagtttgc cagtgataca catggggatc agcaatcgcg 4680catatgaaat cacgccatgt agtgtattga ccgattcctt gcggtccgaa tgggccgaac 4740ccgctcgtct ggctaagatc ggccgcagcg atcgcatcca tggcctccgc gaccggctgc 4800agaacagcgg gcagttcggt ttcaggcagg tcttgcaacg tgacaccctg tgcacggcgg 4860gagatgcaat aggtcaggct ctcgctgaat tccccaatgt caagcacttc cggaatcggg 4920agcgcggccg atgcaaagtg ccgataaaca taacgatctt tgtagaaacc atcggcgcag 4980ctatttaccc gcaggacata tccacgccct cctacatcga agctgaaagc acgagattct 5040tcgccctccg agagctgcat caggtcggag acgctgtcga acttttcgat cagaaacttc 5100tcgacagacg tcgcggtgag ttcaggcttt ttcatggttt aataagaaga gaaaagagtt 5160cttttgttat ggctgaagta atagagaaat gagctcgagc gtgtcctctc caaatgaaat 5220gaacttcctt atatagagga agggtcttgc gaaggatagt gggattgtgc gtcatccctt 5280acgtcagtgg agatgtcaca tcaatccact tgctttgaag acgtggttgg aacgtcttct 5340ttttccacga tgctcctcgt gggtgggggt ccatctttgg gaccactgtc ggcagaggca 5400tcttgaatga tagcctttcc tttatcgcaa tgatggcatt tgtaggagcc accttccttt 5460tctactgtcc tttcgatgaa gtgacagata gctgggcaat ggaatccgag gaggtttccc 5520gaaattatcc tttgttgaaa agtctcaata gccctttggt cttctgagac tgtatctttg 5580acatttttgg agtagaccag agtgtcgtgc tccaccatgt tgacgaagat tttcttcttg 5640tcattgagtc gtaaaagact ctgtatgaac tgttcgccag tcttcacggc gagttctgtt 5700agatcctcga tttgaatctt agactccatg catggcctta gattcagtag gaactacctt 5760tttagagact ccaatctcta ttacttgcct tggtttatga agcaagcctt gaatcgtcca 5820tactggaata gtacttctga tcttgagaaa tatgtctttc tctgtgttct tgatgcaatt 5880agtcctgaat cttttgactg catctttaac cttcttggga aggtatttga tctcctggag 5940attgttactc gggtagatcg tcttgatgag acctgctgcg taggcctctc taaccatctg 6000tgggtcagca ttctttctga aattgaagag gctaaccttc tcattatcag tggtgaacat 6060agtgtcgtca ccttcacctt cgaacttcct tcctagatcg taaagataga ggaaatcgtc 6120cattgtaatc tccggggcaa aggagatctc ttttggggct ggatcactgc tgggcctttt 6180ggttcctagc gtgagccagt gggctttttg ctttggtggg cttgttaggg ccttagcaaa 6240gctcttgggc ttgagttgag cttctccttt ggggatgaag ttcaacctgt ctgtttgctg 6300acttgttgtg tacgcgtcag ctgctgctct tgcctctgta atagtggcaa atttcttgtg 6360tgcaactccg ggaacgccgt ttgttgccgc ctttgtacaa ccccagtcat cgtatatacc 6420ggcatgtgga ccgttataca caacgtagta gttgatatga gggtgttgaa tacccgattc 6480tgctctgaga ggagcaactg tgctgttaag ctcagatttt tgtgggattg gaattggatc 6540gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct 6600agaaataatt ttgtttaact ttaagaagga gatataccca tggaaaagcc tgaactcacc 6660gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag 6720ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc 6780ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt 6840gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg 6900acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa 6960ctgcccgctg ttctgcagcc ggtcgcggag gctatggatg cgatcgctgc ggccgatctt 7020agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg 7080cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac 7140gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac 7200tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac 7260aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac 7320gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc 7380tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc 7440cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct 7500tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca 7560caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat 7620agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagtgagg tacagcttgg 7680atcgatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag 7740caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa 7800ggaggaacta tatccggatg atcgggcgcg ccgtcgacgg atccgtacga gatccggccg 7860gccagatcct gcaggatata atgagccgta aacaaagatg attaagtagt aattaatacg 7920tactagtaaa agtggcaaaa gataacgaga aagaaccaat ttctttgcat tcggccttag 7980cggaaggcat atataagctt tgattatttt atttagtgta atgatttcgt acaaccaaag 8040catttattta gtactctcac acttgtgtcg cggccgcatc gatgaaggtg atccagggtt 8100atactgggac agcagccaac tgagcaatct tccccaaata tccaagcaat tgtgcaacac 8160cagtatgcat cggcggagct ctgtagggca ggttgtgctt ctggcacaat ttctcaacct 8220caaaggaaac tgctttcaaa tggtgtcttg gaaggttggg ccacagatga tgctcaatct 8280ggtaattcaa acctccaaag aaccagtcag tgatgagtcc gcgttggatg ttcatggttg 8340tgaggatctg cccagcacag aaaccgtgac catcccaaac ggattcctca accttatcca 8400gcggatagtg gttcaaaaac accacaatcg cgatcccaaa gccgcccaga cactccgaga 8460taaggaaaaa cgccaggcca gtgaggaaac ttggcatgta acagaacaag aaaacgctct 8520tcagaatcca gtgcagcgcc agaccggctg cctctctccg gtactgccgt ttgtaataga 8580gattctngga gtctgtcttg tatgacatca cccccagaat gctttggaaa caccagatga 8640accgaaggat ggcgatggta gggaggaaat aatactgctg gtacttgatg atattgcggg 8700ttccaggtcc ggcattcttg acgtcctctg gcgaccaagc caaaacgggg agattatcaa 8760tgtcggggtc atgtccttgc acgtttgtgg cggcgtgatg gccgttgtgc ctgtccttcc 8820accaagtcaa agaatatcct tggagaacgt ngccaaagaa aagcccacca agattgccat 8880aatcacggtt cgtgaaaacc tggtgatgcg cgtagtcatg cgccacccaa cccagctgtt 8940ggtagcaaag tcccaaaaac atggctgaaa cgtagtacca ctgcaggtac ataaccatga 9000gaacacccac ggcgccaagg gcgacagtgg tcagtgattt gtacacataa aacagagggg 9060acgcgttgaa catacccttc tcaaccatac tgttgcggag cttgcggaaa tcctnggaaa 9120tgtcgtcact tgggctcttg ggagtaagtg gtgaagaggg ctccataaca ggcatccgct 9180tcaacttgtt gagcgcctgc tgtgaatgca tgaccatgaa cacgtcggta gcatctcggt 9240tccggtagtt ttcaatgatt tgagcccctc cagggtgatg gtttacccaa gcggaaacgt 9300cgtacgtggt gccatcaatt gtcagaggca atgcgtcccg tttaggtgcc atggtttgcg 9360gccgcttggg gggctatgga agactttctt agttagttgt gtgaataagc aatgttggga 9420gaatcgggac tacttatagg ataggaataa aacagaaaag tattaagtgc taatgaaata 9480tttagactga taattaaaat cttcacgtat gtccacttga tataaaaacg tcaggaataa 9540aggaagtaca gtagaattta aaggtactct ttttatatat acccgtgttc tctttttggc 9600tagctagttg cataaaaaat aatctatatt tttatcatta ttttaaatat cttatgagat 9660ggtaaatatt tatcataatt ttttttacta ttatttatta tttgtgtgtg taatacatat 9720agaagttaat tacaaatttt atttactttt tcattatttt gatatgattc accattaatt 9780tagtgttatt atttataata gttcatttta atctttttgt atatattatg cgtgcagtac 9840ttttttccta catataacta ctattacatt ttatttatat aatattttta ttaatgaatt 9900ttcgtgataa tatgtaatat tgttcattat tatttcagat tttttaaaaa tatttgtgtt 9960attatttatg aaatatgtaa tttttttagt atttgatttt atgatgataa agtgttctaa 10020attcaaaaga agggggaaag cgtaaacatt aaaaaacgtc atcaaacaaa aacaaaatct 10080tgttaataaa gataaaactg tttgttttga tcactgttat ttcgtaatat aaaaacatta 10140tttatattta tattgttgac aaccaaattt gcctatcaaa tctaaccaat ataatgcatg 10200cgtggcaggt aatgtactac catgaactta agtcatgaca taataaaccg tgaatctgac 10260caatgcatgt acctanctaa attgtatttg tgacacgaag caaatgattc aattcacaat 10320ggagatggga aacaaataat gaagaaccca gaactaagaa agcttttctg aaaaataaaa 10380taaaggcaat gtcaaaagta tactgcatca tcagtccaga aagcacatga tattttttta 10440tcagtatcaa tgcagctagt tttattttac aatatcgata tagctagttt aaatatattg 10500cagctagatt tataaatatt tgtgttatta tttatcattt gtgtaatcct gtttttagta 10560ttttagttta tatatgatga taatgtattc caaatttaaa agaagggaaa taaatttaaa 10620caagaaaaaa agtcatcaaa caaaaaacaa atgaaagggt ggaaagatgt taccatgtaa 10680tgtgaatgtt acagtatttc ttttattata gagttaacaa attaactaat atgattttgt 10740taataatgat aaaatatttt ttttattatt atttcataat ataaaaatag tttacttaat 10800ataaaaaaaa ttctatcgtt cacaacaaag ttggccacct aatttaacca tgcatgtacc 10860catggaccat attaggtaac catcaaacct gatgaagaga taaagagatg aagacttaag 10920tcataacaca aaaccataaa aaacaaaaat acaatcaacc gtcaatctga ccaatgcatg 10980aaaaagctgc aatagtgagt ggcgacacaa agcacatgat tttcttacaa cggagataaa 11040accaaaaaaa tatttcatga acaacctaga acaaataaag cttttatata ataaatatat 11100aaataaataa aggctatgga ataatatact tcaatatatt tggattaaat aaattgttgg 11160cggggttgat atatttatac acacctaaag tcacttcaat ctcattttca cttaactttt 11220attttttttt tctttttatt tatcataaag agaatattga taatatactt tttaacatat 11280ttttatgaca ttttttattg gtgaaaactt attaaaaatc ataaattttg taagttagat 11340ttatttaaag agttcctctt cttattttaa attttttaat aaatttttaa ataactaaaa 11400tttgtgttaa aaatgttaaa aaatgtgtta ttaacccttc tcttcgagga cgtacgtcta 11460gagtcgacct gca 114731344710DNAArtificial SequencepKR1117 134ctagaggatc ccccgggctg caggaattca ctggccgtcg ttttacaacg tcgtgactgg 60gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg 120cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc 180gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata 240tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg 300ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa 360gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc 420gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg 480gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 540tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt 600caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc 660ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa 720gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt 780aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt 840ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc 900atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg 960gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg 1020gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac 1080atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca 1140aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta 1200actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat 1260aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa 1320tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag 1380ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat 1440agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt 1500tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg 1560aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 1620gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 1680atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 1740gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 1800gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 1860tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 1920accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 1980ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 2040cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 2100agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 2160ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 2220tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 2280ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 2340cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 2400gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt 2460tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag 2520cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg 2580cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc 2640tatgaccatg attacgccaa gcttgcatgc ctgcaggcta gcctaagtac gtactcaaaa 2700tgccaacaaa taaaaaaaaa gttgctttaa taatgccaaa acaaattaat aaaacactta 2760caacaccgga ttttttttaa ttaaaatgtg ccatttagga taaatagtta atatttttaa 2820taattattta aaaagccgta tctactaaaa tgatttttat ttggttgaaa atattaatat 2880gtttaaatca acacaatcta tcaaaattaa actaaaaaaa aaataagtgt acgtggttaa 2940cattagtaca gtaatataag aggaaaatga gaaattaaga aattgaaagc gagtctaatt 3000tttaaattat gaacctgcat atataaaagg aaagaaagaa tccaggaaga aaagaaatga 3060aaccatgcat ggtcccctcg tcatcacgag tttctgccat ttgcaataga aacactgaaa 3120cacctttctc tttgtcactt aattgagatg ccgaagccac ctcacaccat gaacttcatg 3180aggtgtagca cccaaggctt ccatagccat gcatactgaa gaatgtctca agctcagcac 3240cctacttctg tgacgtgtcc ctcattcacc ttcctctctt ccctataaat aaccacgcct 3300caggttctcc gcttcacaac tcaaacattc tctccattgg tccttaaaca ctcatcagtc 3360atcaccgcgg ccgcaaacca tggcacccaa gcgagaggcc ttgcccatca cgattgatgg 3420cacaacctat gatgtgtccg catgggtgaa ccatcacccc gggggcgcag acatcatgga 3480gaattatcgg aaccgagatg ccacggatgt gttcatggtc atgcactccc acgatgcgtt 3540gaacaagctg aagcgcatgc ctgtgatgga gcccacttcg ccacgaagcc ccaagactcc 3600caacgacgag gttgctgagg acttccgcaa gcttcgaaag gacatgattg caaaaggcat 3660gttcaacgca tcccctctct tctacgtgta caaaagtgcg accacagtag ccctgggcgc 3720cctggctatt ctgatggtga tgcacctgca gtggtactac atcccagcca ttttgttggg 3780actttgctac cagcagctgg ggtggttggc acacgattac tgccaccatc aggtgttctc 3840taaccgggcg tacaacaatt ttgctggact tgtattcggc aatgtgatgc aaggatactc 3900cgggacttgg tggaaggaca ggcacaacgg ccatcacgcc gccacgaacg tgcaagggca 3960cgatcccgac atcgacgacc tcccggtgtt ggcctggtcc ccggaggacg tcaaaaacgc 4020cggtcccacg acgcggaagc tcatcaagtg gcaacaatac tatttcctcc ccaccatcgc 4080aaccctccga ttcatctggt gcttccagag cattctggcg gttatggcat acaagacaga 4140tgcaaggaat atatattacc aacgccagta cgcaaaggag gccgtggggc tggctctgca 4200ttggattctg aaaggggtat tcatgttctg ttacatgccc ggcatactga cgggcttggc 4260cttcttcctc atctcggagt gcctgggcgg gtttgggatt gccattgtcg tgttcttgaa 4320tcactaccca ttggagaagg tggaggagtc cgtgtgggac agccacgggt tttgcgcggg 4380gcagatccac acgacgatga acatccaacg cggggtcatc gttgactggt tctttggagg 4440cctgaactac caaatcgaac accatctgtg gccgacgctg ccccggcatc acttgaaagc 4500tgcttctttt gaggtggaga aaatttgcca gaagcacaaa ttgccataca gagcaccccc 4560catgtccgat ggtgttgctc aattgcttgg cttcttgggg aagattgcta agctggcagc 4620tgtcccagtg taaccctaaa cgtaccacgc ggccgccacc gcggcccgag attccggcct 4680cttcggccgc caagcgaccc gggtggacgt 47101356275DNAArtificial SequencepKR1119 135ggccgcgaca caagtgtgag agtactaaat aaatgctttg gttgtacgaa atcattacac 60taaataaaat aatcaaagct tatatatgcc ttccgctaag gccgaatgca aagaaattgg 120ttctttctcg ttatcttttg ccacttttac tagtacgtat taattactac ttaatcatct 180ttgtttacgg ctcattatat cctgcaggtc tagaggatcc ccgggtaccg agctcgaatt 240cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc 300gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc 360gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg tattttctcc 420ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg 480atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg 540cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt 600gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc 660tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc 720ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc 780cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga 840gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt 900ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag 960tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag 1020aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta 1080ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg 1140agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca 1200gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag 1260gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc 1320gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg 1380tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc 1440ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg 1500cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg 1560gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga 1620cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac 1680tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa 1740aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca 1800aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag 1860gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac 1920cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa 1980ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc 2040accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag 2100tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac 2160cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc 2220gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc 2280ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca 2340cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc 2400tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg 2460ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct 2520ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata 2580ccgctcgccg cagccgaacg

accgagcgca gcgagtcagt gagcgaggaa gcggaagagc 2640gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg 2700acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca 2760ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg 2820tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagcttgcat 2880gcctgcaggt cgactctaga cgtacgtcct cgaagagaag ggttaataac acatttttta 2940acatttttaa cacaaatttt agttatttaa aaatttatta aaaaatttaa aataagaaga 3000ggaactcttt aaataaatct aacttacaaa atttatgatt tttaataagt tttcaccaat 3060aaaaaatgtc ataaaaatat gttaaaaagt atattatcaa tattctcttt atgataaata 3120aaaagaaaaa aaaaataaaa gttaagtgaa aatgagattg aagtgacttt aggtgtgtat 3180aaatatatca accccgccaa caatttattt aatccaaata tattgaagta tattattcca 3240tagcctttat ttatttatat atttattata taaaagcttt atttgttcta ggttgttcat 3300gaaatatttt tttggtttta tctccgttgt aagaaaatca tgtgctttgt gtcgccactc 3360actattgcag ctttttcatg cattggtcag attgacggtt gattgtattt ttgtttttta 3420tggttttgtg ttatgactta agtcttcatc tctttatctc ttcatcaggt ttgatggtta 3480cctaatatgg tccatgggta catgcatggt taaattaggt ggccaacttt gttgtgaacg 3540atagaatttt ttttatatta agtaaactat ttttatatta tgaaataata ataaaaaaaa 3600tattttatca ttattaacaa aatcatatta gttaatttgt taactctata ataaaagaaa 3660tactgtaaca ttcacattac atggtaacat ctttccaccc tttcatttgt tttttgtttg 3720atgacttttt ttcttgttta aatttatttc ccttctttta aatttggaat acattatcat 3780catatataaa ctaaaatact aaaaacagga ttacacaaat gataaataat aacacaaata 3840tttataaatc tagctgcaat atatttaaac tagctatatc gatattgtaa aataaaacta 3900gctgcattga tactgataaa aaaatatcat gtgctttctg gactgatgat gcagtatact 3960tttgacattg cctttatttt atttttcaga aaagctttct tagttctggg ttcttcatta 4020tttgtttccc atctccattg tgaattgaat catttgcttc gtgtcacaaa tacaatttag 4080ntaggtacat gcattggtca gattcacggt ttattatgtc atgacttaag ttcatggtag 4140tacattacct gccacgcatg cattatattg gttagatttg ataggcaaat ttggttgtca 4200acaatataaa tataaataat gtttttatat tacgaaataa cagtgatcaa aacaaacagt 4260tttatcttta ttaacaagat tttgtttttg tttgatgacg ttttttaatg tttacgcttt 4320cccccttctt ttgaatttag aacactttat catcataaaa tcaaatacta aaaaaattac 4380atatttcata aataataaca caaatatttt taaaaaatct gaaataataa tgaacaatat 4440tacatattat cacgaaaatt cattaataaa aatattatat aaataaaatg taatagtagt 4500tatatgtagg aaaaaagtac tgcacgcata atatatacaa aaagattaaa atgaactatt 4560ataaataata acactaaatt aatggtgaat catatcaaaa taatgaaaaa gtaaataaaa 4620tttgtaatta acttctatat gtattacaca cacaaataat aaataatagt aaaaaaaatt 4680atgataaata tttaccatct cataagatat ttaaaataat gataaaaata tagattattt 4740tttatgcaac tagctagcca aaaagagaac acgggtatat ataaaaagag tacctttaaa 4800ttctactgta cttcctttat tcctgacgtt tttatatcaa gtggacatac gtgaagattt 4860taattatcag tctaaatatt tcattagcac ttaatacttt tctgttttat tcctatccta 4920taagtagtcc cgattctccc aacattgctt attcacacaa ctaactaaga aagtcttcca 4980tagcccccca agcggccgca aaccatggca cccaagcgag aggccttgcc catcacgatt 5040gatggcacaa cctatgatgt gtccgcatgg gtgaaccatc accccggggg cgcagacatc 5100atggagaatt atcggaaccg agatgccacg gatgtgttca tggtcatgca ctcccacgat 5160gcgttgaaca agctgaagcg catgcctgtg atggagccca cttcgccacg aagccccaag 5220actcccaacg acgaggttgc tgaggacttc cgcaagcttc gaaaggacat gattgcaaaa 5280ggcatgttca acgcatcccc tctcttctac gtgtacaaaa gtgcgaccac agtagccctg 5340ggcgccctgg ctattctgat ggtgatgcac ctgcagtggt actacatccc agccattttg 5400ttgggacttt gctaccagca gctggggtgg ttggcacacg attactgcca ccatcaggtg 5460ttctctaacc gggcgtacaa caattttgct ggacttgtat tcggcaatgt gatgcaagga 5520tactccggga cttggtggaa ggacaggcac aacggccatc acgccgccac gaacgtgcaa 5580gggcacgatc ccgacatcga cgacctcccg gtgttggcct ggtccccgga ggacgtcaaa 5640aacgccggtc ccacgacgcg gaagctcatc aagtggcaac aatactattt cctccccacc 5700atcgcaaccc tccgattcat ctggtgcttc cagagcattc tggcggttat ggcatacaag 5760acagatgcaa ggaatatata ttaccaacgc cagtacgcaa aggaggccgt ggggctggct 5820ctgcattgga ttctgaaagg ggtattcatg ttctgttaca tgcccggcat actgacgggc 5880ttggccttct tcctcatctc ggagtgcctg ggcgggtttg ggattgccat tgtcgtgttc 5940ttgaatcact acccattgga gaaggtggag gagtccgtgt gggacagcca cgggttttgc 6000gcggggcaga tccacacgac gatgaacatc caacgcgggg tcatcgttga ctggttcttt 6060ggaggcctga actaccaaat cgaacaccat ctgtggccga cgctgccccg gcatcacttg 6120aaagctgctt cttttgaggt ggagaaaatt tgccagaagc acaaattgcc atacagagca 6180ccccccatgt ccgatggtgt tgctcaattg cttggcttct tggggaagat tgctaagctg 6240gcagctgtcc cagtgtaacc ctaaacgtac cacgc 627513611467DNAArtificial SequencepKR1122 136ggagatccaa 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 atcgattcga catcgatcta gtaacataga 3840tgacaccgcg cgcgataatt tatcctagtt tgcgcgctat attttgtttt ctatcgcgta 3900ttaaatgtat aattgcggga ctctaatcat aaaaacccat ctcataaata acgtcatgca 3960ttacatgtta attattacat gcttaacgta attcaacaga aattatatga taatcatcgc 4020aagaccggca acaggattca atcttaagaa actttattgc caaatgtttg aacgatctgc 4080ttcgacgcac tccttcttta ggtacctcac tattcctttg ccctcggacg agtgctgggg 4140cgtcggtttc cactatcggc gagtacttct acacagccat cggtccagac ggccgcgctt 4200ctgcgggcga tttgtgtacg cccgacagtc ccggctccgg atcggacgat tgcgtcgcat 4260cgaccctgcg cccaagctgc atcatcgaaa ttgccgtcaa ccaagctctg atagagttgg 4320tcaagaccaa tgcggagcat atacgcccgg agccgcggcg atcctgcaag ctccggatgc 4380ctccgctcga agtagcgcgt ctgctgctcc atacaagcca accacggcct ccagaagaag 4440atgttggcga cctcgtattg ggaatccccg aacatcgcct cgctccagtc aatgaccgct 4500gttatgcggc cattgtccgt caggacattg ttggagccga aatccgcgtg cacgaggtgc 4560cggacttcgg ggcagtcctc ggcccaaagc atcagctcat cgagagcctg cgcgacggac 4620gcactgacgg tgtcgtccat cacagtttgc cagtgataca catggggatc agcaatcgcg 4680catatgaaat cacgccatgt agtgtattga ccgattcctt gcggtccgaa tgggccgaac 4740ccgctcgtct ggctaagatc ggccgcagcg atcgcatcca tggcctccgc gaccggctgc 4800agaacagcgg gcagttcggt ttcaggcagg tcttgcaacg tgacaccctg tgcacggcgg 4860gagatgcaat aggtcaggct ctcgctgaat tccccaatgt caagcacttc cggaatcggg 4920agcgcggccg atgcaaagtg ccgataaaca taacgatctt tgtagaaacc atcggcgcag 4980ctatttaccc gcaggacata tccacgccct cctacatcga agctgaaagc acgagattct 5040tcgccctccg agagctgcat caggtcggag acgctgtcga acttttcgat cagaaacttc 5100tcgacagacg tcgcggtgag ttcaggcttt ttcatggttt aataagaaga gaaaagagtt 5160cttttgttat ggctgaagta atagagaaat gagctcgagc gtgtcctctc caaatgaaat 5220gaacttcctt atatagagga agggtcttgc gaaggatagt gggattgtgc gtcatccctt 5280acgtcagtgg agatgtcaca tcaatccact tgctttgaag acgtggttgg aacgtcttct 5340ttttccacga tgctcctcgt gggtgggggt ccatctttgg gaccactgtc ggcagaggca 5400tcttgaatga tagcctttcc tttatcgcaa tgatggcatt tgtaggagcc accttccttt 5460tctactgtcc tttcgatgaa gtgacagata gctgggcaat ggaatccgag gaggtttccc 5520gaaattatcc tttgttgaaa agtctcaata gccctttggt cttctgagac tgtatctttg 5580acatttttgg agtagaccag agtgtcgtgc tccaccatgt tgacgaagat tttcttcttg 5640tcattgagtc gtaaaagact ctgtatgaac tgttcgccag tcttcacggc gagttctgtt 5700agatcctcga tttgaatctt agactccatg catggcctta gattcagtag gaactacctt 5760tttagagact ccaatctcta ttacttgcct tggtttatga agcaagcctt gaatcgtcca 5820tactggaata gtacttctga tcttgagaaa tatgtctttc tctgtgttct tgatgcaatt 5880agtcctgaat cttttgactg catctttaac cttcttggga aggtatttga tctcctggag 5940attgttactc gggtagatcg tcttgatgag acctgctgcg taggcctctc taaccatctg 6000tgggtcagca ttctttctga aattgaagag gctaaccttc tcattatcag tggtgaacat 6060agtgtcgtca ccttcacctt cgaacttcct tcctagatcg taaagataga ggaaatcgtc 6120cattgtaatc tccggggcaa aggagatctc ttttggggct ggatcactgc tgggcctttt 6180ggttcctagc gtgagccagt gggctttttg ctttggtggg cttgttaggg ccttagcaaa 6240gctcttgggc ttgagttgag cttctccttt ggggatgaag ttcaacctgt ctgtttgctg 6300acttgttgtg tacgcgtcag ctgctgctct tgcctctgta atagtggcaa atttcttgtg 6360tgcaactccg ggaacgccgt ttgttgccgc ctttgtacaa ccccagtcat cgtatatacc 6420ggcatgtgga ccgttataca caacgtagta gttgatatga gggtgttgaa tacccgattc 6480tgctctgaga ggagcaactg tgctgttaag ctcagatttt tgtgggattg gaattggatc 6540gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct 6600agaaataatt ttgtttaact ttaagaagga gatataccca tggaaaagcc tgaactcacc 6660gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag 6720ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc 6780ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt 6840gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg 6900acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa 6960ctgcccgctg ttctgcagcc ggtcgcggag gctatggatg cgatcgctgc ggccgatctt 7020agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg 7080cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac 7140gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac 7200tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac 7260aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac 7320gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc 7380tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc 7440cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct 7500tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca 7560caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat 7620agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagtgagg tacagcttgg 7680atcgatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag 7740caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa 7800ggaggaacta tatccggatg atcgggcgcg ccgtcgacgg atccgtacga gatccggccg 7860gccagatcct gcaggatata atgagccgta aacaaagatg attaagtagt aattaatacg 7920tactagtaaa agtggcaaaa gataacgaga aagaaccaat ttctttgcat tcggccttag 7980cggaaggcat atataagctt tgattatttt atttagtgta atgatttcgt acaaccaaag 8040catttattta gtactctcac acttgtgtcg cggccgcgtg gtacgtttag ggttacactg 8100ggacagctgc cagcttagca atcttcccca agaagccaag caattgagca acaccatcgg 8160acatgggggg tgctctgtat ggcaatttgt gcttctggca aattttctcc acctcaaaag 8220aagcagcttt caagtgatgc cggggcagcg tcggccacag atggtgttcg atttggtagt 8280tcaggcctcc aaagaaccag tcaacgatga ccccgcgttg gatgttcatc gtcgtgtgga 8340tctgccccgc gcaaaacccg tggctgtccc acacggactc ctccaccttc tccaatgggt 8400agtgattcaa gaacacgaca atggcaatcc caaacccgcc caggcactcc gagatgagga 8460agaaggccaa gcccgtcagt atgccgggca tgtaacagaa catgaatacc cctttcagaa 8520tccaatgcag agccagcccc acggcctcct ttgcgtactg gcgttggtaa tatatattcc 8580ttgcatctgt cttgtatgcc ataaccgcca gaatgctctg gaagcaccag atgaatcgga 8640gggttgcgat ggtggggagg aaatagtatt gttgccactt gatgagcttc cgcgtcgtgg 8700gaccggcgtt tttgacgtcc tccggggacc aggccaacac cgggaggtcg tcgatgtcgg 8760gatcgtgccc ttgcacgttc gtggcggcgt gatggccgtt gtgcctgtcc ttccaccaag 8820tcccggagta tccttgcatc acattgccga atacaagtcc agcaaaattg ttgtacgccc 8880ggttagagaa cacctgatgg tggcagtaat cgtgtgccaa ccaccccagc tgctggtagc 8940aaagtcccaa caaaatggct gggatgtagt accactgcag gtgcatcacc atcagaatag 9000ccagggcgcc cagggctact gtggtcgcac ttttgtacac gtagaagaga ggggatgcgt 9060tgaacatgcc ttttgcaatc atgtcctttc gaagcttgcg gaagtcctca gcaacctcgt 9120cgttgggagt cttggggctt cgtggcgaag tgggctccat cacaggcatg cgcttcagct 9180tgttcaacgc atcgtgggag tgcatgacca tgaacacatc cgtggcatct cggttccgat 9240aattctccat gatgtctgcg cccccggggt gatggttcac ccatgcggac acatcatagg 9300ttgtgccatc aatcgtgatg ggcaaggcct ctcgcttggg tgccatggtt tgcggccgct 9360tggggggcta tggaagactt tcttagttag ttgtgtgaat aagcaatgtt gggagaatcg 9420ggactactta taggatagga ataaaacaga aaagtattaa gtgctaatga aatatttaga 9480ctgataatta aaatcttcac gtatgtccac ttgatataaa aacgtcagga ataaaggaag 9540tacagtagaa tttaaaggta ctctttttat atatacccgt gttctctttt tggctagcta 9600gttgcataaa aaataatcta tatttttatc attattttaa atatcttatg agatggtaaa 9660tatttatcat aatttttttt actattattt attatttgtg tgtgtaatac atatagaagt 9720taattacaaa ttttatttac tttttcatta ttttgatatg attcaccatt aatttagtgt 9780tattatttat aatagttcat tttaatcttt ttgtatatat tatgcgtgca gtactttttt 9840cctacatata actactatta cattttattt atataatatt tttattaatg aattttcgtg 9900ataatatgta atattgttca ttattatttc agatttttta aaaatatttg tgttattatt 9960tatgaaatat gtaatttttt tagtatttga ttttatgatg ataaagtgtt ctaaattcaa 10020aagaaggggg aaagcgtaaa cattaaaaaa cgtcatcaaa caaaaacaaa atcttgttaa 10080taaagataaa actgtttgtt ttgatcactg ttatttcgta atataaaaac attatttata 10140tttatattgt tgacaaccaa atttgcctat caaatctaac caatataatg catgcgtggc 10200aggtaatgta ctaccatgaa cttaagtcat gacataataa accgtgaatc tgaccaatgc 10260atgtacctan ctaaattgta tttgtgacac gaagcaaatg attcaattca caatggagat 10320gggaaacaaa taatgaagaa cccagaacta agaaagcttt tctgaaaaat aaaataaagg 10380caatgtcaaa agtatactgc atcatcagtc cagaaagcac atgatatttt tttatcagta 10440tcaatgcagc tagttttatt ttacaatatc gatatagcta gtttaaatat attgcagcta 10500gatttataaa tatttgtgtt attatttatc atttgtgtaa tcctgttttt agtattttag 10560tttatatatg atgataatgt attccaaatt taaaagaagg gaaataaatt taaacaagaa 10620aaaaagtcat caaacaaaaa acaaatgaaa gggtggaaag atgttaccat gtaatgtgaa 10680tgttacagta tttcttttat tatagagtta acaaattaac taatatgatt ttgttaataa 10740tgataaaata ttttttttat tattatttca taatataaaa atagtttact taatataaaa 10800aaaattctat cgttcacaac aaagttggcc acctaattta accatgcatg tacccatgga 10860ccatattagg taaccatcaa acctgatgaa gagataaaga gatgaagact taagtcataa 10920cacaaaacca taaaaaacaa aaatacaatc aaccgtcaat ctgaccaatg catgaaaaag 10980ctgcaatagt gagtggcgac acaaagcaca tgattttctt acaacggaga taaaaccaaa 11040aaaatatttc atgaacaacc tagaacaaat aaagctttta tataataaat atataaataa 11100ataaaggcta tggaataata tacttcaata tatttggatt aaataaattg ttggcggggt 11160tgatatattt atacacacct aaagtcactt caatctcatt ttcacttaac ttttattttt 11220tttttctttt tatttatcat aaagagaata ttgataatat actttttaac atatttttat 11280gacatttttt attggtgaaa acttattaaa aatcataaat tttgtaagtt

agatttattt 11340aaagagttcc tcttcttatt ttaaattttt taataaattt ttaaataact aaaatttgtg 11400ttaaaaatgt taaaaaatgt gttattaacc cttctcttcg aggacgtacg tctagagtcg 11460acctgca 114671375250DNAArtificial SequencepKR393 137gatcccccgg gctgcaggaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac 60cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat 120agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg 180cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatggtgc 240actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca 300cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg 360accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga 420cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct 480tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 540taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 600tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt 660gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 720gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 780cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 840tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac 900tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 960atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 1020ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 1080gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 1140gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc 1200gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt 1260gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 1320gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc 1380cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag 1440atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca 1500tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 1560ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 1620gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 1680tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 1740ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt 1800ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 1860gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 1920ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 1980tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 2040ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 2100agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 2160agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 2220gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 2280tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 2340accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca 2400gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg 2460attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac 2520gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg 2580gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac 2640catgattacg ccaagcttgc atgcctgcag gctagcctaa gtacgtactc aaaatgccaa 2700caaataaaaa aaaagttgct ttaataatgc caaaacaaat taataaaaca cttacaacac 2760cggatttttt ttaattaaaa tgtgccattt aggataaata gttaatattt ttaataatta 2820tttaaaaagc cgtatctact aaaatgattt ttatttggtt gaaaatatta atatgtttaa 2880atcaacacaa tctatcaaaa ttaaactaaa aaaaaaataa gtgtacgtgg ttaacattag 2940tacagtaata taagaggaaa atgagaaatt aagaaattga aagcgagtct aatttttaaa 3000ttatgaacct gcatatataa aaggaaagaa agaatccagg aagaaaagaa atgaaaccat 3060gcatggtccc ctcgtcatca cgagtttctg ccatttgcaa tagaaacact gaaacacctt 3120tctctttgtc acttaattga gatgccgaag ccacctcaca ccatgaactt catgaggtgt 3180agcacccaag gcttccatag ccatgcatac tgaagaatgt ctcaagctca gcaccctact 3240tctgtgacgt gtccctcatt caccttcctc tcttccctat aaataaccac gcctcaggtt 3300ctccgcttca caactcaaac attctctcca ttggtcctta aacactcatc agtcatcacc 3360gcggccgcca attcaggtgc ccatgatgtt ggccgcaggc tatcttctag tgctctcggc 3420cgctcgccag agcttccagc aggacattga caaccccaac ggggcctact cgacctcgtg 3480gactggcctg cccattgtga tgtctgtggt ctatctcagc ggtgtgtttg ggctcacaaa 3540gtacttcgag aaccggaagc ccatgacggg gctgaaggac tacatgttca cttacaatct 3600ctaccaggtg atcatcaacg tgtggtgcgt ggtggccttt ctcctggagg tgcggcgtgc 3660gggcatgtca ctcatcggca ataaggtgga ccttgggccc aactccttca ggctcggctt 3720cgtcacgtgg gtgcactaca acaacaagta cgtggagctc ctcgacaccc tatggatggt 3780gctgcgcaag aagacgcagc aggtctcctt cctccacgtc tatcatcacg tgcttctgat 3840gtgggcctgg ttcgttgtcg tcaagctcgg caatggtggt gacgcatatt ttggcggtct 3900catgaactcg atcatccacg tgatgatgta ttcctactac accatggcgc tcctgggctg 3960gtcatgcccc tggaagcgct acctcacgca ggcacagctc gtgcagtttt gcatctgcct 4020cgcccactcc acatgggcgg cagtaacggg tgcctacccg tggcgaattt gcttggtgga 4080ggtgtgggtg atggtgtcca tgctggtgct cttcacacgc ttctaccgcc aggcctatgc 4140caaggaggcg aaggccaagg aggcgaaaaa gctcgcacag gaggcatcac aggccaaggc 4200ggtcaaggcg gagtaagtca ctggaggtgg accgcacatg caccacgggc ccggcgagca 4260gcatggttcg gcgagtcagg cccggtcatg cgtcatggtt ggagtttgca gggcggcagg 4320tgatcgcctc cgccatgcac ggccacaggc acagccggtc ctctggacgt cccaactttc 4380aaccgtggtg caaagcacgc tggcgaccgc gagcagcagt cagcgcagcg tgttatcaca 4440gtgtcgctgg ctgcacgtgc tctctccatc gcggccgcat ttcgcaccaa atcaatgaaa 4500gtaataatga aaagtctgaa taagaatact taggcttaga tgcctttgtt acttgtgtaa 4560aataacttga gtcatgtacc tttggcggaa acagaataaa taaaaggtga aattccaatg 4620ctctatgtat aagttagtaa tacttaatgt gttctacggt tgtttcaata tcatcaaact 4680ctaattgaaa ctttagaacc acaaatctca atcttttctt aatgaaatga aaaatcttaa 4740ttgtaccatg tttatgttaa acaccttaca attggttgga gaggaggacc aaccgatggg 4800acaacattgg gagaaagaga ttcaatggag atttggatag gagaacaaca ttctttttca 4860cttcaataca agatgagtgc aacactaagg atatgtatga gactttcaga agctacgaca 4920acatagatga gtgaggtggt gattcctagc aagaaagaca ttagaggaag ccaaaatcga 4980acaaggaaga catcaagggc aagagacagg accatccatc tcaggaaaag gagctttggg 5040atagtccgag aagttgtaca agaaattttt tggagggtga gtgatgcatt gctggtgact 5100ttaactcaat caaaattgag aaagaaagaa aagggagggg gctcacatgt gaatagaagg 5160gaaacgggag aattttacag ttttgatcta atgggcatcc cagctagtgg taacatattc 5220accatgttta accttcacgt acgtctagag 52501384140DNAArtificial SequencepKR407 138ggccgcattt 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 300tggttggaga ggaggaccaa ccgatgggac aacattggga gaaagagatt caatggagat 360ttggatagga gaacaacatt ctttttcact tcaatacaag atgagtgcaa cactaaggat 420atgtatgaga ctttcagaag ctacgacaac atagatgagt gaggtggtga ttcctagcaa 480gaaagacatt agaggaagcc aaaatcgaac aaggaagaca tcaagggcaa gagacaggac 540catccatctc aggaaaagga gctttgggat agtccgagaa gttgtacaag aaattttttg 600gagggtgagt gatgcattgc tggtgacttt aactcaatca aaattgagaa agaaagaaaa 660gggagggggc tcacatgtga atagaaggga aacgggagaa ttttacagtt ttgatctaat 720gggcatccca gctagtggta acatattcac catgtttaac cttcacgtac gtctagagga 780tcccccgggc tgcaggaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc 840tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag 900cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg 960cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac 1020tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc 1080cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac 1140cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg 1200aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta 1260gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 1320aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 1380ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 1440ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 1500agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct 1560tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg 1620tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 1680ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat 1740gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 1800acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga 1860tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 1920gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga 1980actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 2040aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 2100cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 2160tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 2220cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 2280tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 2340ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 2400ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 2460cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 2520aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct 2580agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 2640tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 2700ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 2760cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 2820atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 2880ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 2940tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 3000gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 3060gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac 3120cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt 3180gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat 3240tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc 3300aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc 3360tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca 3420tgattacgcc aagcttgcat gcctgcaggc tagcctaagt acgtactcaa aatgccaaca 3480aataaaaaaa aagttgcttt aataatgcca aaacaaatta ataaaacact tacaacaccg 3540gatttttttt aattaaaatg tgccatttag gataaatagt taatattttt aataattatt 3600taaaaagccg tatctactaa aatgattttt atttggttga aaatattaat atgtttaaat 3660caacacaatc tatcaaaatt aaactaaaaa aaaaataagt gtacgtggtt aacattagta 3720cagtaatata agaggaaaat gagaaattaa gaaattgaaa gcgagtctaa tttttaaatt 3780atgaacctgc atatataaaa ggaaagaaag aatccaggaa gaaaagaaat gaaaccatgc 3840atggtcccct cgtcatcacg agtttctgcc atttgcaata gaaacactga aacacctttc 3900tctttgtcac ttaattgaga tgccgaagcc acctcacacc atgaacttca tgaggtgtag 3960cacccaaggc ttccatagcc atgcatactg aagaatgtct caagctcagc accctacttc 4020tgtgacgtgt ccctcattca ccttcctctc ttccctataa ataaccacgc ctcaggttct 4080ccgcttcaca actcaaacat tctctccatt ggtccttaaa cactcatcag tcatcaccgc 41401395414DNAArtificial SequencepKR1018 139ggccgcattt 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 300tggttggaga ggaggaccaa ccgatgggac aacattggga gaaagagatt caatggagat 360ttggatagga gaacaacatt ctttttcact tcaatacaag atgagtgcaa cactaaggat 420atgtatgaga ctttcagaag ctacgacaac atagatgagt gaggtggtga ttcctagcaa 480gaaagacatt agaggaagcc aaaatcgaac aaggaagaca tcaagggcaa gagacaggac 540catccatctc aggaaaagga gctttgggat agtccgagaa gttgtacaag aaattttttg 600gagggtgagt gatgcattgc tggtgacttt aactcaatca aaattgagaa agaaagaaaa 660gggagggggc tcacatgtga atagaaggga aacgggagaa ttttacagtt ttgatctaat 720gggcatccca gctagtggta acatattcac catgtttaac cttcacgtac gtctagagga 780tcccccgggc tgcaggaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc 840tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag 900cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg 960cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac 1020tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc 1080cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac 1140cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg 1200aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta 1260gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 1320aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 1380ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 1440ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 1500agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct 1560tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg 1620tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 1680ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat 1740gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 1800acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga 1860tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 1920gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga 1980actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 2040aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 2100cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 2160tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 2220cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 2280tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 2340ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 2400ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 2460cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 2520aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct 2580agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 2640tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 2700ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 2760cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 2820atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 2880ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 2940tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 3000gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 3060gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac 3120cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt 3180gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat 3240tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc 3300aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc 3360tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca 3420tgattacgcc aagcttgcat gcctgcaggc tagcctaagt acgtactcaa aatgccaaca 3480aataaaaaaa aagttgcttt aataatgcca aaacaaatta ataaaacact tacaacaccg 3540gatttttttt aattaaaatg tgccatttag gataaatagt taatattttt aataattatt 3600taaaaagccg tatctactaa aatgattttt atttggttga aaatattaat atgtttaaat 3660caacacaatc tatcaaaatt aaactaaaaa aaaaataagt gtacgtggtt aacattagta 3720cagtaatata agaggaaaat gagaaattaa gaaattgaaa gcgagtctaa tttttaaatt 3780atgaacctgc atatataaaa ggaaagaaag aatccaggaa gaaaagaaat gaaaccatgc 3840atggtcccct cgtcatcacg agtttctgcc atttgcaata gaaacactga aacacctttc 3900tctttgtcac ttaattgaga tgccgaagcc acctcacacc atgaacttca tgaggtgtag 3960cacccaaggc ttccatagcc atgcatactg aagaatgtct caagctcagc accctacttc 4020tgtgacgtgt ccctcattca ccttcctctc ttccctataa ataaccacgc ctcaggttct 4080ccgcttcaca actcaaacat tctctccatt ggtccttaaa cactcatcag tcatcaccgc 4140ggccgcacca tgtctcctaa gcggcaagct ctgccaatca caattgatgg cgcaacttat 4200gatgtgtctg cttgggtcaa tcaccaccct ggaggagctg acattatcga gaactatcgc 4260aaccgcgatg cgaccgatgt cttcatggtg atgcactctc aagaagccgt cgccaagttg 4320aagagaatgc ctgttatgga gccttcctct cctgacacac ctgttgcacc caagcctaag 4380cgtgatgagc cccaggagga tttccgcaag ttgcgggagg aattcatctc caagggtatg 4440ttcgagacga gtttcctttg gtatttttac aagacttcaa ctaccgtcgg tttgatggtc 4500ctttccatct tgatgaccgt gtacacgaat tggtatttca ccgctgcttt ggttcttggc 4560gtgtgctacc aacagctagg ctggttgtcc cacgactatt gccatcacca ggttttcaca 4620aaccgcaaga ttaacgacgc tttcggtctc tttttcggta acgtgatgca gggatactca 4680cagacttggt ggaaggatag gcacaatggt caccatgccg ccaccaatgt ggtcggccat 4740gacccagata ttgataacct ccccatcctg gcttggtctc ccgaagatgt caagagggct 4800actccttcga ctcggaatct catcaagtac cagcagtact acttcattcc caccattgca 4860tcccttaggt tcatctggtg cctccaatcc atcggcggcg tcatgtccta caagagcgag 4920gagaggaacc tgtactacaa gcgccagtac actaaggagg cgattggtct ggccctccac 4980tgggtgctca aggccacttt ctattgcagt gccatgccta gctttgccac cggtttggga 5040tgcttcttga tctccgagct gctcggagga tttggcattg ccatcgttgt gtttctgaat 5100cactatcctt tggacaaggt tgaggagact gtctgggatg agcacgggtt cagcgccagc 5160cagatccacg agacgttgaa cattaagccc ggccttctca ccgattgggt ctttggtggt 5220ctcaactacc agattgagca ccacttgtgg cccaacatgc ccaggcacaa cctcacggca 5280gcttccctgg aggtgcagaa gttgtgcgcc aagcacaacc tgccctacag

ggccccagcc 5340atcatccccg gggttcagaa attggtcagc ttcttaggcg agattgccca gctggctgct 5400gtccctgaat gagc 54141407908DNAArtificial SequencepKR1020R 140ggagatccaa 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 acgagatccg gccggccaga tcctgcagcc cgggggatcc 5160tctagacgta cgtgaaggtt aaacatggtg aatatgttac cactagctgg gatgcccatt 5220agatcaaaac tgtaaaattc tcccgtttcc cttctattca catgtgagcc ccctcccttt 5280tctttctttc tcaattttga ttgagttaaa gtcaccagca atgcatcact caccctccaa 5340aaaatttctt gtacaacttc tcggactatc ccaaagctcc ttttcctgag atggatggtc 5400ctgtctcttg cccttgatgt cttccttgtt cgattttggc ttcctctaat gtctttcttg 5460ctaggaatca ccacctcact catctatgtt gtcgtagctt ctgaaagtct catacatatc 5520cttagtgttg cactcatctt gtattgaagt gaaaaagaat gttgttctcc tatccaaatc 5580tccattgaat ctctttctcc caatgttgtc ccatcggttg gtcctcctct ccaaccaatt 5640gtaaggtgtt taacataaac atggtacaat taagattttt catttcatta agaaaagatt 5700gagatttgtg gttctaaagt ttcaattaga gtttgatgat attgaaacaa ccgtagaaca 5760cattaagtat tactaactta tacatagagc attggaattt caccttttat ttattctgtt 5820tccgccaaag gtacatgact caagttattt tacacaagta acaaaggcat ctaagcctaa 5880gtattcttat tcagactttt cattattact ttcattgatt tggtgcgaaa tgcggccgct 5940cattcaggga cagcagccag ctgggcaatc tcgcctaaga agctgaccaa tttctgaacc 6000ccggggatga tggctggggc cctgtagggc aggttgtgct tggcgcacaa cttctgcacc 6060tccagggaag ctgccgtgag gttgtgcctg ggcatgttgg gccacaagtg gtgctcaatc 6120tggtagttga gaccaccaaa gacccaatcg gtgagaaggc cgggcttaat gttcaacgtc 6180tcgtggatct ggctggcgct gaacccgtgc tcatcccaga cagtctcctc aaccttgtcc 6240aaaggatagt gattcagaaa cacaacgatg gcaatgccaa atcctccgag cagctcggag 6300atcaagaagc atcccaaacc ggtggcaaag ctaggcatgg cactgcaata gaaagtggcc 6360ttgagcaccc agtggagggc cagaccaatc gcctccttag tgtactggcg cttgtagtac 6420aggttcctct cctcgctctt gtaggacatg acgccgccga tggattggag gcaccagatg 6480aacctaaggg atgcaatggt gggaatgaag tagtactgct ggtacttgat gagattccga 6540gtcgaaggag tagccctctt gacatcttcg ggagaccaag ccaggatggg gaggttatca 6600atatctgggt catggccgac cacattggtg gcggcatggt gaccattgtg cctatccttc 6660caccaagtct gtgagtatcc ctgcatcacg ttaccgaaaa agagaccgaa agcgtcgtta 6720atcttgcggt ttgtgaaaac ctggtgatgg caatagtcgt gggacaacca gcctagctgt 6780tggtagcaca cgccaagaac caaagcagcg gtgaaatacc aattcgtgta cacggtcatc 6840aagatggaaa ggaccatcaa accgacggta gttgaagtct tgtaaaaata ccaaaggaaa 6900ctcgtctcga acataccctt ggagatgaat tcctcccgca acttgcggaa atcctcctgg 6960ggctcatcac gcttaggctt gggtgcaaca ggtgtgtcag gagaggaagg ctccataaca 7020ggcattctct tcaacttggc gacggcttct tgagagtgca tcaccatgaa gacatcggtc 7080gcatcgcggt tgcgatagtt ctcgataatg tcagctcctc cagggtggtg attgacccaa 7140gcagacacat cataagttgc gccatcaatt gtgattggca gagcttgccg cttaggagac 7200atggtgcggc cgcggtgatg actgatgagt gtttaaggac caatggagag aatgtttgag 7260ttgtgaagcg gagaacctga ggcgtggtta tttataggga agagaggaag gtgaatgagg 7320gacacgtcac agaagtaggg tgctgagctt gagacattct tcagtatgca tggctatgga 7380agccttgggt gctacacctc atgaagttca tggtgtgagg tggcttcggc atctcaatta 7440agtgacaaag agaaaggtgt ttcagtgttt ctattgcaaa tggcagaaac tcgtgatgac 7500gaggggacca tgcatggttt catttctttt cttcctggat tctttctttc cttttatata 7560tgcaggttca taatttaaaa attagactcg ctttcaattt cttaatttct cattttcctc 7620ttatattact gtactaatgt taaccacgta cacttatttt ttttttagtt taattttgat 7680agattgtgtt gatttaaaca tattaatatt ttcaaccaaa taaaaatcat tttagtagat 7740acggcttttt aaataattat taaaaatatt aactatttat cctaaatggc acattttaat 7800taaaaaaaat ccggtgttgt aagtgtttta ttaatttgtt ttggcattat taaagcaact 7860ttttttttat ttgttggcat tttgagtacg tacttaggct agcctgca 790814118662DNAArtificial SequencepKR1022R 141cgcgccagat cctctagagt cgacctgcag gcatgcaagc ttggcgtaat catggtcata 60gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag 120cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg 180ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca 240acgcgcgggg agaggcggtt tgcgtattgg atcgatccct gaaagcgacg ttggatgtta 300acatctacaa attgcctttt cttatcgacc atgtacgtaa gcgcttacgt ttttggtgga 360cccttgagga aactggtagc tgttgtgggc ctgtggtctc aagatggatc attaatttcc 420accttcacct acgatggggg gcatcgcacc ggtgagtaat attgtacggc taagagcgaa 480tttggcctgt agacctcaat tgcgagcttt ctaatttcaa actattcggg cctaactttt 540ggtgtgatga tgctgactgg caggatatat accgttgtaa tttgagctcg tgtgaataag 600tcgctgtgta tgtttgtttg attgtttctg ttggagtgca gcccatttca ccggacaagt 660cggctagatt gatttagccc tgatgaactg ccgaggggaa gccatcttga gcgcggaatg 720ggaatggatt tcgttgtaca acgagacgac agaacaccca cgggaccgag cttcgcgagc 780ttttgtatcc gtggcatcct tggtccgggc gatttgttca cgtccatgag gcgctctcca 840aaggaacgca tattttccgg tgcaaccttt ccggttcttc ctctactcga cctcttgaag 900tcccagcatg aatgttcgac cgctccgcaa gcggatcttt ggcgcaacca gccggtttcg 960cacgtcgatt ctcgcgagcc tgcatacttt ggcaagattg ctgaatgacg ctgatgcttc 1020atcgcaatct gcgataatgg ggtaagtatc cggtgaaggc cgcaggtcag gccgcctgag 1080cactcagtgt cttggatgtc cagttccacg gcagctgttg ctcaagcctg ctgatcggag 1140cgtccgcaag gtcggcgcgg acgtcggcaa gccaggcctg cggatcgatg ttattgagct 1200tggcgctcat gatcagtgtc gccatgaacg ccgcacgttc agcacaacga tccgatccgg 1260caaacagcca tgacttcctg ccgagtacat agcctctgag cgttcgttcg gcagcattgt 1320tcgtcaggca aatcgggccg tcatcgagga atgacgtaat gccatcccat cgcttgagca 1380tgtaatttat cgcctcggcg acgggagaac tgcgcgacaa tttcccccgc tcggtttcga 1440gccaatcatg cagctcttcg gcgagtgacc ttgatcaggc caccgccacg accgcggaag 1500acgaacagat gcctgcgcat cggatcgcgc ttcagcgtct cttgcaccat cagcgacaaa 1560ccgggaaagc ctttgcgcat gtccgtactt atgtcgccac ttgggagggc ttcgtctacg 1620tggccttcgt gatcgacgtc ttcgcccgtc gcattgtcgg atggcgggcg agccggacag 1680cacatgcagg ctttgtcctc gatgccctcg aggaggctca tcatgatcgg cgtcccgctc 1740atggcggcct agtgcatcac tcggatcgcg gtgttcaata cgtgtccttt cgctattccg 1800agcggttggc agaagcaggt atcgagccat ctatcggaag cgtcggcgac agcacgacaa 1860cgccctcgca gaagcgatca acggtcttta caaggccgag gtcattcatc ggcgtggacc 1920atggaggagc ttcgaagcgg tcgagttcgc taccttggaa tggatagact ggttcaacca 1980cggcggcttt tgaagcccat cggcaatata ccgccagccg aagacgagga tcagtattac 2040gccatgctgg acgaagcagc catggctgcg cattttaacg aaatggcctc cggcaaaccc 2100ggtgcggttc acttgttgcg tgggaaagtt cacgggactc cgcgcacgag ccttcttcgt 2160aatagccata tcgaccgaat tgacctgcag gggggggggg gaaagccacg ttgtgtctca 2220aaatctctga tgttacattg cacaagataa aaatatatca tcatgaacaa taaaactgtc 2280tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcttg 2340ctcgaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg 2400cgataatgtc gggcaatcag gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc 2460agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt 2520cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac 2580tcctgatgat gcatggttac tcaccactgc gatccccggg aaaacagcat tccaggtatt 2640agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg 2700gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc 2760tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg 2820taatggctgg cctgttgaac aagtctggaa agaaatgcat aagcttttgc cattctcacc 2880ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa 2940attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc 3000catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa 3060atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt 3120tttctaatca gaattggtta attggttgta acactggcag agcattacgc tgacttgacg 3180ggacggcggc tttgttgaat aaatcgaact tttgctgagt tgaaggatca gatcacgcat 3240cttcccgaca acgcagaccg ttccgtggca aagcaaaagt tcaaaatcac caactggtcc 3300acctacaaca aagctctcat caaccgtggc tccctcactt tctggctgga tgatggggcg 3360attcaggcct ggtatgagtc agcaacacct tcttcacgag gcagacctca gcgccccccc 3420ccccctgcag gtcttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 3480tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 3540acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 3600aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 3660cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 3720gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 3780cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 3840tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 3900tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 3960ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 4020tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 4080gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 4140ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 4200tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 4260agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 4320aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 4380cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt 4440agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 4500tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 4560gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 4620gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 4680ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 4740gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 4800ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 4860ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 4920acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 4980gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 5040cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca 5100tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc 5160gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc 5220gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg 5280gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agggtgcctt 5340gatgtgggcg ccggcggtcg agtggcgacg gcgcggcttg tccgcgccct ggtagattgc 5400ctggccgtag gccagccatt tttgagcggc cagcggccgc gataggccga cgcgaagcgg 5460cggggcgtag ggagcgcagc gaccgaaggg taggcgcttt ttgcagctct tcggctgtgc 5520gctggccaga cagttatgca caggccaggc gggttttaag agttttaata agttttaaag 5580agttttaggc ggaaaaatcg ccttttttct cttttatatc agtcacttac atgtgtgacc 5640ggttcccaat gtacggcttt gggttcccaa tgtacgggtt ccggttccca atgtacggct 5700ttgggttccc aatgtacgtg ctatccacag gaaagagacc ttttcgacct ttttcccctg 5760ctagggcaat ttgccctagc atctgctccg tacattagga accggcggat gcttcgccct 5820cgatcaggtt gcggtagcgc atgactagga tcgggccagc ctgccccgcc tcctccttca 5880aatcgtactc cggcaggtca tttgacccga tcagcttgcg cacggtgaaa cagaacttct 5940tgaactctcc ggcgctgcca ctgcgttcgt agatcgtctt gaacaaccat ctggcttctg 6000ccttgcctgc ggcgcggcgt gccaggcggt agagaaaacg gccgatgccg ggatcgatca 6060aaaagtaatc ggggtgaacc gtcagcacgt ccgggttctt gccttctgtg atctcgcggt 6120acatccaatc agctagctcg atctcgatgt actccggccg cccggtttcg ctctttacga 6180tcttgtagcg gctaatcaag gcttcaccct cggataccgt caccaggcgg ccgttcttgg 6240ccttcttcgt acgctgcatg gcaacgtgcg tggtgtttaa ccgaatgcag gtttctacca 6300ggtcgtcttt ctgctttccg ccatcggctc gccggcagaa cttgagtacg tccgcaacgt 6360gtggacggaa cacgcggccg ggcttgtctc ccttcccttc ccggtatcgg ttcatggatt 6420cggttagatg ggaaaccgcc atcagtacca ggtcgtaatc ccacacactg gccatgccgg 6480ccggccctgc ggaaacctct acgtgcccgt ctggaagctc gtagcggatc acctcgccag 6540ctcgtcggtc acgcttcgac agacggaaaa cggccacgtc catgatgctg cgactatcgc 6600gggtgcccac gtcatagagc atcggaacga aaaaatctgg ttgctcgtcg cccttgggcg 6660gcttcctaat cgacggcgca ccggctgccg gcggttgccg ggattctttg cggattcgat 6720cagcggccgc ttgccacgat tcaccggggc gtgcttctgc ctcgatgcgt tgccgctggg 6780cggcctgcgc ggccttcaac ttctccacca ggtcatcacc cagcgccgcg ccgatttgta 6840ccgggccgga tggtttgcga ccgctcacgc cgattcctcg ggcttggggg ttccagtgcc 6900attgcagggc cggcagacaa

cccagccgct tacgcctggc caaccgcccg ttcctccaca 6960catggggcat tccacggcgt cggtgcctgg ttgttcttga ttttccatgc cgcctccttt 7020agccgctaaa attcatctac tcatttattc atttgctcat ttactctggt agctgcgcga 7080tgtattcaga tagcagctcg gtaatggtct tgccttggcg taccgcgtac atcttcagct 7140tggtgtgatc ctccgccggc aactgaaagt tgacccgctt catggctggc gtgtctgcca 7200ggctggccaa cgttgcagcc ttgctgctgc gtgcgctcgg acggccggca cttagcgtgt 7260ttgtgctttt gctcattttc tctttacctc attaactcaa atgagttttg atttaatttc 7320agcggccagc gcctggacct cgcgggcagc gtcgccctcg ggttctgatt caagaacggt 7380tgtgccggcg gcggcagtgc ctgggtagct cacgcgctgc gtgatacggg actcaagaat 7440gggcagctcg tacccggcca gcgcctcggc aacctcaccg ccgatgcgcg tgcctttgat 7500cgcccgcgac acgacaaagg ccgcttgtag ccttccatcc gtgacctcaa tgcgctgctt 7560aaccagctcc accaggtcgg cggtggccca tatgtcgtaa gggcttggct gcaccggaat 7620cagcacgaag tcggctgcct tgatcgcgga cacagccaag tccgccgcct ggggcgctcc 7680gtcgatcact acgaagtcgc gccggccgat ggccttcacg tcgcggtcaa tcgtcgggcg 7740gtcgatgccg acaacggtta gcggttgatc ttcccgcacg gccgcccaat cgcgggcact 7800gccctgggga tcggaatcga ctaacagaac atcggccccg gcgagttgca gggcgcgggc 7860tagatgggtt gcgatggtcg tcttgcctga cccgcctttc tggttaagta cagcgataac 7920ttcatgcgtt cccttgcgta tttgtttatt tactcatcgc atcatatacg cagcgaccgc 7980atgacgcaag ctgttttact caaatacaca tcaccttttt agacggcggc gctcggtttc 8040ttcagcggcc aagctggccg gccaggccgc cagcttggca tcagacaaac cggccaggat 8100ttcatgcagc cgcacggttg agacgtgcgc gggcggctcg aacacgtacc cggccgcgat 8160catctccgcc tcgatctctt cggtaatgaa aaacggttcg tcctggccgt cctggtgcgg 8220tttcatgctt gttcctcttg gcgttcattc tcggcggccg ccagggcgtc ggcctcggtc 8280aatgcgtcct cacggaaggc accgcgccgc ctggcctcgg tgggcgtcac ttcctcgctg 8340cgctcaagtg cgcggtacag ggtcgagcga tgcacgccaa gcagtgcagc cgcctctttc 8400acggtgcggc cttcctggtc gatcagctcg cgggcgtgcg cgatctgtgc cggggtgagg 8460gtagggcggg ggccaaactt cacgcctcgg gccttggcgg cctcgcgccc gctccgggtg 8520cggtcgatga ttagggaacg ctcgaactcg gcaatgccgg cgaacacggt caacaccatg 8580cggccggccg gcgtggtggt gtcggcccac ggctctgcca ggctacgcag gcccgcgccg 8640gcctcctgga tgcgctcggc aatgtccagt aggtcgcggg tgctgcgggc caggcggtct 8700agcctggtca ctgtcacaac gtcgccaggg cgtaggtggt caagcatcct ggccagctcc 8760gggcggtcgc gcctggtgcc ggtgatcttc tcggaaaaca gcttggtgca gccggccgcg 8820tgcagttcgg cccgttggtt ggtcaagtcc tggtcgtcgg tgctgacgcg ggcatagccc 8880agcaggccag cggcggcgct cttgttcatg gcgtaatgtc tccggttcta gtcgcaagta 8940ttctacttta tgcgactaaa acacgcgaca agaaaacgcc aggaaaaggg cagggcggca 9000gcctgtcgcg taacttagga cttgtgcgac atgtcgtttt cagaagacgg ctgcactgaa 9060cgtcagaagc cgactgcact atagcagcgg aggggttgga ccacaggacg ggtgtggtcg 9120ccatgatcgc gtagtcgata gtggctccaa gtagcgaagc gagcaggact gggcggcggc 9180caaagcggtc ggacagtgct ccgagaacgg gtgcgcatag aaattgcatc aacgcatata 9240gcgctagcag cacgccatag tgactggcga tgctgtcgga atggacgata tcccgcaaga 9300ggcccggcag taccggcata accaagccta tgcctacagc atccagggtg acggtgccga 9360ggatgacgat gagcgcattg ttagatttca tacacggtgc ctgactgcgt tagcaattta 9420actgtgataa actaccgcat taaagctagc ttgcttggtc gttccgcgtg aacgtcggct 9480cgattgtacc tgcgttcaaa tactttgcga tcgtgttgcg cgcctgcccg gtgcgtcggc 9540tgatctcacg gatcgactgc ttctctcgca acgccatccg acggatgatg tttaaaagtc 9600ccatgtggat cactccgttg ccccgtcgct caccgtgttg gggggaaggt gcacatggct 9660cagttctcaa tggaaattat ctgcctaacc ggctcagttc tgcgtagaaa ccaacatgca 9720agctccaccg ggtgcaaagc ggcagcggcg gcaggatata ttcaattgta aatggcttca 9780tgtccgggaa atctacatgg atcagcaatg agtatgatgg tcaatatgga gaaaaagaaa 9840gagtaattac caattttttt tcaattcaaa aatgtagatg tccgcagcgt tattataaaa 9900tgaaagtaca ttttgataaa acgacaaatt acgatccgtc gtatttatag gcgaaagcaa 9960taaacaaatt attctaattc ggaaatcttt atttcgacgt gtctacattc acgtccaaat 10020gggggcttag atgagaaact tcacgatcga tgccttgatt tcgccattcc cagataccca 10080tttcatcttc agattggtct gagattatgc gaaaatatac actcatatac ataaatactg 10140acagtttgag ctaccaattc agtgtagccc attacctcac ataattcact caaatgctag 10200gcagtctgtc aactcggcgt caatttgtcg gccactatac gatagttgcg caaattttca 10260aagtcctggc ctaacatcac acctctgtcg gcggcgggtc ccatttgtga taaatccacc 10320atatcgaatt aattcagact cctttgcccc agagatcaca atggacgact tcctctatct 10380ctacgatcta gtcaggaagt tcgacggaga aggtgacgat accatgttca ccactgataa 10440tgagaagatt agccttttca atttcagaaa gaatgctaac ccacagatgg ttagagaggc 10500ttacgcagca ggtctcatca agacgatcta cccgagcaat aatctccagg agatcaaata 10560ccttcccaag aaggttaaag atgcagtcaa aagattcagg actaactgca tcaagaacac 10620agagaaagat atatttctca agatcagaag tactattcca gtatggacga ttcaaggctt 10680gcttcacaaa ccaaggcaag taatagagat tggagtctct aaaaaggtag ttcccactga 10740atcaaaggcc atggagtcaa agattcaaat agaggaccta acagaactcg ccgtaaagac 10800tggcgaacag ttcatacaga gtctcttacg actcaatgac aagaagaaaa tcttcgtcaa 10860catggtggag cacgacacgc ttgtctactc caaaaatatc aaagatacag tctcagaaga 10920ccaaagggca attgagactt ttcaacaaag ggtaatatcc ggaaacctcc tcggattcca 10980ttgcccagct atctgtcact ttattgtgaa gatagtggaa aaggaaggtg gctcctacaa 11040atgccatcat tgcgataaag gaaaggccat cgttgaagat gcctctgccg acagtggtcc 11100caaagatgga cccccaccca cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc 11160ttcaaagcaa gtggattgat gtgatatctc cactgacgta agggatgacg cacaatccca 11220ctatccttcg caagaccctt cctctatata aggaagttca tttcatttgg agaggacacg 11280ctgaaatcac cagtctccaa gcttgcgggg atcgtttcgc atgattgaac aagatggatt 11340gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca 11400gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct 11460ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct 11520atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc 11580gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct 11640tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga 11700tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg 11760gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc 11820agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac 11880ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat 11940cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga 12000tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc 12060cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg 12120actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat 12180tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg 12240atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccgg atcgatccaa 12300cacttacgtt tgcaacgtcc aagagcaaat agaccacgaa cgccggaagg ttgccgcagc 12360gtgtggattg cgtctcaatt ctctcttgca ggaatgcaat gatgaatatg atactgacta 12420tgaaactttg agggaatact gcctagcacc gtcacctcat aacgtgcatc atgcatgccc 12480tgacaacatg gaacatcgct atttttctga agaattatgc tcgttggagg atgtcgcggc 12540aattgcagct attgccaaca tcgaactacc cctcacgcat gcattcatca atattattca 12600tgcggggaaa ggcaagatta atccaactgg caaatcatcc agcgtgattg gtaacttcag 12660ttccagcgac ttgattcgtt ttggtgctac ccacgttttc aataaggacg agatggtgga 12720gtaaagaagg agtgcgtcga agcagatcgt tcaaacattt ggcaataaag tttcttaaga 12780ttgaatcctg ttgccggtct tgcgatgatt atcatataat ttctgttgaa ttacgttaag 12840catgtaataa ttaacatgta atgcatgacg ttatttatga gatgggtttt tatgattaga 12900gtcccgcaat tatacattta atacgcgata gaaaacaaaa tatagcgcgc aaactaggat 12960aaattatcgc gcgcggtgtc atctatgtta ctagatcgat caaacttcgg tactgtgtaa 13020tgacgatgag caatcgagag gctgactaac aaaaggtaca tcgcgatgga tcgatccatt 13080cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac 13140gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt 13200cccagtcacg acgttgtaaa acgacggcca gtgaattcct gcagcccggg ggatccgccc 13260actcgaggcg cgccgtcgac ggatccgtac gagatccggc cggccagatc ctgcagcccg 13320ggggatcctc tagacgtacg tgaaggttaa acatggtgaa tatgttacca ctagctggga 13380tgcccattag atcaaaactg taaaattctc ccgtttccct tctattcaca tgtgagcccc 13440ctcccttttc tttctttctc aattttgatt gagttaaagt caccagcaat gcatcactca 13500ccctccaaaa aatttcttgt acaacttctc ggactatccc aaagctcctt ttcctgagat 13560ggatggtcct gtctcttgcc cttgatgtct tccttgttcg attttggctt cctctaatgt 13620ctttcttgct aggaatcacc acctcactca tctatgttgt cgtagcttct gaaagtctca 13680tacatatcct tagtgttgca ctcatcttgt attgaagtga aaaagaatgt tgttctccta 13740tccaaatctc cattgaatct ctttctccca atgttgtccc atcggttggt cctcctctcc 13800aaccaattgt aaggtgttta acataaacat ggtacaatta agatttttca tttcattaag 13860aaaagattga gatttgtggt tctaaagttt caattagagt ttgatgatat tgaaacaacc 13920gtagaacaca ttaagtatta ctaacttata catagagcat tggaatttca ccttttattt 13980attctgtttc cgccaaaggt acatgactca agttatttta cacaagtaac aaaggcatct 14040aagcctaagt attcttattc agacttttca ttattacttt cattgatttg gtgcgaaatg 14100cggccgctca ttcagggaca gcagccagct gggcaatctc gcctaagaag ctgaccaatt 14160tctgaacccc ggggatgatg gctggggccc tgtagggcag gttgtgcttg gcgcacaact 14220tctgcacctc cagggaagct gccgtgaggt tgtgcctggg catgttgggc cacaagtggt 14280gctcaatctg gtagttgaga ccaccaaaga cccaatcggt gagaaggccg ggcttaatgt 14340tcaacgtctc gtggatctgg ctggcgctga acccgtgctc atcccagaca gtctcctcaa 14400ccttgtccaa aggatagtga ttcagaaaca caacgatggc aatgccaaat cctccgagca 14460gctcggagat caagaagcat cccaaaccgg tggcaaagct aggcatggca ctgcaataga 14520aagtggcctt gagcacccag tggagggcca gaccaatcgc ctccttagtg tactggcgct 14580tgtagtacag gttcctctcc tcgctcttgt aggacatgac gccgccgatg gattggaggc 14640accagatgaa cctaagggat gcaatggtgg gaatgaagta gtactgctgg tacttgatga 14700gattccgagt cgaaggagta gccctcttga catcttcggg agaccaagcc aggatgggga 14760ggttatcaat atctgggtca tggccgacca cattggtggc ggcatggtga ccattgtgcc 14820tatccttcca ccaagtctgt gagtatccct gcatcacgtt accgaaaaag agaccgaaag 14880cgtcgttaat cttgcggttt gtgaaaacct ggtgatggca atagtcgtgg gacaaccagc 14940ctagctgttg gtagcacacg ccaagaacca aagcagcggt gaaataccaa ttcgtgtaca 15000cggtcatcaa gatggaaagg accatcaaac cgacggtagt tgaagtcttg taaaaatacc 15060aaaggaaact cgtctcgaac atacccttgg agatgaattc ctcccgcaac ttgcggaaat 15120cctcctgggg ctcatcacgc ttaggcttgg gtgcaacagg tgtgtcagga gaggaaggct 15180ccataacagg cattctcttc aacttggcga cggcttcttg agagtgcatc accatgaaga 15240catcggtcgc atcgcggttg cgatagttct cgataatgtc agctcctcca gggtggtgat 15300tgacccaagc agacacatca taagttgcgc catcaattgt gattggcaga gcttgccgct 15360taggagacat ggtgcggccg cggtgatgac tgatgagtgt ttaaggacca atggagagaa 15420tgtttgagtt gtgaagcgga gaacctgagg cgtggttatt tatagggaag agaggaaggt 15480gaatgaggga cacgtcacag aagtagggtg ctgagcttga gacattcttc agtatgcatg 15540gctatggaag ccttgggtgc tacacctcat gaagttcatg gtgtgaggtg gcttcggcat 15600ctcaattaag tgacaaagag aaaggtgttt cagtgtttct attgcaaatg gcagaaactc 15660gtgatgacga ggggaccatg catggtttca tttcttttct tcctggattc tttctttcct 15720tttatatatg caggttcata atttaaaaat tagactcgct ttcaatttct taatttctca 15780ttttcctctt atattactgt actaatgtta accacgtaca cttatttttt ttttagttta 15840attttgatag attgtgttga tttaaacata ttaatatttt caaccaaata aaaatcattt 15900tagtagatac ggctttttaa ataattatta aaaatattaa ctatttatcc taaatggcac 15960attttaatta aaaaaaatcc ggtgttgtaa gtgttttatt aatttgtttt ggcattatta 16020aagcaacttt ttttttattt gttggcattt tgagtacgta cttaggctag cctgcaggag 16080atccaagctt ttgatccatg cccttcattt gccgcttatt aattaatttg gtaacagtcc 16140gtactaatca gttacttatc cttcccccat cataattaat cttggtagtc tcgaatgcca 16200caacactgac tagtctcttg gatcataaga aaaagccaag gaacaaaaga agacaaaaca 16260caatgagagt atcctttgca tagcaatgtc taagttcata aaattcaaac aaaaacgcaa 16320tcacacacag tggacatcac ttatccacta gctgatcagg atcgccgcgt caagaaaaaa 16380aaactggacc ccaaaagcca tgcacaacaa cacgtactca caaaggtgtc aatcgagcag 16440cccaaaacat tcaccaactc aacccatcat gagccctcac atttgttgtt tctaacccaa 16500cctcaaactc gtattctctt ccgccacctc atttttgttt atttcaacac ccgtcaaact 16560gcatgccacc ccgtggccaa atgtccatgc atgttaacaa gacctatgac tataaatagc 16620tgcaatctcg gcccaggttt tcatcatcaa gaaccagttc aatatcctag tacaccgtat 16680taaagaattt aagatatact gcggccgcac catggaggtg gtgaatgaaa tagtctcaat 16740tgggcaggaa gttttaccca aagttgatta tgcccaactc tggagtgatg ccagtcactg 16800tgaggtgctt tacttgtcca tcgcatttgt catcttgaag ttcactcttg gcccccttgg 16860tccaaaaggt cagtctcgta tgaagtttgt tttcaccaat tacaaccttc tcatgtccat 16920ttattcgttg ggatcattcc tctcaatggc atatgccatg tacaccatcg gtgttatgtc 16980tgacaactgc gagaaggctt ttgacaacaa cgtcttcagg atcaccacgc agttgttcta 17040tttgagcaag ttcctggagt atattgactc cttctatttg ccactgatgg gcaagcctct 17100gacctggttg caattcttcc atcatttggg ggcaccgatg gatatgtggc tgttctataa 17160ttaccgaaat gaagctgttt ggatttttgt gctgttgaat ggtttcatcc actggatcat 17220gtacggttat tattggacca gattgatcaa gctgaagttc cccatgccaa aatccctgat 17280tacatcaatg cagatcattc aattcaatgt tggtttctac attgtctgga agtacaggaa 17340cattccctgt tatcgccaag atgggatgag gatgtttggc tggttcttca attactttta 17400tgttggcaca gtcttgtgtt tgttcttgaa tttctatgtg caaacgtata tcgtcaggaa 17460gcacaaggga gccaaaaaga ttcagtgagc ggccgcaagt atgaactaaa atgcatgtag 17520gtgtaagagc tcatggagag catggaatat tgtatccgac catgtaacag tataataact 17580gagctccatc tcacttcttc tatgaataaa caaaggatgt tatgatatat taacactcta 17640tctatgcacc ttattgttct atgataaatt tcctcttatt attataaatc atctgaatcg 17700tgacggctta tggaatgctt caaatagtac aaaaacaaat gtgtactata agactttcta 17760aacaattcta accttagcat tgtgaacgag acataagtgt taagaagaca taacaattat 17820aatggaagaa gtttgtctcc atttatatat tatatattac ccacttatgt attatattag 17880gatgttaagg agacataaca attataaaga gagaagtttg tatccattta tatattatat 17940actacccatt tatatattat acttatccac ttatttaatg tctttataag gtttgatcca 18000tgatatttct aatattttag ttgatatgta tatgaaaggg tactatttga actctcttac 18060tctgtataaa ggttggatca tccttaaagt gggtctattt aattttattg cttcttacag 18120ataaaaaaaa aattatgagt tggtttgata aaatattgaa ggatttaaaa taataataaa 18180taacatataa tatatgtata taaatttatt ataatataac atttatctat aaaaaagtaa 18240atattgtcat aaatctatac aatcgtttag ccttgctgga cgaatctcaa ttatttaaac 18300gagagtaaac atatttgact ttttggttat ttaacaaatt attatttaac actatatgaa 18360attttttttt ttatcagcaa agaataaaat taaattaaga aggacaatgg tgtcccaatc 18420cttatacaac caacttccac aagaaagtca agtcagagac aacaaaaaaa caagcaaagg 18480aaatttttta atttgagttg tcttgtttgc tgcataattt atgcagtaaa acactacaca 18540taaccctttt agcagtagag caatggttga ccgtgtgctt agcttctttt attttatttt 18600tttatcagca aagaataaat aaaataaaat gagacacttc agggatgttt caacaagctt 18660gg 18662

Claims

1. An isolated polynucleotide comprising: (a) a nucleotide sequence encoding a polypeptide having delta-8 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:47, SEQ ID NO:49 or SEQ ID NO:57; (b) a nucleotide sequence encoding a polypeptide having delta-8 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:92, SEQ ID NO:93 or SEQ ID NO:62; (c) a nucleotide sequence encoding a polypeptide having delta-8 desaturase activity, wherein the nucleotide sequence hybridizes under stringent conditions to a nucleotide sequence as set forth in SEQ ID NO:92, SEQ ID NO:93 or SEQ ID NO:62; or (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.

2-45. (canceled)

46. The polynucleotide of claim 1 wherein the nucleotide sequence comprises SEQ ID NO:92, SEQ ID NO:93 or SEQ ID NO:62.

47. The polypeptide of claim 1, wherein the amino acid sequence of the polypeptide comprises (a) SEQ ID NO:47, SEQ ID NO:49 or SEQ ID NO:57; or (b) an amino acid sequence that differs from the amino acid sequences in (a) by at least one conservative amino acid substitution.

48. A recombinant DNA construct comprising the polynucleotide of claim 1 operably linked to at least one regulatory sequence.

49. A cell comprising in its genome the recombinant DNA construct of claim 48.

50. An oilseed plant comprising in its genome the recombinant construct of claim 48.

51. A transgenic seed comprising in its genome the recombinant construct of claim 48.

52. Oil or by-products obtained from the seed of claim 51.

53. Progeny plants obtained from the seed of claim 51.

54. Food or feed comprising an ingredient derived from the processing of the seed of claim 51.

55. Food or feed which incorporates the oil of claim 52.

56. The oil or by-products of claim 52 wherein the by-product is lecithin.

57. A method for producing a transformed plant comprising transforming a plant cell with the polynucleotide of claim 1 and regenerating a plant from the transformed plant cell.

58. A transgenic seed obtained from the plant made by the method of claim 57.

59. Progeny plants obtained from seed of claim 58.

60. Oil or by-products obtained from the seed of claim 58.

61. A method for making long-chain polyunsaturated fatty acids in a cell having a reduced level of by-product fatty acids, said method comprising: (a) transforming a host cell with at least one recombinant DNA construct comprising an isolated polynucleotide encoding at least two delta-8 desaturases operably linked to at least one regulatory sequence; and (b) selecting those transformed host cells obtained having a reduced level of by-product fatty acids, when compared to the level of such metabolic by-product fatty acids in a transformed host cell having at least one recombinant DNA construct comprising an isolated polynucleotide encoding one delta-8 desaturase operably linked to a regulatory sequence.

62. A method for producing at least one polyunsaturated fatty acid in an oilseed plant cell comprising: (a) transforming an oilseed plant cell with a first recombinant DNA construct comprising an isolated polynucleotide encoding at least one delta-8 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; (b) regenerating an oilseed plant from the transformed cell of step (a); and (c) selecting those seeds obtained from the plants of step (b) having an altered level of polyunsaturated fatty acids when compared to the level in seeds obtained from a nontransformed oilseed plant.

63. Oil or by-products obtained from the seeds of claim 62.

64. Food or feed which incorporates the oil of claim 63.

65. The oilseed plant of claim 50 comprising: (a) a first recombinant DNA construct comprising an isolated polynucleotide encoding at least one delta-8 desaturase polypeptide, operably linked to at least one regulatory sequence; and (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.

66. A transgenic seed obtained from the oilseed plant of claim 65.

67. Progeny plants obtained from the seed of claim 66.