Patent application title: DELTA-5 DESATURASE AND ITS USE IN MAKING POLYUNSATURATED FATTY ACIDS
Inventors:
Howard Glenn Damude (Hockessin, DE, US)
Quinn Qun Zhu (West Chester, PA, US)
IPC8 Class: AA23D904FI
USPC Class:
426 33
Class name: Food or edible material: processes, compositions, and products fermentation processes isolated triglyceride other than milk derived
Publication date: 2012-03-29
Patent application number: 20120076894
Abstract:
Isolated nucleic acid fragments and recombinant constructs comprising
such fragments encoding delta-5 desaturase along with a method of making
long chain polyunsaturated fatty acids (PUFAs) using this delta-5
desaturase in plants and oleaginous yeast are disclosed.Claims:
1-21. (canceled)
22. Oil or by-products obtained from a transgenic seed obtained from an oilseed plant produced by a method comprising: (a) transforming an oilseed plant cell with a recombinant DNA construct comprising an isolated polynucleotide comprising: (i) a nucleotide sequence encoding a polypeptide having delta-5 desaturase activity, wherein the polypeptide has at least 80% amino acid identity, based on the Clustal W method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:2; (ii) a nucleotide sequence encoding a polypeptide having delta-5 desaturase activity, wherein the nucleotide sequence has at least 80% sequence identity, based on the BLASTN method of alignment, when compared to a nucleotide sequence as set forth in SEQ ID NO:1 or SEQ ID NO:3; (iii) a nucleotide sequence encoding a polypeptide having delta-5 desaturase activity, wherein the nucleotide sequence selectively hybridizes under stringent conditions to a nucleotide sequence as set forth in SEQ ID NO:1 or SEQ ID NO:3; or (iv) a complement of the nucleotide sequence of (a), (b) or (c), wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary, operably linked to at least one regulatory sequence and at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8 desaturase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase a delta-9 desaturase a delta-9 elongase a C14/16 elongase a C16/18 elongase, a C18/20 elongase and a C20/22 elongase; (b) regenerating an oilseed plant from the transformed cell of step (a); and (c) selecting those seeds obtained from the plants of step (b) having an altered level of polyunsaturated fatty acids when compared to the level in seeds obtained from a nontransformed oilseed plant.
23. The oil or by-products of claim 22, wherein the oilseed plant is selected from the group consisting of soybean, Brassica species, sunflower, maize, cotton, flax, and safflower.
24. (canceled)
25. Food or feed which incorporates the oil of claim 22.
26. Food or feed which incorporates the oil of claim 23.
27-29. (canceled)
30. Food or feed comprising an ingredient derived from the processing of a transgenic seed obtained from an oilseed plant produced by a method comprising: (a) transforming an oilseed plant cell with a recombinant DNA construct comprising an isolated polynucleotide comprising: (i) a nucleotide sequence encoding a polypeptide having delta-5 desaturase activity, wherein the polypeptide has at least 80% amino acid identity, based on the Clustal W method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:2; (ii) a nucleotide sequence encoding a polypeptide having delta-5 desaturase activity, wherein the nucleotide sequence has at least 80% sequence identity, based on the BLASTN method of alignment, when compared to a nucleotide sequence as set forth in SEQ ID NO:1 or SEQ ID NO:3; (iii) a nucleotide sequence encoding a polypeptide having delta-5 desaturase activity, wherein the nucleotide sequence selectively hybridizes under stringent conditions to a nucleotide sequence as set forth in SEQ ID NO:1 or SEQ ID NO:3; or (iv) a complement of the nucleotide sequence of (a), (b) or (c), wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary, operably linked to at least one regulatory sequence and at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8 desaturase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a delta-9 desaturase, a delta-9 elongase, a C14/16 elongase, a C16/18 elongase, a C18/20 elongase and a C20/22 elongase; (b) regenerating an oilseed plant from the transformed cell of step (a); and (c) selecting those seeds obtained from the plants of step (b) having an altered level of polyunsaturated fatty acids when compared to the level in seeds obtained from a nontransformed oilseed plant.
31. The food or feed of claim 30, wherein the oilseed plant is selected from the group consisting of soybean, Brassica species, sunflower, maize, cotton, flax, and safflower.
32. The oil or by-products of claim 22 wherein the by-product is lecithin.
33. The oil or by-products of claim 23 wherein the by-product is lecithin.
34-36. (canceled)
37. The oil or by-products of claim 22, wherein the nucleotide sequence comprises SEQ ID NO:1 or SEQ ID NO:3.
38. The oil or by-products of claim 22, wherein the amino acid sequence of the polypeptide comprises (a) SEQ ID NO:2; or (b) an amino acid sequence that differs from the amino acid sequences in (a) by at least one conservative amino acid substitution.
39. The oil or by-products of claim 30, wherein the nucleotide sequence comprises SEQ ID NO:1 or SEQ ID NO:3.
40. The oil or by-products of claim 30, wherein the amino acid sequence of the polypeptide comprises (a) SEQ ID NO:2; or (b) an amino acid sequence that differs from the amino acid sequences in (a) by at least one conservative amino acid substitution.
Description:
[0001] This application claims the benefit of U.S. Provisional Patent
Application 60/801,119, filed May 17, 2006, the entire contents of which
are herein incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention is in the field of biotechnology. More specifically, this invention pertains to the identification of nucleic acid fragments encoding a delta-5 fatty acid desaturase enzyme and the use of this desaturase in making long chain polyunsaturated fatty acids (PUFAs).
BACKGROUND OF THE INVENTION
[0003] The importance of PUFAs is undisputed. For example, certain PUFAs are important biological components of healthy cells and are recognized as: "essential" fatty acids that cannot be synthesized de novo in mammals and instead must be obtained either in the diet or derived by further desaturation and elongation of linoleic acid (LA;18:2 omega-6) or α-linolenic acid (ALA; 18:3 omega-3); constituents of plasma membranes of cells, where they may be found in such forms as phospholipids or triacylglycerols; necessary for proper development (particularly in the developing infant brain) and for tissue formation and repair; and, precursors to several biologically active eicosanoids of importance in mammals (e.g., prostacyclins, eicosanoids, leukotrienes, prostaglandins). Additionally, a high intake of long-chain omega-3 PUFAs produces cardiovascular protective effects (Dyerberg, J. et al., Amer. J. Clin. Nutr., 28:958-966 (1975); Dyerberg, J. et al., Lancet, 2(8081)117-119 (July 15, 1978); Shimokawa, H., World Rev. Nutr. Diet, 88:100-108 (2001); von Schacky, C. and Dyerberg, J., World Rev. Nutr. Diet, 88:90-99 (2001)). And, numerous other studies document wide-ranging health benefits conferred by administration of omega-3 and/or omega-6 PUFAs against a variety of symptoms and diseases (e.g., asthma, psoriasis, eczema, diabetes, cancer).
[0004] A variety of different hosts including plants, algae, fungi and yeast are being investigated as means for commercial PUFA production. Genetic engineering has demonstrated that the natural abilities of some hosts (even those natively limited to LA and ALA fatty acid production) can be substantially altered to result in high-level production of various long-chain omega-3/omega-6 PUFAs. Whether this is the result of natural abilities or recombinant technology, production of arachidonic acid (ARA; 20:4 omega-6), eicosapentaenoic acid (EPA; 20:5 omega-3) and docosahexaenoic acid (DHA; 22:6 omega-3) may all require expression of a delta-5 desaturase.
[0005] Most delta-5 desaturase enzymes identified so far have the primary ability to convert dihomo-gamma-linolenic acid (DGLA; 20:3 omega-6) to ARA, with secondary activity in converting eicosatetraenoic acid (ETA; 20:4 omega-3) to EPA (where DNA is subsequently synthesized from EPA following reaction with an additional C20/22 elongase and a delta-4 desaturase), The delta-5 desaturase has a role in both the delta-6 desaturase/delta-6 elongase pathway (which is predominantly found in algae, mosses, fungi, nematodes and humans and which is characterized by the production of gamma-linolenic acid (GLA; 18:3 omega-6) omegaand/or stearidonic acid (STA; 18:4 omega-3))omega and the delta-9 elongase/delta-8 desaturase pathway (which operates in some organisms, such as euglenoid species and which is characterized by the production of eicosadienoic acid (EDA; 20:2 omega-6)omega and/or eicosatrienoic acid (ETrA; 20:3 omega-3)) omega (FIG. 1).
[0006] Based on the role delta-5 desaturase enzymes play in the synthesis of e.g., ARA, EPA and DHA, there has been considerable effort to identify and characterize these enzymes from various sources. As such, numerous delta-5 desaturases have been disclosed in both the open literature (e.g., GenBank Accession No. AF199596, No. AF226273, No. AF320509, No. AB072976, No. AF489588, No. AJ510244, No. AF419297, No. AF07879, No. AF067654 and No. AB022097) and the patent literature (e.g., U.S. Pat. Nos. 5,972,664 and 6,075,183). Also, commonly owned, co-pending application having Provisional Application No. 60/801,172 (filed May 17, 2006) discloses amino acid and nucleic acid sequences for a delta-5 desaturase enzyme from Euglena gracilis, while commonly owned, co-pending application having Provisional Application No. 60/915733 (BB1614) (filed May 3, 2007) discloses amino acid and nucleic acid sequences for a delta-5 desaturase enzyme from Euglena anabaena.
[0007] The instant invention concerns the identification and isolation of additional genes encoding delta-5 desaturases from Peridinium sp. CCMP626 that would be suitable for heterologous expression in a variety of host organisms for use in the production of omega-3/omega-6 fatty acids.
SUMMARY OF THE INVENTION
[0008] The present invention concerns an isolated polynucleotide comprising: [0009] (a) a nucleotide sequence encoding a polypeptide having delta-5 desaturase activity, wherein the polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 01100% amino acid identity, based on the Clustal W method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:2; [0010] (b) a nucleotide sequence encoding a polypeptide having delta-5 desaturase activity, wherein the nucleotide sequence has at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity, based on the BLASTN method of alignment, when compared to a nucleotide sequence as set forth in SEQ ID NO:1 or SEQ ID NO:3;
[0011] (c) a nucleotide sequence encoding a polypeptide having delta-5 desaturase activity, wherein the nucleotide sequence hybridizes under stringent conditions to a nucleotide sequence as set forth in SEQ ID NO:1 or SEQ ID NO:3; or [0012] (d) a complement of the nucleotide sequence of (a), (b) or (c), wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.
[0013] In a second embodiment, the invention concerns a recombinant DNA construct comprising any of the isolated polynucleotides of the invention operably linked to at least one regulatory sequence.
[0014] In a third embodiment, the invention concerns a cell comprising in its genome the recombinant DNA construct of the invention. Such cells can be plant cells or yeast cells.
[0015] In a fourth embodiment, the invention concerns a method for transforming a cell, comprising transforming a cell with a recombinant construct of the invention or an isolated polynucleotide of the invention and selecting those cells transformed with the recombinant construct or the isolated polynucleotide.
[0016] In a fifth embodiment, the invention concerns transgenic seed comprising in its genome the recombinant construct of the invention or a transgenic seed obtained from a plant made by a method of the invention. Also of interest is oil or by-products obtained from such transgenic seeds. In a sixth embodiment, the invention concerns a method for making long-chain polyunsaturated fatty acids in a plant cell comprising: [0017] (a) transforming a cell with the recombinant construct of the invention; and [0018] (b) selecting those transformed cells that make long-chain polyunsaturated fatty acids.
[0019] In a seventh embodiment, the invention concerns a method for producing at least one polyunsaturated fatty acid in an oilseed plant cell comprising: [0020] (a) transforming an oilseed plant cell with a first recombinant DNA construct comprising an isolated polynucleotide encoding at least one delta-5 desaturase polypeptide, operably linked to at least one regulatory sequence and at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8 desaturase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a delta-9 desaturase, a delta-9 elongase, a C14/16 elongase, a C16/18 elongase, a C18/20 elongase and a C20/22 elongase; [0021] (b) regenerating an oilseed plant from the transformed cell of step (a); and [0022] (c) selecting those seeds obtained from the plants of step (b) having an altered level of polyunsaturated fatty acids when compared to the level in seeds obtained from a nontransformed oilseed plant.
[0023] In an eighth embodiment, the invention concerns an oilseed plant comprising in its genome the recombinant construct of the invention. Suitable oilseed plants include, but are not limited to, soybean, Brassica species, sunflower, maize, cotton, flax and safflower.
[0024] In a ninth embodiment, the invention concerns an oilseed plant comprising: [0025] (a) a first recombinant DNA construct comprising an isolated polynucleotide encoding at least one delta-5 desaturase polypeptide, operably linked to at least one regulatory sequence; and [0026] (b) at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8 desaturase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a delta-9 desaturase, a delta-9 elongase, a C14/16 elongase, a C16/18 elongase, a C16/20 elongase and a C20/22 elongase.
[0027] Also of interest are transgenic seeds obtained from such oilseed plants as well as oil or by-products obtained from these transgenic seeds. A preferred by-product is lecithin.
[0028] In a tenth embodiment, the invention concerns food or feed incorporating an oil or seed of the invention or food or feed comprising an ingredient derived from the processing of the seeds.
[0029] In an eleventh embodiment, the invention concerns progeny plants obtained from obtained from a plant made by the method of the invention or an oilseed plant of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS
[0030] FIG. 1 illustrates the omega-3/omega-6 fatty acid biosynthetic pathway.
[0031] FIG. 2 shows a chromatogram of the lipid profile of a Peridinium sp. CCMP626 cell extract as described in Example 1.
[0032] FIG. 3 shows a portion of an alignment between and among delta-5 desaturase proteins and delta-8 desaturase proteins using a Clustal W analysis (MegAlign® program of DNASTAR software).
[0033] FIG. 4 graphically represents the relationship between SEQ ID NOs:1, 2, 4, 5, 6, 8 and 10, each of which relates to the Peridinium sp. CCMP626 delta-5 desaturase.
[0034] FIG. 5A illustrates the cloning strategy utilized for amplification of the Peridinium sp. CCMP626 delta-5 desaturase gene (RD5). FIG. 5B is a plasmid map of pZUF17, while FIG. 5C is a plasmid map of pDMW368.
[0035] FIG. 6 provides plasmid maps for the following: (A) pKUNF12T6E; (B) pRD5S; and, (C) pZURD5S.
[0036] FIGS. 7A and 7B show a comparison of the DNA sequence of the Peridinium sp. CCMP626 delta-5 desaturase gene (designated as "RD5"; SEQ ID NO:1) and the synthetic gene (designated as "RD5S"; SEQ ID NO:3) codon-optimized for expression in Yarrowia lipolytica.
[0037] FIGS. 8A and 8B show a Clustal V alignment (with default parameters) of a Pavlova lutheri delta-8 desaturase (SEQ ID NO:18), a Pavlova salina delta-8 desaturase (SEQ ID NO:66), a Euglena gracilis delta-8 desaturase (SEQ ID NO:16) and two different Rhizopus stolonifer delta-6 fatty acid desaturases (SEQ ID NOs:53 and 65).
[0038] FIG. 9 provides a plasmid map for pY98.
[0039] FIG. 10A provides the fatty acid profiles for Yarrowia lipolytica expressing pY98 (SEQ ID NO:76; comprising a Mortierella alpina delta-5 desaturase gene designated as "MaD5") or pDMW368 (SEQ ID NO:23; comprising the Peridinium sp. CCMP626 delta-5 desaturase gene designated as "RD5") and fed various substrates. FIG. 10B provides a comparison of the omega-3 and omega-6 substrate specificity of MaD5 versus RD5.
[0040] FIG. 11 provides plasmid maps for the following: (A) pKR916; (B) pKR1038; and, (C) pKR328.
[0041] FIG. 12A provides the average fatty acid profiles for ten events having the highest delta-5 desaturase activity when the Mortierella alpina enzyme (MaD5) is transformed into soybean embryos. FIG. 126 provides the average fatty acid profiles for ten events having the highest delta-5 desaturase activity when the Peridinium sp. CCMP626 enzyme (RD5) is transformed into soybean embryos. Fatty acids are identified as 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic acid), LA, ALA, EDA, SCI, DGLA, ARA, ERA, JUP, ETA and EPA. Fatty acids listed as "others" include: 18:2 (5,9), GLA, STA, 20:0, 20:1(11), 20:2 (7,11) or 20:2 (8,11) and DPA. Each of these "other" fatty acids is present at a relative abundance of less than 3.0% of the total fatty acids. Fatty acid compositions for an individual embryo were expressed as the weight percent (wt. %) of total fatty acids and the average fatty acid composition is an average of six individual embryos for each event.
[0042] FIG. 13 provides the activity of the delta-5 desaturase for the "correct' substrates (i.e., DGLA and ETA) versus the "wrong" substrates (i.e., EDA and ERA). The activity of the delta-5 desaturase for the "correct" substrates ("Correct % delta-5 desat") is plotted on the x-axis and the activity of the delta-5 desaturase for the "wrong" substrates ("Wrong % delta-5 desat") is plotted on the y-axis for MaD5(see FIG. 12A) or RD5 (see FIG. 126).
[0043] The invention can be more fully understood from the following detailed description and the accompanying sequence descriptions, which form a part of this application.
[0044] The following sequences comply with 37 C.F.R. §1.821-1.825 ("Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures--the Sequence Rules") and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.
[0045] SEQ ID NOs:1-26, 50, 51, 53-56, 63-72 and 75-76 are ORFs encoding genes or proteins (or portions thereof), or plasmids, as identified in Table 1.
TABLE-US-00001 TABLE 1 Summary Of Nucleic Acid And Protein SEQ ID Numbers Nucleic acid Protein Description and Abbreviation SEQ ID NO. SEQ ID NO. Peridinium sp. CCMP626 delta-5 1 2 desaturase ("RD5") (1392 bp) (463 AA) Synthetic delta-5 desaturase, derived from 3 2 Peridinium sp. CCMP626, codon-- (1392 bp) (463 AA) optimized for expression in Yarrowia lipolytica ("RD5S") Peridinium sp. CCMP626--fragment of 4 5 pT-12-D5 (563 bp) (187 AA) Peridinium sp. CCMP626--fragment of 6 -- pT-RD5-5'C2 (693 bp) Peridinium sp. CCMP626--5' sequence 7 -- relative to SEQ ID NO: 4 (511 bp) Peridinium sp. CCMP626--fragment of 8 -- pT-RD5-5'2nd (358 bp) Peridinium sp. CCMP626--5' sequence 9 -- relative to SEQ ID NO: 6 (161 bp) Peridinium sp. CCMP626--fragment of 10 -- pT-RD5-3' (299 bp) Peridinium sp. CCMP626--3' sequence 11 -- relative to SEQ ID NO: 4 (247 bp) Pythium irregulare delta-5 desaturase -- 12 (GenBank Accession No. AAL13311) (456 AA) Phytophthora megasperma delta-5 -- 13 desaturase (GenBank Accession No. (477 AA) CAD53323) Phaeodactylum tricornutum delta-5 -- 14 desaturase (GenBank Accession No. (469 AA) AAL92562) Dictyostelium discoideum delta-5 -- 15 desaturase (GenBank Accession No. (467 AA) XP_640331) Euglena gracilis delta-8 desaturase (PCT -- 16 Publications No. WO 2006/012325 and (421 AA) No. WO 2006/012326) Pavlova lutheri (CCMP459) delta-8 17 18 desaturase (1269 bp) (423 AA) Conserved Region 1 -- 19 (7 AA) Conserved Region 2 -- 20 (7 AA) Thalassiosira pseudonana delta-8 -- 21 sphingolipid desaturase (GenBank (476 AA) Accession No. AAX14502) Plasmid pZUF17 22 -- (8165 bp) Plasmid pDMW368 23 -- (8480 bp) Plasmid pKUNF12T6E 24 -- (12,649 bp).sup. Synthetic C18/20 elongase gene derived 25 26 from Thraustochytrium aureum (U.S. (819 bp) (272 AA) Pat. No. 6,677,145), codon-optimized for expression in Yarrowia lipolytica ("EL2S") Plasmid pRD5S 50 -- (4112 bp) Plasmid pZURD5S 51 -- (8480 bp) Rhizopus stolonifer delta-6 fatty acid -- 53 desaturase (NCBI Accession No. (459 AA) AAX22052) Pavlova lutheri delta-8 desaturase-- 54 -- portion of cDNA insert from clone (695 bp) eps1c.pk002.f22 (5' end of cDNA insert) Pavlova lutheri delta-8 desaturase--fully 55 -- sequenced EST eps1c.pk002.f22: fis (full (1106 bp) insert sequence) Pavlova lutheri delta-8 desaturase-- -- 56 translation of nucleotides 1-864 of fully (287 AA) sequenced EST eps1c.pk002.f22: fis (full insert sequence; SEQ ID NO: 55) Pavlova lutheri delta-8 desaturase--full 5' 63 -- end sequence from genome walking (1294 bp) Pavlova lutheri delta-8 desaturase-- 64 -- assembled sequence (1927 bp) Rhizopus stolonifer delta-6 fatty acid -- 65 desaturase (NCBI Accession No. (459 AA) ABB96724) Pavlova salina delta-8 desaturase -- 66 (427 AA) Mortierella alpina delta-5 desaturase 67 68 (1338 bp) (446 AA) Plasmid pY5-22 69 -- (6473 bp) Plasmid pY5-22GPD 70 -- (6970 bp) Yarrowia lipolytica glyceraldehyde-3- 71 -- phosphate dehydrogenase promoter (968 bp) (GPD) Plasmid pYZDE2-S 72 -- (8630 bp) Plasmid pKR136 75 -- (6339 bp) Plasmid pY98 76 -- (8319 bp) Euglena gracilis delta-9 elongase 77 -- ("EgD9e") (774 bp) Euglena gracilis delta-8 desaturase 78 -- ("EgD8") (1263 bp) Plasmid pKR906 81 -- (4311 bp) Plasmid pKR72 82 -- (7085 bp) Plasmid pKS102 83 -- (2540 bp) Plasmid pKR197 84 -- (4359 bp) Plasmid pKR911 85 -- (5147 bp) Plasmid pKR680 86 -- (6559 bp) Plasmid pKR913 87 -- (9014 bp) Plasmid pKR767 88 -- (5561 bp) Plasmid pKR916 89 -- (11,889 bp).sup. Plasmid pKR974 90 -- (5661 bp) Saprolegnia diclina delta-5 desaturase 91 -- ("SdD5") (1413 bp) Plasmid pKR1033 92 -- (5621 bp) Plasmid pKR1038 93 -- (11,949 bp).sup. Plasmid pKR328 94 -- (8671 bp)
[0046] SEQ ID NOs:27-29 correspond to AP primer, Smart IV oligonucleotide primer and CDSIII 5' primer, respectively, used for Peridinium sp. CCMP626 cDNA synthesis.
[0047] SEQ ID NOs:30-33 correspond to degenerate oligonucleotide primers 5-1A, 5-1 B, 5-1C and 5-1D, respectively, that encode Conserved Region 1.
[0048] SEQ ID NOs:34-37 correspond to degenerate oligonucleotide primers 5-4AR, 5-4BR, 5-4CR and 5-4DR, respectively, that encode Conserved Region 2.
[0049] SEQ ID NOs:38-42 correspond to primers ODMW520, ODMW521, DNR CDS 5', ODMW541 and ODMW542, respectively, used for 5' RACE.
[0050] SEQ ID NOs:43-45 correspond to primers ODMW523, AUAP and ODMW524, respectively, used for 3' RACE.
[0051] SEQ ID NOs:46-49 correspond to primers YL807, YL810, YL808 and YL809, respectively, used for amplification of the full length cDNA of RD5.
[0052] SEQ ID NO:52 corresponds to primer 17, used for sequencing the Pavlova lutheri (CCMP459) cDNA library.
[0053] SEQ ID NOs:57 and 58 correspond to primers SeqE and SeqW, respectively, used for sequencing Pavlova lutheri (CCMP459) clones.
[0054] SEQ ID NOs:59 and 60 correspond to the universal primer AP1 and primer GSP PvDES, respectively, used for amplification of genomic Pavlova lutheri (CCMP459) DNA.
[0055] SEQ ID NOs:61 and 62 correspond to primers M13-28Rev and PavDES seq, respectively, used for sequencing Pavlova lutheri (CCMP459) genomic inserts.
[0056] SEQ ID NOs:73 and 74 are primers GPDsense and GPDantisense, respectively, used for amplifying the GPD promoter.
[0057] SEQ ID NOs:79 and 80 correspond to primers oEugEL1-1 and oEugEL1-2, respectively, used to amplify a Euglena gracilis delta-9 elongase (EgD9e).
DETAILED DESCRIPTION OF THE INVENTION
[0058] All patents, patent applications, and publications cited herein are incorporated by reference in their entirety. This specifically includes the following Applicants' Assignee's co-pending applications: U.S. Pat. No. 7,125.672, U.S. Pat. No. 7,189,559, U.S. Pat. No. 7,192,762, U.S. Pat. No. 7,198,937, U.S. Pat. No. 7,202,356, U.S. patent applications Ser. No. 10/840,579 and Ser. No. 10/840,325 (filed May 6, 2004), U.S. patent application Ser. No. 10/869,630 (filed Jun. 16, 2004), U.S. patent application Ser. No. 10/882,760 (filed Jul. 1, 2004), U.S. patent applications Ser. No. 10/985,254 and Ser. No. 10/985,691 (filed Nov. 10, 2004), U.S. patent application Ser. No. 11/024,544 (filed Dec. 29, 2004), U.S. patent application Ser. No. 11/166,993 (filed Jun. 24, 2005), U.S. patent application Ser. No. 11/183,664 (filed Jul. 18, 2005), U.S. patent application Ser. No. 11/185,301 (filed Jul. 20, 2005), U.S. patent application Ser. No. 11/190,750 (filed Jul. 27, 2005), U.S. patent application Ser. No. 11/198,975 (filed Aug. 8, 2005), U.S. patent application Ser. No. 11/225,354 (filed Sep. 13, 2005), U.S. patent application Ser. No. 11/253,882 (filed Oct. 19, 2005), U.S. patent applications Ser. No. 11/264784 and Ser. No. 11/264,737 (filed Nov. 1, 2005), U.S. patent application Ser. No. 11/265761 (filed Nov. 2, 2005), U.S. patent application Ser. No. 11/737,772 (filed Apr. 20, 2007), U.S. patent application Ser. No. 11/787,772 (filed Apr. 17, 2007), U.S. patent application Ser. No. 11/740,298 (filed Apr. 26, 2007), U.S. Patent Applications No. 60/801,172 and No. 60/801,119 (filed May 17, 2006), U.S. Patent Application No. 60/853,563 (filed Oct. 23, 2006), U.S. Patent Application No. 60/855,177 (filed Oct. 30, 2006), U.S. patent applications Ser. No. 11/601,563 and Ser. No. 11/601,564 (filed Nov. 16, 2006), U.S. patent application Ser. No. 11/635,258 (filed Dec. 7, 2006), U.S. patent application Ser. No. 11/613,420 (filed Dec. 20, 2006), U.S. Patent Application No. 60/909,790 (filed Apr. 3, 2007), U.S. Patent Application No. 60/911,925 (filed Apr. 16, 2007), U.S. Patent Application No. 60/910,831 (filed Apr. 10, 2007) and U.S. Patent Application No. 60/915,733 (BB1614) (filed May 3, 2007). This additionally includes the following Applicants' Assignee's co-pending applications: PCT Publication No. US 2004/0172682, concerning the production of PUFAs in plants; and, U.S. Pat. No. 7,129,089, concerning annexin promoters and their use in expression of transgenes in plants.
[0059] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a plant" includes a plurality of such plants, reference to "a cell" includes one or more cells and equivalents thereof known to those skilled in the art, and so forth.
[0060] In accordance with the subject invention, Applicants identify a novel Peridinium sp. CCMP626 delta-5 desaturase enzyme and gene encoding the same that may be used for the manipulation of biochemical pathways for the production of healthful PUFAs. Thus, the subject invention finds many applications.
[0061] PUFAs, or derivatives thereof, made by the methodology disclosed herein can be used as dietary substitutes, or supplements, particularly infant formulas, for patients undergoing intravenous feeding or for preventing or treating malnutrition. Alternatively, the purified PUFAs (or derivatives thereof) may be incorporated into cooking oils, fats or margarines formulated so that in normal use the recipient would receive the desired amount for dietary supplementation. The PUFAs may also be incorporated into infant formulas, nutritional supplements or other food products and may find use as anti-inflammatory or cholesterol lowering agents. Optionally, the compositions may be used for pharmaceutical use (human or veterinary).
[0062] Supplementation of humans or animals with PUFAs produced by recombinant means can result in increased levels of the added PUFAs, as well as their metabolic progeny. For example, treatment with EPA can result not only in increased levels of EPA, but also downstream products of EPA such as eicosanoids (i.e., prostaglandins, leukotrienes, thromboxanes). Complex regulatory mechanisms can make it desirable to combine various PUFAs, or add different conjugates of PUFAs, in order to prevent, control or overcome such mechanisms to achieve the desired levels of specific PUFAs in an individual.
Definitions
[0063] In this disclosure, a number of terms and abbreviations are used. The following definitions are provided.
[0064] "Open reading frame" is abbreviated ORF.
[0065] "Polymerase chain reaction" is abbreviated PCR.
[0066] "American Type Culture Collection" is abbreviated ATCC.
[0067] "Polyunsaturated fatty acid(s)" is abbreviated PUFA(s).
[0068] "Triacylglycerols" are abbreviated TAGS.
[0069] The term "invention" or "present invention" as used herein is not meant to be limiting to any one specific embodiment of the invention but applies generally to any and all embodiments of the invention as described in the claims and specification.
[0070] The term "fatty acids" refers to long chain aliphatic acids (alkanoic acids) of varying chain lengths, from about C12 to C22 (although both longer and shorter chain-length acids are known). The predominant chain lengths are between C16 and C22. The structure of a fatty acid is represented by a simple notation system of "X:Y", where X is the total number of carbon (C) atoms in the particular fatty acid and Y is the number of double bonds. Additional details concerning the differentiation between "saturated fatty acids" versus "unsaturated fatty acids", "monounsaturated fatty acids" versus "polyunsaturated fatty acids" (or "PUFAs"), and "omega-6 fatty acids" (omega-6 or n-6) versus "omega-3 fatty acids" (omega-3 or n-3) are provided in U.S. Patent Publication No, 2005/0136519.
[0071] Fatty acids are described herein by a simple notation system of "X:Y", wherein X is number of carbon (C) atoms in the particular fatty acid and Y is the number of double bonds. The number following the fatty acid designation indicates the position of the double bond from the carboxyl end of the fatty acid with the "c" affix for the cis-configuration of the double bond (e.g., palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1, 9c), petroselinic acid (18:1, 6c), LA (18:2, 9c,12c), GLA (18:3, 6c,9c,12c) and ALA (18:3, 9c,12c,15c)). Unless otherwise specified, 18:1, 18:2 and 18:3 refer to oleic, LA and ALA fatty acids, respectively. If not specifically written as otherwise, double bonds are assumed to be of the cis configuration. For instance, the double bonds in 18:2 (9,12) would be assumed to be in the cis configuration.
[0072] Nomenclature used to describe PUFAs in the present disclosure is shown below in Table 2. In the column titled "Shorthand Notation", the omega-reference system is used to indicate the number of carbons, the number of double bonds and the position of the double bond closest to the omega carbon, counting from the omega carbon (which is numbered 1 for this purpose). The remainder of the Table summarizes the common names of omega-3 and omega-6 fatty acids and their precursors, the abbreviations that will be used throughout the specification and each compounds' chemical name.
TABLE-US-00002 TABLE 2 Nomenclature of Polyunsaturated Fatty Acids And Precursors Shorthand Common Name Abbreviation Chemical Name Notation Myristic -- tetradecanoic 14:0 Palmitic Palmitate hexadecanoic 16:0 Palmitoleic -- 9-hexadecenoic 16:1 Stearic -- octadecanoic 18:0 Oleic -- cis-9-octadecenoic 18:1 Linoleic LA cis-9,12-octadecadienoic 18:2 omega-6 γ-Linoleic GLA cis-6,9,12- 18:3 omega-6 octadecatrienoic Eicosadienoic EDA cis-11,14-eicosadienoic 20:2 omega-6 Dihomo-γ- DGLA cis-8,11,14- 20:3 omega-6 Linoleic eicosatrienoic Arachidonic ARA cis-5,8,11,14- 20:4 omega-6 eicosatetraenoic α-Linolenic ALA cis-9,12,15- 18:3 omega-3 octadecatrienoic Stearidonic STA cis-6,9,12,15- 18:4 omega-3 octadecatetraenoic Eicosatrienoic ETrA or cis-11,14,17- 20:3 omega-3 ERA eicosatrienoic Sciadonic SCI cis-5,11,14-eicosatrienoic 20:3b omega-6 Juniperonic JUP cis-5,11,14,17- 20:4b omega-3 eicosatetraenoic Eicosa- ETA cis-8,11,14,17- 20:4 omega-3 tetraenoic eicosatetraenoic Eicosa- EPA cis-5,8,11,14,17- 20:5 omega-3 pentaenoic eicosapentaenoic Docosa- DPA cis-7,10,13,16,19- 22:5 omega-3 pentaenoic docosapentaenoic Docosa- DHA cis-4,7,10,13,16,19- 22:6 omega-3 hexaenoic docosahexaenoic
[0073] The terms "triacylglycerol", "oil" and "TAGs" refer to neutral lipids composed of three fatty acyl residues esterified to a glycerol molecule (and such terms will be used interchangeably throughout the present disclosure herein). Such oils can contain long chain PUFAs, as well as shorter saturated and unsaturated fatty acids and longer chain saturated fatty acids. Thus, "oil biosynthesis" generically refers to the synthesis of TAGs in the cell.
[0074] "Percent (%) PUFAs in the total lipid and oil fractions" refers to the percent of PUFAs relative to the total fatty acids in those fractions. The term "total lipid fraction" or "lipid fraction" both refer to the sum of all lipids (i.e., neutral and polar) within an oleaginous organism, thus including those lipids that are located in the phosphatidylcholine (PC) fraction, phosphatidyletanolamine (PE) fraction and triacylglycerol (TAG or oil) fraction. However, the terms "lipid" and "oil" will be used interchangeably throughout the specification.
[0075] A metabolic pathway, or biosynthetic pathway, in a biochemical sense, can be regarded as a series of chemical reactions occurring within a cell, catalyzed by enzymes, to achieve either the formation of a metabolic product to be used or stored by the cell, or the initiation of another metabolic pathway (then called a flux generating step). Many of these pathways are elaborate.sub.; and involve a step by step modification of the initial substance to shape it into a product having the exact chemical structure desired.
[0076] The term "PUFA biosynthetic pathway" refers to a metabolic process that converts oleic acid to LA, EDA, GLA, DGLA, ARA, ALA, STA, ETrA, ETA, EPA, DPA and DHA. This process is well described in the literature (e.g., see PCT Publication No. WO 2006/052870). Briefly, this process involves elongation of the carbon chain through the addition of carbon atoms and desaturation of the molecule through the addition of double bonds, via a series of special desaturation and elongation enzymes (i.e., "PUFA biosynthetic pathway enzymes") present in the endoplasmic reticulin membrane. More specifically, "PUFA biosynthetic pathway enzymes" refer to any of the following enzymes (and genes which encode said enzymes) associated with the biosynthesis of a PUFA, including: a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a delta-9 desaturase, a delta-8 desaturase, a delta-9 elongase, a C14/16 elongase, a C16/18 elongase, a C18/20 elongase and/or a C20/22 elongase.
[0077] The term "omega-3/omega-6 fatty acid biosynthetic pathway" refers to a set of genes which, when expressed under the appropriate conditions encode enzymes that catalyze the production of either or both omega-3 and omega-6 fatty acids. Typically the genes involved in the omega-3/omega-6 fatty acid biosynthetic pathway encode PUFA biosynthetic pathway enzymes. A representative pathway is illustrated in FIG. 1, providing for the conversion of myristic acid through various intermediates to DHA, which demonstrates how both omega-3 and omega-6 fatty acids may be produced from a common source. The pathway is naturally divided into two portions where one portion will generate omega-3 fatty acids and the other portion, only omega-6 fatty acids. That portion that only generates omega-3 fatty acids will be referred to herein as the omega-3 fatty acid biosynthetic pathway, whereas that portion that generates only omega-6 fatty acids will be referred to herein as the omega-6 fatty acid biosynthetic pathway.
[0078] The term "functional" as used herein in context with the omega-3/omega-6 fatty acid biosynthetic pathway means that some (or all) of the genes in the pathway express active enzymes, resulting in in vivo catalysis or substrate conversion. It should be understood that "omega-3/omega-6 fatty acid biosynthetic pathway" or "functional omega-3/omega-6 fatty acid biosynthetic pathway" does not imply that all the genes listed in the above paragraph are required, as a number of fatty acid products will only require the expression of a subset of the genes of this pathway.
[0079] The term "delta-6 desaturase/delta-6 elongase pathway" will refer to a PUFA biosynthetic pathway that minimally includes at least one delta-6 desaturase and at least one C18/20 elongase, thereby enabling biosynthesis of DGLA and/or ETA from LA and ALA, respectively, with GLA and/or STA as intermediate fatty acids. With expression of other desaturases and elongases, ARA, EPA, DPA and DHA may also be synthesized.
[0080] The term "delta-9 elongase/delta-8 desaturase pathway" will refer to a PUFA biosynthetic pathway that minimally includes at least one delta-9 elongase and at least one delta-8 desaturase, thereby enabling biosynthesis of DGLA and/or ETA from LA and ALA, respectively, with EDA and/or ETrA as intermediate fatty acids. With expression of other desaturases and elongases, ARA, EPA, DPA and DHA may also be synthesized.
[0081] The term "intermediate fatty acid" refers to any fatty acid produced in a fatty acid metabolic pathway that can be further converted to an intended product fatty acid in this pathway by the action of other metabolic pathway enzymes. For instance, when EPA is produced using the delta-9 elongase/delta-8 desaturase pathway, EDA, ETrA, DGLA, ETA and ARA can be produced and are considered "intermediate fatty acids" since these fatty acids can be further converted to EPA via action of other metabolic pathway enzymes.
[0082] The term "by-product fatty acid" refers to any fatty acid produced in a fatty acid metabolic pathway that is not the intended fatty acid product of the pathway nor an "intermediate fatty acid" of the pathway. For instance, when EPA is produced using the delta-9 elongase/delta-8 desaturase pathway, sciadonic acid (SGI) and juniperonic acid (JUP) also can be produced by the action of a delta-5 desaturase on either EDA or ETrA, respectively. They are considered to be "by-product fatty acids" since neither can be further converted to EPA by the action of other metabolic pathway enzymes.
[0083] The term "desaturase" refers to a polypeptide that can desaturate, i.e., introduce a double bond, in one or more fatty acids to produce a fatty acid or precursor of interest. Despite use of the omega-reference system throughout the specification to refer to specific fatty acids, it is more convenient to indicate the activity of a desaturase by counting from the carboxyl end of the substrate using the delta-system. Of particular interest herein are delta-5 desaturases that catalyze the conversion of DGLA to ARA and/or ETA to EPA. Other desaturases include: 1.) delta-17 desaturases that desaturate a fatty acid between the 17th and 18th carbon atom numbered from the carboxyl-terminal end of the molecule and which, for example, catalyze the conversion of ARA to EPA and/or DGLA to ETA; 2.) delta-6 desaturases that catalyze the conversion of LA to GLA and/or ALA to STA; 3.) delta-12 desaturases that catalyze the conversion of oleic acid to LA; 4.) delta-15 desaturases that catalyze the conversion of LA to ALA and/or GLA to STA; 5.) delta-4 desaturases that catalyze the conversion of DPA to DHA; 6.) delta-8 desaturases that catalyze the conversion of EDA to DGLA and/or ETrA to ETA; and, 7.) delta-9 desaturases that catalyze the conversion of palmitate to palmitoleic acid (16:1) and/or stearate to oleic acid. In the art, delta-15 and delta-17 desaturases are also occasionally referred to as "omega-3 desaturases", "w-3 desaturases", and/or "omega-3 desaturases", based on their ability to convert omega-6 fatty acids into their omega-3 counterparts (e.g., conversion of LA into ALA and ARA into EPA, respectively). In some embodiments, it is most desirable to empirically determine the specificity of a particular fatty acid desaturase by transforming a suitable host with the gene for the fatty acid desaturase and determining its effect on the fatty acid profile of the host.
[0084] The term "delta-5 desaturase" refers to an enzyme that desaturates a fatty acid between the fifth and sixth carbon atom numbered from the carboxyl-terminal end of the molecule. Preferably, a delta-5 desaturase converts dihomo-gamma-linolenic acid [20:3, DGLA] to arachidonic acid [20:4, ARA] or converts eicosatetraenoic acid [20:4, ETA] to eicosapentaenoic acid [20:5, EPA].
[0085] For the purposes herein, the term "RD5" refers to a delta-5 desaturase enzyme (SEQ ID NO:2) isolated from Peridinium sp. CCMP626, encoded by SEQ ID NO:1 herein. Similarly, the term "RD5S" refers to a synthetic delta-5 desaturase derived from Peridinium sp. CCMP626 that is codon-optimized for expression in Yarrowia lipolytica (i.e., SEQ ID NOs:3 and 2).
[0086] The terms "conversion efficiency" and "percent substrate conversion" refer to the efficiency by which a particular enzyme (e.g., a desaturase) can convert substrate to product. The conversion efficiency is measured according to the following formula: ([product]/[substrate+product])*100, where `product` includes the immediate product and all products in the pathway derived from it.
[0087] The term "elongase" refers to a polypeptide that can elongate a fatty acid carbon chain to produce an acid that is 2 carbons longer than the fatty acid substrate that the elongase acts upon. This process of elongation occurs in a multi-step mechanism in association with fatty acid synthase, as described in U.S. Patent Publication No. 2005/0132442 and PCT Publication No. WO 2005/047480. Examples of reactions catalyzed by elongase systems are the conversion of GLA to DGLA, STA to ETA and EPA to DPA. In general, the substrate selectivity of elongases is somewhat broad but segregated by both chain length and the degree and type of unsaturation. For example, a C14/16 elongase will utilize a C14 substrate (e.g., myristic acid), a C16/18 elongase will utilize a C16 substrate (e.g., palmitate), a C18/20 elongase (also known as a delta-6 elongase as the terms can be used interchangeably) will utilize a C16 substrate (e.g., GLA, STA) and a C20/22 elongase will utilize a C20 substrate (e.g., EPA). in like manner, a delta-9 elongase is able to catalyze the conversion of LA and ALA to EDA and ETrA, respectively. It is important to note that some elongases have broad specificity and thus a single enzyme may be capable of catalyzing several elongase reactions (e.g., thereby acting as both a C16/18 elongase and a C18/20 elongase).
[0088] The term "oleaginous" refers to those organisms that tend to store their energy source in the form of lipid (Weete, In: Fungal Lipid Biochemistry, 2nd Ed., Plenum, 1980). The term "oleaginous yeast" refers to those microorganisms classified as yeasts that can make oil. Generally, the cellular oil or TAG content of oleaginous microorganisms follows a sigmoid curve, wherein the concentration of lipid increases until it reaches a maximum at the late logarithmic or early stationary growth phase and then gradually decreases during the late stationary and death phases (Yongmanitchai and Ward, Appl. Environ. Microbial., 57:419-25 (1991)). It is not uncommon for oleaginous microorganisms to accumulate in excess of about 25% of their dry cell weight as oil. Examples of oleaginous yeast include, but are no means limited to, the following genera: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.
[0089] The term "conservative amino acid substitution" refers to a substitution of an amino acid residue in a given protein with another amino acid, without altering the chemical or functional nature of that protein. For example, it is well known in the art that alterations in a gene that result in the production of a chemically equivalent amino acid at a given site (but that do not affect the structural and functional properties of the encoded, folded protein) are common. For the purposes of the present invention, "conservative amino acid substitutions" are defined as exchanges within one of the following five groups: [0090] 1. small aliphatic, nonpolar or slightly polar residues: Ala [A], Ser [S], Thr [T] (Pro [P], Gly [G]); [0091] 2. polar, negatively charged residues and their amides: Asp [D], Asn [N], Glu [E], Gln [Q]; [0092] 3. polar, positively charged residues: His [H], Arg [R], Lys [K]; [0093] 4. large aliphatic, nonpolar residues: Met [M], Leu [I], lie [I], Val M (Cys [C]) ; and, [0094] 5. large aromatic residues: Phe [F], Tyr [Y], Trp [W]. Conservative amino acid substitutions generally maintain: 1) the structure of the polypeptide backbone in the area of the substitution; 2) the charge or hydrophobicity of the molecule at the target site; or 3) the bulk of the side chain, Additionally, in many cases, alterations of the N-terminal and C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein.
[0095] As used herein, "nucleic acid" means a polynucleotide and includes single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases. Nucleic acids may also include fragments and modified nucleotides. Thus, the terms "polynucleotide", "nucleic acid sequence", "nucleotide sequence" or "nucleic acid fragment" are used interchangeably and is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. Nucleotides (usually found in their 5'-monophosphate form) are referred to by their single letter designation as follows: "A" for adenylate or deoxyadenylate (for RNA or DNA, respectively), "C" for cytidylate or deosycytidylate, "G" for guanylate or deoxyguanylate, "U" for uridlate, "T" for deosythymidylate, "R" for purines (A or G), "Y" for pyrimidiens (C or T), "K" for G or T, "H" for A or C or T, "I" for inosine, and "N" for any nucleotide.
[0096] The terms "subfragment that is functionally equivalent" and "functionally equivalent subfragment" are used interchangeably herein. These terms refer to a portion or subsequence of an isolated nucleic acid fragment in which the ability to alter gene expression or produce a certain phenotype is retained whether or not the fragment or subfragment encodes an active enzyme. For example, the fragment or subfragment can be used in the design of chimeric genes to produce the desired phenotype in a transformed plant. Chimeric genes can be designed for use in suppression by linking a nucleic acid fragment or subfragment thereof, whether or not it encodes an active enzyme, in the sense or antisense orientation relative to a plant promoter sequence.
[0097] The term "conserved domain" or "motif" means a set of amino acids conserved at specific positions along an aligned sequence of evolutionarily related proteins. While amino acids at other positions can vary between homologous proteins, amino acids that are highly conserved at specific positions indicate amino acids that are essential in the structure, the stability, or the activity of a protein. Because they are identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers, or "signatures", to determine if a protein with a newly determined sequence belongs to a previously identified protein family.
[0098] The terms "homology", "homologous", "substantially similar" and "corresponding substantially" are used interchangeably herein. They refer to nucleic acid fragments wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype. These terms also refer to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotides that do not substantially alter the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment. It is therefore understood, as those skilled in the art will appreciate, that the invention encompasses more than the specific exemplary sequences.
[0099] Moreover, the skilled artisan recognizes that substantially similar nucleic acid sequences encompassed by this invention are also defined by their ability to hybridize (under moderately stringent conditions, e.g., 0.5×SSC, 0.1% SDS, 60° C.) with the sequences exemplified herein, or to any portion of the nucleotide sequences disclosed herein and which are functionally equivalent to any of the nucleic acid sequences disclosed herein. Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions.
[0100] The term "selectively hybridizes" includes reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids. Selectively hybridizing sequences typically have about at least 80% sequence identity, or 90% sequence identity, up to and including 100% sequence identity (i.e., fully complementary) with each other.
[0101] The term "stringent conditions" or "stringent hybridization conditions" includes reference to conditions under which a probe will selectively hybridize to its target sequence. Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, optionally less than 500 nucleotides in length.
[0102] Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SOS (sodium dodecyl sulphate) at 37° C., and a wash in 1X to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SOS at 37° C., and a wash in 0.1×SSC at 60 to 65° C.,
[0103] Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth et al., Anal. Biochem. 138:267-284 (1984): Tm=81.5° 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 Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1° C. for each 1% of mismatching; thus, Tm, hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with ≧90% identity are sought, the Tm can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) 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° C. lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, 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 Tm of less than 45° C. (aqueous solution) or 32° C. (formamide solution) it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, Part I,
[0104] Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995). Hybridization and/or wash conditions can be applied for at least 10, 30, 60, 90, 120, or 240 minutes.
[0105] A "substantial portion" of an amino acid or nucleotide sequence is that portion comprising enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Basic
[0106] Local Alignment Search Tool; Altschul, S. F., et al., J. Mol. Biol., 215:403-410 (1993)). In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers, Accordingly, a "substantial portion" of a nucleotide sequence comprises enough of the sequence to specifically identify and/or isolate a nucleic acid fragment comprising the sequence. The instant specification teaches the complete amino acid and nucleotide sequence encoding encoding particular algal proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.
[0107] The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the invention herein also includes isolated nucleic acid fragments that are complementary to the complete sequences as reported in the accompanying Sequence Listing, as well as those substantially similar nucleic acid sequences.
[0108] The terms "homology" and "homologous" are used interchangeably herein. They refer to nucleic acid fragments wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype. These terms also refer to modifications of the nucleic acid fragments of the present invention such as deletion or insertion of one or more nucleotides that do not substantially alter the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment. It is therefore understood, as those skilled in the art will appreciate, that the invention encompasses more than the specific exemplary sequences.
[0109] Moreover, the skilled artisan recognizes that homologous nucleic acid sequences encompassed by this invention are also defined by their ability to hybridize, under moderately stringent conditions (e.g., 0.5 ×SSC, 0.1% SDS, 60° C.) with the sequences exemplified herein, or to any portion of the nucleotide sequences disclosed herein and which are functionally equivalent to any of the nucleic acid sequences disclosed herein.
[0110] "Codon degeneracy" refers to the nature in the genetic code permitting 3Q variation of the nucleotide sequence without effecting the amino acid sequence of an encoded polypeptide. Accordingly, the instant invention relates to any nucleic acid fragment that encodes all or a substantial portion of the amino acid sequence encoding the instant algal polypeptide as set forth in SEQ ID NO:2. The skilled artisan is well aware of the "codon-bias" exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.
[0111] "Chemically synthesized", as related to a sequence of DNA, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of DNA may be accomplished using well-established procedures or, automated chemical synthesis can be performed using one of a number of commercially available machines. "Synthetic genes" can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form gene segments that are then enzymatically assembled to construct the entire gene. Accordingly, the genes can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell, where sequence information is available.
[0112] "Gene" refers to a nucleic acid fragment that expresses a specific protein, and that may refer to the coding region alone or may include regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. "Native gene" refers to a gene as found in nature with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. "Endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign" gene refers to a gene that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, native genes introduced into a new location within the native host, or chimeric genes. A "transgene" is a gene that has been introduced into the genome by a transformation procedure. A "codon-optimized gene" is a gene having its frequency of codon usage designed to mimic the frequency of preferred codon usage of the host cell.
[0113] "Coding sequence refers to a DNA sequence that codes for a specific amino acid sequence. "Suitable regulatory sequences" refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures.
[0114] The term "allele" refers to one of several alternative forms of a gene occupying a given locus on a chromosome. When all the alleles present at a given locus on a chromosome are the same, then that plant is homozygous at that locus. If the alleles present at a given locus on a chromosome differ, then that plant is heterozygous at that locus.
[0115] "Promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
[0116] A promoter sequence may consist of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancer" is a DNA sequence that can stimulate promoter activity, and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro, J. K., and Goldberg, R. B., Biochemistry of Plants, 15:1-82 (1989).
[0117] "Translation leader sequence" refers to a polynucleotide sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner, R. and Foster, G. D., Mol. Biotechnol., 3:225-236 (1995)).
[0118] The terms "3' non-coding sequences" and "transcription terminator" refer to DNA sequences located downstream of a coding sequence. This includes . polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor. The 3' region can influence the transcription, RNA processing or stability, or translation of the associated coding sequence.
[0119] "RNA transcript" refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from post-transcriptional processing of the primary transcript and is referred to as the mature RNA. "Messenger RNA" or "mRNA" refers to the RNA that is without introns and that can be translated into protein by the cell. "cDNA" refers to a double-stranded DNA that is complementary to, and derived from, mRNA. "Sense" RNA refers to RNA transcript that includes the mRNA and so can be translated into protein by the cell. "Antisense RNA" refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (U.S. Pat. No. 5,107,065; PCT Publication No. WO 99/28508). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5' non-coding sequence, 3' non-coding sequence, or the coding sequence. "Functional RNA" refers to antisense RNA, ribozyme RNA, or other RNA that is not translated and yet has an effect on cellular processes.
[0120] The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
[0121] The term "expression", as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragments of the invention. Expression may also refer to translation of mRNA into a polypeptide.
[0122] "Mature" protein refers to a post-translationally processed polypeptide, i.e., one from which any pre- or propeptides present in the primary translation product have been removed. `Precursor" protein refers to the primary product of translation of mRNA, i.e., with pre- and propeptides still present. Pre- and propeptides may be (but are not limited to) intracellular localization signals.
[0123] "Transformation" refers to the transfer of a nucleic acid molecule into a host organism, resulting in genetically stable inheritance. The nucleic acid molecule may be a plasmid that replicates autonomously, for example, or, it may integrate into the genome of the host organism. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" or "recombinant" or "transformed" organisms.
[0124] The terms "plasmid", "vector" and "cassette" refer to an extra chromosomal element often carrying genes that are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA fragments. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell. "Expression cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.
[0125] The term "percent identity", as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in: 1.) Computational Molecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.) Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.) Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and, 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991).
[0126] Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the MegAlign® program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences is performed using the "Clustal method of alignment" which encompasses several varieties of the algorithm including the "Clustal V method of alignment" corresponding to the alignment method labeled Clustal V (described by Higgins and Sharp, CABIOS, 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in the MegAlign® program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). For multiple alignments, the default values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal V method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences using the Clustal V program, it is possible to obtain a "percent identity" by viewing the "sequence distances" table in the same program. Additionally the "Clustal W method of alignment" is available and corresponds to the alignment method labeled Clustal W (described by Higgins and Sharp, CABIOS, 5:151-153 (1989); Higgins, D. G. et al., Comput Appl. Biosci., 8:189-191(1992)) and found in the MegAlign® v6.1 program of the LASERGENE bioinforrnatics computing suite (DNASTAR Inc.). Default parameters for multiple alignment correspond to GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergen Seqs (%)=30, DNA Transition VVeight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB. After alignment of the sequences using the Clustal W program, it is possible to obtain a "percent identity" by viewing the "sequence distances" table in the same program.
[0127] "BLASTN method of alignment" is an algorithm provided by the National Center for Biotechnology Information (NGBI) to compare nucleotide sequences using default parameters.
[0128] It is well understood by one skilled in the art that many levels of sequence identity are useful in identifying polypeptides, from other species, wherein such polypeptides have the same or similar function or activity. Suitable nucleic acid fragments (isolated polynucleotides of the present invention) encode polypeptides that are at least about 70% identical, preferably at least about 75% identical, and more preferably at least about 80% identical to the amino acid sequences reported herein. Preferred nucleic acid fragments encode amino acid sequences that are at least about 85% identical to the amino acid sequences reported herein. More preferred nucleic acid fragments encode amino acid sequences that are at least about 90% identical to the amino acid sequences reported herein. Most preferred are nucleic acid fragments that encode amino acid sequences that are at least about 95% identical to the amino acid sequences reported herein, Although preferred ranges are described above, any integer amino acid identity from 67% to 100% may be useful in describing the present invention, such as 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
[0129] Suitable nucleic acid fragments not only have the above homologies but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250 amino acids.
[0130] The term "conserved domain" or "motif" means a set of amino acids conserved at specific positions along an aligned sequence of evolutionarily related proteins. While amino acids at other positions can vary between homologous proteins, amino acids that are highly conserved at specific positions indicate amino acids that are essential in the structure, the stability, or the activity of a protein. Because they are identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers, or "signatures", to determine if a protein with a newly determined sequence belongs to a previously identified protein family.
[0131] The term "sequence analysis software" refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. "Sequence analysis software" may be commercially available or independently developed. Typical sequence analysis software will include, but is not limited to: 1.) the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol., 215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.) Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTA program incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the "default values" of the program referenced, unless otherwise specified. As used herein "default values" will mean any set of values or parameters that originally load with the software when first initialized. With regard to the BLASTP algorithm used herein default parameters will include the Robinson and Robinson amino acid frequencies (Robinson A. B., Robinson L. R., Proc. Natl Acad. Sci. U.S.A., 88:8880-8884 (1991)), the BLOSUM62 scoring matrix and the gap cost Δ(g)=11+g.
[0132] The term "plant parts" includes differentiated and undifferentiated tissues including, but not limited to the following: roots, stems, shoots, leaves, pollen, seeds, tumor tissue and various forms of cells and culture (e.g., single cells, protoplasts, embryos and callus tissue). The plant tissue may be in plant or in a plant organ, tissue or cell culture.
[0133] The term "plant organ" refers to plant tissue or group of tissues that constitute a morphologically and functionally distinct part of a plant The term "genome" refers to the following: (1) the entire complement of genetic material (genes and non-coding sequences) present in each cell of an organism, or virus or organelle; (2) a complete set of chromosomes inherited as a (haploid) unit from one parent.
[0134] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989) (hereinafter "Maniatis"); by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., Current Protocols in Molecular Biolociy, published by Greene Publishing Assoc. and Wiley-Interscience, Hoboken, N.J. (1987).
[0135] The terms "recombinant construct", "expression construct", "chimeric construct", "construct", and "recombinant DNA construct" are used interchangeably herein. A recombinant construct comprises an artificial combination of nucleic acid fragments, e.g., regulatory and coding sequences that are not found together in nature. For example, a chimeric construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. Such a construct may be used by itself or may be used in conjunction with a vector. If a vector is used, then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art. For example, a plasmid vector can be used. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells comprising any of the isolated nucleic acid fragments of the invention. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al., EMBO J. 4:2411-2418 (1985); De Almeida et al., Mol. Gen. Genetics 218:78-86 (1989)), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, immunoblotting analysis of protein expression, or phenotypic analysis, among others.
[0136] The term "expression", as used herein, refers to the production of a functional end-product (e.g., a mRNA or a protein [either precursor or mature]).
[0137] The term "introduced" means providing a nucleic acid (e.g., expression construct) or protein into a cell. Introduced includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell, and includes reference to the transient provision of a nucleic acid or protein to the cell. introduced includes reference to stable or transient transformation methods, as well as sexually crossing. Thus, "introduced" in the context of inserting a nucleic acid fragment (e.g., a recombinant DNA construct/expression construct) into a cell, means "transfection" or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid fragment into a eukaryotic or prokaryotic cell where the nucleic acid fragment may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
[0138] "Mature" protein refers to a post-translationally processed polypeptide (i.e., one from which any pre- or propeptides present in the primary translation product have been removed). "Precursor" protein refers to the primary product of translation of mRNA (i.e., with pre- and propeptides still present). Pre- and propeptides may be but are not limited to intracellular localization signals.
[0139] "Stable transformation" refers to the transfer of a nucleic acid fragment into a genome of a host organism, including both nuclear and organellar genomes, resulting in genetically stable inheritance. In contrast, "transient transformation" refers to the transfer of a nucleic acid fragment into the nucleus, or DNA-containing organelle, of a host organism resulting in gene expression without integration or stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" organisms.
[0140] As used herein, "transgenic" refers to a plant or a cell which comprises within its genome a heterologous polynucleotide. Preferably, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of an expression construct. Transgenic is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. The term "transgenic" as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.
[0141] "Antisense inhibition" refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein. "Co-suppression" refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Pat. No. 5,231,020). Co-suppression constructs in plants previously have been designed by focusing on overexpression of a nucleic acid sequence having homology to an endogenous mRNA, in the sense orientation, which results in the reduction of all RNA having homology to the overexpressed sequence (Vaucheret et al., Plant J. 16:651-659 (1998); Gura, Nature 404:804-808 (2000)). The overall efficiency of this phenomenon is low, and the extent of the RNA reduction is widely variable. More recent work has described the use of "hairpin" structures that incorporate all, or part, of an mRNA encoding sequence in a complementary orientation that results in a potential "stem-loop" structure for the expressed RNA (PCT Publication No. WO 99/53050, published Oct. 21, 1999; PCT Publication No. WO 02100904, published Jan. 3, 2002). This increases the frequency of co-suppression in the recovered transgenic plants. Another variation describes the use of plant viral sequences to direct the suppression, or "silencing", of proximal mRNA encoding sequences (PCT Publication No. WO 98/36083, published Aug. 20, 1998). Both of these co-suppressing phenomena have not been elucidated mechanistically, although genetic evidence has begun to unravel this complex situation (Elmayan et al., Plant Cell 10:1747-1757 (1998)).
An Overview: Microbial Biosynthesis of Fatty Acids and Triacylglycerols
[0142] In general, lipid accumulation in oleaginous microorganisms is triggered in response to the overall carbon to nitrogen ratio present in the growth medium. This process, leading to the de novo synthesis of free palmitate (16:0) in oleaginous microorganisms, is described in detail in PCT Publication No. WO 2004/101757. Palmitate is the precursor of longer-chain saturated and unsaturated fatty acid derivates, which are formed through the action of elongases and desaturases (FIG. 1).
[0143] TAGs (the primary storage unit for fatty acids) are formed by a series of reactions that involve: 1.) the esterification of one molecule of acyl-CoA to glycerol-3-phosphate via an acyltransferase to produce lysophosphatidic acid; 2.) the esterification of a second molecule of acyl-CoA via an acyltransferase to yield 1,2-diacylglycerol phosphate (commonly identified as phosphatidic acid); 3.) removal of a phosphate by phosphatidic acid phosphatase to yield 1,2-diacylglycerol (DAG); and, 4.) the addition of a third fatty acid by the action of an acyltransferase to form TAG. A wide spectrum of fatty acids can be incorporated into TAGs, including saturated and unsaturated fatty acids and short-chain and long-chain fatty acids.
Biosynthesis of Omega Fatty Acids
[0144] The metabolic process wherein oleic acid is converted to omega-3/omega-6 fatty acids involves elongation of the carbon chain through the addition of carbon atoms and desaturation of the molecule through the addition of double bonds. This requires a series of special desaturation and elongation enzymes present in the endoplasmic reticulim membrane. However, as seen in FIG. 1 and as described below, there are often multiple alternate pathways for production of a specific omega-3/omega-6 fatty acid.
[0145] Specifically, all pathways require the initial conversion of oleic acid to LA, the first of the omega-6 fatty acids, by a delta-12 desaturase. Then, using the "delta-6 desaturase/delta-6 elongase pathway", omega-6 fatty acids are formed as follows: (1) LA is converted to GLA by a delta-6 desaturase; (2) GLA is converted to DGLA by a C18/20 elongase; and, (3) DGLA is converted to ARA by a delta-5 desaturase.
[0146] Alternatively, the "delta-6 desaturase/delta-6 elongase pathway" can be utilized for formation of omega-3 fatty acids as follows: (1) LA is converted to ALA, the first of the omega-3 fatty acids, by a delta-15 desaturase; (2) ALA is converted to STA by a delta-6 desaturase; (3) STA is converted to ETA by a C18/20 elongase; (4) ETA is converted to EPA by a delta-5 desaturase; (5) EPA is converted to DPA by a C20/22 elongase; and, (6) DPA is converted to DHA by a delta-4 desaturase. Optionally, omega-6 fatty acids may be converted to omega-3 fatty acids; for example, ETA and EPA are produced from DGLA and ARA, respectively, by delta-17 desaturase activity.
[0147] Alternate pathways for the biosynthesis of omega-3/omega-6 fatty acids utilize a delta-9 elongase and delta-8 desaturase. More specifically, LA and ALA may be converted to EDA and ETrA, respectively, by a delta-9 elongase; then, a delta-8 desaturase converts EDA to DGLA and/or ETrA to ETA.
[0148] It is contemplated that the particular functionalities required to be expressed in a specific host organism for production of omega-3/omega-6 fatty acids will depend on the host cell (and its native PUFA profile and/or desaturase/elongase profile), the availability of substrate, and the desired end product(s). One skilled in the art will be able to identify various candidate genes encoding each of the enzymes desired for omega-3/omega-6 fatty acid biosynthesis. Useful desaturase and elongase sequences may be derived from any source, e.g., isolated from a natural source (from bacteria, algae, fungi, plants, animals, etc.), produced via a semi-synthetic route or synthesized de novo. Although the particular source of the desaturase and elongase genes introduced into the host is not critical, considerations for choosing a specific polypeptide having desaturase or elongase activity include: 1.) the substrate specificity of the polypeptide; 2.) whether the polypeptide or a component thereof is a rate-limiting enzyme; 3.) whether the desaturase or elongase is essential for synthesis of a desired PUFA; and/or, 4.) co-factors required by the polypeptide. The expressed polypeptide preferably has parameters compatible with the biochemical environment of its location in the host cell (see PCT Publication No. WO 2004/101757 for additional details).
[0149] In additional embodiments, it will also be useful to consider the conversion efficiency of each particular desaturase and/or elongase. More specifically, since each enzyme rarely functions with 100% efficiency to convert substrate to product, the final lipid profile of un-purified oils produced in a host cell will typically be a mixture of various PUFAs consisting of the desired omega-3/omega-6 fatty acid, as well as various upstream intermediary PUFAs. Thus, each enzyme's conversion efficiency is also a variable to consider, when optimizing biosynthesis of a desired fatty acid.
[0150] With each of the considerations above in mind, candidate genes having the appropriate desaturase and elongase activities (e.g., delta-6 desaturases, C18/20 elongases, delta-5 desaturases, delta-17 desaturases, delta-15 desaturases, delta-9 desaturases, delta-12 desaturases, C14/16 elongases, C16/18 elongases, delta-9 elongases, delta-8 desaturases, delta-4 desaturases and C20/22 elongases) can be identified according to publicly available literature (e.g., GenBank), the patent literature, and experimental analysis of organisms having the ability to produce PUFAs. These genes will be suitable for introduction into a specific host organism, to enable or enhance the organism's synthesis of PUFAs.
Sequence Identification of a Novel Peridinium sp. CCMP626 Delta-5 Desaturase
[0151] In the present invention, a nucleotide sequence (SEQ ID NO:1) has been isolated from Peridinium sp. CCMP626 encoding a delta-5 desaturase (SEQ ID NO:2), designated herein as "RD5".
[0152] Comparison of the RD5 nucleotide base and deduced amino acid sequences to public databases reveals that the most similar known sequences are about 67% identical to the amino acid sequence of RD5 reported herein over a length of 463 amino acids using a Clustal W alignment method. More preferred amino acid fragments are at least about 70%-80% identical to the sequences herein, where those sequences that are at least about 80%-90% identical are particularly suitable and those sequences that are at feast about 90%-95% identical are most preferred. Similarly, preferred RD5 encoding nucleic acid sequences corresponding to the instant ORF are those encoding active proteins and which are at least about 70%-80% identical to the nucleic acid sequences of RD5 reported herein, where those sequences that are at least about 80%-90% identical are particularly suitable and those sequences that are at least about 90%-95% identical are most preferred.
[0153] In alternate embodiments, the instant RD5 desaturase sequence can be codon-optimized for expression in a particular host organism. As is well known in the art, this can be a useful means to further optimize the expression of the enzyme in the alternate host, since use of host-preferred codons can substantially enhance the expression of the foreign gene encoding the polypeptide. In general, host-preferred codons can be determined within a particular host species of interest by examining codon usage in proteins (preferably those expressed in the largest amount) and determining which codons are used with highest frequency. Then, the coding sequence for a polypeptide of interest having e.g., desaturase activity can be synthesized in whole or in part using the codons preferred in the host species.
[0154] In one preferred embodiment of the invention herein, RD5 was codon-optimized for expression in Yarrowia lipolytica. This was possible by first determining the Y. lipolytica codon usage profile (see PCT Publication No. WO 04/101757 and U.S. Pat. No. 7,125,672) and identifying those codons that were preferred. Then, for further optimization of gene expression in Y. lipolytica, the consensus sequence around the `ATG` initiation codon was determined. This optimization resulted in modification of 247 by of the 1392 by coding region (17.7%) and optimization of 229 codons of the total 463 codons (49.4%). None of the modifications in the codon-optimized gene ("RD5S"; SEQ ID NO:3) changed the amino acid sequence of the encoded protein (SEQ ID NO:2), As described in Example 11, the codon-optimized gene was 8.9% more efficient desaturating DGLA to ARA than the wildtype gene, when expressed in Y. lipolytica.
[0155] One skilled in the art would be able to use the teachings herein to create various other codon-optimized delta-5 desaturase proteins suitable for optimal expression in alternate hosts (i.e., other than Yarrowia lipolytica), based on the wildtype RD5 sequence. Accordingly, the instant invention relates to any codon-optimized delta-5 desaturase protein that is derived from the wildtype RD5 (i.e., encoded by SEQ ID NO:2). This includes, but is not limited to, the nucleotide sequence set forth in SEQ ID NO:3, which encodes a synthetic delta-5 desaturase protein (i.e., RD5S) that was codon-optimized for expression in Yarrowia lipolytica.
Identification and Isolation of Homologs
[0156] Any of the instant desaturase sequences (i.e., RD5, RD5S) or portions thereof may be used to search for delta-5 desaturase homologs in the same or other bacterial, algal, fungal, or plant species using sequence analysis software. In general, such computer software matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.
[0157] Alternatively, any of the instant desaturase sequences or portions thereof may also be employed as hybridization reagents for the identification of delta-5 homologs. The basic components of a nucleic acid hybridization test include a probe, a sample suspected of containing the gene or gene fragment of interest and a specific hybridization method. Probes of the present invention are typically single-stranded nucleic acid sequences that are complementary to the nucleic acid sequences to be detected. Probes are "hybridizable" to the nucleic acid sequence to be detected. Although the probe length can vary from 5 bases to tens of thousands of bases, typically a probe length of about 15 bases to about 30 bases is suitable. Only part of the probe molecule need be complementary to the nucleic acid sequence to be detected. In addition, the complementarity between the probe and the target sequence need not be perfect. Hybridization does occur between imperfectly complementary molecules with the result that a certain fraction of the bases in the hybridized region are not paired with the proper complementary base.
[0158] Hybridization methods are well defined. Typically the probe and sample must be mixed under conditions that will permit nucleic acid hybridization. This involves contacting the probe and sample in the presence of an inorganic or organic salt under the proper concentration and temperature conditions. The probe and sample nucleic acids must be in contact for a long enough time that any possible hybridization between the probe and sample nucleic acid may occur. The concentration of probe or target in the mixture will determine the time necessary for hybridization to occur. The higher the probe or target concentration, the shorter the hybridization incubation time needed. Optionally, a chaotropic agent may be added (e.g., guanidinium chloride, guanidinium thiocyanate, sodium thiocyanate, lithium tetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate, potassium iodide, cesium trifluoroacetate). If desired, one can add formamide to the hybridization mixture, typically 30-50% (v/v).
[0159] Various hybridization solutions can be employed. Typically, these comprise from about 20 to 60% volume, preferably 30%, of a polar organic solvent. A common hybridization solution employs about 30-50% v/v formamide, about 0.15 to 1 M sodium chloride, about 0.05 to 0.1 M buffers (e.g., sodium citrate, Tris-HCl, PIPES or HEPES (pH range about 6-9)), about 0.05 to 0.2% detergent (e.g., sodium dodecylsulfate), or between 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kdal), polyvinylpyrrolidone (about 250-500 kdal), and serum albumin. Also included in the typical hybridization solution will be unlabeled carrier nucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g., calf thymus or salmon sperm DNA, or yeast RNA), and optionally Thorn about 0.5 to 2% wt/vol glycine. Other additives may also be included, such as volume exclusion agents that include a variety of polar water-soluble or swellable agents (e.g., polyethylene glycol), anionic polymers (e.g., polyacrylate or polymethylacrylate) and anionic saccharidic polymers (e.g., dextran sulfate).
[0160] Nucleic acid hybridization is adaptable to a variety of assay formats. One of the most suitable is the sandwich assay format. The sandwich assay is particularly adaptable to hybridization under non-denaturing conditions. A primary component of a sandwich-type assay is a solid support. The solid support has adsorbed to it or covalently coupled to it immobilized nucleic acid probe that is unlabeled and complementary to one portion of the sequence.
[0161] In additional embodiments, any of the delta-5 desaturase nucleic acid fragments described herein (or any homologs identified thereof) may be used to isolate genes encoding homologous proteins from the same or other bacterial, algal, fungal, or plant species. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to: 1.) methods of nucleic acid hybridization; 2.) methods of DNA and RNA amplification, as exemplified by various uses of nucleic acid amplification technologies [e.g., polymerase chain reaction (PCR), Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR), Tabor, S. et al., Proc. Natl. Acad. Sci. U.S.A., 82:1074 (1985); or strand displacement amplification (SDA), Walker, et al., Proc. Natl. Acad. Sci. U.S.A., 89:392 (1992)]; and 3.) methods of library construction and screening by complementation.
[0162] For example, genes encoding similar proteins or polypeptides to the delta-5 desaturases described herein could be isolated directly by using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from e.g., any desired yeast or fungus using methodology well known to those skilled in the art (wherein those organisms producing ARA [or derivatives thereof] would be preferred). Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis, supra). Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan (e.g., random primers DNA labeling, nick translation or end-labeling techniques), or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part of (or full-length of) the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full-length DNA fragments under conditions of appropriate stringency.
[0163] Typically, in PCR-type amplification techniques, the primers have different sequences and are not complementary to each other. Depending on the desired test conditions, the sequences of the primers should be designed to provide for both efficient and faithful replication of the target nucleic acid. Methods of PCR primer design are common and well known in the art (Thein and Wallace, "The use of oligonucleotides as specific hybridization probes in the Diagnosis of Genetic Disorders", in Human Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986) pp 33-50, IRL: Herndon, Va.; and Rychlik, W., in Methods in Molecular Biology, White, B. A. Ed., (1993) Vol. 15, pp 31-39, PCR Protocols: Current Methods and Applications. Humania: Totowa, N.J.).
[0164] Generally two short segments of the instant sequences may be used in PCR protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. PCR may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the instant nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3' end of the mRNA precursor encoding eukaryotic genes.
[0165] Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al., Proc. Natl. Acad. Sci. U.S.A., 85:8998 (1988)) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3' or 5' end. Primers oriented in the 3' and 5' directions can be designed from the instant sequences. Using commercially available 3' RACE or 5' RACE systems (GibcoIBRL, Gaithersburg, Md.), specific 3' or 5' cDNA fragments can be isolated (Ohara et al., Proc. Natl. Acad. Sci. U.S.A., 86:5673 (1989); Loh et al., Science, 243:217 (1989)).
[0166] In other embodiments, any of the delta-5 desaturase nucleic acid fragments described herein (or any homologs identified thereof) may be used for creation of new and improved fatty acid desaturases. As is well known in the art, in vitro mutagenesis and selection, chemical mutagenesis, "gene shuffling" methods or other means can be employed to obtain mutations of naturally occurring desaturase genes. Alternatively, improved fatty acids may be synthesized by domain swapping, wherein a functional domain from any of the delta-5 desaturase nucleic acid fragments described herein are exchanged with a functional domain in an alternate desaturase gene to thereby result in a novel protein.
Methods for Production of Various Omega-3 and/or Omega-6 Fatty Acids
[0167] It is expected that introduction of chimeric genes encoding the delta-5 desaturases described herein. (i.e., RD5, RD5S or other mutant enzymes, codon-optimized enzymes or homologs thereof), under the control of the appropriate promoters will result in increased production of ARA and/or EPA in the transformed host organism, respectively. As such, the present invention encompasses a method for the direct production of PUFAs comprising exposing a fatty acid substrate (i.e., DGLA or ETA) to the desaturase enzymes described herein (e.g., RD5, RD5S), such that the substrate is converted to the desired fatty acid product (i.e., ARA or EPA, respectively).
[0168] More specifically, it is an object of the present invention to provide a method for the production of ARA in a host cell (e.g., oleaginous yeast, soybean), wherein the host cell comprises: [0169] (i) an isolated nucleotide molecule encoding a delta-5 desaturase polypeptide having at least 67% identity when compared to a polypeptide having an amino acid sequence as set forth in SEQ ID NO:2, based on BLASTP algorithms or Clustal W alignment methods; and, [0170] (ii) a source of dihomo-γ-linoleic acid; wherein the host cell is grown under conditions such that the delta-5 desaturase is expressed and the DGLA is converted to ARA, and wherein the ARA is optionally recovered.
[0171] The person of skill in the art will recognize that the broad substrate range of the delta-5 desaturase may additionally allow for the use of the enzyme for the conversion of ETA to EPA. Accordingly the invention provides a method for the production of EPA, wherein the host cell comprises: [0172] (i) an isolated nucleotide molecule encoding a delta-5 desaturase polypeptide having at least 67% identity when compared to a polypeptide having an amino acid sequence as set forth in SEQ ID NO:2, based on BLASTP algorithms or Clustal W alignment methods; and, [0173] (ii) a source of ETA; wherein the host cell is grown under conditions such that the delta-5 desaturase is expressed and the ETA is converted to EPA, and wherein the EPA is optionally recovered.
[0174] Alternatively, each delta-5 desaturase gene and its corresponding enzyme product described herein can be used indirectly for the production of omega-3 fatty acids (see U.S. Patent Publication No 2005/0136519). Indirect production of omega-3/omega-6 PUFAs occurs wherein the fatty acid substrate is converted indirectly into the desired fatty acid product, via means of an intermediate step(s) or pathway intermediate(s). Thus, it is contemplated that the delta-5 desaturases described herein (e.g., RDS, RD5S or other mutant enzymes, codon-optimized enzymes or homologs thereof) may be expressed in conjunction with additional genes encoding enzymes of the PUFA biosynthetic pathway (e,g., delta-6 desaturases, C18/20 elongases, delta-17 desaturases, delta-15 desaturases, delta-9 desaturases, delta-12 desaturases, C14/16 elongases, G16/18 elongases, delta-9 elongases, delta-8 desaturases, delta-4 desaturases, C20/22 elongases) to result in higher levels of production of longer-chain omega-3 fatty acids (e.g., EPA, DPA and DHA). The particular genes included within a particular expression cassette will depend on the host cell (and its PUFA profile and/or desaturase/elongase profile), the availability of substrate and the desired end product(s).
[0175] In alternative embodiments, it may be useful to disrupt a host organism's native delta-5 desaturase, based on the complete sequences described herein, the complement of those complete sequences, substantial portions of those sequences, codon-optimized desaturases derived therefrom and those sequences that are substantially homologous thereto.
Plant Expression Systems, Cassettes and Vectors, and Transformation
[0176] In one embodiment, this invention concerns a recombinant construct comprising any one of the delta-5 desaturase polynucleotides of the invention operably linked to at least one regulatory sequence suitable for expression in a plant. A promoter is a DNA sequence that directs cellular machinery of a plant to produce RNA from the contiguous coding sequence downstream (3') of the promoter. The promoter region influences the rate, developmental stage, and cell type in which the RNA transcript of the gene is made. The RNA transcript is processed to produce mRNA which serves as a template for translation of the RNA sequence into the amino acid sequence of the encoded polypeptide. The 5' non-translated leader sequence is a region of the mRNA upstream of the protein coding region that may play a role in initiation and translation of the mRNA. The 3' transcription term ination/polyadenylation signal is a non-translated region downstream of the protein coding region that functions in the plant cell to cause termination of the RNA transcript and the addition of polyadenylate nucleotides to the 3' end of the RNA.
[0177] The origin of the promoter chosen to drive expression of the delta-5 desaturase coding sequence is not important as long as it has sufficient transcriptional activity to accomplish the invention by expressing translatable mRNA for the desired nucleic acid fragments in the desired host tissue at the right time. Either heterologous or non-heterologous (i.e., endogenous) promoters can be used to practice the invention. For example, suitable promoters include, but are not limited to: the alpha prime subunit of beta conglycinin promoter, the Kunitz trypsin inhibitor 3 promoter, the annexin promoter, the glycinin Gyl promoter, the beta subunit of beta conglycinin promoter, the P34/Gly Bd m 30K promoter, the albumin promoter, the Leg Al promoter and the Leg A2 promoter.
[0178] The annexin, or P34, promoter is described in PCT Publication No. WO 2004/071178 (published Aug. 26, 2004). The level of activity of the annexin promoter is comparable to that of many known strong promoters, such as: (1) the CaMV 355 promoter (Atanassova et al., Plant Mol. Biol. 37:275-285 (1998); Battraw and Hall, Plant Mol. Biol. 15:527-538 (1990); Holtorl et al., Plant Mol. Biol. 29:637-646 (1995); Jefferson et al., EMBO J. 6:3901-3907 (1987); Wilmink et al., Plant Mol. Biol. 28:949-955 (1995)); (2) the Arabidopsis oleosin promoters (Plant et al., Plant Mol. Biol. 25:193-205 (1994); Li, Texas A&M University Ph.D. dissertation, pp. 107-128 (1997)); (3) the Arabidopsis ubiquitin extension protein promoters (Callis et al., J Biol. Chem. 265(21):12485-93 (1990)); (4) a tomato ubiquitin gene promoter (Rollfinke et al., Gene. 211(2):267-76 (1998)); (5) a soybean heat shock protein promoter (Schoffl et al., Mol Gen Genet. 217(2-3):246-53 (1989)); and, (6) a maize H3 histone gene promoter (Atanassova at al., Plant Mol Biol. 37(2):275-85 (1989)).
[0179] Another useful feature of the annexin promoter is its expression profile in developing seeds. The annexin promoter is most active in developing seeds at early stages (before 10 days after pollination) and is largely quiescent in later stages. The expression profile of the annexin promoter is different from that of many seed-specific promoters, e.g., seed storage protein promoters, which often provide highest activity in later stages of development (Chen et al., Day. Genet. 10:112-122 (1989); Ellerstrom et al., Plant Mol. Biol. 32:1019-1027 (1996); Keddie at al., Plant Mol. Biol. 24:327-340 (1994); Plant et al., (supra); Li, (supra)). The annexin promoter has a more conventional expression profile but remains distinct from other known seed specific promoters. Thus, the annexin promoter will be a very attractive candidate when overexpression, or suppression, of a gene in embryos is desired at an early developing stage. For example, it may be desirable to overexpress a gene regulating early embryo development or a gene involved in the metabolism prior to seed maturation.
[0180] Following identification of an appropriate promoter suitable for expression of a specific delta-5 desaturase coding sequence, the promoter is then operably linked in a sense orientation using conventional means well known to those skilled in the art.
[0181] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J. at al., In Molecular Cloning: A Laboratory Manual; 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y., 1989 (hereinafter "Sambrook et al., 1989") or Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl, K., Eds.; In Current Protocols in Molecular Biology; John Wiley and Sons: New York, 1990 (hereinafter "Ausubel et al., 1990").
[0182] Once the recombinant construct has been made, it may then be introduced into a plant cell of choice by methods well known to those of ordinary skill in the art (e.g., transfection, transformation and electroporation). Oilseed plant cells are the preferred plant cells. The transformed plant cell is then cultured and regenerated under suitable conditions permitting expression of the long-chain PUFA which is then optionally recovered and purified.
[0183] The recombinant constructs of the invention may be introduced into one plant cell; or, alternatively, each construct may be introduced into separate plant cells. Expression in a plant cell may be accomplished in a transient or stable fashion as is described above.
[0184] The desired long-chain PUFAs can be expressed in seed. Also within the scope of this invention are seeds or plant parts obtained from such transformed plants.
[0185] Plant parts include differentiated and undifferentiated tissues including, but not limited to the following: roots, sterns, shoots, leaves, pollen, seeds, tumor tissue and various forms of cells and culture (e.g., single cells, protoplasts, embryos and callus tissue). The plant tissue may be in plant or in a plant organ, tissue or cell culture.
[0186] The term "plant organ" refers to plant tissue or a group of tissues that constitute a morphologically and functionally distinct part of a plant. The term "genome" refers to the following: (1) the entire complement of genetic material (genes and non-coding sequences) that is present in each cell of an organism, or virus or organelle; and/or (2) a complete set of chromosomes inherited as a (haploid) unit from one parent.
[0187] Thus, this invention also concerns a method for transforming a cell, comprising transforming a cell with the recombinant construct of the invention and selecting those cells transformed with the recombinant construct of the invention.
[0188] Also of interest is a method for producing a transformed plant comprising transforming a plant cell with the delta-5 desaturase polynucleotides of the instant invention and regenerating a plant from the transformed plant cell.
[0189] Methods for transforming dicots (primarily by use of Agrobacterium tumefaciens) and obtaining transgenic plants have been published, among others, for; cotton (U.S. Pat. No. 5,004,863; U.S. Pat. No. 5,159,135); soybean (U.S. Pat. No. 5,569,834; U.S. Pat. No. 5,416,011); Brassica (U.S. Pat. No. 5,463,174); peanut (Cheng et al. Plant Cell Rep. 15:653-657 (1996); McKently et al. Plant. Cell Rep. 14:699-703 (1995)); papaya (Ling, K. et al. Bio/technology 9:752-758 (1991)); and pea (Grant at al. Plant Cell Rep. 15:254-258 (1995)). For a review of other commonly used methods of plant transformation see Newell, C. A. (Mol. Biotechnol. 16:53-65 (2000)). One of these methods of transformation uses Agrobacterium rhizogenes (Tepfler, M. and Casse-Delbart, F. Microbiol. Sci. 4:24-28 (1987)). Transformation of soybeans using direct delivery of DNA has been published using PEG fusion (PCT Publication No. WO 92/17598), electroporation (Chowrira, G. M. et al., Mol. Biotechnol. 3:17-23 (1995); Christou, P. et al., Proc. Natl. Acad. Sci. U.S.A. 84:3962-3966 (1987)), microinjection and particle bombardement (McCabe, D. E. et. al., Bio/Technology 6:923 (1988); Christou et al., Plant Physiol. 87:671-674 (1988)).
[0190] There are a variety of methods for the regeneration of plants from plant tissue. The particular method of regeneration will depend on the starting plant tissue and the particular plant species to be regenerated. The regeneration, development and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (Weissbach and Weissbach, In: Methods for Plant Molecular Biology, (Eds.), Academic: San Diego, Calif. (1988)). This regeneration and growth process typically includes the steps of selection of transformed cells and culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants, Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants. A transgenic plant of the present invention containing a desired polypeptide is cultivated using methods well known to one skilled in the art.
[0191] In addition to the above discussed procedures, practitioners are familiar with the standard resource materials which describe specific conditions and procedures for: the construction, manipulation and isolation of macromolecules (e.g., DNA molecules, plasmids, etc.); the generation of recombinant DNA fragments and recombinant expression constructs; and, the screening and isolating of clones. See, for example: Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor: NY (1989); Maliga et al., Methods in Plant Molecular Biology, Cold Spring Harbor: NY (1995); Birren et al., Genome Analysis: Detecting Genes, Vol.1, Cold Spring Harbor: NY (1998); Birren et al., Genome Analysis: Analyzing DNA, Vol. 2, Cold Spring Harbor: NY (1998); Plant Molecular Biology: A Laboratory Manual, eds. Clark, Springer: NY (1997).
[0192] Examples of oilseed plants include, but are not limited to: soybean, Brassica species, sunflower, maize, cotton, flax and safflower.
[0193] Examples of PUFAs having at least twenty carbon atoms and four or more carbon-carbon double bonds include, but are not limited to, omega-3 fatty acids such as EPA, DPA and DHA and the omega-6 fatty acid ARA. Seeds obtained from such plants are also within the scope of this invention as well as oil obtained from such seeds.
[0194] Thus, in one embodiment this invention concerns an oilseed plant comprising:
[0195] (a) a first recombinant DNA construct comprising an isolated polynucleotide encoding a delta-5 desaturase polypeptide, operably linked to at least one regulatory sequence; and,
[0196] (b) at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8 desaturase, a delta-9 desaturase, a delta-9 elongase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a C14/16 elongase, a C16/18 elongase, a C18/20 elongase and a C20/22 elongase.
[0197] Additional desaturases are discussed, for example, in U.S. Pat. Nos. 6,075,183, 5,968,809, 6,136,574, 5,972,664, 6,051,754, 6,410,288 and PCT Publication Nos. WO 98/46763, WO 98/46764, WO 00/12720 and WO 00/40705.
[0198] The choice of combination of cassettes used depends in part on the PUFA profile and/or desaturase/elongase profile of the oilseed plant cells to be transformed and the long-chain PUFA which is to be expressed.
[0199] In another aspect, this invention concerns a method for making long-chain PUFAs in a plant cell comprising: [0200] (a) transforming a cell with the recombinant construct of the invention; and, [0201] (b) selecting those transformed cells that make long-chain PUFAs.
[0202] In still another aspect, this invention concerns a method for producing at least one PUFA in a soybean cell comprising: [0203] (a) transforming a soybean cell with a first recombinant DNA construct comprising: [0204] (i) an isolated polynucleotide encoding a delta-5 desaturase polypeptide, operably linked to at least one regulatory sequence; and, [0205] (ii) at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8 desaturase, a delta-9 desaturase, a delta-9 elongase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a C14/16 elongase, a C16/18 elongase, a C18/20 elongase and a C20/22 elongase; [0206] (b) regenerating a soybean plant from the transformed cell of step (a); and, [0207] (c) selecting those seeds obtained from the plants of step (b) having an altered level of PUFAs when compared to the level in seeds obtained from a nontransforrned soybean plant. In other preferred embodiments, the at least one additional recombinant DNA construct encodes a polypeptide having delta-9 elongase activity, e.g., the delta-9 elongase isolated or derived from Isochrysis galbana (GenBank Accession No. AF390174; IgD9e) or the delta-9 elongase isolated or derived from Euglena gracilis.
[0208] In other preferred embodiments, the at least one additional recombinant DNA construct encodes a polypeptide having delta-8 desaturase activity. For example, PCT Publication No. WO 2005/103263 (published Apr. 22, 2005) discloses amino acid and nucleic acid sequences for a delta-8 desaturase enzyme from Pavlova salina (see also U.S. Publication No. 2005/0273885). Sayanova et al. (FEBS Lett. 580:1946-1952 (2006)) describes the isolation and characterization of a cDNA from the free living soil amoeba Acanthamoeba castellanii that, when expressed in Arabidopsis, encodes a C20 delta-8 desaturase. Also, Applicants' Assignee's co-pending application having Provisional Application No. 60/795,810 filed Apr. 28, 2006 (Attorney Docket No. BB-1566) discloses amino acid and nucleic acid sequences for a delta-8 desaturase enzyme from Pavlova lutheri (CCMP459). U.S Provisional Application No. 60/853,563 (filed Oct. 23, 2006; Attorney Docket No. BB-1574) discloses amino acid and nucleic add sequences for a delta-8 desaturase enzyme from Tetruetreptia pomquetensis CCMP1491, Eutreptiella sp. CCMP389 and Eutreptiella cf--gymnastica CCMP1594.
Microbial Expression Systems, Cassettes and Vectors, and Transformation
[0209] The delta-5 desaturase genes and gene products described herein (i.e., the Peridinium sp. CCMP626 delta-5 desaturase, or other mutant enzymes, codon-optimized enzymes or homologs thereof) may also be produced in heterologous microbial host cells, particularly in the cells of oleaginous yeasts (e.g., Yarrowia lipolytica).
[0210] Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct chimeric genes for production of any of the gene products of the instant sequences. These chimeric genes could then be introduced into appropriate microorganisms via transformation to provide high-level expression of the encoded enzymes. Vectors or DNA cassettes useful for the transformation of suitable microbial host cells are well known in the art. The specific choice of sequences present in the construct is dependent upon the desired expression products (supra), the nature of the host cell and the proposed means of separating transformed cells versus non-transformed cells. Typically, however, the vector or cassette contains sequences directing transcription and translation of the relevant gene(s), a selectable marker and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5' of the gene that controls transcriptional initiation (e.g., a promoter) and a region 3' of the DNA fragment that controls transcriptional termination (i.e., a terminator). It is most preferred when both control regions are derived from genes from the transformed microbial host cell, although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a production host.
[0211] Initiation control regions or promoters which are useful to drive expression of the instant delta-5 desaturase ORFs in the desired microbial host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of directing expression of these genes in the selected host cell is suitable for the present invention. Expression in a microbial host cell can be accomplished in a transient or stable fashion. Transient expression can be accomplished by inducing the activity of a regulatable promoter operably linked to the gene of interest. Stable expression can be achieved by the use of a constitutive promoter operably linked to the gene of interest. As an example, when the host cell is yeast, transcriptional and translational regions functional in yeast cells are provided, particularly from the host species (e.g., see PCT Publication Nos. WO 2004/101757 and WO 2006/052870 for preferred transcriptional initiation regulatory regions for use in Yarrowia lipolytica). Any one of a number of regulatory sequences can be used, depending upon whether constitutive or induced transcription is desired, the efficiency of the promoter in expressing the ORF of interest, the ease of construction and the like.
[0212] Nucleotide sequences surrounding the translational initiation codon `ATG` have been found to affect expression in yeast cells. If the desired polypeptide is poorly expressed in yeast, the nucleotide sequences of exogenous genes can be modified to include an efficient yeast translation initiation sequence to obtain optimal gene expression. For expression in yeast, this can be done by site-directed mutagenesis of an inefficiently expressed gene by fusing it in-frame to an endogenous yeast gene, preferably a highly expressed gene. Alternatively, one can determine the consensus translation initiation sequence in the host and engineer this sequence into heterologous genes for their optimal expression in the host of interest.
[0213] The termination region can be derived from the 3' region of the gene from which the initiation region was obtained or from a different gene. A large number of termination regions are known and function satisfactorily in a variety of hosts (when utilized both in the same and different genera and species from where they were derived). The termination region usually is selected more as a matter of convenience rather than because of any particular property, Preferably, when the microbial host is a yeast cell, the termination region is derived from a yeast gene (particularly Saccharomyces, Schizosaccharomyces, Candida, Yarrowia or Kluyveromyces). The 3'-regions of mammalian genes encoding γ-interferon and α-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: ˜100 by of the 3' region of the Yarrowia lipolytica extracellular protease (XPR; GenBank Accession Na M17741); the acyl-coA oxidase (Aco3: GenBank Accession No. AJ001301 and No. CAA04661; Pox3: GenBank Accession No, XP--503244) terminators; the Pex20 (GenBank Accession No. AF054613) terminator; the Pex16 (GenBank Accession No. U75433) terminator; the Lip1 (GenBank Accession No. 250020) terminator; the Lip2 (GenBank Accession No. AJ012632) terminator; and the 3-oxoacyl-coA thiolase (OCT; GenBank Accession No. X69988) terminator.
[0214] As one of skill in the art is aware, merely inserting a gene into a cloning vector does not ensure that it will be successfully expressed at the level needed. In response to the need for a high expression rate, many specialized expression vectors have been created by manipulating a number of different genetic elements that control aspects of transcription, translation, protein stability, oxygen limitation and secretion from the microbial host cell. More specifically, some of the molecular features that have been manipulated to control gene expression include: (1) the nature of the relevant transcriptional promoter and terminator sequences; (2) the number of copies of the cloned gene and whether the gene is plasmid-borne or integrated into the genome of the host cell; (3) the final cellular location of the synthesized foreign protein; (4) the efficiency of translation and correct folding of the protein in the host organism; (5) the intrinsic stability of the mRNA and protein of the cloned gene within the host cell; and (6) the codon usage within the cloned gene, such that its frequency approaches the frequency of preferred codon usage of the host cell. Each of these types of modifications are encompassed in the present invention, as means to further optimize expression of the delta-5 desaturase described herein.
[0215] Once the DNA encoding a polypeptide suitable for expression in an appropriate microbial host cell (e.g., oleaginous yeast) has been obtained (e.g., a chimeric gene comprising a promoter, ORF and terminator), it is placed in a plasmid vector capable of autonomous replication in a host cell, or it is directly integrated into the genome of the host cell. Integration of expression cassettes can occur randomly within the host genome or can be targeted through the use of constructs containing regions of homology with the host genome sufficient to target recombination within the host locus. Where constructs are targeted to an endogenous locus, all or some of the transcriptional and translational regulatory regions can be provided by the endogenous locus.
[0216] In the present invention, the preferred method of expressing genes in Yarrowia lipolytica is by integration of linear DNA into the genome of the host; and, integration into multiple locations within the genome can be particularly useful when high level expression of genes are desired [e.g., in the Ura3 locus (GenBank Accession No. AJ306421), the Leu2 gene locus (GenBank Accession No. AF260230), the Lys5 gene (GenBank Accession No. M34929), the Aco2 gene locus (GenBank Accession No. AJ001300), the Pox3 gene locus (Pox3: GenBank Accession No. XP--503244; or, Aco3: GenBank Accession No. AJ001301), the delta-12 desaturase gene locus (PCT Publication No. WO2004/104167), the bpi gene locus (GenBank Accession No. Z50020) and/or the Lip2 gene locus (GenBank Accession No. AJ012632)].
[0217] Advantageously, the Ura3 gene can be used repeatedly in combination with 5-fluoroorotic acid (5-fluorouracil-6-carboxylic acid monohydrate; "5-FOA") selection (infra), to readily permit genetic modifications to be integrated into the Yarrowia genome in a facile manner.
[0218] Where two or more genes are expressed from separate replicating vectors, it is desirable that each vector has a different means of selection and should lack homology to the other construct(s) to maintain stable expression and prevent reassortment of elements among constructs. Judicious choice of regulatory regions, selection means and method of propagation of the introduced construct(s) can be experimentally determined so that all introduced genes are expressed at the necessary levels to provide for synthesis of the desired products.
[0219] Constructs comprising the gene of interest may be introduced into a microbial host cell by any standard technique. These techniques include transformation (e.g., lithium acetate transformation [Methods in Enzymology, 194:186-187 (1991)]), protoplast fusion, bolistic impact, electroporation, microinjection, or any other method that introduces the gene of interest into the host cell. More specific teachings applicable for oleaginous yeasts (i.e., Yarrowia lipolytica) include U.S. Pat. No. 4,880,741 and U.S. Pat. No. 5,071,764 and Chen, D. C. et al. (Appl. Microbiol. Biotechnol., 48(2)232-235 (1997)).
[0220] For convenience, a host cell that has been manipulated by any method to take up a DNA sequence (e.g., an expression cassette) will be referred to as "transformed" or "recombinant" herein. Thus, the term "transformed" and "recombinant" are used interchangeably herein. The transformed host will have at least one copy of the expression construct and may have two or more, depending upon whether the gene is integrated into the genome, amplified or is present on an extrachromosomal element having multiple copy numbers.
[0221] The transformed host cell can be identified by various selection techniques, as described in PCT Publication Nos. WO 2004/101757 and WO 2006/052870. Preferred selection methods for use herein are resistance to kanamycin, hygromycin and the amino glycoside G418, as well as ability to grow on media lacking uracil, leucine, lysine, tryptophan or histidine. In alternate embodiments, 5-FOA is used for selection of yeast Ura- mutants. The compound is toxic to yeast cells that possess a functioning URA3 gene encoding orotidine 5'-monophosphate decarboxylase (OMP decarboxylase); thus, based on this toxicity, 5-FOA is especially useful for the selection and identification of Ura.sup.- mutant yeast strains (Bartel, P. L. and Fields, S., Yeast 2-Hybrid System, Oxford University: New York, v. 7, pp 109-147, 1997). More specifically, one can first knockout the native Ura3 gene to produce a strain having a Ura+ phenotype, wherein selection occurs based on 5-FOA resistance. Then, a cluster of multiple chimeric genes and a new Ura3 gene can be integrated into a different locus of the Yarrowia genome to thereby produce a new strain having a Ura+ phenotype. Subsequent integration produces a new Ura3- strain (again identified using 5-FOA selection), when the introduced Ura3 gene is knocked out. Thus, the Ura3 gene (in combination with 5-FOA selection) can be used as a selection marker in multiple rounds of transformation.
[0222] Following transformation, substrates suitable for the instant delta-5 desaturase (and, optionally other PUFA enzymes that are co-expressed within the host cell) may be produced by the host either naturally or transgenically, or they may be provided exogenously.
[0223] Microbial host cells for expression of the instant genes and nucleic acid fragments may include hosts that grow on a variety of feedstocks, including simple or complex carbohydrates, fatty acids, organic acids, oils and alcohols, and/or hydrocarbons over a wide range of temperature and pH values. Based on the needs of the Applicants' Assignee, the genes described in the instant invention will be expressed in an oleaginous yeast (and in particular Yarrowia lipolytica); however, it is contemplated that because transcription, translation and the protein biosynthetic apparatus is highly conserved, any bacteria, yeast, algae and/or fungus will be a suitable microbial host for expression of the present nucleic acid fragments. Preferred microbial hosts, however, are oleaginous yeasts. These organisms are naturally capable of oil synthesis and accumulation, wherein the oil can comprise greater than about 25% of the cellular dry weight, more preferably greater than about 30% of the cellular dry weight, and most preferably greater than about 40% of the cellular dry weight. Genera typically identified as oleaginous yeast include, but are not limited to: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces. More specifically, illustrative oil-synthesizing yeasts include: Rhodosporidium toruloides, Lipomyces starkeyii, L. lipoferus, Candida revkaufi, C. pulcherrima, C. tropicalis, C. utilis, Trichosporon pullans, T. cutaneum, Rhodotorula glutinus, R. graminis, and Yarrowia lipolytica (formerly classified as Candida lipolytica).
[0224] Most preferred is the oleaginous yeast Yarrowia lipolytica; and, in a further embodiment, most preferred are the Y. lipolytica strains designated as ATCC #20362, ATCC #8862, ATCC #18944, ATCC #76982 and/or LGAM S(7)1 (Papanikolaou S., and Aggelis G., Bioresour. Technol. 82(1):43-9 (2002)).
[0225] Historically, various strains of Y. lipolytica have been used for the manufacture and production of: isocitrate lyase; lipases; polyhydroxyalkanoates; citric acid; erythritol; 2-oxoglutaric acid; γ-decalactone; γ-dodecalatone; and pyruvic acid. Specific teachings applicable for engineering ARA, EPA and DHA production in Y. lipolytica are provided in U.S. patent application Ser. No. 11/264,784 (WO 2006/055322), U.S. patent application Ser. No. 11/265,761 (WO 2006/052870) and U.S. patent application Ser. No. 11/264,737 (WO 2006/052871), respectively.
[0226] Other preferred microbial hosts include oleaginous bacteria, algae and other fungi; and, within this broad group of microbial hosts, of particular interest are microorganisms that synthesize omega-3/omega-6 fatty acids (or those that can be genetically engineered for this purpose [e.g., other yeast such as Saccharomyces cerevisiae]). Thus, for example, transformation of Mortierella alpina (which is commercially used for production of ARA) with any of the present delta-5 desaturase genes under the control of inducible or regulated promoters could yield a transformant organism capable of synthesizing increased quantities of DGLA. The method of transformation of M. alpina is described by Mackenzie et al. (Appl. Environ. Microbial., 66:4655 (2000)). Similarly, methods for transformation of Thraustochytriales microorganisms are disclosed in U.S. Pat. No. 7,001,772.
[0227] Based on the teachings described above, in one embodiment this invention is drawn to a method of producing either ARA or EPA, respectively, comprising: [0228] (a) providing an oleaginous yeast comprising: [0229] (i) a first recombinant DNA construct comprising an isolated polynucleotide encoding a delta-5 desaturase polypeptide, operably linked to at least one regulatory sequence; and, [0230] (ii) a source of desaturase substrate consisting of either DGLA or ETA, respectively; and, [0231] (b) growing the yeast of step (a) in the presence of a suitable fermentable carbon source wherein the gene encoding the delta-5 desaturase polypeptide is expressed and DGLA is converted to ARA or ETA is converted to EPA, respectively; and, [0232] (c) optionally recovering the ARA or EPA, respectively, of step (b). Substrate feeding may be required.
[0233] Of course, since naturally produced PUFAs in oleaginous yeast are limited to 18:2 fatty acids (i.e., LA), and less commonly, 18:3 fatty acids (i.e., ALA), in more preferred embodiments of the present invention the oleaginous yeast will be genetically engineered to express multiple enzymes necessary for long-chain PUFA biosynthesis (thereby enabling production of e.g., ARA, EPA, DPA and DHA), in addition to the delta-5 desaturases described herein.
[0234] Specifically, in one embodiment this invention concerns an oleaginous yeast comprising:
[0235] (a) a first recombinant DNA construct comprising an isolated polynucleotide encoding a delta-5 desaturase polypeptide, operably linked to at least one regulatory sequence; and,
[0236] (b) at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of: a delta-4 desaturase, a delta-5 desaturase, delta-6 desaturase, a delta-9 desaturase, a delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase, a delta-9 elongase, a C14/16 elongase, a C16/18 elongase, a C18/20 elongase and a C20/22 elongase.
[0237] In particularly preferred embodiments, the at least one additional recombinant
[0238] DNA construct encodes a polypeptide having delta-9 elongase activity, e.g., the delta-9 elongase isolated or derived from Isochrysis galbana (GenBank Accession No. AF390174; IgD9e or IgD9eS) or the delta-9 elongase isolated or derived from Euglena gracilis.
Metabolic Engineering of Omega-3 and/or Omega-6 Fatty Acid Biosynthesis in Microbes
[0239] Methods for manipulating biochemical pathways are well known to those skilled in the art; and, it is expected that numerous manipulations will be possible to maximize omega-3 and/or omega-6 fatty acid biosynthesis in oleaginous yeasts, and particularly, in Yarrowia lipolytica. This manipulation may require metabolic engineering directly within the PUFA biosynthetic pathway or additional coordinated manipulation of various other metabolic pathways.
[0240] In the case of manipulations within the PUFA biosynthetic pathway, it may be desirable to increase the production of LA to enable increased production of omega-6 and/or omega-3 fatty acids. Introducing and/or amplifying genes encoding delta-9 and/or delta-12 desaturases may accomplish this. To maximize production of omega-6 unsaturated fatty acids, it is well known to one skilled in the art that production is favored in a host microorganism that is substantially free of ALA; thus, preferably, the host is selected or obtained by removing or inhibiting delta-15 or omega-3 type desaturase activity that permits conversion of LA to ALA. Alternatively, it may be desirable to maximize production of omega-3 fatty acids (and minimize synthesis of omega-6 fatty acids). In this example, one could utilize a host microorganism wherein the delta-12 desaturase activity that permits conversion of oleic acid to LA is removed or inhibited; subsequently, appropriate expression cassettes would be introduced into the host, along with appropriate substrates (e.g., ALA) for conversion to omega-3 fatty acid derivatives of ALA (e.g., STA, ETrA, ETA, EPA, DPA, DHA).
[0241] In alternate embodiments, biochemical pathways competing with the omega-3 and/or omega-6 fatty acid biosynthetic pathways for energy or carbon, or native PUFA biosynthetic pathway enzymes that interfere with production of a particular PUFA end-product, may be eliminated by gene disruption or down-regulated by other means (e.g., antisense mRNA).
[0242] Detailed discussion of manipulations within the PUFA biosynthetic pathway as a means to increase ARA, EPA or DHA (and associated techniques thereof) are presented in PCT Publication Nos. WO 2006/055322, WO 2006/052870 and WO 2006/052871, respectively, as are desirable manipulations in the TAG biosynthetic pathway and the TAG degradation pathway (and associated techniques thereof).
[0243] Within the context of the present invention, it may be useful to modulate the expression of the fatty acid biosynthetic pathway by any one of the strategies described above. For example, the present invention provides methods whereby genes encoding key enzymes in the delta-9 elongase/delta-8 desaturase biosynthetic pathway are introduced into oleaginous yeasts for the production of omega-3 and/or omega-6 fatty acids. It will be particularly useful to express the present the delta-5 desaturase genes in oleaginous yeasts that do not naturally possess omega-3 and/or omega-6 fatty acid biosynthetic pathways and coordinate the expression of these genes, to maximize production of preferred PUFA products using various means for metabolic engineering of the host organism.
Microbial Fermentation Processes for PUFA Production
[0244] The transformed host cell is grown under conditions that optimize expression of chimeric desaturase genes and produce the greatest and the most economical yield of desired PUFAs. In general, media conditions that may be optimized include the type and amount of carbon source, the type and amount of nitrogen source, the carbon-to-nitrogen ratio, the amount of different mineral ions, the oxygen level, growth temperature, pH, length of the biomass production phase, length of the oil accumulation phase and the time and method of cell harvest. Yarrowia lipolytica are generally grown in complex media (e.g., yeast extract-peptone-dextrose broth (YPD)) or a defined minimal media that lacks a component necessary for growth and thereby forces selection of the desired expression cassettes (e.g., Yeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.)).
[0245] Fermentation media in the present invention must contain a suitable carbon source. Suitable carbon sources are taught in PCT Publication No. WO 2004/101757. Although it is contemplated that the source of carbon utilized in the present invention may encompass a wide variety of carbon-containing sources, preferred carbon sources are sugars, glycerol, and/or fatty acids. Most preferred is glucose and/or fatty acids containing between 10-22 carbons. Nitrogen may be supplied from an inorganic (e.g., (NH4)2SO4) 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+2, Co+2, Zn+2, Mg+2) that promote synthesis of lipids and PUFAs (Nakahara, T, et al., Ind. Appl. Single Cell Oils, D. J. Kyle and R. Colin, eds. pp 61-97 (1992)).
[0246] Preferred growth media in the present invention are common commercially prepared media, such as Yeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.). Other defined or synthetic growth media may also be used and the appropriate medium for growth of the transformant host cells will be known by one skilled in the art of microbiology or fermentation science. A suitable pH range for the fermentation is typically between about pH 4.0 to pH 8.0, wherein pH 5.5 to pH 7.5 is preferred as the range for the initial growth conditions. The fermentation may be conducted under aerobic or anaerobic conditions, wherein microaerobic conditions are preferred.
[0247] Typically, accumulation of high levels of PUFAs in oleaginous yeast cells requires a two-stage process, since the metabolic state must be "balanced" between growth and synthesis/storage of fats. Thus, most preferably, a two-stage fermentation process is necessary for the production of PUFAs in oleaginous yeast (e.g., Yarrowia lipolytica). This approach is described in PCT Publication No. WO 2004/101757, as are various suitable fermentation process designs (i.e., batch, fed-batch
and continuous) and considerations during growth. Purification and Processing of PUFA Oils
[0248] PUFAs may be found in the host microorganisms and plants as free fatty acids or in esterified forms such as acylglycerols, phospholipids, sulfolipids or glycolipids, and may be extracted from the host cells through a variety of means well-known in the art. One review of extraction techniques, quality analysis and acceptability standards for yeast lipids is that of Z. Jacobs (Critical Reviews in Biotechnology, 12(5/6):463-491 (1992)). A brief review of downstream processing is also available by A. Singh and O. Ward (Adv. Appl. Microbiol., 45:271-312 (1997)).
[0249] In general, means for the purification of PUFAs may include extraction with organic solvents, sonication, supercritical fluid extraction (e.g., using carbon dioxide), saponification and physical means such as presses, or combinations thereof. One is referred to the teachings of PCT Publication No. WO 2004/101757 for additional details. Methods of isolating seed oils are well known in the art: (Young et al., Processing of Fats and Oils, In The Lipid Handbook, Gunstone et al., eds., Chapter 5 pp 253-257; Chapman & Hall: London (1994)). For example, soybean oil is produced using a series of steps involving the extraction and purification of an edible oil product from the oil-bearing seed. Soybean oils and soybean byproducts are produced using the generalized steps shown in Table 3.
TABLE-US-00003 TABLE 3 Generalized Steps for Soybean Oil and Byproduct Production Process Impurities Removed and/or Step Process By-Products Obtained # 1 soybean seed # 2 oil extraction meal # 3 degumming lecithin # 4 alkali or physical refining gums, free fatty acids, pigments # 5 water washing soap # 6 bleaching color, soap, metal # 7 (hydrogenation) # 8 (winterization) stearine # 9 deodorization free fatty acids, tocopherols, sterols, volatiles # 10 oil products
[0250] More specifically, soybean seeds are cleaned, tempered, dehulled and flaked, thereby increasing the efficiency of oil extraction. Oil extraction is usually accomplished by solvent (e.g., hexane) extraction but can also be achieved by a combination of physical pressure and/or solvent extraction. The resulting oil is called crude oil. The crude oil may be degummed by hydrating. phospholipids and other polar and neutral lipid complexes that facilitate their separation from the nonhydrating, triglyceride fraction (soybean oil). The resulting lecithin gums may be further processed to make commercially important lecithin products used in a variety of food and industrial products as emulsification and release (i.e., antisticking) agents. Degummed oil may be further refined for the removal of impurities (primarily free fatty acids, pigments and residual gums). Refining is accomplished by the addition of a caustic agent that reacts with free fatty acid to form soap and hydrates phosphatides and proteins in the crude oil. Water is used to wash out traces of soap formed during refining. The soapstock byproduct may be used directly in animal feeds or acidulated to recover the free fatty acids. Color is removed through adsorption with a bleaching earth that removes most of the chlorophyll and carotenoid compounds. The refined oil can be hydrogenated, thereby resulting in fats with various melting properties and textures. Winterization (fractionation) may be used to remove stearine from the hydrogenated oil through crystallization under carefully controlled cooling conditions. Deodorization (principally via steam distillation under vacuum) is the last step and is designed to remove compounds which impart odor or flavor to the oil. Other valuable byproducts such as tocopherols and sterols may be removed during the deodorization process. Deodorized distillate containing these byproducts may be sold for production of natural vitamin E and other high-value pharmaceutical products. Refined, bleached, (hydrogenated, fractionated) and deodorized oils and fats may be packaged and sold directly or further processed into more specialized products. A more detailed reference to soybean seed processing, soybean oil production and byproduct utilization can be found in Erickson, Practical Handbook of Soybean Processing and Utilization, The American Oil Chemists' Society and United Soybean Board (1995). Soybean oil is liquid at room temperature because it is relatively low in saturated fatty acids when compared with oils such as coconut, palm, palm kernel and cocoa butter.
[0251] Plant and microbial oils containing PUFAs that have been refined andfor purified can be hydrogenated, to thereby result in fats with various melting properties and textures. Many processed fats (including spreads, confectionary fats, hard butters, margarines, baking shortenings, etc.) require varying degrees of solidity at room temperature and can only be produced through alteration of the source oil's physical properties. This is most commonly achieved through catalytic hydrogenation.
[0252] Hydrogenation is a chemical reaction in which hydrogen is added to the unsaturated fatty acid double bonds with the aid of a catalyst such as nickel. For example, high oleic soybean oil contains unsaturated oleic, LA and linolenic fatty acids and each of these can be hydrogenated. Hydrogenation has two primary effects. First, the oxidative stability of the oil is increased as a result of the reduction of the unsaturated fatty acid content. Second, the physical properties of the oil are changed because the fatty acid modifications increase the melting point resulting in a semi-liquid or solid fat at room temperature.
[0253] There are many variables which affect the hydrogenation reaction, which in turn alter the composition of the final product. Operating conditions including pressure, temperature, catalyst type and concentration, agitation and reactor design are among the more important parameters that can be controlled. Selective hydrogenation conditions can be used to hydrogenate the more unsaturated fatty acids in preference to the less unsaturated ones, Very light or brush hydrogenation is often employed to increase stability of liquid oils. Further hydrogenation converts a liquid oil to a physically solid fat. The degree of hydrogenation depends on the desired performance and melting characteristics designed for the particular end product. Liquid shortenings (used in the manufacture of baking products, solid fats and shortenings used for commercial frying and roasting operations) and base stocks for margarine manufacture are among the myriad of possible oil and fat products achieved through hydrogenation. A more detailed description of hydrogenation and hydrogenated products can be found in Patterson, H. B. W., Hydrogenation of Fats and Oils: Theory and Practice. The American Oil Chemists' Society (1994).
[0254] Hydrogenated oils have become somewhat controversial due to the presence of trans-fatty acid isomers that result from the hydrogenation process. Ingestion of large amounts of trans-isomers has been linked with detrimental health effects including increased ratios of low density to high density lipoproteins in the blood plasma and increased risk of coronary heart disease.
PUFA-Containing Oils for Use in Foodstuffs
[0255] The market place currently supports a large variety of food and feed products, incorporating omega-3 and/or omega-6 fatty acids (particularly ARA, EPA and DHA). It is contemplated that the plant/seed oils, altered seeds and microbial oils of the invention comprising PUFAs will function in food and feed products to impart the health benefits of current formulations. Compared to other vegetable oils, the oils of the invention are believed to function similarly to other oils in food applications from a physical standpoint (for example, partially hydrogenated oils such as soybean oil are widely used as ingredients for soft spreads, margarine and shortenings for baking and frying).
[0256] Plant/seed oils, altered seeds and microbial oils containing omega-3 and/or omega-6 fatty acids as described herein will be suitable for use in a variety of food and feed products including, but not limited to: food analogs, meat products, cereal products, baked foods, snack foods and dairy products. Additionally, the present plant/seed oils, altered seeds and microbial oils may be used in formulations to impart health benefit in medical foods including medical nutritionals, dietary supplements, infant formula as well as pharmaceutical products. One of skill in the art of food processing and food formulation will understand how the amount and composition of the plant and microbial oils may be added to the food or feed product. Such an amount will be referred to herein as an "effective" amount and will depend on the food or feed product, the diet that the product is intended to supplement or the medical condition that the medical food or medical nutritional is intended to correct or treat.
[0257] Food analogs can be made using processes well known to those skilled in the art. There can be mentioned meat analogs, cheese analogs, milk analogs and the like. Meat analogs made from soybeans contain soy protein or tofu and other ingredients mixed together to simulate various kinds of meats. These meat alternatives are sold as frozen, canned or dried foods. Usually, they can be used the same way as the foods they replace. Meat alternatives made from soybeans are excellent sources of protein, iron and B vitamins. Examples of meat analogs include, but are not limited to: ham analogs, sausage analogs, bacon analogs, and the like.
[0258] Food analogs can be classified as imitation or substitutes depending on their functional and compositional characteristics. For example, an imitation cheese need only resemble the cheese it is designed to replace. However, a product can generally be called a substitute cheese only if it is nutritionally equivalent to the cheese it is replacing and meets the minimum compositional requirements for that cheese. Thus, substitute cheese will often have higher protein levels than imitation cheeses and be fortified with vitamins and minerals.
[0259] Milk analogs or nondairy food products include, but are not limited to, imitation milks and nondairy frozen desserts (e.g., those made from soybeans and/or soy protein products).
[0260] Meat products encompass a broad variety of products. In the United States "meat" includes "red meats" produced from cattle, hogs and sheep. In addition to the red meats there are poultry items which include chickens, turkeys, geese, guineas, ducks and the fish and shellfish. There is a wide assortment of seasoned and processed meat products: fresh, cured and fried, and cured and cooked. Sausages and hot dogs are examples of processed meat products. Thus, the term "meat products" as used herein includes, but is not limited to, processed meat products.
[0261] A cereal food product is a food product derived from the processing of a cereal grain. A cereal grain includes any plant from the grass family that yields an edible grain (seed). The most popular grains are barley, corn, millet, oats, quinoa, rice, rye, sorghum, triticale, wheat and wild rice. Examples of a cereal food product include, but are not limited to: whole grain, crushed grain, grits, flour, bran, germ, breakfast cereals, extruded foods, pastas, and the like.
[0262] A baked goods product comprises any of the cereal food products mentioned above and has been baked or processed in a manner comparable to baking (i.e., to dry or harden by subjecting to heat). Examples of a baked good product include, but are not limited to: bread, cakes, doughnuts, bars, pastas, bread crumbs, baked snacks, mini-biscuits, mini-crackers, mini-cookies, and mini-pretzels. As was mentioned above, oils of the invention can be used as an ingredient.
[0263] A snack food product comprises any of the above or below described food products.
[0264] A fried food product comprises any of the above or below described food products that has been fried.
[0265] A health food product is any food product that imparts a health benefit. Many oilseed-derived food products may be considered as health foods.
[0266] A beverage can be in a liquid or in a dry powdered form.
[0267] For example, there can be mentioned non-carbonated drinks such as fruit juices, fresh, frozen, canned or concentrate; flavored or plain milk drinks, etc. Adult and infant nutritional formulas are well known in the art and commercially available (e,g, Similace, Ensure®, Jevity®, and Alimentum® from Ross Products Division, Abbott Laboratories).
[0268] Infant formulas are liquids or reconstituted powders fed to infants and young children. "Infant formula" is defined herein as an enteral nutritional product which can be substituted for human breast milk in feeding infants and typically is composed of a desired percentage of fat mixed with desired percentages of carbohydrates and proteins in an aquous solution (e.g., see U.S. Pat. No. 4,670,285). Based on the worldwide composition studies, as well as levels specified by expert groups, average human breast milk typically contains about 0.20% to 0.40% of total fatty acids (assuming about 50% of calories from fat); and, generally 25. the ratio of DHA to ARA would range from about 1:1 to 1:2 (see, e.g., formulations of Enfamil LIPIL® (Mead Johnson & Company) and Similac Advance® (Ross Products Division, Abbott Laboratories)). Infant formulas have a special role to play in the diets of infants because they are often the only source of nutrients for infants; and, although breast-feeding is still the best nourishment for infants, infant formula is a close enough second that babies not only survive but thrive.
[0269] A dairy product is a product derived from milk. A milk analog or nondairy product is derived from a source other than milk, for example, soymilk as was discussed above. These products include, but are not limited to: whole milk, skim milk, fermented milk products such as yogurt or sour milk, cream, butter, condensed milk, dehydrated milk, coffee whitener, coffee creamer, ice cream, cheese, etc.
[0270] Additional food products into which the PUFA-containing oils of the invention could be included are, for example, chewing gums, confections and frostings, gelatins and puddings, hard and soft candies, jams and jellies, white granulated sugar, sugar substitutes, sweet sauces, toppings and syrups, and dry-blended powder mixes.
PUFA-Containing Oils for Use in Health Food Products and Pharmaceuticals
[0271] A health food product is any food product that imparts a health benefit and include functional foods, medical foods, medical nutritionals and dietary supplements. Additionally, the plant/seed oils, altered seeds and microbial oils of the invention may be used in standard pharmaceutical compositions (e.g., the long-chain PUFA containing oils could readily be incorporated into the any of the above mentioned food products, to thereby produce a functional or medical food). More concentrated formulations comprising PUFAs include capsules, powders, tablets, softgels, gelcaps, liquid concentrates and emulsions which can be used as a dietary supplement in humans or animals other than humans.
PUFA-Containing Oils for Use in Animal Feeds
[0272] Animal feeds are generically defined herein as products intended for use as feed or for mixing in feed for animals other than humans. The plant/seed oils, altered seeds and microbial oils of the invention can be used as an ingredient in various animal feeds.
[0273] More specifically, although not limited therein, it is expected that the oils of the invention can be used within pet food products, ruminant and poultry food products and aquacultural food products. Pet food products are those products intended to be fed to a pet (e.g., dog, cat, bird, reptile, rodent). These products can include the cereal and health food products above, as well as meat and meat byproducts, soy protein products, grass and hay products (e.g., alfalfa, timothy, oat or brome grass, vegetables). Ruminant and poultry food products are those wherein the product is intended to be fed to an animal (e.g., turkeys, chickens, cattle, swine). As with the pet foods above, these products can include cereal and health food products, soy protein products, meat and meat byproducts, and grass and hay products as listed above. Aquacultural food products (or "aquafeeds") are those products intended to be used in aquafarming, i.e., which concerns the propagation, cultivation or farming of aquatic organisms and/or animals in fresh or marine waters.
EXAMPLES
[0274] The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.
General Methods
[0275] Standard recombinant DNA and molecular cloning techniques used in the Examples are well known in the art and are described by: 1.) Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989) (Maniatis); 2.) T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1984); and 3.) Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience Hoboken, N.J. (1987).
[0276] Materials and methods suitable for the maintenance and growth of microbial cultures are well known in the art. Techniques suitable for use in the following examples may be found as set out in Manual of Methods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, Eds), American Society for Microbiology: Washington, D.C. (1994)); or by Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, 2nd ed., Sinauer Associates: Sunderland, Mass. (1989). All reagents, restriction enzymes and materials used for the growth and maintenance of microbial cells were obtained from Aldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.), unless otherwise specified. E. coli (XL1-Blue) competent cells were purchased from the Stratagene Company (San Diego, Calif.). E. coli strains were typically grown at 37° C. on Luria Bertani (LB) plates.
[0277] General molecular cloning was performed according to standard methods (Sambrook et al., supra). DNA sequence was generated on an ABI Automatic sequencer using dye terminator technology (U.S. Pat. No. 5,366,860; EP 272,007) using a combination of vector and insert-specific primers. Sequence editing was performed in Sequencher (Gene Codes Corporation, Ann Arbor, Mich.). All sequences represent coverage at least two times in both directions. Unless otherwise indicated herein comparisons of genetic sequences were accomplished using DNASTAR software (DNASTAR Inc., Madison, Wis.).
[0278] The meaning of abbreviations is as follows: "sec" means second(s), "min" means minute(s), "h" means hour(s), "d" means day(s), "μL`" means microliter(s), "mL" means milliliter(s), "L" means liter(s), "μM" means micromolar, "mM" means millimolar, "M" means molar, "mmol" means millimole(s), "μmole" mean micromole(s), "g" means gram(s), "μg" means microgram(s), "ng" means nanogram(s), "U" means unit(s), "bp" means base pair(s) and "kB" means kilobase(s)
Transformation and Cultivation of Yarrowia lipolytica
[0279] Yarrowia lipolytica strain ATCC #20362 was purchased from the American Type Culture Collection (Rockville, Md.). Y. lipolytica strains were usually grown at 28° C. on YPD agar (1% yeast extract, 2% bactopeptone, 2% glucose, 2% agar).
[0280] Transformation of Y. lipolytica was performed according to the method of Chen, D. C. et al. (Appl. Microbial Biotechnol., 48(2):232-235 (1997)), unless otherwise noted. Briefly, Yarrowia was streaked onto a YPD plate and grown at 30° C. for approximately 18 hr. Several large loopfuls of cells were scraped from the plate and resuspended in 1 mL of transformation buffer containing: 2.25 mL of 50% PEG, average MW 3350; 0.125 mL of 2 M Li acetate, pH 6.0; 0.125 mL of 2 M DTT; and 50 μg sheared salmon sperm DNA. Then, approximately 500 ng of linearized plasmid DNA was incubated in 100 μl of resuspended cells, and maintained at 39° C. for 1 hr with vortex mixing at 15 min intervals. The cells were plated onto selection media plates and maintained at 30° C. for 2 to 3 days. For selection of transformants, minimal medium ("MM") was generally used; the composition of MM is as follows: 0.17% yeast nitrogen base (DIFCO Laboratories, Detroit, Mich.) without ammonium sulfate or amino acids, 2% glucose, 0.1% praline, pH 6.1). Supplements of uracil were added as appropriate to a final concentration of 0.01% (thereby producing "MMU" selection media, prepared with 20 g/L agar).
[0281] Alternatively, transformants were selected on 5-fluoroorotic acid ("FOA"; also 5-fluorouracil-6-carboxylic acid monohydrate) selection media, comprising: 0.17% yeast nitrogen base (DIFCO Laboratories) without ammonium sulfate or amino acids, 2% glucose, 0.1% proline, 75 mg/L uracil, 75 mg/L uridine, 900 mg/L FOA (Zymo Research Corp., Orange, Cali.f) and 20 g/L agar.
Fatty Acid Analysis of Yarrowia lipolytica
[0282] For fatty acid analysis, cells were collected by centrifugation and lipids were extracted as described in Bligh, E. G. & Dyer, W. J. (Can. J. Biochem. Physiol., 37:911-917 (1959)). Fatty acid methyl esters were prepared by transesterification of the lipid extract with sodium methoxide (Roughen, G., and Nishida I., Arch Biochem Biophys., 276(1):38-46 (1990)) and subsequently analyzed with a Hewlett-Packard 6890 GC fitted with a 30-m×0.25 mm (i.d.) HP-INNOWAX (Hewlett-Packard) column. The oven temperature was from 170° C. (25 min hold) to 185° C. at 3.5° C./min.
[0283] For direct base transesterification, Yarrowia culture (3 mL) was harvested, washed once in distilled water, and dried under vacuum in a Speed-Vac for 5-10 min. Sodium methoxide (100 μl 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 μl hexane, the sample was vortexed and spun. The upper layer was removed and analyzed by GC as described above.
Example 1
Peridinium sp. CCMP626 Lipid Profile and RNA Isolation
[0284] Peridinium sp. CCMP626 (red algae) was purchased from The Provasoli-Guillard National Center for Culture of Marine Phytoplankton (Boothbay Harbor, Me.). About 200 mg cells were dried for 5 min under vacuum, resuspended in 100 μL 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 μL injected from hexane layer) were separated and quantified using a Hewlett-Packard 6890 Gas Chromatograph fitted with an Omegawax 320 fused silica capillary column (Catalog #24152, Supelco Inc., Bellefonte, Pa.). The oven temperature was programmed to hold at 220° C. for 2.7 min, increase to 240° C. at 20° C./min and then hold for an additional 2.3 min. Carrier gas was supplied by a Whatman hydrogen generator. Retention times were compared to those for methyl esters of standards commercially available (Catalog #U-99-A, Nu-Chek Prep, Inc., Elysian, Minn.) and the resulting chromatogram is shown in FIG. 2.
[0285] The GC profile demonstrated that Peridinium sp. CCMP626 cells produced large amounts of both ARA and EPA, thus suggesting the presence of a delta-5 desaturase with high efficiency. Since only a trace amount of GLA was detected, it was hypothesized that Peridinium sp. CCMP626 utilizes a delta-9 elongase/delta-8 desaturase pathway for PUFA production.
[0286] Total RNA was extracted from CCMP626 cells. Specifically, cells from 1 L of culture were collected by centrifugation, quick frozen in liquid N2 and stored at -80° C. The cell pellet was resuspended in 1 mL of Trizol reagent (Invitrogen, Carlsbad, Calif.), mixed with 0.6 mL of glass beads (0.5 mm), and the mixture was homogenized at the highest setting on a Biospec Mini leadbeater (Bartlesville, Okla.) for 3 min. Total RNA was then isolated, according to Invitrogen's protocol for Trizol. In this way, total RNA (34 μg) was obtained from 1 L culture. The total RNA sample was used for preparation of cDNA.
Example 2
Peridinium sp. CCMP626 cDNA Synthesis
[0287] cDNA was synthesized directly from the Peridinium sp. CCMP626 total RNA as follows. Specifically, the total RNA was primered with adapter primer AP (SEQ ID NO:27) from Invitrogen's 3'-RACE kit (Carlsbad, Calif.), in the presence of the Smart IV oligonucleotide (SEQ ID NO:28) from the BD-Clontech Creator® Smart® cDNA library kit (Mississauga, ON, Canada). The reverse transcription was done with Superscript II reverse transcriptase from the 3'-RACE kit according to the protocol of the Creator® Smart® cDNA library kit.
[0288] The 1st strand cDNA synthesis mixture was used as template for PCR amplification, using primer AP (SEQ ID NO:27) as the 3' primer and CDSIII 5' primer (SEQ ID NO:29) as the 5' primer (supplied with the BD-Clontech Creator Smart® cDNA library kit). Amplification was carried out with Clontech Advantage cDNA polymerase mix at 94° C. for 30 sec, followed by 20 cycles of 94° C. for 10 sec and 68° C. for 6 min. A final extension at 68° C. for 7 min was performed.
Example 3
Isolation of a Portion of the Coding Region of the Peridinium sp. CCMP626 Delta-5 Desaturase Gene
[0289] The present Example describes the identification of a portion of the Peridinium sp. CCMP626 gene encoding delta-5 desaturase (designated herein as "RD5" [i.e., red algae D5] and corresponding to SECS ID NOs:1 and 2), by use of primers derived from conserved regions of other known delta-5 and delta-8 desaturase sequences.
[0290] Various considerations were made when evaluating which desaturases might enable design of degenerate primers suitable to isolate the Peridinium sp. CCMP626 delta-5 desaturase. Specifically, the Applicants knew that only delta-5, delta-6 and delta-8 desaturase sequences comprise a conserved `HPGG` motif at their N-terminus (wherein the `HPGG` domain is part of the well-known cytochrome B5 domain); in contrast, delta-9 desaturases possess a `HPGG` motif of the cytochrome B5 domain at their C-terminus, while both delta-17 and delta-12 desaturases lack the cytochrome B5 domain. Based on the GC results described in FIG. 2, it was assumed that a delta-9 elongase/delta-8 desaturase pathway operated in Peridinium sp. CCMP626; thus, among the desaturases sharing the N-terminal conserved `HPGG` motif, only delta-5 and delta-8 desaturases were expected within the organism. Finally, although only a few delta-8 desaturase sequences are known, numerous delta-5 desaturase are publicly available. The Applicants selected those delta-5 desaturase sequences that possessed lower homology to "traditional" delta-5 desaturase genes and that also shared high homology to one another.
[0291] Based on the above, the four delta-5 desaturases and two delta-8 desaturases shown below in Table 3 were aligned, using the method of Clustal W (slow, accurate, Gonnet option; Thompson et al., Nucleic Acids Res., 22:4673-4680 (1994)) of the MegAlign® program of DNASTAR software.
Table 3
Delta-5 and Delta-8 Desaturases Aligned to Identify Regions of Conserved
TABLE-US-00004 [0292] Amino Acids SEQ ID Desaturase Organism Reference NO: delta-5 Pythium irregulare GenBank Accession No. 12 AAL13311 delta-5 Phytophthora GenBank Accession No. 13 megasperma CAD53323 delta-5 Phaeodactylum GenBank Accession No. 14 tricornutum AAL92562 delta-5 Dictyostelium GenBank Accession No. 15 discoideum XP_640331 delta-8 Euglena gracilis PCT Publications No. WO 16 2006/012325 and No. WO 2006/012326 delta-8 Pavlova lutheri Example 12 (infra) 18
[0293] FIG. 3 shows a portion of the resulting alignment, containing several stretches of conserved amino acid sequence among the 6 different organisms. Based on this alignment, two sets of degenerate oligonucleotides were designed to amplify a portion of the coding region of the delta-5 desaturase gene from Peridinium sp. CCMP626, corresponding to the regions of FIG. 3 that are labeled as "Conserved Region 1" and "Conserved Region 2". Specifically, the conserved amino acid sequence GHH(IN)YTN (SEQ ID NO:19) was designed to correspond to Conserved Region 1, while the conserved amino acid sequence NFQ(V/A)(S/N)HV (SEQ ID NO:20) was designed to correspond to Conserved Region 2. In order to reduce the degeneracy of the oligonucleotides, 4 sets of oligonucleotides (i.e., 5-1A, 5-1B, 5-1C and 5-1D) were designed to encode Conserved Region 1; and, 4 sets of oligonucleotides (i.e., 5-4AR, 5-4BR, 5-4CR and 5-4DR) were designed to encode the anti-sense strand of Conserved Region 2.
TABLE-US-00005 TABLE 4 Degenerate Oligonucleotides Used To Amplify The Delta-5 Desaturase Gene From Peridinium sp. CCMP626 Oligo- nucleotide Name Sequence SEQ ID NO 5-1A GGHCAYCAYRTBTAYACAAA SEQ ID NO: 30 5-1B GGHCAYCAYRTBTAYACCAA SEQ ID NO: 31 5-1C GGHCAYCAYRTBTAYACGAA SEQ ID NO: 32 5-1D GGHCAYCAYRTBTAYACTAA SEQ ID NO: 33 5-4AR ACRTGRYTNACYTGRAAGTT SEQ ID NO: 34 5-4BR ACRTGRYTNACYTGRAAATT SEQ ID NO: 35 5-4CR ACRTGNGANACYTGRAAGTT SEQ ID NO: 36 5-4DR ACRTGNGANACYTGRAAATT SEQ ID NO: 37 [Note: The nucleic acid degeneracy code used for SEQ ID NOs: 30 to 37 was as follows: R = A/G; Y = C/T; B = G/T/C; and H = A/C/T.]
[0294] Based on the full-length sequences of the delta-5 sequences of FIG. 3, it was hypothesized that the Peridinium sp. CCMP626 delta-5 gene fragment amplified as described above would be about 630 by in length (lacking about 210 amino acids at its N-terminal and 70 amino acids at its C-terminal).
[0295] A total of sixteen different PCR amplifications were conducted, as all combinations of the primers were tested (i.e., primer 5-1A was used with each of 5-4AR, 5-4BR, 5-4CR and 5-4DR, individually; similarly, primer 5-1 B was used with each 5-4AR, 5-4BR, 5-4CR and 5-4DR; etc.). The PCR amplifications were carried out in a 50 μl total volume comprising: PCR buffer (containing 10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-HCl (pH 8.75), 2 mM MgSO4, 0.1% Triton X-100), 100 μg/mL BSA (final concentration), 200 μM each deoxyribonucleotide triphosphate, 10 pmole of each primer, 10 ng cDNA of Peridinium sp. CCMP626 and 1 μl of Taq DNA polymerase (Epicentre Technologies, Madison, Wis.). The thermocycler conditions were set for 35 cycles at 95° C. for 1 min, 56° C. for 30 sec and 72° C. for 1 min, followed by a final extension at 72° C. for 10 min.
[0296] The PCR products were purified using a Qiagen PCR purification kit (Valencia, Calif.). One fragment of the approximate expected size was then further purified following gel electrophoresis in 1% (w/v) agarose and then cloned into the pGEM-T-easy vector (Promega, Madison, Wis.). The ligated DNA was used to transform cells of E. coli DH10B and transformants were selected on LB (1% bacto-tryptone, 0.5% bacto-yeast extract and 1% NaCl) agar containing ampicillin (100 μg/mL). Analysis of the plasmid DNA from a group of 12 transformants confirmed the presence of the insert with the expected size (plasmids were designated as "pT-12-D1", "pT-12-D2", "pT-12-D3", etc. to "pT-12-D12").
[0297] Sequence analyses showed that pT-12-D5 contained a 563 by fragment (SEQ ID NO:4), which encoded 187 amino acids (SEQ ID NO:5) (including amino acids that corresponded to Conserved Region 1 and 2). Identity of the Peridinium sp. CCMP626 sequence was determined by conducting BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., J. Mol. Biol., 215:403-410 (1993)) searches for similarity to sequences contained in the BLAST "nr" database (comprising all non-redundant GenBank GDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the SWISS-PROT protein sequence database, EMBL and DDBJ databases). The sequence was analyzed for similarity to all publicly available DNA sequences contained in the "nr" database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). SEQ 1D NO:4 was compared for similarity to all publicly available protein sequences contained in the "nr" database, using the BLASTX algorithm (Gish, W. and States, D. J., Nature Genetics, 3:266-272 (1993)) provided by the NCBI.
[0298] The results of the BLASTX comparison summarizing the sequence to which SEQ ID NO:4 has the most similarity are reported according to the % identity, % similarity and Expectation value. "% Identity" is defined as the percentage of amino acids that are identical between the two proteins. "% Similarity" is defined as the percentage of amino acids that are identical or conserved between the two proteins. "Expectation value" estimates the statistical significance of the match, specifying the number of matches, with a given score, that are expected in a search of a database of this size absolutely by chance. Thus, the translated amino acid sequence of SEQ ID NO:4 (i.e., SEQ ID NO:5) had 64% identity and 78% similarity with the amino acid sequence of the delta-8-sphingolipid desaturase of Thalassiosira pseudonana (GenBank Accession No, AAX14502; SEQ ID NO:21), with an Expectation value of 2E-64; additionally, the partial fragment of SEQ ID NO:4 had 65% identity and 78% similarity with the delta-5 fatty acid desaturase of Phaeodactylum tricornutum (GenBank Accession No. AAL92562; SEQ ID NO:14), with an Expectation value of 7E-62.
Example 4
Isolation of the 5` Coding Region of the Peridinium sp. CCMP626 Delta-5 Desaturase Gene
[0299] To isolate the N-terminal portion of the putative delta-5 desaturase identified in Example 3, a modified 5' RACE technique based on RACE protocols from two different companies (i.e., Invitrogen and BD-Clontech) was utilized.
[0300] Briefly, the double-stranded cDNA of Peridinium sp. CCMP626 (Example 2) was used as the template in a 5' RACE experiment, comprising two separate.rounds of PCR amplification. In the first round of PCR amplification, the oligonucleotide primers consisted of a gene specific oligonucleotide (i.e., ODMW520; SEQ ID NO:38) and the generic oligonucleotide CDSIII 5' primer (SEQ ID NO:29) from the BD-Clontech Creator® Smart® cDNA library kit. The PCR amplifications were carried out in a 50 μl total volume, comprising: 25 μl of LA Tag® pre-mix (TakeRa Bio Inc., Otsu, Shiga, 520-2193, Japan), 10 pmole of each primer and 1 μl of Taq DNA polymerase (Epicentre Technologies, Madison, Wis.). The thermocycler conditions were set for 35 cycles at 95° C. for 1 min, 56° C. for 30 sec and 72° C. for 1 min, followed by a final extension at 72° C. for 10 min.
[0301] The second round of PCR amplification used 1 μl of the product from the first round PCR reaction as template. Primers consisted of a gene specific oligonucleotide (i.e., ODMW521; SEQ ID NO:39) and the generic oligonucleotide DNR CDS 5' (SEQ ID NO:40), supplied with BD-Clontech's Creator® Smart® cDNA library kit. Amplification was conducted as described above.
[0302] The products of the second round PCR reaction were electrophoresed in 1% (w/v) agarose. Products between 400 by and 800 by were then purified from the gel and cloned into the pGEM-T-easy vector (Promega, Madison, Wis.). The ligated DNA was used to transform E. coli DH10B and transformants were selected on LB agar containing ampicillin (100 μg/mL).
[0303] Analysis of the plasmid DNA from one transformant comprising the 5' region of the putative delta-5 desaturase gene confirmed the presence of the expected plasmid, designated pT-RD5-5'C2. Sequence analyses showed that pT-RD5-5'C2 contained a fragment of 693 by (SEQ 1D NO:6), which over with 182 by from the 5' end of the 563 by fragment of pT-12-D5 (Example 3; SEQ ID NO:4) and additionally provided 511 by of 5' upstream sequence (SEQ ID NO:7) (FIG. 4). The sequence of pT-RD5-5'C2 also corrected the sequence corresponding to Conserved Region 1, resulting from use of a degenerate oligonucleotide for initial PCR amplification of the 563 by fragment in pT-12-D5 (Example 3). However, there was no translation initiation codon in the extended 693 by fragment of SEQ ID NO:6.
[0304] A second round of the modified 5' RACE was carried out as described above, except that oligonucleotides ODMW541 (SEQ ID NO:41) and ODMW542 (SEQ ID NO:42) were used as gene-specific primers. Products between 200 by and 400 bp were then purified from a gel and cloned into the pGEM-T-easy vector (Promega, Madison, Wis.). The ligated DNA was transformed into E. coli DH10B and transformants were selected on LB agar containing ampicillin (100 μg/mL).
[0305] Analysis of the plasmid DNA from one transformant comprising the 5' region of the putative delta-5 desaturase gene confirmed the presence of the expected plasmid, designated pT-RD5-5'2nd . Sequence analyses showed that pT-RD5-5'2nd contained a fragment of 358 by (SEQ ID NO:8), which over-lapped with 197 by of the 5' end of the DNA fragment in pT-RD5-5'C2 described above and additionally provided 161 by of 5' upstream sequence (SEQ ID NO:9). One-hundred sixteen (116) by of the 5' extended fragment encoded the N-terminal portion of the putative delta-5 desaturase gene, including the translation initiation codon, thus providing the complete 5' sequence of the gene.
Example 5
Isolation of the 3' Coding Region of the Peridinium sp. CCMP626 Delta-5 Desaturase Gene
[0306] To isolate the C-terminal portion of the putative delta-5 desaturase identified in Example 3, a 3' RACE technique was utilized. The methodology was described above in Example 4; however, the primers used on both the first and second round of PCR amplification were as shown below in Table 5.
TABLE-US-00006 TABLE 5 Oligonucleotide Primers Used For 3' RACE PCR Gene Specific Generic Amplification Oligonucleotide Oligonucleotide 1st Round ODMW523 AUAP (SEQ ID NO: 43) (SEQ ID NO: 44) 2nd Round ODMW524 AUAP (SEQ ID NO: 45) (SEQ ID NO: 44) *Primer AUAP was supplied in Invitrogen's 3'-RACE kit (Carlsbad, CA).
[0307] Following isolation and purification of products (Le., 200-500 bp), the fragments were cloned into the pGEM-T-easy vector (Promega) and transformed into E. coli DH10B, as in Example 4.
[0308] Analysis of the plasmid DNA from one transformant comprising the 3' region of the delta-5 desaturase gene confirmed the presence of the expected plasmid, designated pT-RD5-3'. Sequence analyses showed that pT-RD5-3' contained a fragment of 299 by (SEQ ID NO:10), which over-lapped with 52 by from the 3' end of the 563 by fragment of pT-12-D5 (Example 3, SEQ ID NO:4) and provided 247 by of additional 3' downstream sequence (SEQ ID NO:11). The first 202 by of the extended 247 by fragment included within pT-RD5-3' encoded the C-terminal coding region (including the translation stop codon) of the putative delta-5 desaturase gene. The sequence of pT-RD5-3' also corrected the sequence corresponding to Conserved Region 2, resulting from use of a degenerate oligonucleotide for initial PCR amplification of the 563 by fragment in pT-12-D5 (Example 3):
[0309] After 2 rounds of 5' RACE and one round of 3' RACE, the DNA sequence of the entire putative Peridinium sp. CCMP626 delta-5 desaturase (RD5) coding region was determined. As shown in FIG. 4, the RD5 CDS was 1392 by in length (SEQ ID NO:1) and encoded a polypeptide with 463 amino acids (SEQ ID NO:2), based on alignment of SEQ ID NOs:4, 6, 8 and 10. The results of BLASTP sequence analysis algorithms using the full length RD5 gene as the query sequence showed that it shared 67% identity and 81% similarity with the delta-5 fatty acid desaturase of Phaeodactylum tricornutum (GenBank Accession No. AAL92562; SEQ ID NO:14), with an Expectation value of 0.0. Additionally, the full length RD5 gene shared 64% identity and 76% similarity with the delta-8-sphingolipid desaturase of Thalassiosira pseudonana (GenBank Accession No. AAX14502; SEQ ID NO:21), with an Expectation value of 2E-178.
Example 6
Generation of Construct pDMW368, Comprising RD5
[0310] The present Example describes the generation of pDMW368, comprising a chimeric FBAIN::RD5::Pex20 gene (FIG. 5C). This was designed to integrate the chimeric gene into the genome of Yarrowia lipolytica and then study the function of the Peridinium sp. CCMP626 delta-5 desaturase in Yarrowia lipolytica.
[0311] Based on the full length cDNA of RD5 (SEQ ID NO:1), oligonucleotides YL807 and YL810 (SEQ ID NOs:46 and 47, respectively) were used as primers to amplify the first portion of RD5 (FIG. 5A). Primer YL807 contained a NcoI site and primer YL810 contained a BamH1 site. Then, primers YL808 and YL809 (SEQ ID NOs:48 and 49, respectively) were used as primers to amplify the second portion of RD5. Primer YL809 contained a BamH1 site, while primer YL808 contained a NotI site. The PCR reactions, using primer pairs YL807/YL810 or YL808/YL809, with Peridinium sp. CCMP626 cDNA (Example 2) as template, were individually carried out in a 50 μl total volume comprising: PCR buffer (containing 10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-HCl (pH 8.75), 2 mM MgSO4, 0.1% Triton X-100), 100 μg/mL BSA (final concentration), 200 μM each deoxyribonucleotide triphosphate, 10 pmole of each primer and 1 μl of Pfu DNA polymerase (Stratagene, San Diego, Calif.). The thermocycler conditions were set for 35 cycles at 95° C. for 1 min, 56° C. for 30 sec and 72° C. for 1 min, followed by a final extension at 72° C. for 10 min.
[0312] The individual PCR products were purified using a Qiagen PCR purification kit. The PCR products from the reaction amplified with primers YL807/YL810 were digested with NcoI and BamH1, while the PCR products from the reaction amplified with primers YL808/YL809 were digested with BamHI and NotI. The NcoI/BamH1- and the BamH1/NotI-digested DNA fragments were purified following gel electrophoresis in 1% (w/v) agarose, and then directionally ligated with NcoI/NotI-digested pZUF17 (FIG. 5B; SEQ ID NO:22; comprising a synthetic delta-17 desaturase gene ["D17st"] derived from Saprolegnia diclina (U.S. Patent Publication No. 200310196217 A1), codon-optimized for expression in Yarrowia lipolytica (PCT Publication No. WO 2004/101757)). The product of this ligation was pDMW368 (FIG. 5C; SEQ ID NO:23, which thereby contained the following components:
TABLE-US-00007 TABLE 6 Components Of Plasmid pDMW368 RE Sites And Nucleotides Within SEQ ID Description Of Fragment NO: 23 And Chimeric Gene Components EcoR I/BsiW I FBAIN::RD5::Pex20, comprising: (6063-318) FBAIN: Yarrowia lipolytica FBAIN promoter (PCT Publication No. WO 2005/049805; U.S. Pat. No. 7,202,356) RD5: Peridinium sp. CCMP626 delta-5 desaturase (SEQ ID NO: 1 described herein) Pex20: Pex20 terminator sequence of Yarrowia Pex20 gene (GenBank Accession No. AF054613) 1354-474 ColE1 plasmid origin of replication 2284-1424 ampicillin-resistance gene (AmpR) for selection in E. coli 3183-4487 Yarrowia autonomous replication sequence (ARS18; GenBank Accession No. A17608) 6020-4533 Yarrowia Ura 3 gene (GenBank Accession No. AJ306421)
The term "FBAIN promoter" or "FBAIN promoter region" refers to the 5' upstream untranslated region in front of the `ATG` translation initiation codon of the Yarrowia lipolytica fructose-bisphosphate aldolase enzyme (E.C. 4.1.2.13) encoded by the fba1 gene and that is necessary for expression, plus a portion of 5' coding region that has an intron of the fba1 gene.
Example 7
Generation of Yarrowia lipolytica Strain M4 to Produce About 8% DGLA of Total Lipids
[0313] The present Example describes the construction of strain M4, derived from Yarrowia lipolytica ATCC #20362, capable of producing 8% DGLA relative to the total lipids. This strain was engineered to express the delta-6 desaturase/delta-6 elongase pathway, via introduction of construct pKUNF12T6E (FIG. 6A; SEQ ID NO:24). This construct was generated to integrate four chimeric genes (comprising a delta-12 desaturase, a delta-6 desaturase and two C18/20 elongases) into the Ura3 loci of wild type Yarrowia strain ATCC #20362, to thereby enable production of DGLA. Thus, pKUNF12T6E contained the following components:
TABLE-US-00008 TABLE 7 Description of Plasmid pKUNF12T6E (SEQ ID NO: 24) RE Sites And Nucleotides Within SEQ ID Description Of Fragment NO: 24 And Chimeric Gene Components AscI/BsiWI 784 bp 5' portion of Yarrowia Ura3 gene (GenBank (9420-8629) Accession No. AJ306421) SphI/PacI 516 bp 3' portion of Yarrowia Ura3 gene (GenBank (12128-1) Accession No. AJ306421) SwaI/BsiWI FBAIN::EL1S::Pex20, comprising: (6380-8629) FBAIN: Yarrowia lipolytica FBAIN promoter (PCT Publication No. WO 2005/049805; U.S. Pat. No. 7,202,356; labeled as "Fba1 + intron" in Figure) EL1S: codon-optimized elongase 1 gene (PCT Publication No. WO 2004/101753), derived from Mortierella alpina (GenBank Accession No. AX464731) Pex20: Pex20 terminator sequence from Yarrowia Pex20 gene (GenBank Accession No. AF054613) BglII/SwaI TEF::delta-6S::Lip1, comprising: (4221-6380) TEF: Yarrowia lipolytica TEF promoter (GenBank Accession No. AF054508) delta-6S: codon-optimized delta-6 desaturase gene (PCT Publication No. WO 2004/101753), derived from Mortierella alpina (GenBank Accession No. AF465281) Lip1: Lip1 terminator sequence from Yarrowia Lip1 gene (GenBank Accession No. Z50020) PmeI/ClaI FBA::F.delta-12::Lip2, comprising: (4207-1459) FBA: Yarrowia lipolytica FBA promoter (PCT Publication No. WO 2005/049805; U.S. Pat. No. 7,202,356; labeled as "FBA1" in Figure) F.delta-12: Fusarium moniliforme delta-12 desaturase gene (PCT Publication No. WO 2005/047485) Lip2: Lip2 terminator sequence from Yarrowia Lip2 gene (GenBank Accession No. AJ012632) ClaI/PacI TEF::EL2Syn::XPR2, comprising: (1459-1) TEF: Yarrowia lipolytica TEF promoter (GenBank Accession No. AF054508) EL2Syn: codon-optimized elongase gene (SEQ ID NO: 25), derived from Thraustochytrium aureum (U.S. Pat. No. 6,677,145) XPR2: ~100 bp of the 3' region of the Yarrowia Xpr gene (GenBank Accession No. M17741)
[0314] Plasmid pKUNF12T6E was digested with AscI/SphI, and then used for transformation of wild type V. lipolytica ATCC #20362 according to the General Methods. The transformant cells were plated onto FOA selection media plates and maintained at 30° C. for 2 to 3 days. The FOA resistant colonies were picked and streaked onto MM and MMU selection plates. The colonies that could grow on MMU plates but not on MM plates were selected as Ura- strains. Single colonies of Ura- strains were then inoculated into liquid MMU at 30° C. and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
[0315] GC analyses showed the presence of DGLA in the transformants containing the 4 chimeric genes of pKUNF12T6E, but not in the wild type Yarrowia control strain. Most of the selected 32 Ura.sup.- strains produced about 6% DGLA of total lipids. There were 2 strains strains M4 and 13-8) that produced about 8% DGLA of total lipids.
Example 8
Functional Analysis of RD5 Gene in Yarrowia lipolytica Strain M4
[0316] Plasmid pDMW368 (Example 6; comprising a chimeric FBAIN::RD5::Pex20 gene was transformed into strain M4 (Example 7), as described in the General Methods. The transformants were selected on MM plates. After 2 days grown at 30° C., 3 transformants grown on the MM plates were picked and re-streaked onto fresh MM plates. Once grown, these strains were individually inoculated into 3 mL liquid MM at 30° C. and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
[0317] GC analyses showed that there were about 4.2% DGLA and 2.2% ARA of total lipids produced in all three transformants, wherein the conversion efficiency of DGLA to ARA in these three strains was determined to be about 35% (average). The conversion efficiency was measured according to the following formula: ([product]/[substrate+product])*100, where `product` includes the immediate product and all products in the pathway derived from it. Thus, this experimental data demonstrated that the cloned Peridinium sp. CCMP626 delta-5 desaturase, described herein as SEQ ID NOs:1 and 2, efficiently desaturated DGLA to ARA.
Example 9
Synthesis of a Codon-Optimized Delta-5 Desaturase Gene ("RD5S'') for Expression in Yarrowia lipolytica
[0318] The codon usage of the delta-5 desaturase gene of Peridinium sp. CCMP626 (SEQ ID NOs:1 and 2; RD5) was optimized for expression in Yarrowia lipolytica, in a manner similar to that described in PCT Publication No. WO 2004/101753 and U.S. Pat. No. 7,125,672. Specifically, a codon-optimized delta-5 desaturase gene (designated "RD5S", SEQ ID NO:3) was designed based on the coding sequence of the delta-5 desaturase gene of RD5 (SEQ ID NO:2), according to the Yarrowia codon usage pattern (PCT Publication No. WO 2004/101753), the consensus sequence around the `ATG` translation initiation codon, and the general rules of RNA stability (Guhaniyogi, G. and J. Brewer, Gene, 265(1-2):11-23 (2001)). In addition to modification of the translation initiation site, 247 by of the 1392 by coding region was modified (171%, FIGS. 7A and 7B) and 229 codons were optimized (49.4%). The GC content was increased from 49.3% within the wild type gene (i.e., RD5) to 54.2% within the synthetic gene (Le., RD5S). A NcoI site and NotI sites were incorporated around the translation initiation codon and after the stop codon of RD5S (SEQ ID NO:3), respectively. None of the modifications in the codon-optimized gene changed the amino acid sequence of the encoded protein (SEQ ID NO:2). The designed RD5S gene was synthesized by GenScript Corporation (Piscataway, N.J.) and cloned into pUC57 (GenBank Accession No. Y14837) to generate pRD5S (SEQ ID NO:50; FIG. 6B) (RD5S labeled as "RD5S(626)" in Figure).
Example 10
Generation of Construct pZURD5S, Comprising RD5S
[0319] The present Example describes the construction of plasmid pZURD5S comprising a chimeric FBAIN:RD5S::Pex20 gene. Plasmid pZURD5S (SEQ ID NO:51; FIG. 6C) was constructed by replacing the Nco I/Not I fragment of pZUF17 (FIG. 5B; SEQ ID NO:22) with the Nco I/Not I RD5S fragment from pRD5S (SEQ ID NO:50; FIG. 6B). The product of this ligation was pZURD5S (FIG. 6C; SEQ ID NO:51), which thereby contained the following components:
TABLE-US-00009 TABLE 8 Components Of Plasmid pZURD5S RE Sites And Nucleotides Within SEQ ID Description Of Fragment NO: 51 And Chimeric Gene Components EcoR I/BsiW I FBAIN::RD5S::Pex20, comprising: (7458-1713) FBAIN: Yarrowia lipolytica FBAIN promoter (PCT Publication No. WO 2005/049805; U.S. Pat. No. 7,202,356) RD5S: codon-optimized delta-5 desaturase (SEQ ID NO: 3, described herein as RD5S), derived from Peridinium sp. CCMP626 (labeled as "RD5S(626)" in Figure) Pex20: Pex20 terminator sequence of Yarrowia Pex20 gene (GenBank Accession No. AF054613) 2749-1869 ColE1 plasmid origin of replication 3679-2819 ampicillin-resistance gene (AmpR) for selection in E. coli 4578-5882 Yarrowia autonomous replication sequence (ARS18; GenBank Accession No. A17608) 7415-5928 Yarrowia Ura 3 gene (GenBank Accession No. AJ306421)
Example 11
Expression of the Codon-Optimized Delta-5 Desaturase ("RD5S") in Yarrowia lipolytica Strain M4
[0320] Plasmid pZURD5S (Example 10; comprising a chimeric FBAIN::RD5S::Pex20 gene) was used for transformation into strain M4 (Example 7), as described in the General Methods. Eight (8) transformants grown on the MM plates were picked and re-streaked onto fresh MM plates. Once grown, these strains were individually inoculated into 3 mL liquid MM at 30° C. and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
[0321] The GC results showed that there were about 5.4% DGLA and 3.3% ARA of total lipids produced in all 8 transformants. The conversion efficiency whereby RD5S converted DGLA to ARA in these 8 strains was at an average rate of about 38%. The conversion efficiency was measured according to the following formula: ([product]/[substrate+product]*100, where `product` includes the immediate product and all products in the pathway derived from it. Thus, this experimental data demonstrated that the codon-optimized delta-5 desaturase gene (ROSS, as set forth in SEQ ID NO:3) derived from Peridinium sp. CCMP626 was about 8.9% more efficient converting DGLA to ARA than the wild type RD5 (Example 8).
Example 12
Isolation of a Pavlova lutheri (CCMP459) Delta-8 Desaturase
[0322] The present example describes the isolation of the Pavlova lutheri (CCMP459) delta-8 desaturase utilized in Example 3 and in FIG. 3 (also described in U.S. patent application Ser. No. 11/737,772, filed Apr. 20, 2007). This required: synthesis of Pavlova lutheri (CCMP459) cDNA; library construction and sequencing; identification of delta-8 desaturase homologs; and, cloning of a full-length delta-8 desaturase from genomic DNA.
Pavlova lutheri (CCMP459) cDNA Synthesis, Library Construction and Sequencing
[0323] A cDNA library of Pavlova lutheri (CCMP459) was synthesized as described in PCT Publication No. WO 20041071467 (published Aug. 26, 2004). Briefly, frozen pellets of Pav459 were obtained from the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (COMP, West Boothbay Harbor, Me.). These pellets were crushed in liquid nitrogen and total RNA was extracted from Pav459 by using the Qiagen RNeasy® Maxi Kit (Qiagen, Valencia, Calif.), per the manufacturer's instructions. From this total RNA, mRNA was isolated using oligo dT cellulose resin, which was then used for the construction of a cDNA library using the pSport1 vector (Invitrogen, Carlsbad, Calif.). The cDNA thus produced was directionally cloned (5' Sal/I/3' NotI into pSport1 vector. The Pav459 library contained approximately 6.1×105 clones per mL, each with an average insert size of approximately 1200 bp. The Pavlova lutheri library was named eps1c.
[0324] For sequencing, clones first were recovered from archived glycerol cultures grown/frozen in 384-well freezing media plates, and inoculated with an automatic QPix® colony picker (Genetix) in 96-well deep-well plates containing LB+100 mg/mL ampicillin. After growing 20 hrs at 37° C., cells were pelleted by centrifugation and stored at -20° C. Plasmids then were isolated on an Eppendorf 5Prime robot, using a modified 96-well format alkaline lysis miniprep method (Eppendorf PerfectPrep®). Briefly, a filter and vacuum manifold was used to facilitate removal of cellular debris after acetate precipitation. Plasmid DNA was then bound on a second filter plate directly from the filtrate, washed, dried and eluted.
[0325] Plasmids were end-sequenced in 384-well plates, using vector-primed T7 primer (SEQ ID NO:52) and the ABI BigDye version 3 Prism sequencing kit. For the sequencing reaction, 100-200 ng of template and 6.4 pmoL of primer were used, and the following reaction conditions were repeated 25 times: 96° C. for 10 sec, 50° C. for 5 sec and 60° C. for 4 min. After ethanol-based cleanup, cycle sequencing reaction products were resolved and detected on Perkin-Elmer MI 3700 automated sequencers.
Identification of Delta-8 Desaturase Enzyme Homologs from Pavlova lutheri cDNA Library eps1c
[0326] cDNA clones encoding Pavlova lutheri delta-8 desaturase homologs (hereby called delta-8 desaturases) were identified by conducting BLAST searches for similarity to sequences contained in the BLAST "nr" database (as described in Example 3). The P-value (probability) of observing a match of a cDNA sequence to a sequence contained in the searched databases merely by chance as calculated by BLAST are reported herein as "pLog" values, which represent the negative of the logarithm of the reported P-value. Accordingly, the greater the pLog value, the greater the likelihood that the cDNA sequence and the BLAST "hit" represent homologous proteins.
[0327] The BLASTX search using the nucleotide sequence from clone epslc.pk002.f22 revealed similarity of the protein encoded by the cDNA to the delta-6 desaturase from Rhizopus stolonifer (SEQ ID NO:53) (NCBI Accession No. AAX22052 (GI 60499699), locus AAX22052, CDS AY795076; Lu et al., unpublished). The sequence of a portion of the cDNA insert from clone eps1c.pk002.f22 is shown in SEQ ID NO:54 (5' end of cDNA insert). Subsequently, the full insert sequence (eps1c.pk002.f22:fis) was obtained and is shown in SEQ ID NO:55. Sequence for the deduced amino acid sequence (from nucleotide 1 of SEQ ID NO:55 to the first stop codon at nucleotide 864 of SEQ ID NO:55) is shown in SEQ ID NO:56. Full insert sequencing was carried out using a modified transposition protocol. Clones identified for full insert sequencing were recovered from archived glycerol stocks as single colonies, and plasmid DNA was isolated via alkaline lysis. Plasmid templates were transposed via the Template Generation System (TGS II) transposition kit (Finnzymes Oy, Espoo, Finland), following the manufacturer's protocol. The transposed DNA was transformed into EH10B electro-competent cells (Edge BioSystems, Gaithersburg, Md.) via electroporation. Multiple transformants were randomly selected from each transposition reaction, plasmid DNA was prepared, and templates were sequenced as above (ABI BigDye v3.1) outward from the transposition event site, utilizing unique primers SeqE (SEQ ID NO:57) and SeqW (SEQ ID NO:58).
[0328] Sequence data was collected (ABI Prism Collections software) and assembled using the Phrap sequence assembly program (P. Green, University of Washington, Seattle). Assemblies were viewed by the Consed sequence editor (D. Gordon, University of Washington, Seattle) for final editing.
[0329] The amino acid sequence set forth in SEQ ID NO:56 was evaluated by BLASTP, yielding a pLog value of 19.52 (E value of 3e-20) versus the delta-6 desaturase from Mortierella alpine (NCBI Accession No. BAC82361 (GI 34221934), locus BAC82361, CDS AB070557; Sakuradani and Shimizu, Biosci. Biotechnol. Biochem., 67:704-711 (2003)). Based on the results from the BLASTP comparison to the Mortierella alpine and other fatty acid desaturases, the Pavlova lutheri delta-8 desaturase was not full length and was lacking sequence at the 5' end.
Cloning a Full-Length Delta-8 Desaturase from Pavlova lutheri Genomic DNA
[0330] Genomic DNA was isolated from Pavlova lutheri (CCMP459) using the Qiagen DNeasy® Plant Maxi Prep Kit according to the manufacturer's protocol. Using 1 maxi column per 1 gm of frozen cell pellet, a total of 122 pg of genomic DNA was isolated from 4 gm of Pavlova lutheri culture. The final concentration of genomic DNA was 22.8 ng/μL. GenomeWalker libraries were synthesized using the Universal GenomeWalker® kit (BD Biosciences Clonetech, Palo Alto, Calif.) following the manufacturer's protocol (Prot #PT3042-1, version PRO3300). Briefly, four restriction digests were set up as per the protocol using 300 ng of genomic DNA per reaction. After phenol clean up, pellets were dissolved in 4 μL of water and adapters were ligated as per the protocol.
[0331] For the primary PCR, the Advantage®-GC Genornic PCR kit (BD Biosciences Clonetech) was used following the manufacturer's protocol (Prot #PT3090-1, version PR1X433). For each restriction digest, 1 μL of library was combined with 22.8 μL of PCR grade water, 10 μL of 5× GC Genornic PCR Reaction Buffer, 2.2 μL of 25 mM Mg(CH3CO2)2, 10 μL of GC-Melt (5 M), 1 μL of 50× dNTP mix (10 mM each), 1 μL of Advantage-GC Genomic Pol. Mix (50×), 1 μL of Universal GenomeWalker® primer API (10 μM, SEQ ID NO:59) and 1 μL of GSP PvDES (10 μM, SEQ ID NO:60). After denaturation at 95° C., the following reaction conditions were repeated 35 times: 94° C. for 30 sec, 68° C. for 6 min. After these reaction conditions, an additional extension at 68° C. was carried out for 6 min followed by cooling to 15° C. until removed.
[0332] The primary PCR reaction for each library was analyzed by agarose gel electrophoresis and DNA bands with molecular weights around 6 kB, 3.5 kb, 2.5 kB and 1.2 kB were observed. DNA bands for each library were purified using the Zymocleae® Gel DNA Recovery Kit (Zymo Research, Orange, Calif.) following the manufacturer's protocol. The resulting DNA was cloned into the pGEM®-T Easy Vector (Promega) following the manufacturer's protocol and inserts were sequenced using the T7 (SEQ ID NO:52) and M13-28Rev (SEQ ID NO:61) primers as described above. Additional sequence was then obtained using a gene-specific sequencing primer PavDES seq (SEQ ID NO:62) that was derived from the newly acquired sequence data. The full 5' end sequence obtained by genome walking is shown in SEQ ID NO:63. The sequence of the overlapping regions of the genomic sequence (SEQ ID NO:63) and the fully sequenced EST eps1c.pk002.f22:fis (SEQ ID NO:55) were aligned using Sequencher® (Version 4.2, Gene Codes Corporation, Ann Arbor, Mich.) using the Large Gap assembly algorithm. Interestingly, the comparison showed that the EST that was originally sequenced (SEQ ID NO:55) was lacking 459 by when compared to the genomic sequence (SEQ ID NO:63). This missing sequence in the EST appeared to be a deletion rather than an intron as no clear intron splice sites were identified in the genomic DNA at the 5' end of the gene. The genomic sequence for the 5' end (SEQ ID NO:63) was combined with the 3' end of the EST sequence (SEQ ID NO:55) to yield SEQ ID NO:64. Using EditSeq® 6.1 sequence analysis software (DNASTAR Inc., Madison, Wis.), an ORE was identified (SEQ ID NO:17). The amino acid sequence coded for by SEQ ID NO:17 is shown in SEQ ID NO:18.
[0333] The amino acid sequence set forth in SEQ ID NO:18 was evaluated by BLASTP, yielding a pLog value of 35.10 (E value of 8e-36) versus the delta-6 desaturase from Rhizopus stolonifer (SEQ ID NO:65) (NCBI Accession No. ABB96724 (GI 83027409), locus ABB96724, CDS DQ291156; Zhang et al., unpublished). Furthermore, the Pavlova lutheri delta-8 desaturase is 78.0% identical to the Pavlova salina delta-8 desaturase sequence (SEQ ID NO:66) disclosed in PCT Publication No. WO 2005/103253 (published Apr. 22, 2005) using the Jotun Hein method. Sequence percent identity calculations performed by the Jotun Hein method (Hein, J. J., Meth. Enz.,183:626-645 (1990)) were done using the MegAlign® v6.1 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.) with the default parameters for pairwise alignment (KTUPLE=2). The Pavlova lutheri delta-8 desaturase is 76.4% identical to the Pavlova salina delta-8 desaturase sequence using the Clustal V method. Sequence percent identity calculations performed by the Clustal V method (Higgins, D. G. and Sharp, P. M., Comput. Appl. Biosci., 5:151-153 (1989); Higgins et al., Comput. Appl. Biosci., 8:189-191 (1992)) were done using the MegAlign® v6.1 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.) with the default parameters for pairwise alignment (KTUPLE=1, GAP PENALTY=3, WINDOW=5, DIAGONALS SAVED=5 and GAP LENGTH PENALTY=10). BLAST scores and probabilities indicate that the fragment of SEQ ID NO:17 encodes an entire Pavlova lutheri delta-8 desaturase.
[0334] FIGS. 8A and 8B show a Clustal V alignment (with default parameters) of SEQ ID NO:18 (the amino acid sequence of the Pavlova lutheri delta-8 desaturase), SEQ ID NO:66 (the amino acid sequence of Pavlova salina delta-8 desaturase sequence, supra), SEQ ID NO:16 (the amino acid sequence of Euglena gracilis delta-8 desaturase sequence disclosed as SEQ ID NO:2 in PCT Publication No. WO 2006/012325; published Feb. 2, 2006), SEQ ID NO:65 (the amino acid sequence for the Rhizopus stolonifer delta-6 fatty acid desaturase (NCBI Accession No. ABB96724, supra)) and SEQ ID NO:53 (the amino acid sequence for the Rhizopus stolonifer delta-6 fatty acid desaturase (NCBI Accession No. AAX22052, supra)). The results of the Clustal V alignment show that SEQ ID NO:18 is 76.4%, 22.6%, 22.2% and 22.2% identical to SEQ ID NO:66, SEQ 1D NO:16, SEQ ID NO:65 and SEQ ID NO:53, respectively.
Example 13
Transformation of Somatic Soybean Embryo Cultures
Culture Conditions
[0335] Soybean embryogenic suspension cultures (cv. Jack) are maintained in 35 mL liquid medium SB196 (infra) on a rotary shaker, 150 rpm, 26° C. with cool white fluorescent lights on 16:8 hr day/night photoperiod at light intensity of 60-85 μE/m2/s. Cultures are subcultured every 7 days to 2 weeks by inoculating approximately 35 mg of tissue into 35 mL of fresh liquid SB196 (the preferred subculture interval is every 7 days).
[0336] Soybean embryogenic suspension cultures are transformed with soybean expression plasmids by the method of particle gun bombardment (Klein et al., Nature, 327:70 (1987)) using a DuPont Biolistic PDS1000/HE instrument (helium retrofit) for all transformations.
Soybean Embryogenic Suspension Culture Initiation:
[0337] Soybean cultures are initiated twice each month with 5-7 days between each initiation. Pods with immature seeds from available soybean plants 45-55 days after planting are picked, removed from their shells and placed into a sterilized magenta box. The soybean seeds are sterilized by shaking them for 15 min in a 5% Clorox solution with 1 drop of ivory soap (i.e., 95 mL of autoclaved distilled water plus 5 mL Clorox and 1 drop of soap, mixed well). Seeds are rinsed using 2 1-liter bottles of sterile distilled water and those less than 4 mm are placed on individual microscope slides. The small end of the seed is cut and the cotyledons pressed out of the seed coat. Cotyledons are transferred to plates containing SB1 medium (25-30 cotyledons per plate). Plates are wrapped with fiber tape and stored for 8 weeks. After this time secondary embryos are cut and placed into SB196 liquid media for 7 days.
Preparation of DNA for Bombardment:
[0338] Either an intact plasmid or a DNA plasmid fragment containing the delta-5 desaturase genes of interest and the selectable marker gene are used for bombardment. Fragments from soybean expression plasmids comprising the delta-5 desaturase of the present invention are obtained by gel isolation of digested plasmids. The resulting DNA fragments are separated by gel electrophoresis on 1% SeaPlaque GTG agarose (BioWhitaker Molecular Applications) and the DNA fragments containing gene cassettes are cut from the agarose gel. DNA is purified from the agarose using the GELase digesting enzyme following the manufacturers protocol.
[0339] A 50 μL aliquot of sterile distilled water containing 3 mg of gold particles is added to 5 μL of a 1 μg/μL DNA solution (either intact plasmid or DNA fragment prepared as described above), 50 μL 2.5 M CaCl2 and 20 μL of 0.1 M spermidine. The mixture is shaken 3 min on level 3 of a vortex shaker and spun for 10 sec in a bench microfuge. After a wash with 400 μL of 100% ethanol, the pellet is suspended by sonication in 40 μL of 100% ethanol. DNA suspension (5 μL) is dispensed to each flying disk of the Biolistic PDS1000/HE instrument disk. Each 5 μL aliquot contains approximately 0.375 mg gold particles per bombardment (i.e., per disk).
Tissue Preparation and Bombardment with DNA:
[0340] Approximately 150-200 mg of 7 day old embryonic suspension cultures is placed in an empty, sterile 60×15 mm petri dish and the dish is covered with plastic mesh. Tissue is bombarded 1 or 2 shots per plate with membrane rupture pressure set at 1100 PSI and the chamber is evacuated to a vacuum of 27-28 inches of mercury. Tissue is placed approximately 3.5 inches from the retaining/stopping screen.
Selection of Transformed Embryos:
[0341] Transformed embryos ate selected using hygromycin as the selectable marker. Specifically, following bombardment, the tissue is placed into fresh SB196 media and cultured as described above. Six days post-bombardment, the SB196 is exchanged with fresh SB196 containing 30 mg/L hygromycin. The selection media is refreshed weekly. Four to six weeks post-selection, green, transformed tissue is observed growing from untransformed, necrotic embryogenic clusters. Isolated, green tissue is removed and inoculated into multiwell plates to generate new, clonally propagated, transformed embryogenic suspension cultures.
Embryo Maturation:
[0342] Embryos are cultured for 4-6 weeks at 26° 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 μE/m2s. After this time embryo clusters are removed to a solid agar media, SB166, for 1-2 weeks. Clusters are then subcultured to medium SB103 for 3 weeks. During this period, individual embryos are removed from the clusters and screened for alterations in their fatty acid compositions as described supra.
Media Recipes:
SB 196--FN Lite Liquid Proliferation Medium (Per Liter)
TABLE-US-00010 [0343] SB 196 - FN Lite Liquid Proliferation Medium (per liter) MS FeEDTA - 100x Stock 1 10 mL MS Sulfate - 100x Stock 2 10 mL FN Lite Halides - 100x Stock 3 10 mL FN Lite P, B, Mo - 100x Stock 4 10 mL B5 vitamins (1 mL/L) 1.0 mL 2,4-D (10 mg/L final concentration) 1.0 mL KNO3 2.83 gm (NH4)2SO4 0.463 gm asparagine 1.0 gm sucrose (1%) 10 gm pH 5.8
FN Lite Stock Solutions
TABLE-US-00011 [0344] Stock Number 1000 mL 500 mL 1 MS Fe EDTA 100x Stock Na2 EDTA* 3.724 g 1.862 g FeSO4--7H2O 2.784 g 1.392 g 2 MS Sulfate 100x stock MgSO4--7H2O 37.0 g 18.5 g MnSO4--H2O 1.69 g 0.845 g ZnSO4--7H2O 0.86 g 0.43 g CuSO4--5H2O 0.0025 g 0.00125 g 3 FN Lite Halides 100x Stock CaCl2--2H2O 30.0 g 15.0 g KI 0.083 g 0.0715 g CoCl2--6H2O 0.0025 g 0.00125 g 4 FN Lite P, B, Mo 100x Stock KH2PO4 18.5 g 9.25 g H3BO3 0.62 g 0.31 g Na2MoO4--2H2O 0.025 g 0.0125 g *Add first, dissolve in dark bottle while stirring
SB1 Solid Medium (Per Liter)
[0345] 1 package MS salts (Gibco/ BRL--Cat. No. 11117-066) [0346] 1 mL B5 vitamins 1000× stock [0347] 31.5 g sucrose [0348] 2 mL 2,4-D (20 mg/L final concentration) [0349] pH 5.7 [0350] 8 g TC agar
SB 166 Solid Medium (Per Liter)
[0350] [0351] 1 package MS salts (Gibco/ BRL--Cat. No. 11117-066) [0352] 1 mL B5 vitamins 1000× stock [0353] 60 g maltose [0354] 750 mg MgCl2 hexahydrate [0355] 5 g activated charcoal [0356] pH 5.7 [0357] 2 g gelrite
SB 103 Solid Medium (Per Liter)
[0357] [0358] 1 package MS salts (Gibco/BRL--Cat. No. 11117-066) [0359] 1 mL B5 vitamins 1000× stock [0360] 60 g maltose [0361] 750 mg MgCl2 hexahydrate [0362] pH 5.7 [0363] 2 g gelrite
SB 71-4 Solid Medium (Per Liter)
[0363] [0364] 1 bottle Gamborg's B5 salts with sucrose (Gibco/BRL--Cat. No. 21153-036) [0365] pH 5.7 [0366] 5 g TC agar
2,4-D Stock
[0366] [0367] Obtain prernade from Phytotech Cat. No D 295 concentration 1 mg/mL
B5 Vitamins Stock (Per 100 mL)
[0367] [0368] Store aliquots at -20° C. [0369] 10 g myo-inositol [0370] 100 mg nicotinic acid [0371] 100 mg pyridoxine HCl [0372] 1 g thiamine If the solution does not dissolve quickly enough, apply a low level of heat via the hot stir plate.
Example 14
Functional Analysis of Delta-5 Desaturase (SEQ ID NOs:1 and 2) in Somatic Soybean Embryos
[0373] Mature somatic soybean embryos are a good model for zygotic embryos. While in the globular embryo state in liquid culture, somatic soybean embryos contain very low amounts of triacylglycerol (TAG) or storage proteins typical of maturing, zygotic soybean embryos. At this developmental stage, the ratio of total triacylglyceride to total polar lipid (phospholipids and glycolipid) is about 1:4, as is typical of zygotic soybean embryos at the developmental stage from which the somatic embryo culture was initiated. At the globular stage as well, the mRNAs for the prominent seed proteins, α'-subunit of β-conglycinin, kunitz trypsin inhibitor 3, and seed lectin are essentially absent. Upon transfer to hormone-free media to allow differentiation to the maturing somatic embryo state, TAG becomes the most abundant lipid class. As well, mRNAs for α'-subunit of β-conglycinin, kunitz trypsin inhibitor 3 and seed lectin become very abundant messages in the total mRNA population, On this basis, the somatic soybean embryo system behaves very similarly to maturing zygotic soybean embryos in vivo, and is thus a good and rapid model system for analyzing the phenotypic effects of modifying the expression of genes in the fatty acid biosynthesis pathway (see PCT Publication No. WO 2002/00904, Example 3). Most importantly, the model system is also predictive of the fatty acid composition of seeds from plants derived from transgenic embryos.
[0374] Transgenic somatic soybean embryos containing the delta-5 desaturase of the present invention are analyzed in the following way. Fatty acid methyl esters are prepared from single, matured, somatic soy embryos by transesterification. Individual embryos are placed in a vial containing 50 μL of trimethylsulfonium hydroxide (TMSH) and 0.5 mL of hexane and incubated for 30 min at room temperature while shaking. Fatty acid methyl esters (5 μL injected from hexane layer) are separated and quantified using a Hewlett-Packard 6890 Gas Chromatograph fitted with an Omegawax 320 fused silica capillary column (Catalog #24152, Supelco Inc.). The oven temperature are programmed to hold at 220° C. for 2.6 min, increase to 240° C. at 20° C./min and then hold for an additional 2.4 min. Carrier gas is supplied by a Whatman hydrogen generator. Retention times are compared to those for methyl esters of standards commercially available (Nu-Chek Prep, Inc.). Routinely, 5-10 embryos per event are analyzed by GC, using the methodology described above.
Example 15
Co-Expressing Other Promoter/Gene/Terminator Cassette Combinations in Somatic Soybean Embryos
[0375] In addition to the genes, promoters, terminators and gene cassettes described herein, one skilled in the art can appreciate that other promoter/gene/terminator cassette combinations can be synthesized in a way similar to, but not limited to, that described herein. For'instance, PCT Publications No. WO 2004/071467 and No. WO 2004/071178 describe the isolation of a number of promoter and transcription terminator sequences for use in embryo-specific expression in soybean. Furthermore, PCT Publications No. WO 2004/071467, No. WO 2005/047479 and No. WO 2006/012325 describe the synthesis of multiple promoter/gene/terminator cassette combinations by ligating individual promoters, genes and transcription terminators together in unique combinations. Generally, a NotI site flanked by the suitable promoter (e.g., those listed in, but not limited to, Table 9) and a transcription terminator (e.g., those listed in, but not limited to, Table 10) is used to clone the desired gene. NotI sites can be added to a gene of interest such as those listed in, but not limited to, Table 11 using PCR amplification with oligonucleotides designed to introduce NotI sites at the 5' and 3' ends of the gene. The resulting PCR product is then digested with NotI and cloned into a suitable promoter/NotI/terminator cassette.
[0376] In addition, PCT Publications No. WO 2004/071467, No. WO 2005/047479 and No. WO 2006/012325 describe the further linking together of individual gene cassettes in unique combinations, along with suitable selectable marker cassettes, in order to obtain the desired phenotypic expression. Although this is done mainly using different restriction enzymes sites, one skilled in the art can appreciate that a number of techniques can be utilized to achieve the desired promoter/gene/transcription terminator combination. In so doing, any combination of embryo-specific promoter/gene/ transcription terminator cassettes can be achieved. One skilled in the art can also appreciate that these cassettes can be located on individual DNA fragments or on multiple fragments where co-expression of genes is the outcome of co-transformation of multiple DNA fragments.
TABLE-US-00012 TABLE 9 Seed-specific Promoters Promoter Organism Promoter Reference β-conglycinin α'-subunit soybean Beachy et al., EMBO J., 4: 3047-3053 (1985) kunitz trypsin inhibitor soybean Jofuku et al., Plant Cell, 1: 1079-1093 (1989) Annexin soybean PCT Publication No. WO 2004/071467 glycinin Gy1 soybean PCT Publication No. WO 2004/071467 albumin 2S soybean U.S. Pat. No. 6,177,613 legumin A1 pea Rerie et al., Mol. Gen. Genet., 225: 148-157 (1991) β-conglycinin β-subunit soybean PCT Publication No. WO 2004/071467 BD30 (also called P34) soybean PCT Publication No. WO 2004/071467 legumin A2 pea Rerie et al., Mol. Gen. Genet., 225: 148-157 (1991)
TABLE-US-00013 TABLE 10 Transcription Terminators Transcription Terminator Organism Reference phaseolin 3' bean PCT Publication No. WO 2004/071467 kunitz trypsin inhibitor 3' soybean PCT Publication No. WO 2004/071467 BD30 (also called P34) 3' soybean PCT Publication No. WO 2004/071467 legumin A2 3' pea PCT Publication No. WO 2004/071467 albumin 2S 3' soybean PCT Publication No. WO 2004/071467
TABLE-US-00014 TABLE 11 PUFA Biosynthetic Pathway Genes Gene Organism Reference delta-6 desaturase Saprolegnia diclina PCT Publication No. WO 2002/081668 delta-6 desaturase Mortierella alpina U.S. Pat. No. 5,968,809 elongase Mortierella alpina PCT Publication No. WO 2000/12720; U.S. Pat. No. 6,403,349 delta-5 desaturase Mortierella alpina U.S. Pat. No. 6,075,183 delta-5 desaturase Saprolegnia diclina PCT Publication No. WO 2002/081668 delta-15 desaturase Fusarium PCT Publication No. WO moniliforme 2005/047479 delta-17 desaturase Saprolegnia diclina PCT Publication No. WO 2002/081668 elongase Thraustochytrium PCT Publication No. WO aureum 2002/08401; U.S. Pat. No. 6,677,145 elongase Pavlova sp. Pereira et al., Biochem. J., 384: 357-366 (2004) delta-4 desaturase Schizochytrium PCT Publication No. WO aggregatum 2002/090493 delta-9 elongase Isochrysis galbana PCT Publication No. WO 2002/077213 delta-9 elongase Euglena gracilis U.S. patent application No. 11/601563 delta-8 desaturase Euglena gracilis PCT Publication No. WO 2000/34439; U.S. Pat. No. 6,825,017; PCT Publication No. WO 2004/057001; PCT Publication No. WO 2006/012325 delta-8 desaturase Acanthamoeba Sayanova et al., FEBS Lett., castellanii 580: 1946-1952 (2006) delta-8 desaturase Pavlova salina PCT Publication No. WO 2005/103253 delta-8 desaturase Pavlova lutheri U.S. patent application No. 11/737,772
Example 16
Chlorsulfuron Selection (ALS) and Plant Regeneration
Chlorsulfuron (ALS) Selection:
[0377] Following bombardment, the plant tissue is divided between 2 flasks with fresh SB196 media and cultured as described in Example 13. Six to seven days post-bombardment, the SB196 is exchanged with fresh SB196 containing selection agent of 100 ng/mL chlorsulfuron. The selection media is refreshed weekly. Four to six weeks post selection, green, transformed tissue is observed growing from untransformed, necrotic embryogenic clusters. Isolated, green tissue is removed and inoculated into multiwell plates containing SB196 to generate new, clonally propagated, transformed embryogenic suspension cultures.
Regeneration of Soybean Somatic Embryos into Plants:
[0378] In order to obtain whole plants from embryogenic suspension cultures, the tissue must be regenerated. Embyros are matured as described in Example 13. After subculturing on medium SB103 for 3 weeks, individual embryos are removed from the clusters and screened for alterations in their fatty acid compositions as described in Example 14. It should be noted that any detectable phenotype, resulting from the expression of the genes of interest, can be screened at this stage. This would include, but not be limited to: alterations in fatty acid profile, protein profile and content, carbohydrate content, growth rate, viability, or the ability to develop normally into a soybean plant.
[0379] Matured individual embryos are desiccated by placing them into an empty, small petri dish (35×10 mm) for approximately 4 to 7 days. The plates are sealed with fiber tape (creating a small humidity chamber). Desiccated embryos are planted into SB71-4 medium where they are left to germinate under the same culture conditions described above. Germinated plantlets are removed from germination medium and rinsed thoroughly with water and then are planted in Redi-Earth in a 24-cell pack tray, covered with a clear plastic dome. After 2 weeks the dome is removed and plants hardened off for a further week. if plantlets look hardy, they are transplanted to 10'' pots of Redi-Earth with up to 3 plantlets per pot. After 10 to 16 weeks, mature seeds are harvested, chipped and analyzed for fatty acids as described in Example 14.
[0380] Media recipes can be found in Example 13 and chlorsulfuron stock is 1 mg/mL in 0.01 N ammonium hydroxide.
Example 17
Comparing the Substrate Specificity of the Mortierella alpina Delta-5 Desaturase (MaD5) with the Peridinium sp. CCMP626 Delta-5 Desaturase (RD5) in Yarrowia Lipolytica
[0381] The present Example describes comparison of the substrate specificity of a Mortierella alpina delta-5 desaturase (MaD5; SEQ ID NOs:67 and 68), which is described in U.S. Pat. No. 6,075,183 and PCT Publication Nos. WO 2004/071467 and No. WO 2005/047479) to that of RD5 (SEQ ID NO:2) in Yarrowia lipolytica.
[0382] This work included the following steps: (1) construction of Yarrowia expression vector pY98 comprising MaD5; (2) transformation of pY98 and pDMW368 into Yarrowia strain Y2224; and, 3.) comparison of lipid profiles within transformant organisms comprising pY98 or pDMW368 after feeding fatty acid substrates.
Construction of Yarrowia Expression Vector pY98, Comprising MaD5
[0383] Plasmid pY5-22 (SEQ ID NO:69) is a shuttle plasmid that can replicate both in E. coli and Yarrowia lipolytica, containing the following: a Yarrowia autonomous replication sequence (ARS18; GenBank Accession No. M91600); a ColE1 plasmid origin of replication; an ampicillin-resistance gene (AmpR) for selection in E. coli; a Yarrowia URA3 gene (GenBank Accession No. AJ306421) for selection in Yarrowia; and, a chimeric TEF::NcoI/NotI::XPR cassette, wherein "XPR" was ˜100 by of the 3' region of the Yarrowia Xpr gene (GenBank Accession No. M17741). Although the construction of plasmid pY5-22 is not described herein in detail, it was derived from pY5 (previously described in PCT Publication No. WO 2004/101757).
[0384] Plasmid pY5-22GPD (SEQ ID NO:70) was created from pY5-22 (SEQ ID NO:69), by replacing the TEF promoter with the Yarrowia lipolytica GPD promoter (SEQ ID NO:71) using techniques well known to one skilled in the art. The Yarrowia "GPD promoter" refers to the 5' upstream untranslated region in front of the `ATG` translation initiation codon of a protein encoded by the Yarrowia lipolytica glyceraldehyde-3-phosphate dehydrogenase (GPD) gene and that is necessary for expression (PCT Publication No. WO 2005/003310). More specifically, the Yarrowia lipolytica GPD promoter was amplified from plasmid pYZDE2-S (SEQ ID NO:72: which was previously described in U.S. patent application Ser. No. 11/737,772 (the contents of which are hereby incorporated by reference)) using oligonucleotides GPDsense (SEQ ID NO:73) and GPDantisense (SEQ ID NO:74). The resulting DNA fragment was digested with SalI/NotI and cloned into the SalI/NotI fragment of pY5-22 (SEQ ID NO:69), thus replacing the TEF promoter and NcoI/NotI site with the GPD promoter and a unique NotI site, and thereby producing pY5-22GPD (SEQ ID NO:70).
[0385] The Mortierella alpina delta-5 desaturase gene (SEQ ID NO:67) was released from pKR136 (SEQ ID NO:75; which was previously described in PCT Publication No WO 2004/071467 (the contents of which are hereby incorporated by reference)) by digestion with NotI and cloned into the NotI site of pY5-22GPD to produce pY98 (SEQ ID NO:76; FIG. 9).
Transformation of pY98 (Comprising MaD5) and pDMW368 (Comprising RD5) into Yarrowia Strain Y2224 and Comparison of Lipid Profiles
[0386] Strain Y2224 was isolated in the following manner: Yarrowia lipolytica ATCC #20362 cells from a YPD agar plate (1% yeast extract, 2% bactopeptone, 2% glucose, 2% agar) were streaked onto a MM plate (75 mg/L each of uracil and uridine, 6.7 g/L YNB with ammonia sulfate, without amino acid, and 20 g/L glucose) containing 250 mg/L 5-FOA (Zymo Research). Plates were incubated at 28° C. and four of the resulting colonies were patched separately onto MM plates containing 200 mg/mL 5-FOA and MM plates lacking uracil and uridine to confirm uracil Ura3 auxotrophy.
[0387] Strain Y2224 was transformed with pY98 (SEQ ID NO:76, FIG. 9) and pDMW368 (SEQ ID NO:23; FIG. 5C; Example 6) as described in the General Methods.
[0388] Single colonies of transformant Yarrowia lipolytica containing pY98 (SEQ ID NO:76) or pDMW368 (SEQ ID NO:23) were grown in 3 mL MM lacking uracil supplemented with 0.2% tergitol at 30° C. for 1 day. After this, 0.1 mL was transferred to 3 rnL of the same medium supplemented with either EDA, ETrA, DGLA, ETA or no fatty acid. These were incubated for 16 h at 30° 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 μL of 1% sodium methoxide for 3D min at 50° C. after which 500 μL of 1M NaCl and 100 μL of heptane were added. After thorough mixing and centrifugation, fatty acid methyl esters (FAMEs) were analyzed by GC.
[0389] FAMEs (5 μL injected from hexane layer) were separated and quantified using a Hewlett-Packard 6890 Gas Chromatograph fitted with an Omegawax 320 fused silica capillary column (Catalog No. 24152, Supelco Inc.). The oven temperature was programmed to hold at 220° C. for 2.6 min, increase to 240° C. at 20° C./min and then hold for an additional 2.4 min. Carrier gas was supplied by a Whatman hydrogen generator. Retention times were compared to those for methyl esters of standards commercially available (Nu-Chek Prep, Inc.).
[0390] The fatty acid profiles for Yarrowia lipolytica expressing pY98 (SEQ ID NO:76) or pDMW368 (SEQ ID NO:23) and fed various substrates are shown in FIG. 10A. In FIG. 10A shading indicates the substrates fed and products produced; fatty acids are identified as 16:0 (palmitate), 16:1, 18:0 (stearic acid), 18:1 (oleic acid), LA, GLA, ALA, STA, EDA, SCI (sciadonic acid or cis-5,11,14-eicosatrienoic acid; 20:3 omega-6), DGLA, ARA, ETrA, JUP (juniperonic acid or cis-5,11,14,17-eicosatrienoic acid; 20:4 omega-3), ETA and EPA. Fatty acid compositions were expressed as the weight percent (wt. %) of total fatty acids.
[0391] Percent delta-5 desaturation ("% delta-5 desat") of RD5 and MaD5 for each substrate is shown in FIG. 10B and was calculated by dividing the wt. % for product (either SCI, JUP, ARA or EPA) by the sum of the wt. % for the substrate and product (either EDA and SCI, ETrA and JUP, DGLA and ARA, or ETA and EPA, respectively) and multiplying by 100 to express as a %, depending on which substrate was fed.
[0392] The activities of MaD5 and RD5 are compared using the ratio of the percent delta-5 desaturation ("Ratio Desat R/Ma") in FIG. 10B and are calculated by dividing the percent delta-5 desaturation for RD5 on a particular substrate by the percent delta-5 desaturation for MaD5 on the same substrate.
[0393] The substrate specificity of RD5 and MaD5 for the correct omega-6 fatty acid substrate (i.e., DGLA) versus the by-product fatty acid (i.e., SCI) or the correct omega-3 fatty acid substrate (i.e., ETA) versus the by-product fatty acid (i.e., JUP) is also shown in FIG. 10B. Specifically, the substrate specificity ("Ratio Prod/By-Prod") for omega-6 substrates was calculated by dividing the percent delta-5 desaturation (% delta-5 desat) for DGLA by the percent delta-5 desaturation (% delta-5 desat) for EDA and is shown on the same lines as the results for DGLA.
[0394] The substrate specificity ("Ratio Prod/By-Prod") for omega-3 substrates was calculated by dividing the percent delta-5 desaturation (% delta-5 desat) for ETA by the percent delta-5 desaturation (% delta-5 desat) for ETrA and is shown on the same lines as the results for ETA. Furthermore, the ratio of substrate specificity ("Ratio Prod/By-Prod R/Ma") for omega-6 substrates was determined by dividing the substrate specificity for RD5 on the omega-6 substrates (i.e., DGLA/EDA) by that for MaD5. The ratio of substrate specificity ("Ratio Prod/By-Prod R/Ma") for omega-3 substrates was calculated by dividing the substrate specificity for RD5 on the omega-3 substrates (i.e., ETA/ETrA) by that for MaD5.
[0395] The preference of RD5 and MaD5 for omega-6 or omega-3 substrates is compared using the ratio of the percent delta-5 desaturation ("Ratio n-6/n-3") in FIG. 10B and is calculated by dividing the percent delta-5 desaturation for RD5 and MaD5 on a particular omega-6 substrate (either DGLA or EDA) by the percent delta-5 desaturation on the corresponding omega-3 substrate (either ETA or ETrA, respectively).
[0396] From the results in FIG. 10B, it is clear that RD5 is approximately 3.0- to 9.7-fold more active in Yarrowia than MaD5 when DGLA, EDA, ETrA and ETA are used as substrates. The substrate specificity of RD5 compared to MaD5 (RD5/MaD5) for the correct omega-6 substrate (i.e., DGLA versus EDA) is approximately 0.4 and for the omega-3 substrate (i.e., ETA versus ETrA) is approximately 0.6. RD5 also has an approximate 1.4-fold preference for omega-6 substrates EDA and DGLA) over the omega-3 substrates (i.e., ETrA and ETA), respectively.
Example 18
Construction of Soybean Expression Vector pKR916 for Co-Expression of the Mortierella alpina Delta-5 Desaturase (MaD5) with a Delta-9 Elongase Derived From Euglena gracilis (EgD9e) and a Delta-8 Desaturase Derived from Euglena gracilis (EgD8)
[0397] The present Example describes construction of a soybean vector for co-expression of MaD5 (SEQ ID NO:67, Example 17) with EgD9e (SEQ ID NO:77; which is described in U.S. application Ser. No. 11/601,563 (filed Nov. 16, 2006; Attorney Docket No. BB-1562) and EgD8 (SEQ ID NO:78; described as Eg5 in PCT Publication No. WO 2006/012325).
Euglena gracilis Delta-9 Elongase (EgD9e):
[0398] A clone from the Euglena cDNA library (eeg1c), called eeg1c.pk001.n5f, containing the Euglena gracilis delta-9 elongase (EgD9e; SEQ ID NO:77) was used as template to amplifiy EgD9e with oligonucleotide primers oEugEL1-1 (SEQ ID NO:79) and oEugEL1-2 (SEQ ID NO:80) using the VentR® 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® cloning vector using the Zero Blunt® PCR Cloning Kit (Invitrogen Corporation), following the manufacturer's protocol, to produce pKR906 (SEQ ID NO:81).
[0399] A starting plasmid pKR72 (ATCC Accession No. PTA-6019; SEQ ID NO:82, 7085 by sequence), a derivative of pKS123 which was previously described in PCT Publication No. WO 02/008269 (the contents of which are hereby incorporated by reference), contains the hygromycin B phosphotransferase gene (HPT) (Gritz, L. and Davies, J., Gene, 25:179-188 (1983)), flanked by the T7 promoter and transcription terminator (i.e., a T7prom/HPT/T7term cassette), and a bacterial origin of replication (ori) for selection and replication in bacteria (e.g., E. coli). In addition, pKR72 also contains HPT, flanked by the 35S promoter (Odell et al., Nature, 313:810-812 (1985)) and NOS 3' transcription terminator (Depicker et al., J. Mol. Appl. Genet, 1:561-570 (1982)) (i.e., a 35S/HPT/NOS3' cassette) for selection in plants such as soybean. pKR72 also contains a NotI restriction site, flanked by the promoter for the α' subunit of β-conglycinin (Beachy et al., EMBO J., 4:3047-3053 (1985)) and the 3' transcription termination region of the phaseolin gene (Doyle et al., J. Biol. Chem., 261:9228-9238 (1986)), thus allowing for strong tissue-specific expression in the seeds of soybean of genes cloned into the NotI site.
[0400] The AscI fragment from plasmid pKS102 (SEQ ID NO:83), previously described in PCT Publication No. WO 02/00905 (the contents of which are hereby incorporated by reference), containing a T7prom/hpt/T7term cassette and bacterial ori, was combined with the AscI fragment of plasmid pKR72 (SEQ ID NO:82), containing a βcon/NotI/Phas cassette to produce pKR197 (SEQ ID NO:84), previously described in PCT Publication No. WO 04/071467 (the contents of which are hereby incorporated by reference).
[0401] The gene for the Euglena gracilis delta-9 elongase was released from pKR906 (SEQ ID NO:81) by digestion with NotI and cloned into the NotI site of pKR197 (SEQ ID NO:84) to produce intermediate cloning vector pKR911 (SEQ ID NO:85).
Euglena gracilis Delta-8 Desaturase (EgD8):
[0402] Plasmid pKR680 (SEQ ID NO:86), which was previously described in PCT Publication No. WO 2006/012325 (the contents of which are hereby incorporated by reference), contains the Euglena gracilis delta-8 desaturase (EgD8; SEQ ID NO:78; described as Eg5 in WO 2006/012325) flanked by the Kunitz soybean Trypsin Inhibitor (KTi) promoter (Jofuku et al., Plant Cell, 1:1079-1093 (1989)) and the KTi 3' termination region, the isolation of which is described in U.S. Pat. No. 6,372,965, followed by the soy albumin transcription terminator, which was previously described in PCT Publication No. WO 2004/071467 (i.e., a Kti/NotI/Kti3'Salb3' cassette).
[0403] Plasmid pKR680 (SEQ ID NO:86) was digested with BsiWI and the fragment containing EgD8 was cloned into the BsiWI site of pKR911 (SEQ ID NO:85) to produce pKR913 (SEQ ID NO:87).
Mortierella alpina Delta-5 Desaturase (MaD5):
[0404] Plasmid pKR767 (SEQ ID NO:88), which was previously described in PCT Publication No WO 2006/012325 (the contents of which are hereby incorporated by reference), contains the Mortierella alpina delta-5 desaturase (MaD5; SEQ ID NO:67) flanked by the promoter for the soybean glycinin Gyl gene and the pea legumin A2 3' transcription termination region (i.e. a Gy1/MaD5/legA2 cassette; the construction of which is described in WO 2006/012325).
[0405] The Gy1/Mad5/legA2 cassette was released from pKR767 (SEQ 1D NO:88) by digestion with SbfI and the resulting fragment was cloned into the SbfI site of pKR913 (SEQ ID NO:87) to produce pKR916 (SEQ ID NO:89). A schematic depiction of pKR916 is shown in FIG. 11A. In this way, the Euglena gracilis delta-9 elongase (labeled "eug el1" in FIG. 11A) was co-expressed with the Euglena gracilis delta-8 desaturase (labeled "eug d8-sq5" in FIG, 11A) and the Mortierella alpina delta-5 desaturase (labeled "DELTA 5 DESATURASE M ALPINA" in FIG. 11A) behind strong, seed specific promoters.
Example 19
Construction of Soybean Expression Vector pKR1038 for Co-Expression of the Peridinium sp. CCMP626 Delta-5 Desaturase (RD5) with a Delta-9 Elongase Derived from Euglena gracilis (EqD9e) and a Delta-8 Desaturase Derived from Euglena gracilis (EgD8)
[0406] The present Example describes construction of a soybean vector for co-expression of RD5 (SEQ ID NO:1, Example 5) with EgD9e (SEQ ID NO:77, Example 18) and EgD8 (SEQ ID NO:78, Example 18).
[0407] Starting plasmid pKR974 (SEQ ID NO:90) is identical to pKR767 (SEQ ID NO:88, Example 18) except the NotI fragment containing MaD5 was replaced with a NotI fragment containing the Saprolegnia diclina delta-5 desaturase (SdD5; SEQ ID NO:91, which is described in PCT Publication No. WO 2004/071467). In addition, a MfeI site in the legA2 terminator of pKR767 (SEQ ID NO:88) was removed by digestion with MfeI, filling the MfeI site and religating (i.e., CAATTG converted to CAATTAATTG) and therefore, the legA2 terminator of pKR974 (SEQ ID NO:90) is 770 by versus 766 by for pKR767 (SEQ ID NO:88).
[0408] In order to clone RD5 into a soybean expression vector, a NotI restriction site needed to be introduced at the 5' end of the gene. One skilled in the art will realized that there are many ways to introduce restriction sites into genes such as, but not limited to PCR or by subcloning into vectors containing the appropriate sites. In this case, in order to introduce a NotI site at the 5' end of RD5 (SEQ ID NO:1), pDMW368 (SEQ ID NO:23) was digested with MfeI and then partially digested with NcoI. The NcoI/MfeI fragment containing a full length RD5 (SEQ ID NO:1) was cloned into the NcoI/MfeI site of an intermediate cloning vector having a NotI site directly upstream of the NcoI site (i.e., GCGGCCGCAAACCATGG). The resulting plasmid was then digested with NotI and the fragment containing RD5 (SEQ ID NO:1) was cloned into the NotI site of pKR974 (SEQ ID NO:90) to produce pKR1033 (SEQ ID NO:92).
[0409] The Gy1/EgD5/legA2 cassette was released from pKR1033 (SEQ ID NO:92) by digestion with SbfI and the resulting fragment was cloned into the SbfI site of pKR913 (SEQ ID NO:87) to produce pKR1038 (SEQ ID NO:93). A schematic depiction of pKR1038 (SEQ ID NO:93) is shown in FIG. 11B. In this way, the Euglena gracilis delta-9 elongase (labeled "eug ell" in FIG. 1113) could be co-expressed with the Euglena gracilis delta-8 desaturase (labeled "eug d8-sq5" in FIG. 11B) and the Pendinium sp. CCMP626 delta-5 desaturase (labeled "CCMP626 d5 DS" in FIG. 11B) behind strong, seed specific promoters.
Example 20
Co-Expression of the Euglena gracilis Delta-9 Elongase, the Euglena gracilis Delta-8 Desaturase and the Saprolegnia diclina Delta-17 Desaturase With Either the Mortierella alpina Delta-5 Desaturase (pKR916 & pKR328) or The Peridinium CCMP626 Delta-5 Desaturase (pKR1038 & pKR328) In Soybean Somatic Embryos
[0410] The present Example describes the transformation and expression in soybean somatic embryos of pKR916 (SEQ ID NO:89, Example 18; containing EgD9e, EgD8 and MaD5) with pKR328 (SEQ ID NO:94, FIG. 11C, previously described in PCT Publication No. WO 04/071467), containing the Saprolegnia diclina delta-17 desaturase (SdD17) and the hygromycin phosphotransferase gene for selection on hygromycin. The present Example further describes the transformation and expression in soybean somatic embryos of pKR1038 (SEQ ID NO:93, Example 19; containing EgD9e, EgD8 and RD5) with pKR328 (SEQ ID NO:94, FIG. 11C).
[0411] Soybean embryogenic suspension culture (cv. Jack) was transformed with the AscI fragment containing the expression cassette of pKR916 (SEQ ID NO:89) and intact plasmid pKR328 (SEQ ID NO:94), or with the AscI fragment containing the expression cassette of pK1038 (SEQ ID NO:93) and intact plasmid pKR328 (SEQ ID NO:94), as described in Example 13.
[0412] Embryos were matured in soybean histodifferentiation and maturation liquid medium (SHaM liquid media; Schmidt et al., Cell Biology and Morphogenesis, 24:393 (2005)) using a modified procedure. Briefly, after 4 weeks of selection in SB196 as described in Example 13, embryo clusters were removed to 35 mL of SB228 (SHaM liquid media) in a 250 mL Erlenmeyer flask. Tissue was maintained in SHaM liquid media on a rotary shaker at 130 rpm and 26° C. with cool white fluorescent lights on a 16:8 hr day/night photoperiod at a light intensity of 60-85 μE/m2/s for 2 weeks as embryos matured, Embryos grown for 2 weeks in SHaM liquid media were equivalent in size and fatty acid content to embryos cultured on SB166/SB103 for 5-8 weeks as described in Example 13.
Media Recipes:
SB 228--Soybean Histodifferentiation & Maturation (SHaM) (Per Liter)
TABLE-US-00015 [0413] DDI H2O 600 mL FN-Lite Macro Salts for SHaM 10X 100 mL MS Micro Salts 1000x 1 mL MS FeEDTA 100x 10 mL CaCl 100x 6.82 mL B5 Vitamins 1000x 1 mL L-Methionine 0.149 g Sucrose 30 g Sorbitol 30 g Adjust volume to 900 mL pH 5.8 Autoclave Add to cooled media (≦30° C.): *Glutamine (final concentration 30 mM) 4% 110 mL *Note: Final volume will be 1010 mL after glutamine addition.
Since glutamine degrades relatively rapidly, it may be preferable to add immediately prior to using media. Expiration 2 weeks after glutamine is added; base media can be kept longer without glutamine.
FN-Lite Macro for SHAM 10×--Stock #1 (Per Liter)
TABLE-US-00016 [0414] (NH4)2SO4 (ammonium sulfate) 4.63 g KNO3 (potassium nitrate) 28.3 g MgSO4*7H20 (magnesium sulfate heptahydrate) 3.7 g KH2PO4 (potassium phosphate, monobasic) 1.85 g Bring to volume Autoclave
MS Micro 1000×--Stock #2 (Per 1 Liter)
TABLE-US-00017 [0415] H3BO3 (boric acid) 6.2 g MnSO4*H2O (manganese sulfate monohydrate) 16.9 g ZnSO4*7H20 (zinc sulfate heptahydrate) 8.6 g Na2MoO4*2H20 (sodium molybdate dihydrate) 0.25 g CuSO4*5H20 (copper sulfate pentahydrate) 0.025 g CoCl2*6H20 (cobalt chloride hexahydrate) 0.025 g KI (potassium iodide) 0.8300 g Bring to volume Autoclave
FeEDTA 100×--Stock #3 (Per Liter)
TABLE-US-00018 [0416] Na2EDTA* (sodium EDTA) 3.73 g FeSO4*7H20 (iron sulfate heptahydrate) 2.78 g Bring to Volume Solution is photosensitive. Bottle(s) should be wrapped in foil to omit light. Autoclave *EDTA must be completely dissolved before adding iron.
Ca 100×--Stock #4 (Per Liter)
TABLE-US-00019 [0417] CaCl2*2H20 (calcium chloride dihydrate) 44 g Bring to Volume Autoclave
B5 Vitamin 1000×--Stock #5 (Per Liter)
TABLE-US-00020 [0418] Thiamine*HCl 10 g Nicotinic Acid 1 g Pyridoxine*HCl 1 g Myo-Inositol 100 g Bring to Volume Store frozen
4% Glutamine--Stock #6 (Per Liter)
TABLE-US-00021 [0419] DDI water heated to 30° C. 900 mL L-Glutamine 40 g Gradually add while stirring and applying low heat. Do not exceed 35° C. Bring to Volume Filter Sterilize Store frozen* *Note: Warm thawed stock in 31° C. bath to fully dissolve crystals.
[0420] After maturation in SHaM liquid media, individual embryos were removed from the clusters, dried and screened for alterations in their fatty acid compositions as described supra.
[0421] A subset of soybean embryos (i.e., six embryos per event) transformed with either pKR916 (SEQ ID NO:89) and pKR328 (SEQ ID NO:94), or pKR1038 (SEQ ID NO:93) and pKR328 (SEQ ID NO:94), were harvested and picked into glass GC vials and fatty acid methyl esters were prepared by transesterification. For transesterification, 50 μL 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 μL injected from hexane layer) were separated and quantified using a Hewlett-Packard 6890 Gas Chromatograph fitted with an Omegawax 320 fused silica capillary column (Catalog No. 24152, Supelco Inc.). The oven temperature was programmed to hold at 220° C. for 2.6 min, increase to 240° C. at 20° C./min and then hold for an additional 2.4 min. Carrier gas was supplied by a Whatman hydrogen generator. Retention times were compared to those for methyl esters of standards commercially available (Nu-Chek Prep, Inc.).
[0422] In this way, 60 events transformed with pKR916 (SEQ ID NO:89) and pKR328 (SEQ ID NO:94) and 31 events transformed with pKR1038 (SEQ ID NO:93) and pKR328 (SEQ ID NO:94) were analyzed. The average fatty acid profiles for the ten events having the highest delta-5 desaturase activity for each transformation (pKR916 and pKR328, pKR1038 and pKR328) are shown in FIG. 12A and FIG. 12B, respectively.
[0423] In FIGS. 12A and 12B, fatty acids are identified as 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic acid), LA, ALA, EDA, SCI, DGLA, ARA, ERA, JUP, ETA and EPA. Fatty acids listed as "others" include: 18:2 (5,9), GLA, STA, 20:0, 20:1(11), 20:2 (7,11) or 20:2 (8,11) and DPA. Each of these "other" fatty acids is present at a relative abundance of less than 3.0% of the total fatty acids. Fatty acid compositions for an individual embryo were expressed as the weight percent (wt. %) of total fatty acids and the average fatty acid composition is an average of six individual embryos for each event.
[0424] The activity of the delta-5 desaturase for the "correct" substrates (i.e., DGLA and ETA) is expressed as percent delta-5 desaturation ("Correct % delta-5 desat"), calculated according to the following formula: product]/[substrate+product])*100.
[0425] More specifically, the percent delta-5 desaturation for the "correct" substrates was determined as: ([ARA+EPA]/[DGLA+ETA+ARA+EPA])*100.
[0426] The activity of the delta-5 desaturase for the "wrong" substrates (i.e., EDA and ERA) is also expressed as percent delta-5 desaturation ("Wrong % delta-5 desat"), calculated as: ([SCI+JUP]/[EDA+ERA+SCI+JUP])*100.
[0427] The substrate specificities of MaD5 and RD5 for the "correct" substrates (i.e., DGLA and ETA) versus the "wrong" substrates (i.e., EDA and ERA) were compared and the comparison is shown in FIG. 13. In FIG. 13, the activity of the delta-5 desaturase for the "correct" substrates ("Correct % delta-5 desat") is plotted on the x-axis and the activity of the delta-5 desaturase for the "wrong" substrates ("Wrong % delta-5 desat") is plotted on the y-axis for MaD5 (data from FIG. 12A) and RD5 (data from FIG. 12B).
Sequence CWU
1
SEQUENCE LISTING
<160> NUMBER OF SEQ ID NOS: 94
<210> SEQ ID NO 1
<211> LENGTH: 1392
<212> TYPE: DNA
<213> ORGANISM: Peridinium sp. CCMP626
<220> FEATURE:
<221> NAME/KEY: misc_feature
<223> OTHER INFORMATION: delta-5 desaturase
<400> SEQUENCE: 1
atggctccag atgcggacaa gttgagacag cgcaaggcgc aatcgattca agacacggct 60
gattcgcaag ctaccgaact caagattggc accctgaagg gcttgcaggg gacagaaatc 120
gtcattgatg gagacattta cgatataaaa gactttgatc accccggtgg tgaatccatc 180
atgacttttg ggggaaacga tgtcaccgcc acgtacaaga tgatccaccc ctaccactct 240
aagcaccatt tggagaagat gaagaaagtg ggacgagttc cggactacac ctcggaatac 300
aagtttgata ctccctttga gcgtgaaatc aagcaagagg tcttcaagat tgtgcgacga 360
ggccgcgagt ttggaacacc tggatacttc ttccgggctt tctgctacat tggacttttc 420
ttttacttgc agtatttgtg ggtcacgact cccactacct ttgccttggc gatcttctat 480
ggtgtttcgc aagctttcat tggtttgaac gtacaacatg atgccaacca cggagctgcc 540
tccaagaagc cttggatcaa taacttgcta ggattggggg ctgactttat cggaggttcc 600
aaatggttgt ggatgaacca gcactggacg caccacacat acaccaacca ccatgagaag 660
gatcccgatg ccttgggcgc tgaaccaatg ttgttgttca atgattatcc cttgggtcac 720
ccaaagcgta ctttgattca ccacttccag gccttctatt accttttcgt cttggccgga 780
tactgggtct cttcggtctt caaccctcaa attttggact tgcaacaccg cggtgctcaa 840
gcggttggaa tgaaaatgga gaacgattac attgccaaaa gccgaaagta tgccatcttc 900
ttgcgtctct tgtatattta caccaacatt gtcgctccga tccaaaacca aggcttctcg 960
ttgaccgtgg tcgcccacat tttgaccatg ggcgtcgctt ccagtttgac tttggcgact 1020
ctttttgcct tgtcgcacaa ttttgaaaac gcggatcgcg atcccactta cgaggcccgc 1080
aagggaggag agcctgtttg ttggttcaag tcgcaagtcg aaacctcgtc aacttacgga 1140
ggtttcatct cgggttgctt gacgggcgga ctcaacttcc aagtggaaca ccacttgttc 1200
cctcgtatga gttcggcctg gtacccctac attgccccta ctgttcgaga ggtttgcaaa 1260
aagcacggag tcaagtacgc atactatccc tgggtctggc aaaacttgat ttcaactgtc 1320
aagtatctgc atcaaagcgg aactggatcc aactggaaga atggcgccaa cccctactcg 1380
ggaaaattgt aa 1392
<210> SEQ ID NO 2
<211> LENGTH: 463
<212> TYPE: PRT
<213> ORGANISM: Peridinium sp. CCMP626
<400> SEQUENCE: 2
Met Ala Pro Asp Ala Asp Lys Leu Arg Gln Arg Lys Ala Gln Ser Ile
1 5 10 15
Gln Asp Thr Ala Asp Ser Gln Ala Thr Glu Leu Lys Ile Gly Thr Leu
20 25 30
Lys Gly Leu Gln Gly Thr Glu Ile Val Ile Asp Gly Asp Ile Tyr Asp
35 40 45
Ile Lys Asp Phe Asp His Pro Gly Gly Glu Ser Ile Met Thr Phe Gly
50 55 60
Gly Asn Asp Val Thr Ala Thr Tyr Lys Met Ile His Pro Tyr His Ser
65 70 75 80
Lys His His Leu Glu Lys Met Lys Lys Val Gly Arg Val Pro Asp Tyr
85 90 95
Thr Ser Glu Tyr Lys Phe Asp Thr Pro Phe Glu Arg Glu Ile Lys Gln
100 105 110
Glu Val Phe Lys Ile Val Arg Arg Gly Arg Glu Phe Gly Thr Pro Gly
115 120 125
Tyr Phe Phe Arg Ala Phe Cys Tyr Ile Gly Leu Phe Phe Tyr Leu Gln
130 135 140
Tyr Leu Trp Val Thr Thr Pro Thr Thr Phe Ala Leu Ala Ile Phe Tyr
145 150 155 160
Gly Val Ser Gln Ala Phe Ile Gly Leu Asn Val Gln His Asp Ala Asn
165 170 175
His Gly Ala Ala Ser Lys Lys Pro Trp Ile Asn Asn Leu Leu Gly Leu
180 185 190
Gly Ala Asp Phe Ile Gly Gly Ser Lys Trp Leu Trp Met Asn Gln His
195 200 205
Trp Thr His His Thr Tyr Thr Asn His His Glu Lys Asp Pro Asp Ala
210 215 220
Leu Gly Ala Glu Pro Met Leu Leu Phe Asn Asp Tyr Pro Leu Gly His
225 230 235 240
Pro Lys Arg Thr Leu Ile His His Phe Gln Ala Phe Tyr Tyr Leu Phe
245 250 255
Val Leu Ala Gly Tyr Trp Val Ser Ser Val Phe Asn Pro Gln Ile Leu
260 265 270
Asp Leu Gln His Arg Gly Ala Gln Ala Val Gly Met Lys Met Glu Asn
275 280 285
Asp Tyr Ile Ala Lys Ser Arg Lys Tyr Ala Ile Phe Leu Arg Leu Leu
290 295 300
Tyr Ile Tyr Thr Asn Ile Val Ala Pro Ile Gln Asn Gln Gly Phe Ser
305 310 315 320
Leu Thr Val Val Ala His Ile Leu Thr Met Gly Val Ala Ser Ser Leu
325 330 335
Thr Leu Ala Thr Leu Phe Ala Leu Ser His Asn Phe Glu Asn Ala Asp
340 345 350
Arg Asp Pro Thr Tyr Glu Ala Arg Lys Gly Gly Glu Pro Val Cys Trp
355 360 365
Phe Lys Ser Gln Val Glu Thr Ser Ser Thr Tyr Gly Gly Phe Ile Ser
370 375 380
Gly Cys Leu Thr Gly Gly Leu Asn Phe Gln Val Glu His His Leu Phe
385 390 395 400
Pro Arg Met Ser Ser Ala Trp Tyr Pro Tyr Ile Ala Pro Thr Val Arg
405 410 415
Glu Val Cys Lys Lys His Gly Val Lys Tyr Ala Tyr Tyr Pro Trp Val
420 425 430
Trp Gln Asn Leu Ile Ser Thr Val Lys Tyr Leu His Gln Ser Gly Thr
435 440 445
Gly Ser Asn Trp Lys Asn Gly Ala Asn Pro Tyr Ser Gly Lys Leu
450 455 460
<210> SEQ ID NO 3
<211> LENGTH: 1392
<212> TYPE: DNA
<213> ORGANISM: Peridinium sp. CCMP626
<220> FEATURE:
<221> NAME/KEY: misc_feature
<223> OTHER INFORMATION: synthetic delta-5 desaturase
(codon-optimized)
for Yarrowia lipolytica
<400> SEQUENCE: 3
atggctcccg acgccgacaa gctgcgacag cgaaaggctc agtccatcca ggacactgcc 60
gattctcagg ctaccgagct caagattggc accctgaagg gtctccaagg caccgagatc 120
gtcattgatg gcgacatcta cgacatcaaa gacttcgatc accctggagg cgaatccatc 180
atgacctttg gtggcaacga cgttactgcc acctacaaga tgattcatcc ctaccactcg 240
aagcatcacc tggagaagat gaaaaaggtc ggtcgagtgc ccgactacac ctccgagtac 300
aagttcgata ctcccttcga acgagagatc aaacaggagg tcttcaagat tgtgcgaaga 360
ggtcgagagt ttggaacacc tggctacttc tttcgagcct tctgctacat cggtctcttc 420
ttttacctgc agtatctctg ggttaccact cctaccactt tcgcccttgc tatcttctac 480
ggtgtgtctc aggccttcat tggcctgaac gtccagcacg acgccaacca cggagctgcc 540
tccaaaaagc cctggatcaa caatttgctc ggcctgggtg ccgactttat cggaggctcc 600
aagtggctct ggatgaacca gcactggacc catcacactt acaccaacca tcacgagaag 660
gatcccgacg ccctgggtgc agagcctatg ctgctcttca acgactatcc cttgggtcac 720
cccaagcgaa ccctcattca tcacttccaa gccttctact atctgtttgt ccttgctggc 780
tactgggtgt cttcggtgtt caaccctcag atcctggacc tccagcaccg aggtgcccag 840
gctgtcggca tgaagatgga gaacgactac attgccaagt ctcgaaagta cgctatcttc 900
ctgcgactcc tgtacatcta caccaacatt gtggctccca tccagaacca aggcttttcg 960
ctcaccgtcg ttgctcacat tcttactatg ggtgtcgcct ccagcctgac cctcgctact 1020
ctgttcgccc tctcccacaa cttcgagaac gcagatcggg atcccaccta cgaggctcga 1080
aagggaggcg agcctgtctg ttggttcaag tcgcaggtgg aaacctcctc tacttacggt 1140
ggcttcattt ccggttgcct tacaggcgga ctcaactttc aggtcgagca tcacctgttt 1200
cctcgaatgt cctctgcctg gtacccctac atcgctccta ccgttcgaga ggtctgcaaa 1260
aagcacggcg tcaagtacgc ctactatccc tgggtgtggc agaacctcat ctcgaccgtc 1320
aagtacctgc atcagtccgg aactggctcg aactggaaga acggtgccaa tccctactct 1380
ggcaagctgt aa 1392
<210> SEQ ID NO 4
<211> LENGTH: 563
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: 563 bp fragment of pT-12-D5
<400> SEQUENCE: 4
ggtcatcatg tttataccaa ccaccatgag aaggatcccg atgccttggg cgctgaacca 60
atgttgttgt tcaatgatta tcccttgggt cacccaaagc gtactttgat tcaccacttc 120
caggccttct attacctttt cgtcttggcc ggatactggg tctcttcggt cttcaaccct 180
caaattttgg acttgcaaca ccgcggtgct caagcggttg gaatgaaaat ggagaacgat 240
tacattgcca aaagccgaaa gtatgccatc ttcttgcgtc tcttgtatat ttacaccaac 300
attgtcgctc cgatccaaaa ccaaggcttc tcgttgaccg tggtcgccca cattttgacc 360
atgggcgtcg cttccagttt gactttggcg actctttttg cctcgtcgca caattttgaa 420
aacgcggatc gcgatcccac ttacgaggcc cgcaaggggg gagagcctgt ttgttggttc 480
aagtcgcaag tcgaaacctc gtcaacttac ggaggtttca tctcgggttg cttgacgggc 540
ggactcaact tccaagtatc aca 563
<210> SEQ ID NO 5
<211> LENGTH: 187
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: translation of 563 bp fragment of pT-12-D5
<400> SEQUENCE: 5
Gly His His Val Tyr Thr Asn His His Glu Lys Asp Pro Asp Ala Leu
1 5 10 15
Gly Ala Glu Pro Met Leu Leu Phe Asn Asp Tyr Pro Leu Gly His Pro
20 25 30
Lys Arg Thr Leu Ile His His Phe Gln Ala Phe Tyr Tyr Leu Phe Val
35 40 45
Leu Ala Gly Tyr Trp Val Ser Ser Val Phe Asn Pro Gln Ile Leu Asp
50 55 60
Leu Gln His Arg Gly Ala Gln Ala Val Gly Met Lys Met Glu Asn Asp
65 70 75 80
Tyr Ile Ala Lys Ser Arg Lys Tyr Ala Ile Phe Leu Arg Leu Leu Tyr
85 90 95
Ile Tyr Thr Asn Ile Val Ala Pro Ile Gln Asn Gln Gly Phe Ser Leu
100 105 110
Thr Val Val Ala His Ile Leu Thr Met Gly Val Ala Ser Ser Leu Thr
115 120 125
Leu Ala Thr Leu Phe Ala Ser Ser His Asn Phe Glu Asn Ala Asp Arg
130 135 140
Asp Pro Thr Tyr Glu Ala Arg Lys Gly Gly Glu Pro Val Cys Trp Phe
145 150 155 160
Lys Ser Gln Val Glu Thr Ser Ser Thr Tyr Gly Gly Phe Ile Ser Gly
165 170 175
Cys Leu Thr Gly Gly Leu Asn Phe Gln Val Ser
180 185
<210> SEQ ID NO 6
<211> LENGTH: 693
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: 693 bp fragment of pT-RD5-5'C2
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (10)..(10)
<223> OTHER INFORMATION: n is a, c, g, or t
<400> SEQUENCE: 6
aatcgtcatn gatggagaca tttacgatat aaaagacttt gatcaccccg gtggtgaatc 60
catcatgact tttgggggaa acgatgtcac cgccacgtac aagatgatcc acccctacca 120
ctctaagcac catttggaga agatgaagaa agtgggacga gttccggact acacctcgga 180
atacaagttt gatactccct ttgagcgtga aatcaagyaa gaggtcttca agattgtgcg 240
acgaggccgc gagtttggaa cacctggata cttcttccgg gctttctgct acattggact 300
tttcttttac ttgcagtatt tgtgggtcac gactcccact acctttgcct tggcgatctt 360
ctatggtgtt tcgcaagctt tcattggttt gaacgtacaa catgatgcca accacggagc 420
tgcctccaag aagccttgga tcaataactt gctaggattg ggggctgact ttatcggagg 480
ttccaaatgg ttgtggatga accagcactg gacgcaccac acatacacca accaccatga 540
gaaggatccc gatgccttgg gcgctgaacc aatgttgttg ttcaatgatt atcccttggg 600
tcacccaaag cgtactttga ttcaccactt ccaggccttc tattaccttt tcgtcttggc 660
cggatactgg gtctcttcgg tcttcaaccc tca 693
<210> SEQ ID NO 7
<211> LENGTH: 511
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: 511 bp 5' extended fragment of pT-RD5-5'C2
<400> SEQUENCE: 7
aatcgtcatt gatggagaca tttacgatat aaaagacttt gatcaccccg gtggtgaatc 60
catcatgact tttgggggaa acgatgtcac cgccacgtac aagatgatcc acccctacca 120
ctctaagcac catttggaga agatgaagaa agtgggacga gttccggact acacctcgga 180
atacaagttt gatactccct ttgagcgtga aatcaagyaa gaggtcttca agattgtgcg 240
acgaggccgc gagtttggaa cacctggata cttcttccgg gctttctgct acattggact 300
tttcttttac ttgcagtatt tgtgggtcac gactcccact acctttgcct tggcgatctt 360
ctatggtgtt tcgcaagctt tcattggttt gaacgtacaa catgatgcca accacggagc 420
tgcctccaag aagccttgga tcaataactt gctaggattg ggggctgact ttatcggagg 480
ttccaaatgg ttgtggatga accagcactg g 511
<210> SEQ ID NO 8
<211> LENGTH: 358
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: 358 bp fragment of pT-RD5-5'2nd
<400> SEQUENCE: 8
gatttctttc gttggcattt ttcgttggga aagactcttg caacgatggc tccagatgcg 60
gacaagttga gacagcgcaa ggcgcaatcg attcaagaca cggctgattc gcaagctacc 120
gaactcaaga ttggcaccct gaagggcttg caggggacag aaatcgtcat tgatggagac 180
atttacgata taaaagactt tgatcacccc ggtggtgaat ccatcatgac ttttggagga 240
aacgatgtca ctgccacgta caagatgatc cacccctacc actctaagca ccatttggag 300
aagatgaaga aagtgggacg agttctggac tacacctcgg aatacaagtt tgatactc 358
<210> SEQ ID NO 9
<211> LENGTH: 161
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: 161 bp 5' extended fragment of pT-RD5-5'2nd
<400> SEQUENCE: 9
gatttctttc gttggcattt ttcgttggga aagactcttg caacgatggc tccagatgcg 60
gacaagttga gacagcgcaa ggcgcaatcg attcaagaca cggctgattc gcaagctacc 120
gaactcaaga ttggcaccct gaagggcttg caggggacag a 161
<210> SEQ ID NO 10
<211> LENGTH: 299
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: 299 bp fragment of pT-RD5-3'
<400> SEQUENCE: 10
gaggtttcat ctcgggttgt ttgacgggcg gactcaactt tcaagtggaa caccacttgt 60
tccctcgtat gagttcggcc tggtacccct acattgcccc tgctgttcga gaggtttgca 120
aaaagcacgg agtcaagtac gcatactatc cctgggtctg gcaaaacttg atttcaactg 180
tcaagtatct gcatcaaagc ggaactggat ccaactggaa gaatggcgcc aacccctact 240
cgggaaaatt gtaaatgaat tctagtcaag atgggtcact gcattcaaaa aaaaaaaaa 299
<210> SEQ ID NO 11
<211> LENGTH: 247
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: 247 bp of 3' downstream sequence of
pT-RD5-3'
<400> SEQUENCE: 11
ccacttgttc cctcgtatga gttcggcctg gtacccctac attgcccctg ctgttcgaga 60
ggtttgcaaa aagcacggag tcaagtacgc atactatccc tgggtctggc aaaacttgat 120
ttcaactgtc aagtatctgc atcaaagcgg aactggatcc aactggaaga atggcgccaa 180
cccctactcg ggaaaattgt aaatgaattc tagtcaagat gggtcactgc attcaaaaaa 240
aaaaaaa 247
<210> SEQ ID NO 12
<211> LENGTH: 456
<212> TYPE: PRT
<213> ORGANISM: Pythium irregulare
<220> FEATURE:
<221> NAME/KEY: MISC_FEATURE
<223> OTHER INFORMATION: delta-5 desaturase
<300> PUBLICATION INFORMATION:
<308> DATABASE ACCESSION NUMBER: GenBank Accession No. AAL13311
<400> SEQUENCE: 12
Met Gly Thr Asp Gln Gly Lys Thr Phe Thr Trp Gln Glu Val Ala Lys
1 5 10 15
His Asn Thr Ala Lys Ser Ala Trp Val Ile Ile Arg Gly Glu Val Tyr
20 25 30
Asp Val Thr Glu Trp Ala Asp Lys His Pro Gly Gly Ser Glu Leu Ile
35 40 45
Val Leu His Ser Gly Arg Glu Cys Thr Asp Thr Phe Tyr Ser Tyr His
50 55 60
Pro Phe Ser Asn Arg Ala Asp Lys Ile Leu Ala Lys Tyr Lys Ile Gly
65 70 75 80
Lys Leu Val Gly Gly Tyr Glu Phe Pro Val Phe Lys Pro Asp Ser Gly
85 90 95
Phe Tyr Lys Glu Cys Ser Glu Arg Val Ala Glu Tyr Phe Lys Thr Asn
100 105 110
Asn Leu Asp Pro Lys Ala Ala Phe Ala Gly Leu Trp Arg Met Val Phe
115 120 125
Val Phe Ala Val Ala Ala Leu Ala Tyr Met Gly Met Asn Glu Leu Ile
130 135 140
Pro Gly Asn Val Tyr Ala Gln Tyr Ala Trp Gly Val Val Phe Gly Val
145 150 155 160
Phe Gln Ala Leu Pro Leu Leu His Val Met His Asp Ser Ser His Ala
165 170 175
Ala Cys Ser Ser Ser Pro Ala Met Trp Gln Ile Ile Gly Arg Gly Val
180 185 190
Met Asp Trp Phe Ala Gly Ala Ser Met Val Ser Trp Leu Asn Gln His
195 200 205
Val Val Gly His His Ile Tyr Thr Asn Val Ala Gly Ala Asp Pro Asp
210 215 220
Leu Pro Val Asp Phe Glu Ser Asp Val Arg Arg Ile Val His Arg Gln
225 230 235 240
Val Leu Leu Pro Ile Tyr Lys Phe Gln His Ile Tyr Leu Pro Pro Leu
245 250 255
Tyr Gly Val Leu Gly Leu Lys Phe Arg Ile Gln Asp Val Phe Glu Thr
260 265 270
Phe Val Ser Leu Thr Asn Gly Pro Val Arg Val Asn Pro His Pro Val
275 280 285
Ser Asp Trp Val Gln Met Ile Phe Ala Lys Ala Phe Trp Thr Phe Tyr
290 295 300
Arg Ile Tyr Ile Pro Leu Val Trp Leu Lys Ile Thr Pro Ser Thr Phe
305 310 315 320
Trp Gly Val Phe Phe Leu Ala Glu Phe Thr Thr Gly Trp Tyr Leu Ala
325 330 335
Phe Asn Phe Gln Val Ser His Val Ser Thr Glu Cys Glu Tyr Pro Cys
340 345 350
Gly Asp Ala Pro Ser Ala Glu Val Gly Asp Glu Trp Ala Ile Ser Gln
355 360 365
Val Lys Ser Ser Val Asp Tyr Ala His Gly Ser Pro Leu Ala Ala Phe
370 375 380
Leu Cys Gly Ala Leu Asn Tyr Gln Val Thr His His Leu Tyr Pro Gly
385 390 395 400
Ile Ser Gln Tyr His Tyr Pro Ala Ile Ala Pro Ile Ile Ile Asp Val
405 410 415
Cys Lys Lys Tyr Asn Ile Lys Tyr Thr Val Leu Pro Thr Phe Thr Glu
420 425 430
Ala Leu Leu Ala His Phe Lys His Leu Lys Asn Met Gly Glu Leu Gly
435 440 445
Lys Pro Val Glu Ile His Met Gly
450 455
<210> SEQ ID NO 13
<211> LENGTH: 477
<212> TYPE: PRT
<213> ORGANISM: Phytophthora megasperma
<220> FEATURE:
<221> NAME/KEY: MISC_FEATURE
<223> OTHER INFORMATION: delta-5 desaturase
<300> PUBLICATION INFORMATION:
<308> DATABASE ACCESSION NUMBER: GenBank Accession No. CAD53323
<400> SEQUENCE: 13
Met Ala Pro Ile Glu Thr Val Lys Asp Ala Asn Glu Gly Leu His Gln
1 5 10 15
Arg Lys Gly Ala Ala Ala Ala Ser Lys Asp Thr Thr Thr Phe Thr Trp
20 25 30
Gln Asp Val Ala Lys His Asn Thr Ala Lys Ser Ala Trp Val Thr Ile
35 40 45
Arg Gly Val Val Tyr Asp Val Thr Glu Trp Ala Asp Arg His Pro Gly
50 55 60
Gly Arg Glu Leu Val Leu Leu His Ser Gly Arg Glu Cys Thr Asp Thr
65 70 75 80
Phe Asp Ser Tyr His Pro Phe Ser Asp Arg Ala Asp Lys Ile Leu Ala
85 90 95
Lys Tyr Ala Ile Gly Lys Leu Val Gly Gly Ser Glu Phe Pro Thr Tyr
100 105 110
Lys Pro Asp Thr Gly Phe Tyr Lys Glu Cys Cys Asp Arg Val Asn Gln
115 120 125
Tyr Phe Lys Asp Asn Lys Leu Asp Pro Arg Ser Pro Tyr Ser Gly Leu
130 135 140
Trp Arg Met Ile Leu Val Ala Ile Val Gly Ala Val Ala Tyr Met Gly
145 150 155 160
Met Asn Gln Leu Leu Pro Gly Asn Ile Tyr Ala His Tyr Ala Trp Gly
165 170 175
Ala Leu Phe Gly Val Cys Gln Ala Leu Pro Leu Leu His Val Met His
180 185 190
Asp Ala Ser His Ala Ala Ile Thr Ser Ser Pro Thr Gly Trp Arg Leu
195 200 205
Ile Gly Arg Leu Ala Met Asp Trp Val Ala Gly Ala Asn Met Val Ser
210 215 220
Trp Leu Asn Gln His Val Val Gly His His Ile Tyr Thr Asn Val Ala
225 230 235 240
Gly Ala Asp Pro Asp Leu Pro Val Asp Phe Lys Ser Asp Val Arg Arg
245 250 255
Ile Val Tyr Arg Gln Val Leu Leu Pro Ile Tyr Lys Tyr Gln His Leu
260 265 270
Tyr Leu Pro Pro Leu Tyr Gly Val Leu Gly Leu Lys Phe Arg Val Gln
275 280 285
Asp Val Phe Glu Thr Phe Val Thr Leu Thr Asn Gly Pro Leu Arg Val
290 295 300
Asn Pro Leu Ser Val Gly Asp Trp Ala Glu Met Ile Leu Ser Lys Ala
305 310 315 320
Phe Trp Val Phe Tyr Arg Ile Tyr Leu Pro Leu Ala Val Leu Gln Val
325 330 335
Asp Pro Ala Arg Phe Trp Gly Val Phe Phe Leu Ala Glu Phe Ser Thr
340 345 350
Gly Trp Tyr Leu Ala Phe Asn Phe Gln Val Ser His Val Ser Thr Ala
355 360 365
Cys Glu Tyr Pro Gly Gly Asp Glu Glu Val Thr Ser Ile Asp Asp Glu
370 375 380
Trp Ala Ile Ser Gln Val Lys Ser Ser Val Asp Tyr Gly His Gly Ser
385 390 395 400
Phe Ile Thr Thr Phe Leu Thr Gly Ala Leu Asn Tyr Gln Val Thr His
405 410 415
His Leu Phe Pro Gly Val Ser Gln Tyr His Tyr Pro Ala Ile Ala Pro
420 425 430
Leu Ile Leu Asp Val Cys His Lys Tyr Lys Val Lys Tyr Asn Val Leu
435 440 445
Pro Asp Phe Thr Ala Ala Met Ala Gly His Phe Asp His Leu Val Ile
450 455 460
Met Gly Lys Met Gly Lys Arg Val Thr Ile His Met Gly
465 470 475
<210> SEQ ID NO 14
<211> LENGTH: 469
<212> TYPE: PRT
<213> ORGANISM: Phaeodactylum tricornutum
<220> FEATURE:
<221> NAME/KEY: MISC_FEATURE
<223> OTHER INFORMATION: delta-5 desaturase
<300> PUBLICATION INFORMATION:
<308> DATABASE ACCESSION NUMBER: GenBank Accession No. AAL92562
<400> SEQUENCE: 14
Met Ala Pro Asp Ala Asp Lys Leu Arg Gln Arg Gln Thr Thr Ala Val
1 5 10 15
Ala Lys His Asn Ala Ala Thr Ile Ser Thr Gln Glu Arg Leu Cys Ser
20 25 30
Leu Ser Ser Leu Lys Gly Glu Glu Val Cys Ile Asp Gly Ile Ile Tyr
35 40 45
Asp Leu Gln Ser Phe Asp His Pro Gly Gly Glu Thr Ile Lys Met Phe
50 55 60
Gly Gly Asn Asp Val Thr Val Gln Tyr Lys Met Ile His Pro Tyr His
65 70 75 80
Thr Glu Lys His Leu Glu Lys Met Lys Arg Val Gly Lys Val Thr Asp
85 90 95
Phe Val Cys Glu Tyr Lys Phe Asp Thr Glu Phe Glu Arg Glu Ile Lys
100 105 110
Arg Glu Val Phe Lys Ile Val Arg Arg Gly Lys Asp Phe Gly Thr Leu
115 120 125
Gly Trp Phe Phe Arg Ala Phe Cys Tyr Ile Ala Ile Phe Phe Tyr Leu
130 135 140
Gln Tyr His Trp Val Thr Thr Gly Thr Ser Trp Leu Leu Ala Val Ala
145 150 155 160
Tyr Gly Ile Ser Gln Ala Met Ile Gly Met Asn Val Gln His Asp Ala
165 170 175
Asn His Gly Ala Thr Ser Lys Arg Pro Trp Val Asn Asp Met Leu Gly
180 185 190
Leu Gly Ala Asp Phe Ile Gly Gly Ser Lys Trp Leu Trp Gln Glu Gln
195 200 205
His Trp Thr His His Ala Tyr Thr Asn His Ala Glu Met Asp Pro Asp
210 215 220
Ser Phe Gly Ala Glu Pro Met Leu Leu Phe Asn Asp Tyr Pro Leu Asp
225 230 235 240
His Pro Ala Arg Thr Trp Leu His Arg Phe Gln Ala Phe Phe Tyr Met
245 250 255
Pro Val Leu Ala Gly Tyr Trp Leu Ser Ala Val Phe Asn Pro Gln Ile
260 265 270
Leu Asp Leu Gln Gln Arg Gly Ala Leu Ser Val Gly Ile Arg Leu Asp
275 280 285
Asn Ala Phe Ile His Ser Arg Arg Lys Tyr Ala Val Phe Trp Arg Ala
290 295 300
Val Tyr Ile Ala Val Asn Val Ile Ala Pro Phe Tyr Thr Asn Ser Gly
305 310 315 320
Leu Glu Trp Ser Trp Arg Val Phe Gly Asn Ile Met Leu Met Gly Val
325 330 335
Ala Glu Ser Leu Ala Leu Ala Val Leu Phe Ser Leu Ser His Asn Phe
340 345 350
Glu Ser Ala Asp Arg Asp Pro Thr Ala Pro Leu Lys Lys Thr Gly Glu
355 360 365
Pro Val Asp Trp Phe Lys Thr Gln Val Glu Thr Ser Cys Thr Tyr Gly
370 375 380
Gly Phe Leu Ser Gly Cys Phe Thr Gly Gly Leu Asn Phe Gln Val Glu
385 390 395 400
His His Leu Phe Pro Arg Met Ser Ser Ala Trp Tyr Pro Tyr Ile Ala
405 410 415
Pro Lys Val Arg Glu Ile Cys Ala Lys His Gly Val His Tyr Ala Tyr
420 425 430
Tyr Pro Trp Ile His Gln Asn Phe Leu Ser Thr Val Arg Tyr Met His
435 440 445
Ala Ala Gly Thr Gly Ala Asn Trp Arg Gln Met Ala Arg Glu Asn Pro
450 455 460
Leu Thr Gly Arg Ala
465
<210> SEQ ID NO 15
<211> LENGTH: 467
<212> TYPE: PRT
<213> ORGANISM: Dictyostelium discoideum
<220> FEATURE:
<221> NAME/KEY: MISC_FEATURE
<223> OTHER INFORMATION: delta-5 desaturase
<300> PUBLICATION INFORMATION:
<308> DATABASE ACCESSION NUMBER: GenBank Accession No. XP_640331
<400> SEQUENCE: 15
Met Met Glu Thr Asn Asn Glu Asn Lys Glu Lys Leu Lys Leu Tyr Thr
1 5 10 15
Trp Asp Glu Val Ser Lys His Asn Gln Lys Asn Asp Leu Trp Ile Ile
20 25 30
Val Asp Gly Lys Val Tyr Asn Ile Thr Lys Trp Val Pro Leu His Pro
35 40 45
Gly Gly Glu Asp Ile Leu Leu Leu Ser Ala Gly Arg Asp Ala Thr Asn
50 55 60
Leu Phe Glu Ser Tyr His Pro Met Thr Asp Lys His Tyr Ser Leu Ile
65 70 75 80
Lys Gln Tyr Glu Ile Gly Tyr Ile Ser Ser Tyr Glu His Pro Lys Tyr
85 90 95
Val Glu Lys Ser Glu Phe Tyr Ser Thr Leu Lys Gln Arg Val Arg Lys
100 105 110
His Phe Gln Thr Ser Ser Gln Asp Pro Lys Val Ser Val Gly Val Phe
115 120 125
Thr Arg Met Val Leu Ile Tyr Leu Phe Leu Phe Val Thr Tyr Tyr Leu
130 135 140
Ser Gln Phe Ser Thr Asp Arg Phe Trp Leu Asn Cys Ile Phe Ala Val
145 150 155 160
Leu Tyr Gly Val Ala Asn Ser Leu Phe Gly Leu His Thr Met His Asp
165 170 175
Ala Cys His Thr Ala Ile Thr His Asn Pro Met Thr Trp Lys Ile Leu
180 185 190
Gly Ala Thr Phe Asp Leu Phe Ala Gly Ala Ser Phe Tyr Ala Trp Cys
195 200 205
His Gln His Val Ile Gly His His Leu Tyr Thr Asn Val Arg Asn Ala
210 215 220
Asp Pro Asp Leu Gly Gln Gly Glu Ile Asp Phe Arg Val Val Thr Pro
225 230 235 240
Tyr Gln Ala Arg Ser Trp Tyr His Lys Tyr Gln His Ile Tyr Ala Pro
245 250 255
Ile Leu Tyr Gly Val Tyr Ala Leu Lys Tyr Arg Ile Gln Asp His Glu
260 265 270
Ile Phe Thr Lys Lys Ser Asn Gly Ala Ile Arg Tyr Ser Pro Ile Ser
275 280 285
Thr Ile Asp Thr Ala Ile Phe Ile Leu Gly Lys Leu Val Phe Ile Ile
290 295 300
Ser Arg Phe Ile Leu Pro Leu Ile Tyr Asn His Ser Phe Ser His Leu
305 310 315 320
Ile Cys Phe Phe Leu Ile Ser Glu Leu Val Leu Gly Trp Tyr Leu Ala
325 330 335
Ile Ser Phe Gln Val Ser His Val Val Glu Asp Leu Gln Phe Met Ala
340 345 350
Thr Pro Glu Ile Phe Asp Gly Ala Asp His Pro Leu Pro Thr Thr Phe
355 360 365
Asn Gln Asp Trp Ala Ile Leu Gln Val Lys Thr Thr Gln Asp Tyr Ala
370 375 380
Gln Asp Ser Val Leu Ser Thr Phe Phe Ser Gly Gly Leu Asn Leu Gln
385 390 395 400
Val Ile His His Cys Phe Pro Thr Ile Ala Gln Asp Tyr Tyr Pro Gln
405 410 415
Ile Val Pro Ile Leu Lys Glu Val Cys Lys Glu Tyr Asn Val Thr Tyr
420 425 430
His Tyr Lys Pro Thr Phe Thr Glu Ala Ile Lys Ser His Ile Asn Tyr
435 440 445
Leu Tyr Lys Met Gly Asn Asp Pro Asp Tyr Val Arg Lys Pro Val Asn
450 455 460
Lys Asn Asp
465
<210> SEQ ID NO 16
<211> LENGTH: 421
<212> TYPE: PRT
<213> ORGANISM: Euglena gracilis
<220> FEATURE:
<221> NAME/KEY: MISC_FEATURE
<223> OTHER INFORMATION: delta-8 desaturase
<300> PUBLICATION INFORMATION:
<302> TITLE: DELTA-8 DESATURASE AND ITS USE IN MAKING
POLYUNSATURATED
FATTY ACIDS
<310> PATENT DOCUMENT NUMBER: WO2006012325
<311> PATENT FILING DATE: 2005-06-24
<312> PUBLICATION DATE: 2006-02-02
<313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(421)
<300> PUBLICATION INFORMATION:
<302> TITLE: DELTA-8 DESATURASE AND ITS USE IN MAKING
POLYUNSATURATED
FATTY ACIDS
<310> PATENT DOCUMENT NUMBER: WO2006012326
<311> PATENT FILING DATE: 2005-06-24
<312> PUBLICATION DATE: 2006-02-02
<313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(421)
<400> SEQUENCE: 16
Met Lys Ser Lys Arg Gln Ala Leu Pro Leu Thr Ile Asp Gly Thr Thr
1 5 10 15
Tyr Asp Val Ser Ala Trp Val Asn Phe His Pro Gly Gly Ala Glu Ile
20 25 30
Ile Glu Asn Tyr Gln Gly Arg Asp Ala Thr Asp Ala Phe Met Val Met
35 40 45
His Ser Gln Glu Ala Phe Asp Lys Leu Lys Arg Met Pro Lys Ile Asn
50 55 60
Pro Ser Ser Glu Leu Pro Pro Gln Ala Ala Val Asn Glu Ala Gln Glu
65 70 75 80
Asp Phe Arg Lys Leu Arg Glu Glu Leu Ile Ala Thr Gly Met Phe Asp
85 90 95
Ala Ser Pro Leu Trp Tyr Ser Tyr Lys Ile Ser Thr Thr Leu Gly Leu
100 105 110
Gly Val Leu Gly Tyr Phe Leu Met Val Gln Tyr Gln Met Tyr Phe Ile
115 120 125
Gly Ala Val Leu Leu Gly Met His Tyr Gln Gln Met Gly Trp Leu Ser
130 135 140
His Asp Ile Cys His His Gln Thr Phe Lys Asn Arg Asn Trp Asn Asn
145 150 155 160
Leu Val Gly Leu Val Phe Gly Asn Gly Leu Gln Gly Phe Ser Val Thr
165 170 175
Trp Trp Lys Asp Arg His Asn Ala His His Ser Ala Thr Asn Val Gln
180 185 190
Gly His Asp Pro Asp Ile Asp Asn Leu Pro Leu Leu Ala Trp Ser Glu
195 200 205
Asp Asp Val Thr Arg Ala Ser Pro Ile Ser Arg Lys Leu Ile Gln Phe
210 215 220
Gln Gln Tyr Tyr Phe Leu Val Ile Cys Ile Leu Leu Arg Phe Ile Trp
225 230 235 240
Cys Phe Gln Ser Val Leu Thr Val Arg Ser Leu Lys Asp Arg Asp Asn
245 250 255
Gln Phe Tyr Arg Ser Gln Tyr Lys Lys Glu Ala Ile Gly Leu Ala Leu
260 265 270
His Trp Thr Leu Lys Thr Leu Phe His Leu Phe Phe Met Pro Ser Ile
275 280 285
Leu Thr Ser Leu Leu Val Phe Phe Val Ser Glu Leu Val Gly Gly Phe
290 295 300
Gly Ile Ala Ile Val Val Phe Met Asn His Tyr Pro Leu Glu Lys Ile
305 310 315 320
Gly Asp Ser Val Trp Asp Gly His Gly Phe Ser Val Gly Gln Ile His
325 330 335
Glu Thr Met Asn Ile Arg Arg Gly Ile Ile Thr Asp Trp Phe Phe Gly
340 345 350
Gly Leu Asn Tyr Gln Ile Glu His His Leu Trp Pro Thr Leu Pro Arg
355 360 365
His Asn Leu Thr Ala Val Ser Tyr Gln Val Glu Gln Leu Cys Gln Lys
370 375 380
His Asn Leu Pro Tyr Arg Asn Pro Leu Pro His Glu Gly Leu Val Ile
385 390 395 400
Leu Leu Arg Tyr Leu Ala Val Phe Ala Arg Met Ala Glu Lys Gln Pro
405 410 415
Ala Gly Lys Ala Leu
420
<210> SEQ ID NO 17
<211> LENGTH: 1269
<212> TYPE: DNA
<213> ORGANISM: Pavlova lutheri CCMP459
<220> FEATURE:
<221> NAME/KEY: misc_feature
<223> OTHER INFORMATION: delta-8 desaturase
<400> SEQUENCE: 17
atgggcaagg gtggagacgg cggcgcgcag gcggtgagcg ggaccgacgc gtctctcgct 60
gaggtgagct ccgtcgatag caagagcgtg cacgtcgtgc tctacggcaa gcgcgtggat 120
gtcacaaagt tccagaaggc acacccgggc gggagcaagg tgttccgcat cttccaggag 180
cgcgacgcga cggagcagtt cgagtcttac cactcgccca aggccatcaa gatgatggag 240
ggcatgctca agaagtcgga ggatgcgccc gcttccgtgc ccctgccctc gcggtccacc 300
atgggcacgg agttcaagga gatgattgag cgccacaaga gggctggtct ctacgaccct 360
tgcccgttgg acgagctgtt caagctcacc atcgtccttg cgcccatctt cgtgggcgcc 420
tatctcgtgc ggagcggcgt ctcgcccctc gcgggcgcgc tctccatggg ctttggcttc 480
tacctcgacg gctggcttgc tcacgactac ctgcatcacg cagtcttcaa gggctcggtc 540
aacacgctcg tcaaggcgaa caacgccatg ggatacgccc tcggcttcct ccagggctac 600
gacgtggcct ggtggcgcgc gcgccataac acgcaccacg tgtgcaccaa cgaggatggt 660
tcggacccgg acatcaagac ggcgcccctg ctcatctacg tgcgagagaa cccgtccatt 720
gccaagcggc tcaacttctt ccagcgctgg cagcagtact actatgtgcc gaccatggcc 780
atcctcgacc tctactggcg cctggagtcc atcgcgtacg tggctgtgcg cctgcctaag 840
atgtggatgc aggccgccgc tcttgccgct cactacgcgc tcctgtgctg ggtcttcgca 900
gcgcatctca acctcatccc tctcatgatg gttgcacgcg gcttcgcgac gggcatcgtt 960
gtctttgcaa cccactatgg tgaggacatc ctcgaccgcg agcacgtcga gggcatgacg 1020
ctcgtcgagc agaccgccaa gacctcccgt aacatcacgg gcggctggct agtgaacgtg 1080
ctcacgggct tcatctccct gcagaccgag catcacctct tccccatgat gcccaccggc 1140
aacctaatga ctatccagcc cgaggtacgc gacttcttca agaagcatgg cctcgagtac 1200
cgcgagggca acctcttcca gtgcgtgcac cagaacatca aggctctcgc cttcgagcac 1260
ctcctccac 1269
<210> SEQ ID NO 18
<211> LENGTH: 423
<212> TYPE: PRT
<213> ORGANISM: Pavlova lutheri CCMP459
<400> SEQUENCE: 18
Met Gly Lys Gly Gly Asp Gly Gly Ala Gln Ala Val Ser Gly Thr Asp
1 5 10 15
Ala Ser Leu Ala Glu Val Ser Ser Val Asp Ser Lys Ser Val His Val
20 25 30
Val Leu Tyr Gly Lys Arg Val Asp Val Thr Lys Phe Gln Lys Ala His
35 40 45
Pro Gly Gly Ser Lys Val Phe Arg Ile Phe Gln Glu Arg Asp Ala Thr
50 55 60
Glu Gln Phe Glu Ser Tyr His Ser Pro Lys Ala Ile Lys Met Met Glu
65 70 75 80
Gly Met Leu Lys Lys Ser Glu Asp Ala Pro Ala Ser Val Pro Leu Pro
85 90 95
Ser Arg Ser Thr Met Gly Thr Glu Phe Lys Glu Met Ile Glu Arg His
100 105 110
Lys Arg Ala Gly Leu Tyr Asp Pro Cys Pro Leu Asp Glu Leu Phe Lys
115 120 125
Leu Thr Ile Val Leu Ala Pro Ile Phe Val Gly Ala Tyr Leu Val Arg
130 135 140
Ser Gly Val Ser Pro Leu Ala Gly Ala Leu Ser Met Gly Phe Gly Phe
145 150 155 160
Tyr Leu Asp Gly Trp Leu Ala His Asp Tyr Leu His His Ala Val Phe
165 170 175
Lys Gly Ser Val Asn Thr Leu Val Lys Ala Asn Asn Ala Met Gly Tyr
180 185 190
Ala Leu Gly Phe Leu Gln Gly Tyr Asp Val Ala Trp Trp Arg Ala Arg
195 200 205
His Asn Thr His His Val Cys Thr Asn Glu Asp Gly Ser Asp Pro Asp
210 215 220
Ile Lys Thr Ala Pro Leu Leu Ile Tyr Val Arg Glu Asn Pro Ser Ile
225 230 235 240
Ala Lys Arg Leu Asn Phe Phe Gln Arg Trp Gln Gln Tyr Tyr Tyr Val
245 250 255
Pro Thr Met Ala Ile Leu Asp Leu Tyr Trp Arg Leu Glu Ser Ile Ala
260 265 270
Tyr Val Ala Val Arg Leu Pro Lys Met Trp Met Gln Ala Ala Ala Leu
275 280 285
Ala Ala His Tyr Ala Leu Leu Cys Trp Val Phe Ala Ala His Leu Asn
290 295 300
Leu Ile Pro Leu Met Met Val Ala Arg Gly Phe Ala Thr Gly Ile Val
305 310 315 320
Val Phe Ala Thr His Tyr Gly Glu Asp Ile Leu Asp Arg Glu His Val
325 330 335
Glu Gly Met Thr Leu Val Glu Gln Thr Ala Lys Thr Ser Arg Asn Ile
340 345 350
Thr Gly Gly Trp Leu Val Asn Val Leu Thr Gly Phe Ile Ser Leu Gln
355 360 365
Thr Glu His His Leu Phe Pro Met Met Pro Thr Gly Asn Leu Met Thr
370 375 380
Ile Gln Pro Glu Val Arg Asp Phe Phe Lys Lys His Gly Leu Glu Tyr
385 390 395 400
Arg Glu Gly Asn Leu Phe Gln Cys Val His Gln Asn Ile Lys Ala Leu
405 410 415
Ala Phe Glu His Leu Leu His
420
<210> SEQ ID NO 19
<211> LENGTH: 7
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Conserved Region 1
<220> FEATURE:
<221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (4)..(4)
<223> OTHER INFORMATION: Xaa = Ile or Val
<400> SEQUENCE: 19
Gly His His Xaa Tyr Thr Asn
1 5
<210> SEQ ID NO 20
<211> LENGTH: 7
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Conserved Region 2
<220> FEATURE:
<221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (4)..(4)
<223> OTHER INFORMATION: Xaa = Val or Ala
<220> FEATURE:
<221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (5)..(5)
<223> OTHER INFORMATION: Xaa = Ser or Asn
<400> SEQUENCE: 20
Asn Phe Gln Xaa Xaa His Val
1 5
<210> SEQ ID NO 21
<211> LENGTH: 476
<212> TYPE: PRT
<213> ORGANISM: Thalassiosira pseudonana
<220> FEATURE:
<221> NAME/KEY: MISC_FEATURE
<223> OTHER INFORMATION: delta-8 desaturase
<300> PUBLICATION INFORMATION:
<308> DATABASE ACCESSION NUMBER: GenBank Accession No. AAX14502
<400> SEQUENCE: 21
Met Pro Pro Asn Ala Asp Ile Ser Arg Ile Arg Asn Arg Ile Pro Thr
1 5 10 15
Lys Thr Gly Thr Val Ala Ser Ala Asp Asn Asn Asp Pro Ala Thr Gln
20 25 30
Ser Val Arg Thr Leu Lys Ser Leu Lys Gly Asn Glu Val Val Ile Asn
35 40 45
Gly Thr Ile Tyr Asp Ile Ala Asp Phe Val His Pro Gly Gly Glu Val
50 55 60
Val Lys Phe Phe Gly Gly Asn Asp Val Thr Ile Gln Tyr Asn Met Ile
65 70 75 80
His Pro Tyr His Thr Gly Lys His Leu Glu Lys Met Lys Ala Val Gly
85 90 95
Lys Val Val Asp Trp Gln Ser Asp Tyr Lys Phe Asp Thr Pro Phe Glu
100 105 110
Arg Glu Ile Lys Ser Glu Val Phe Lys Ile Val Arg Arg Gly Arg Glu
115 120 125
Phe Gly Thr Thr Gly Tyr Phe Leu Arg Ala Phe Phe Tyr Ile Ala Leu
130 135 140
Phe Phe Thr Met Gln Tyr Thr Phe Ala Thr Cys Thr Thr Phe Thr Thr
145 150 155 160
Tyr Asp His Trp Tyr Gln Ser Gly Val Phe Ile Ala Ile Val Phe Gly
165 170 175
Ile Ser Gln Ala Phe Ile Gly Leu Asn Val Gln His Asp Ala Asn His
180 185 190
Gly Ala Ala Ser Lys Arg Pro Trp Val Asn Asp Leu Leu Gly Phe Gly
195 200 205
Thr Asp Leu Ile Gly Ser Asn Lys Trp Asn Trp Met Ala Gln His Trp
210 215 220
Thr His His Ala Tyr Thr Asn His Ser Glu Lys Asp Pro Asp Ser Phe
225 230 235 240
Ser Ser Glu Pro Met Phe Ala Phe Asn Asp Tyr Pro Ile Gly His Pro
245 250 255
Lys Arg Lys Trp Trp His Arg Phe Gln Gly Gly Tyr Phe Leu Phe Met
260 265 270
Leu Gly Leu Tyr Trp Leu Pro Thr Val Phe Asn Pro Gln Phe Ile Asp
275 280 285
Leu Arg Gln Arg Gly Ala Gln Tyr Val Gly Ile Gln Met Glu Asn Asp
290 295 300
Phe Ile Val Lys Arg Arg Lys Tyr Ala Val Ala Leu Arg Met Met Tyr
305 310 315 320
Ile Tyr Leu Asn Ile Val Ser Pro Phe Met Asn Asn Gly Leu Ser Trp
325 330 335
Ser Thr Phe Gly Ile Ile Met Leu Met Gly Ile Ser Glu Ser Leu Thr
340 345 350
Leu Ser Val Leu Phe Ser Leu Ser His Asn Phe Ile Asn Ser Asp Arg
355 360 365
Asp Pro Thr Ala Asp Phe Lys Lys Thr Gly Glu Gln Val Cys Trp Phe
370 375 380
Lys Ser Gln Val Glu Thr Ser Ser Thr Tyr Gly Gly Phe Ile Ser Gly
385 390 395 400
Cys Leu Thr Gly Gly Leu Asn Phe Gln Val Glu His His Leu Phe Pro
405 410 415
Arg Met Ser Ser Ala Trp Tyr Pro Tyr Ile Ala Pro Thr Val Arg Glu
420 425 430
Val Cys Lys Lys His Gly Met Ser Tyr Ala Tyr Tyr Pro Trp Ile Gly
435 440 445
Gln Asn Leu Val Ser Thr Phe Lys Tyr Met His Arg Ala Gly Ser Gly
450 455 460
Ala Asn Trp Glu Leu Lys Pro Leu Ser Gly Ser Ala
465 470 475
<210> SEQ ID NO 22
<211> LENGTH: 8165
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Plasmid pZUF17
<400> SEQUENCE: 22
gtacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 60
ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 120
taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 180
tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 240
aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 300
aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 360
ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 420
acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 480
ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 540
tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 600
tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 660
gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 720
agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 780
tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 840
agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 900
tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 960
acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 1020
tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 1080
agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 1140
tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 1200
acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 1260
tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 1320
ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 1380
agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 1440
tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 1500
acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 1560
agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 1620
actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 1680
tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 1740
gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 1800
ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 1860
tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 1920
aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 1980
tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 2040
tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 2100
gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 2160
gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 2220
acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 2280
agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 2340
ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 2400
ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 2460
taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 2520
aacgcgaatt ttaacaaaat attaacgctt acaatttcca ttcgccattc aggctgcgca 2580
actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 2640
gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta 2700
aaacgacggc cagtgaattg taatacgact cactataggg cgaattgggt accgggcccc 2760
ccctcgaggt cgatggtgtc gataagcttg atatcgaatt catgtcacac aaaccgatct 2820
tcgcctcaag gaaacctaat tctacatccg agagactgcc gagatccagt ctacactgat 2880
taattttcgg gccaataatt taaaaaaatc gtgttatata atattatatg tattatatat 2940
atacatcatg atgatactga cagtcatgtc ccattgctaa atagacagac tccatctgcc 3000
gcctccaact gatgttctca atatttaagg ggtcatctcg cattgtttaa taataaacag 3060
actccatcta ccgcctccaa atgatgttct caaaatatat tgtatgaact tatttttatt 3120
acttagtatt attagacaac ttacttgctt tatgaaaaac acttcctatt taggaaacaa 3180
tttataatgg cagttcgttc atttaacaat ttatgtagaa taaatgttat aaatgcgtat 3240
gggaaatctt aaatatggat agcataaatg atatctgcat tgcctaattc gaaatcaaca 3300
gcaacgaaaa aaatcccttg tacaacataa atagtcatcg agaaatatca actatcaaag 3360
aacagctatt cacacgttac tattgagatt attattggac gagaatcaca cactcaactg 3420
tctttctctc ttctagaaat acaggtacaa gtatgtacta ttctcattgt tcatacttct 3480
agtcatttca tcccacatat tccttggatt tctctccaat gaatgacatt ctatcttgca 3540
aattcaacaa ttataataag atataccaaa gtagcggtat agtggcaatc aaaaagcttc 3600
tctggtgtgc ttctcgtatt tatttttatt ctaatgatcc attaaaggta tatatttatt 3660
tcttgttata taatcctttt gtttattaca tgggctggat acataaaggt attttgattt 3720
aattttttgc ttaaattcaa tcccccctcg ttcagtgtca actgtaatgg taggaaatta 3780
ccatactttt gaagaagcaa aaaaaatgaa agaaaaaaaa aatcgtattt ccaggttaga 3840
cgttccgcag aatctagaat gcggtatgcg gtacattgtt cttcgaacgt aaaagttgcg 3900
ctccctgaga tattgtacat ttttgctttt acaagtacaa gtacatcgta caactatgta 3960
ctactgttga tgcatccaca acagtttgtt ttgttttttt ttgttttttt tttttctaat 4020
gattcattac cgctatgtat acctacttgt acttgtagta agccgggtta ttggcgttca 4080
attaatcata gacttatgaa tctgcacggt gtgcgctgcg agttactttt agcttatgca 4140
tgctacttgg gtgtaatatt gggatctgtt cggaaatcaa cggatgctca atcgatttcg 4200
acagtaatta attaagtcat acacaagtca gctttcttcg agcctcatat aagtataagt 4260
agttcaacgt attagcactg tacccagcat ctccgtatcg agaaacacaa caacatgccc 4320
cattggacag atcatgcgga tacacaggtt gtgcagtatc atacatactc gatcagacag 4380
gtcgtctgac catcatacaa gctgaacaag cgctccatac ttgcacgctc tctatataca 4440
cagttaaatt acatatccat agtctaacct ctaacagtta atcttctggt aagcctccca 4500
gccagccttc tggtatcgct tggcctcctc aataggatct cggttctggc cgtacagacc 4560
tcggccgaca attatgatat ccgttccggt agacatgaca tcctcaacag ttcggtactg 4620
ctgtccgaga gcgtctccct tgtcgtcaag acccaccccg ggggtcagaa taagccagtc 4680
ctcagagtcg cccttaggtc ggttctgggc aatgaagcca accacaaact cggggtcgga 4740
tcgggcaagc tcaatggtct gcttggagta ctcgccagtg gccagagagc ccttgcaaga 4800
cagctcggcc agcatgagca gacctctggc cagcttctcg ttgggagagg ggactaggaa 4860
ctccttgtac tgggagttct cgtagtcaga gacgtcctcc ttcttctgtt cagagacagt 4920
ttcctcggca ccagctcgca ggccagcaat gattccggtt ccgggtacac cgtgggcgtt 4980
ggtgatatcg gaccactcgg cgattcggtg acaccggtac tggtgcttga cagtgttgcc 5040
aatatctgcg aactttctgt cctcgaacag gaagaaaccg tgcttaagag caagttcctt 5100
gagggggagc acagtgccgg cgtaggtgaa gtcgtcaatg atgtcgatat gggttttgat 5160
catgcacaca taaggtccga ccttatcggc aagctcaatg agctccttgg tggtggtaac 5220
atccagagaa gcacacaggt tggttttctt ggctgccacg agcttgagca ctcgagcggc 5280
aaaggcggac ttgtggacgt tagctcgagc ttcgtaggag ggcattttgg tggtgaagag 5340
gagactgaaa taaatttagt ctgcagaact ttttatcgga accttatctg gggcagtgaa 5400
gtatatgtta tggtaatagt tacgagttag ttgaacttat agatagactg gactatacgg 5460
ctatcggtcc aaattagaaa gaacgtcaat ggctctctgg gcgtcgcctt tgccgacaaa 5520
aatgtgatca tgatgaaagc cagcaatgac gttgcagctg atattgttgt cggccaaccg 5580
cgccgaaaac gcagctgtca gacccacagc ctccaacgaa gaatgtatcg tcaaagtgat 5640
ccaagcacac tcatagttgg agtcgtactc caaaggcggc aatgacgagt cagacagata 5700
ctcgtcgact caggcgacga cggaattcct gcagcccatc tgcagaattc aggagagacc 5760
gggttggcgg cgtatttgtg tcccaaaaaa cagccccaat tgccccggag aagacggcca 5820
ggccgcctag atgacaaatt caacaactca cagctgactt tctgccattg ccactagggg 5880
ggggcctttt tatatggcca agccaagctc tccacgtcgg ttgggctgca cccaacaata 5940
aatgggtagg gttgcaccaa caaagggatg ggatgggggg tagaagatac gaggataacg 6000
gggctcaatg gcacaaataa gaacgaatac tgccattaag actcgtgatc cagcgactga 6060
caccattgca tcatctaagg gcctcaaaac tacctcggaa ctgctgcgct gatctggaca 6120
ccacagaggt tccgagcact ttaggttgca ccaaatgtcc caccaggtgc aggcagaaaa 6180
cgctggaaca gcgtgtacag tttgtcttaa caaaaagtga gggcgctgag gtcgagcagg 6240
gtggtgtgac ttgttatagc ctttagagct gcgaaagcgc gtatggattt ggctcatcag 6300
gccagattga gggtctgtgg acacatgtca tgttagtgta cttcaatcgc cccctggata 6360
tagccccgac aataggccgt ggcctcattt ttttgccttc cgcacatttc cattgctcgg 6420
tacccacacc ttgcttctcc tgcacttgcc aaccttaata ctggtttaca ttgaccaaca 6480
tcttacaagc ggggggcttg tctagggtat atataaacag tggctctccc aatcggttgc 6540
cagtctcttt tttcctttct ttccccacag attcgaaatc taaactacac atcacacaat 6600
gcctgttact gacgtcctta agcgaaagtc cggtgtcatc gtcggcgacg atgtccgagc 6660
cgtgagtatc cacgacaaga tcagtgtcga gacgacgcgt tttgtgtaat gacacaatcc 6720
gaaagtcgct agcaacacac actctctaca caaactaacc cagctctcca tggctgagga 6780
taagaccaag gtcgagttcc ctaccctgac tgagctgaag cactctatcc ctaacgcttg 6840
ctttgagtcc aacctcggac tctcgctcta ctacactgcc cgagcgatct tcaacgcatc 6900
tgcctctgct gctctgctct acgctgcccg atctactccc ttcattgccg ataacgttct 6960
gctccacgct ctggtttgcg ccacctacat ctacgtgcag ggtgtcatct tctggggttt 7020
ctttaccgtc ggtcacgact gtggtcactc tgccttctcc cgataccact ccgtcaactt 7080
catcattggc tgcatcatgc actctgccat tctgactccc ttcgagtcct ggcgagtgac 7140
ccaccgacac catcacaaga acactggcaa cattgataag gacgagatct tctaccctca 7200
tcggtccgtc aaggacctcc aggacgtgcg acaatgggtc tacaccctcg gaggtgcttg 7260
gtttgtctac ctgaaggtcg gatatgctcc tcgaaccatg tcccactttg acccctggga 7320
ccctctcctg cttcgacgag cctccgctgt catcgtgtcc ctcggagtct gggctgcctt 7380
cttcgctgcc tacgcctacc tcacatactc gctcggcttt gccgtcatgg gcctctacta 7440
ctatgctcct ctctttgtct ttgcttcgtt cctcgtcatt actaccttct tgcatcacaa 7500
cgacgaagct actccctggt acggtgactc ggagtggacc tacgtcaagg gcaacctgag 7560
ctccgtcgac cgatcgtacg gagctttcgt ggacaacctg tctcaccaca ttggcaccca 7620
ccaggtccat cacttgttcc ctatcattcc ccactacaag ctcaacgaag ccaccaagca 7680
ctttgctgcc gcttaccctc acctcgtgag acgtaacgac gagcccatca ttactgcctt 7740
cttcaagacc gctcacctct ttgtcaacta cggagctgtg cccgagactg ctcagatttt 7800
caccctcaaa gagtctgccg ctgcagccaa ggccaagagc gactaagcgg ccgcaagtgt 7860
ggatggggaa gtgagtgccc ggttctgtgt gcacaattgg caatccaaga tggatggatt 7920
caacacaggg atatagcgag ctacgtggtg gtgcgaggat atagcaacgg atatttatgt 7980
ttgacacttg agaatgtacg atacaagcac tgtccaagta caatactaaa catactgtac 8040
atactcatac tcgtacccgg gcaacggttt cacttgagtg cagtggctag tgctcttact 8100
cgtacagtgt gcaatactgc gtatcatagt ctttgatgta tatcgtattc attcatgtta 8160
gttgc 8165
<210> SEQ ID NO 23
<211> LENGTH: 8480
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Plasmid pDMW368
<400> SEQUENCE: 23
ggccgcaagt gtggatgggg aagtgagtgc ccggttctgt gtgcacaatt ggcaatccaa 60
gatggatgga ttcaacacag ggatatagcg agctacgtgg tggtgcgagg atatagcaac 120
ggatatttat gtttgacact tgagaatgta cgatacaagc actgtccaag tacaatacta 180
aacatactgt acatactcat actcgtaccc gggcaacggt ttcacttgag tgcagtggct 240
agtgctctta ctcgtacagt gtgcaatact gcgtatcata gtctttgatg tatatcgtat 300
tcattcatgt tagttgcgta cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 360
gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 420
tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 480
ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 540
cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 600
gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 660
tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 720
agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 780
tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 840
cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 900
ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 960
ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 1020
ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 1080
ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 1140
cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 1200
gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 1260
atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 1320
ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 1380
gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 1440
tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 1500
ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 1560
taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 1620
gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 1680
gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 1740
ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 1800
aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 1860
gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 1920
cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 1980
actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 2040
caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 2100
gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 2160
ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 2220
caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 2280
tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 2340
gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 2400
cccgaaaagt gccacctgac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg 2460
ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct 2520
tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc 2580
ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg 2640
atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt 2700
ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg 2760
tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc 2820
tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttccattc 2880
gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg 2940
ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc 3000
ccagtcacga cgttgtaaaa cgacggccag tgaattgtaa tacgactcac tatagggcga 3060
attgggtacc gggccccccc tcgaggtcga tggtgtcgat aagcttgata tcgaattcat 3120
gtcacacaaa ccgatcttcg cctcaaggaa acctaattct acatccgaga gactgccgag 3180
atccagtcta cactgattaa ttttcgggcc aataatttaa aaaaatcgtg ttatataata 3240
ttatatgtat tatatatata catcatgatg atactgacag tcatgtccca ttgctaaata 3300
gacagactcc atctgccgcc tccaactgat gttctcaata tttaaggggt catctcgcat 3360
tgtttaataa taaacagact ccatctaccg cctccaaatg atgttctcaa aatatattgt 3420
atgaacttat ttttattact tagtattatt agacaactta cttgctttat gaaaaacact 3480
tcctatttag gaaacaattt ataatggcag ttcgttcatt taacaattta tgtagaataa 3540
atgttataaa tgcgtatggg aaatcttaaa tatggatagc ataaatgata tctgcattgc 3600
ctaattcgaa atcaacagca acgaaaaaaa tcccttgtac aacataaata gtcatcgaga 3660
aatatcaact atcaaagaac agctattcac acgttactat tgagattatt attggacgag 3720
aatcacacac tcaactgtct ttctctcttc tagaaataca ggtacaagta tgtactattc 3780
tcattgttca tacttctagt catttcatcc cacatattcc ttggatttct ctccaatgaa 3840
tgacattcta tcttgcaaat tcaacaatta taataagata taccaaagta gcggtatagt 3900
ggcaatcaaa aagcttctct ggtgtgcttc tcgtatttat ttttattcta atgatccatt 3960
aaaggtatat atttatttct tgttatataa tccttttgtt tattacatgg gctggataca 4020
taaaggtatt ttgatttaat tttttgctta aattcaatcc cccctcgttc agtgtcaact 4080
gtaatggtag gaaattacca tacttttgaa gaagcaaaaa aaatgaaaga aaaaaaaaat 4140
cgtatttcca ggttagacgt tccgcagaat ctagaatgcg gtatgcggta cattgttctt 4200
cgaacgtaaa agttgcgctc cctgagatat tgtacatttt tgcttttaca agtacaagta 4260
catcgtacaa ctatgtacta ctgttgatgc atccacaaca gtttgttttg tttttttttg 4320
tttttttttt ttctaatgat tcattaccgc tatgtatacc tacttgtact tgtagtaagc 4380
cgggttattg gcgttcaatt aatcatagac ttatgaatct gcacggtgtg cgctgcgagt 4440
tacttttagc ttatgcatgc tacttgggtg taatattggg atctgttcgg aaatcaacgg 4500
atgctcaatc gatttcgaca gtaattaatt aagtcataca caagtcagct ttcttcgagc 4560
ctcatataag tataagtagt tcaacgtatt agcactgtac ccagcatctc cgtatcgaga 4620
aacacaacaa catgccccat tggacagatc atgcggatac acaggttgtg cagtatcata 4680
catactcgat cagacaggtc gtctgaccat catacaagct gaacaagcgc tccatacttg 4740
cacgctctct atatacacag ttaaattaca tatccatagt ctaacctcta acagttaatc 4800
ttctggtaag cctcccagcc agccttctgg tatcgcttgg cctcctcaat aggatctcgg 4860
ttctggccgt acagacctcg gccgacaatt atgatatccg ttccggtaga catgacatcc 4920
tcaacagttc ggtactgctg tccgagagcg tctcccttgt cgtcaagacc caccccgggg 4980
gtcagaataa gccagtcctc agagtcgccc ttaggtcggt tctgggcaat gaagccaacc 5040
acaaactcgg ggtcggatcg ggcaagctca atggtctgct tggagtactc gccagtggcc 5100
agagagccct tgcaagacag ctcggccagc atgagcagac ctctggccag cttctcgttg 5160
ggagagggga ctaggaactc cttgtactgg gagttctcgt agtcagagac gtcctccttc 5220
ttctgttcag agacagtttc ctcggcacca gctcgcaggc cagcaatgat tccggttccg 5280
ggtacaccgt gggcgttggt gatatcggac cactcggcga ttcggtgaca ccggtactgg 5340
tgcttgacag tgttgccaat atctgcgaac tttctgtcct cgaacaggaa gaaaccgtgc 5400
ttaagagcaa gttccttgag ggggagcaca gtgccggcgt aggtgaagtc gtcaatgatg 5460
tcgatatggg ttttgatcat gcacacataa ggtccgacct tatcggcaag ctcaatgagc 5520
tccttggtgg tggtaacatc cagagaagca cacaggttgg ttttcttggc tgccacgagc 5580
ttgagcactc gagcggcaaa ggcggacttg tggacgttag ctcgagcttc gtaggagggc 5640
attttggtgg tgaagaggag actgaaataa atttagtctg cagaactttt tatcggaacc 5700
ttatctgggg cagtgaagta tatgttatgg taatagttac gagttagttg aacttataga 5760
tagactggac tatacggcta tcggtccaaa ttagaaagaa cgtcaatggc tctctgggcg 5820
tcgcctttgc cgacaaaaat gtgatcatga tgaaagccag caatgacgtt gcagctgata 5880
ttgttgtcgg ccaaccgcgc cgaaaacgca gctgtcagac ccacagcctc caacgaagaa 5940
tgtatcgtca aagtgatcca agcacactca tagttggagt cgtactccaa aggcggcaat 6000
gacgagtcag acagatactc gtcgactcag gcgacgacgg aattcctgca gcccatctgc 6060
agaattcagg agagaccggg ttggcggcgt atttgtgtcc caaaaaacag ccccaattgc 6120
cccggagaag acggccaggc cgcctagatg acaaattcaa caactcacag ctgactttct 6180
gccattgcca ctaggggggg gcctttttat atggccaagc caagctctcc acgtcggttg 6240
ggctgcaccc aacaataaat gggtagggtt gcaccaacaa agggatggga tggggggtag 6300
aagatacgag gataacgggg ctcaatggca caaataagaa cgaatactgc cattaagact 6360
cgtgatccag cgactgacac cattgcatca tctaagggcc tcaaaactac ctcggaactg 6420
ctgcgctgat ctggacacca cagaggttcc gagcacttta ggttgcacca aatgtcccac 6480
caggtgcagg cagaaaacgc tggaacagcg tgtacagttt gtcttaacaa aaagtgaggg 6540
cgctgaggtc gagcagggtg gtgtgacttg ttatagcctt tagagctgcg aaagcgcgta 6600
tggatttggc tcatcaggcc agattgaggg tctgtggaca catgtcatgt tagtgtactt 6660
caatcgcccc ctggatatag ccccgacaat aggccgtggc ctcatttttt tgccttccgc 6720
acatttccat tgctcggtac ccacaccttg cttctcctgc acttgccaac cttaatactg 6780
gtttacattg accaacatct tacaagcggg gggcttgtct agggtatata taaacagtgg 6840
ctctcccaat cggttgccag tctctttttt cctttctttc cccacagatt cgaaatctaa 6900
actacacatc acacaatgcc tgttactgac gtccttaagc gaaagtccgg tgtcatcgtc 6960
ggcgacgatg tccgagccgt gagtatccac gacaagatca gtgtcgagac gacgcgtttt 7020
gtgtaatgac acaatccgaa agtcgctagc aacacacact ctctacacaa actaacccag 7080
ctctccatgg ctccagatgc ggacaagttg agacagcgca aggcgcaatc gattcaagac 7140
acggctgatt cgcaagctac cgaactcaag attggcaccc tgaagggctt gcaggggaca 7200
gaaatcgtca ttgatggaga catttacgat ataaaagact ttgatcaccc cggtggtgaa 7260
tccatcatga cttttggggg aaacgatgtc accgccacgt acaagatgat ccacccctac 7320
cactctaagc accatttgga gaagatgaag aaagtgggac gagttccgga ctacacctcg 7380
gaatacaagt ttgatactcc ctttgagcgt gaaatcaagc aagaggtctt caagattgtg 7440
cgacgaggcc gcgagtttgg aacacctgga tacttcttcc gggctttctg ctacattgga 7500
cttttctttt acttgcagta tttgtgggtc acgactccca ctacctttgc cttggcgatc 7560
ttctatggtg tttcgcaagc tttcattggt ttgaacgtac aacatgatgc caaccacgga 7620
gctgcctcca agaagccttg gatcaataac ttgctaggat tgggggctga ctttatcgga 7680
ggttccaaat ggttgtggat gaaccagcac tggacgcacc acacatacac caaccaccat 7740
gagaaggatc ccgatgcctt gggcgctgaa ccaatgttgt tgttcaatga ttatcccttg 7800
ggtcacccaa agcgtacttt gattcaccac ttccaggcct tctattacct tttcgtcttg 7860
gccggatact gggtctcttc ggtcttcaac cctcaaattt tggacttgca acaccgcggt 7920
gctcaagcgg ttggaatgaa aatggagaac gattacattg ccaaaagccg aaagtatgcc 7980
atcttcttgc gtctcttgta tatttacacc aacattgtcg ctccgatcca aaaccaaggc 8040
ttctcgttga ccgtggtcgc ccacattttg accatgggcg tcgcttccag tttgactttg 8100
gcgactcttt ttgccttgtc gcacaatttt gaaaacgcgg atcgcgatcc cacttacgag 8160
gcccgcaagg gaggagagcc tgtttgttgg ttcaagtcgc aagtcgaaac ctcgtcaact 8220
tacggaggtt tcatctcggg ttgcttgacg ggcggactca acttccaagt ggaacaccac 8280
ttgttccctc gtatgagttc ggcctggtac ccctacattg cccctactgt tcgagaggtt 8340
tgcaaaaagc acggagtcaa gtacgcatac tatccctggg tctggcaaaa cttgatttca 8400
actgtcaagt atctgcatca aagcggaact ggatccaact ggaagaatgg cgccaacccc 8460
tactcgggaa aattgtaagc 8480
<210> SEQ ID NO 24
<211> LENGTH: 12649
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Plasmid pKUNF12T6E
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (2507)..(2507)
<223> OTHER INFORMATION: n is a, c, g, or t
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (2512)..(2515)
<223> OTHER INFORMATION: n is a, c, g, or t
<400> SEQUENCE: 24
taaccctcac taaagggaac aaaagctgga gctccaccgc ggacacaata tctggtcaaa 60
tttcagtttc gttacataaa tcgttatgtc aaaggagtgt gggaggttaa gagaattatc 120
accggcaaac tatctgttaa ttgctaggta cctctagacg tccacccggg tcgcttggcg 180
gccgaagagg ccggaatctc gggccgcggt ggcggccgct tagttggtct tggacttctt 240
gggcttcttc aggtaggact ggacaaagaa gttgccgaac agagcgagca gggtgatcat 300
gtacacgccg agcagctgga ccagagcctg agggtagtcg caggggaaga ggtagtcgta 360
cagggactgc accagcatag ccatgaactg ggtcatctgc agagtggtga tgtagggctt 420
gatgggcttg acgaagccga agccctgaga ggaaaagaag tagtaggcgt acatgacggt 480
gtggacgaag gagttgagga tgacggagaa gtaggcgtcg ccaccaggag cgtacttggc 540
aatagcccac cagatggcga agatggtggc atggtggtac acgtgcagga aggagacctg 600
gttgaacttc ttgcacagga tcatgatagc ggtgtccagg aactcgtagg ccttggagac 660
gtagaacacg tagacgattc gggacatgcc ctgagcgtgg gactcgttgc ccttctccat 720
gtcgttgccg aagaccttgt agccacccag gatagcctgt cggatggtct cgacgcacat 780
gtagagggac agtccgaaga ggaacaggtt gtggagcagc ttgatggtct tcagctcgaa 840
gggcttctcc atctgcttca tgatgggaat gccgaagagc agcatggcca tgtagccgac 900
ctcgaaggcg agcatggtgg agacgtccat catgggcaga ccgtcggtca gagcgtaggg 960
cttagctccg tccatccact ggtcgacacc ggtctcgact cgtccgacca cgtcgtccca 1020
gacagaggag ttggccatgg tgaatgattc ttatactcag aaggaaatgc ttaacgattt 1080
cgggtgtgag ttgacaagga gagagagaaa agaagaggaa aggtaattcg gggacggtgg 1140
tcttttatac ccttggctaa agtcccaacc acaaagcaaa aaaattttca gtagtctatt 1200
ttgcgtccgg catgggttac ccggatggcc agacaaagaa actagtacaa agtctgaaca 1260
agcgtagatt ccagactgca gtaccctacg cccttaacgg caagtgtggg aaccggggga 1320
ggtttgatat gtggggtgaa gggggctctc gccggggttg ggcccgctac tgggtcaatt 1380
tggggtcaat tggggcaatt ggggctgttt tttgggacac aaatacgccg ccaacccggt 1440
ctctcctgaa ttctgcatcg atcgaggaag aggacaagcg gctgcttctt aagtttgtga 1500
catcagtatc caaggcacca ttgcaaggat tcaaggcttt gaacccgtca tttgccattc 1560
gtaacgctgg tagacaggtt gatcggttcc ctacggcctc cacctgtgtc aatcttctca 1620
agctgcctga ctatcaggac attgatcaac ttcggaagaa acttttgtat gccattcgat 1680
cacatgctgg tttcgatttg tcttagagga acgcatatac agtaatcata gagaataaac 1740
gatattcatt tattaaagta gatagttgag gtagaagttg taaagagtga taaatagcgg 1800
ccgcgcctac ttaagcaacg ggcttgataa cagcgggggg ggtgcccacg ttgttgcggt 1860
tgcggaagaa cagaacaccc ttaccagcac cctcggcacc agcgctgggc tcaacccact 1920
ggcacatacg cgcactgcgg tacatggcgc ggatgaagcc acgaggacca tcctggacat 1980
cagcccggta gtgcttgccc atgatgggct taatggcctc ggtggcctcg tccgcgttgt 2040
agaaggggat gctgctgacg tagtggtgga ggacatgagt ctcgatgatg ccgtggagaa 2100
ggtggcggcc gatgaagccc atctcacggt caatggtagc agcggcacca cggacgaagt 2160
tccactcgtc gttggtgtag tggggaaggg tagggtcggt gtgctggagg aaggtgatgg 2220
caacgagcca gtggttaacc cagaggtagg gaacaaagta ccagatggcc atgttgtaga 2280
aaccgaactt ctgaacgagg aagtacagag cagtggccat cagaccgata ccaatatcgc 2340
tgaggacgat gagcttagcg tcactgttct cgtacagagg gctgcgggga tcgaagtggt 2400
taacaccacc gccgaggccg ttatgcttgc ccttgccgcg accctcacgc tggcgctcgt 2460
ggtagttgtg gccggtaaca ttggtgatga ggtagttggg ccagccnacg annnnctcag 2520
taagatgagc gagctcgtgg gtcatctttc cgagacgagt agcctgctgc tcgcgggttc 2580
ggggaacgaa gaccatgtca cgctccatgt tgccagtggc cttgtggtgc tttcggtggg 2640
agatttgcca gctgaagtag gggacaagga gggaagagtg aagaacccag ccagtaatgt 2700
cgttgatgat gcgagaatcg gagaaagcac cgtgaccgca ctcatgggca ataacccaga 2760
gaccagtacc gaaaagaccc tgaagaacgg tgtacacggc ccacagacca gcgcgggcgg 2820
gggtggaggg gatatattcg ggggtcacaa agttgtacca gatgctgaaa gtggtagtca 2880
ggaggacaat gtcgcggagg atataaccgt atcccttgag agcggagcgc ttgaagcagt 2940
gcttagggat ggcattgtag atgtccttga tggtaaagtc gggaacctcg aactggttgc 3000
cgtaggtgtc gagcatgaca ccatactcgg acttgggctt ggcgatatca acctcggaca 3060
tggacgagag cgatgtggaa gaggccgagt ggcggggaga gtctgaagga gagacggcgg 3120
cagactcaga atccgtcaca gtagttgagg tgacggtgcg tctaagcgca gggttctgct 3180
tgggcagagc cgaagtggac gccatggaga gctgggttag tttgtgtaga gagtgtgtgt 3240
tgctagcgac tttcggattg tgtcattaca caaaacgcgt cgtctcgaca ctgatcttgt 3300
cgtggatact cacggctcgg acatcgtcgc cgacgatgac accggacttt cgcttaagga 3360
cgtcagtaac aggcattgtg tgatgtgtag tttagatttc gaatctgtgg ggaaagaaag 3420
gaaaaaagag actggcaacc gattgggaga gccactgttt atatataccc tagacaagcc 3480
ccccgcttgt aagatgttgg tcaatgtaaa ccagtattaa ggttggcaag tgcaggagaa 3540
gcaaggtgtg ggtaccgagc aatggaaatg tgcggaaggc aaaaaaatga ggccacggcc 3600
tattgtcggg gctatatcca gggggcgatt gaagtacact aacatgacat gtgtccacag 3660
accctcaatc tggcctgatg agccaaatcc atacgcgctt tcgcagctct aaaggctata 3720
acaagtcaca ccaccctgct cgacctcagc gccctcactt tttgttaaga caaactgtac 3780
acgctgttcc agcgttttct gcctgcacct ggtgggacat ttggtgcaac ctaaagtgct 3840
cggaacctct gtggtgtcca gatcagcgca gcagttccga ggtagttttg aggcccttag 3900
atgatgcaat ggtgtcagtc gctggatcac gagtcttaat ggcagtattc gttcttattt 3960
gtgccattga gccccgttat cctcgtatct tctacccccc atcccatccc tttgttggtg 4020
caaccctacc catttattgt tgggtgcagc ccaaccgacg tggagagctt ggcttggcca 4080
tataaaaagg ccccccccta gtggcaatgg cagaaagtca gctgtgagtt gttgaatttg 4140
tcatctaggc ggcctggccg tcttctccgg ggcaattgtt cctctatagt actgcgtaca 4200
ctgtttaaac agtgtacgca gatctgcgac gacggaattc ctgcagccca tctgcagaat 4260
tcaggagaga ccgggttggc ggcgtatttg tgtcccaaaa aacagcccca attgccccaa 4320
ttgaccccaa attgacccag tagcgggccc aaccccggcg agagccccct tcaccccaca 4380
tatcaaacct cccccggttc ccacacttgc cgttaagggc gtagggtact gcagtctgga 4440
atctacgctt gttcagactt tgtactagtt tctttgtctg gccatccggg taacccatgc 4500
cggacgcaaa atagactact gaaaattttt ttgctttgtg gttgggactt tagccaaggg 4560
tataaaagac caccgtcccc gaattacctt tcctcttctt ttctctctct ccttgtcaac 4620
tcacacccga aatcgttaag catttccttc tgagtataag aatcattcac catggctgcc 4680
gctccctctg tgcgaacctt tacccgagcc gaggttctga acgctgaggc tctgaacgag 4740
ggcaagaagg acgctgaggc tcccttcctg atgatcatcg acaacaaggt gtacgacgtc 4800
cgagagttcg tccctgacca tcctggaggc tccgtgattc tcacccacgt tggcaaggac 4860
ggcaccgacg tctttgacac ctttcatccc gaggctgctt gggagactct cgccaacttc 4920
tacgttggag acattgacga gtccgaccga gacatcaaga acgatgactt tgccgctgag 4980
gtccgaaagc tgcgaaccct gttccagtct ctcggctact acgactcctc taaggcctac 5040
tacgccttca aggtctcctt caacctctgc atctggggac tgtccaccgt cattgtggcc 5100
aagtggggtc agacctccac cctcgccaac gtgctctctg ctgccctgct cggcctgttc 5160
tggcagcagt gcggatggct ggctcacgac tttctgcacc accaggtctt ccaggaccga 5220
ttctggggtg atctcttcgg agccttcctg ggaggtgtct gccagggctt ctcctcttcc 5280
tggtggaagg acaagcacaa cactcaccat gccgctccca acgtgcatgg cgaggatcct 5340
gacattgaca cccaccctct cctgacctgg tccgagcacg ctctggagat gttctccgac 5400
gtccccgatg aggagctgac ccgaatgtgg tctcgattca tggtcctgaa ccagacctgg 5460
ttctacttcc ccattctctc cttcgctcga ctgtcttggt gcctccagtc cattctcttt 5520
gtgctgccca acggtcaggc tcacaagccc tccggagctc gagtgcccat ctccctggtc 5580
gagcagctgt ccctcgccat gcactggacc tggtacctcg ctaccatgtt cctgttcatc 5640
aaggatcctg tcaacatgct cgtgtacttc ctggtgtctc aggctgtgtg cggaaacctg 5700
ctcgccatcg tgttctccct caaccacaac ggtatgcctg tgatctccaa ggaggaggct 5760
gtcgacatgg atttctttac caagcagatc atcactggtc gagatgtcca tcctggactg 5820
ttcgccaact ggttcaccgg tggcctgaac taccagatcg agcatcacct gttcccttcc 5880
atgcctcgac acaacttctc caagatccag cctgccgtcg agaccctgtg caagaagtac 5940
aacgtccgat accacaccac tggtatgatc gagggaactg ccgaggtctt ctcccgactg 6000
aacgaggtct ccaaggccac ctccaagatg ggcaaggctc agtaagcggc cgcatgagaa 6060
gataaatata taaatacatt gagatattaa atgcgctaga ttagagagcc tcatactgct 6120
cggagagaag ccaagacgag tactcaaagg ggattacacc atccatatcc acagacacaa 6180
gctggggaaa ggttctatat acactttccg gaataccgta gtttccgatg ttatcaatgg 6240
gggcagccag gatttcaggc acttcggtgt ctcggggtga aatggcgttc ttggcctcca 6300
tcaagtcgta ccatgtcttc atttgcctgt caaagtaaaa cagaagcaga tgaagaatga 6360
acttgaagtg aaggaattta aattgccccg gagaagacgg ccaggccgcc tagatgacaa 6420
attcaacaac tcacagctga ctttctgcca ttgccactag gggggggcct ttttatatgg 6480
ccaagccaag ctctccacgt cggttgggct gcacccaaca ataaatgggt agggttgcac 6540
caacaaaggg atgggatggg gggtagaaga tacgaggata acggggctca atggcacaaa 6600
taagaacgaa tactgccatt aagactcgtg atccagcgac tgacaccatt gcatcatcta 6660
agggcctcaa aactacctcg gaactgctgc gctgatctgg acaccacaga ggttccgagc 6720
actttaggtt gcaccaaatg tcccaccagg tgcaggcaga aaacgctgga acagcgtgta 6780
cagtttgtct taacaaaaag tgagggcgct gaggtcgagc agggtggtgt gacttgttat 6840
agcctttaga gctgcgaaag cgcgtatgga tttggctcat caggccagat tgagggtctg 6900
tggacacatg tcatgttagt gtacttcaat cgccccctgg atatagcccc gacaataggc 6960
cgtggcctca tttttttgcc ttccgcacat ttccattgct cggtacccac accttgcttc 7020
tcctgcactt gccaacctta atactggttt acattgacca acatcttaca agcggggggc 7080
ttgtctaggg tatatataaa cagtggctct cccaatcggt tgccagtctc ttttttcctt 7140
tctttcccca cagattcgaa atctaaacta cacatcacac aatgcctgtt actgacgtcc 7200
ttaagcgaaa gtccggtgtc atcgtcggcg acgatgtccg agccgtgagt atccacgaca 7260
agatcagtgt cgagacgacg cgttttgtgt aatgacacaa tccgaaagtc gctagcaaca 7320
cacactctct acacaaacta acccagctct ccatggagtc cattgctccc ttcctgccct 7380
ccaagatgcc tcaggacctg ttcatggacc tcgccagcgc tatcggtgtc cgagctgctc 7440
cctacgtcga tcccctggag gctgccctgg ttgcccaggc cgagaagtac attcccacca 7500
ttgtccatca cactcgaggc ttcctggttg ccgtggagtc tcccctggct cgagagctgc 7560
ctctgatgaa ccccttccac gtgctcctga tcgtgctcgc ctacctggtc accgtgtttg 7620
tgggtatgca gatcatgaag aactttgaac gattcgaggt caagaccttc tccctcctgc 7680
acaacttctg tctggtctcc atctccgcct acatgtgcgg tggcatcctg tacgaggctt 7740
atcaggccaa ctatggactg tttgagaacg ctgccgatca caccttcaag ggtctcccta 7800
tggctaagat gatctggctc ttctacttct ccaagatcat ggagtttgtc gacaccatga 7860
tcatggtcct caagaagaac aaccgacaga tttcctttct gcacgtgtac caccactctt 7920
ccatcttcac catctggtgg ctggtcacct tcgttgctcc caacggtgaa gcctacttct 7980
ctgctgccct gaactccttc atccacgtca tcatgtacgg ctactacttt ctgtctgccc 8040
tgggcttcaa gcaggtgtcg ttcatcaagt tctacatcac tcgatcccag atgacccagt 8100
tctgcatgat gtctgtccag tcttcctggg acatgtacgc catgaaggtc cttggccgac 8160
ctggataccc cttcttcatc accgctctgc tctggttcta catgtggacc atgctcggtc 8220
tcttctacaa cttttaccga aagaacgcca agctcgccaa gcaggccaag gctgacgctg 8280
ccaaggagaa ggccagaaag ctccagtaag cggccgcaag tgtggatggg gaagtgagtg 8340
cccggttctg tgtgcacaat tggcaatcca agatggatgg attcaacaca gggatatagc 8400
gagctacgtg gtggtgcgag gatatagcaa cggatattta tgtttgacac ttgagaatgt 8460
acgatacaag cactgtccaa gtacaatact aaacatactg tacatactca tactcgtacc 8520
cgggcaacgg tttcacttga gtgcagtggc tagtgctctt actcgtacag tgtgcaatac 8580
tgcgtatcat agtctttgat gtatatcgta ttcattcatg ttagttgcgt acgaagtcgt 8640
caatgatgtc gatatgggtt ttgatcatgc acacataagg tccgacctta tcggcaagct 8700
caatgagctc cttggtggtg gtaacatcca gagaagcaca caggttggtt ttcttggctg 8760
ccacgagctt gagcactcga gcggcaaagg cggacttgtg gacgttagct cgagcttcgt 8820
aggagggcat tttggtggtg aagaggagac tgaaataaat ttagtctgca gaacttttta 8880
tcggaacctt atctggggca gtgaagtata tgttatggta atagttacga gttagttgaa 8940
cttatagata gactggacta tacggctatc ggtccaaatt agaaagaacg tcaatggctc 9000
tctgggcgtc gcctttgccg acaaaaatgt gatcatgatg aaagccagca atgacgttgc 9060
agctgatatt gttgtcggcc aaccgcgccg aaaacgcagc tgtcagaccc acagcctcca 9120
acgaagaatg tatcgtcaaa gtgatccaag cacactcata gttggagtcg tactccaaag 9180
gcggcaatga cgagtcagac agatactcgt cgaccttttc cttgggaacc accaccgtca 9240
gcccttctga ctcacgtatt gtagccaccg acacaggcaa cagtccgtgg atagcagaat 9300
atgtcttgtc ggtccatttc tcaccaactt taggcgtcaa gtgaatgttg cagaagaagt 9360
atgtgccttc attgagaatc ggtgttgctg atttcaataa agtcttgaga tcagtttggc 9420
gcgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg 9480
ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 9540
atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 9600
gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 9660
gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 9720
gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 9780
gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 9840
aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 9900
ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 9960
taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 10020
tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 10080
gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt 10140
taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 10200
tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 10260
tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 10320
ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 10380
taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 10440
tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 10500
cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 10560
gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 10620
cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 10680
ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac 10740
aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 10800
atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 10860
tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 10920
gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 10980
aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat 11040
acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 11100
ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 11160
tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 11220
aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 11280
catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 11340
atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 11400
aaaagtgcca cctgatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca 11460
tcaggaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag 11520
ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac 11580
cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga 11640
ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc 11700
accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg 11760
gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa 11820
gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac 11880
caccacaccc gccgcgctta atgcgccgct acagggcgcg tccattcgcc attcaggctg 11940
cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa 12000
gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt 12060
tgtaaaacga cggccagtga attgtaatac gactcactat agggcgaatt gggcccgacg 12120
tcgcatgcag tggtggtatt gtgactgggg atgtagttga gaataagtca tacacaagtc 12180
agctttcttc gagcctcata taagtataag tagttcaacg tattagcact gtacccagca 12240
tctccgtatc gagaaacaca acaacatgcc ccattggaca gatcatgcgg atacacaggt 12300
tgtgcagtat catacatact cgatcagaca ggtcgtctga ccatcataca agctgaacaa 12360
gcgctccata cttgcacgct ctctatatac acagttaaat tacatatcca tagtctaacc 12420
tctaacagtt aatcttctgg taagcctccc agccagcctt ctggtatcgc ttggcctcct 12480
caataggatc tcggttctgg ccgtacagac ctcggccgac aattatgata tccgttccgg 12540
tagacatgac atcctcaaca gttcggtact gctgtccgag agcgtctccc ttgtcgtcaa 12600
gacccacccc gggggtcaga ataagccagt cctcagagtc gcccttaat 12649
<210> SEQ ID NO 25
<211> LENGTH: 819
<212> TYPE: DNA
<213> ORGANISM: Thraustochytrium aureum
<220> FEATURE:
<221> NAME/KEY: misc_feature
<223> OTHER INFORMATION: synthetic elongase (codon-optimized) for
Yarrowia lipolytica
<400> SEQUENCE: 25
atggccaact cctctgtctg ggacgacgtg gtcggacgag tcgagaccgg tgtcgaccag 60
tggatggacg gagctaagcc ctacgctctg accgacggtc tgcccatgat ggacgtctcc 120
accatgctcg ccttcgaggt cggctacatg gccatgctgc tcttcggcat tcccatcatg 180
aagcagatgg agaagccctt cgagctgaag accatcaagc tgctccacaa cctgttcctc 240
ttcggactgt ccctctacat gtgcgtcgag accatccgac aggctatcct gggtggctac 300
aaggtcttcg gcaacgacat ggagaagggc aacgagtccc acgctcaggg catgtcccga 360
atcgtctacg tgttctacgt ctccaaggcc tacgagttcc tggacaccgc tatcatgatc 420
ctgtgcaaga agttcaacca ggtctccttc ctgcacgtgt accaccatgc caccatcttc 480
gccatctggt gggctattgc caagtacgct cctggtggcg acgcctactt ctccgtcatc 540
ctcaactcct tcgtccacac cgtcatgtac gcctactact tcttttcctc tcagggcttc 600
ggcttcgtca agcccatcaa gccctacatc accactctgc agatgaccca gttcatggct 660
atgctggtgc agtccctgta cgactacctc ttcccctgcg actaccctca ggctctggtc 720
cagctgctcg gcgtgtacat gatcaccctg ctcgctctgt tcggcaactt ctttgtccag 780
tcctacctga agaagcccaa gaagtccaag accaactaa 819
<210> SEQ ID NO 26
<211> LENGTH: 272
<212> TYPE: PRT
<213> ORGANISM: Thraustochytrium aureum
<400> SEQUENCE: 26
Met Ala Asn Ser Ser Val Trp Asp Asp Val Val Gly Arg Val Glu Thr
1 5 10 15
Gly Val Asp Gln Trp Met Asp Gly Ala Lys Pro Tyr Ala Leu Thr Asp
20 25 30
Gly Leu Pro Met Met Asp Val Ser Thr Met Leu Ala Phe Glu Val Gly
35 40 45
Tyr Met Ala Met Leu Leu Phe Gly Ile Pro Ile Met Lys Gln Met Glu
50 55 60
Lys Pro Phe Glu Leu Lys Thr Ile Lys Leu Leu His Asn Leu Phe Leu
65 70 75 80
Phe Gly Leu Ser Leu Tyr Met Cys Val Glu Thr Ile Arg Gln Ala Ile
85 90 95
Leu Gly Gly Tyr Lys Val Phe Gly Asn Asp Met Glu Lys Gly Asn Glu
100 105 110
Ser His Ala Gln Gly Met Ser Arg Ile Val Tyr Val Phe Tyr Val Ser
115 120 125
Lys Ala Tyr Glu Phe Leu Asp Thr Ala Ile Met Ile Leu Cys Lys Lys
130 135 140
Phe Asn Gln Val Ser Phe Leu His Val Tyr His His Ala Thr Ile Phe
145 150 155 160
Ala Ile Trp Trp Ala Ile Ala Lys Tyr Ala Pro Gly Gly Asp Ala Tyr
165 170 175
Phe Ser Val Ile Leu Asn Ser Phe Val His Thr Val Met Tyr Ala Tyr
180 185 190
Tyr Phe Phe Ser Ser Gln Gly Phe Gly Phe Val Lys Pro Ile Lys Pro
195 200 205
Tyr Ile Thr Thr Leu Gln Met Thr Gln Phe Met Ala Met Leu Val Gln
210 215 220
Ser Leu Tyr Asp Tyr Leu Phe Pro Cys Asp Tyr Pro Gln Ala Leu Val
225 230 235 240
Gln Leu Leu Gly Val Tyr Met Ile Thr Leu Leu Ala Leu Phe Gly Asn
245 250 255
Phe Phe Val Gln Ser Tyr Leu Lys Lys Pro Lys Lys Ser Lys Thr Asn
260 265 270
<210> SEQ ID NO 27
<211> LENGTH: 37
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Primer AP
<400> SEQUENCE: 27
ggccacgcgt cgactagtac tttttttttt ttttttt 37
<210> SEQ ID NO 28
<211> LENGTH: 39
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Smart IV oligonucleotide primer
<400> SEQUENCE: 28
aagcagtggt atcaacgcag agtggccatt acggccggg 39
<210> SEQ ID NO 29
<211> LENGTH: 23
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Primer CDSIII 5'
<400> SEQUENCE: 29
aagcagtggt atcaacgcag agt 23
<210> SEQ ID NO 30
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Degenerate primer 5-1A
<400> SEQUENCE: 30
gghcaycayr tbtayacaaa 20
<210> SEQ ID NO 31
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Degenerate primer 5-1B
<400> SEQUENCE: 31
gghcaycayr tbtayaccaa 20
<210> SEQ ID NO 32
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Degenerate primer 5-1C
<400> SEQUENCE: 32
gghcaycayr tbtayacgaa 20
<210> SEQ ID NO 33
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Degenerate primer 5-1D
<400> SEQUENCE: 33
gghcaycayr tbtayactaa 20
<210> SEQ ID NO 34
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Degenerate primer 5-4AR
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (9)..(9)
<223> OTHER INFORMATION: n is a, c, g, or t
<400> SEQUENCE: 34
acrtgrytna cytgraagtt 20
<210> SEQ ID NO 35
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Degenerate primer 5-4BR
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (9)..(9)
<223> OTHER INFORMATION: n is a, c, g, or t
<400> SEQUENCE: 35
acrtgrytna cytgraaatt 20
<210> SEQ ID NO 36
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Degenerate primer 5-4CR
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (6)..(6)
<223> OTHER INFORMATION: n is a, c, g, or t
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (9)..(9)
<223> OTHER INFORMATION: n is a, c, g, or t
<400> SEQUENCE: 36
acrtgngana cytgraagtt 20
<210> SEQ ID NO 37
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Degenerate primer 5-4DR
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (6)..(6)
<223> OTHER INFORMATION: n is a, c, g, or t
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (9)..(9)
<223> OTHER INFORMATION: n is a, c, g, or t
<400> SEQUENCE: 37
acrtgngana cytgraaatt 20
<210> SEQ ID NO 38
<211> LENGTH: 25
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Primer ODMW520
<400> SEQUENCE: 38
cgttctccat tttcattcca accgc 25
<210> SEQ ID NO 39
<211> LENGTH: 28
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Primer ODMW521
<400> SEQUENCE: 39
ggttgaagac cgaagagacc cagtatcc 28
<210> SEQ ID NO 40
<211> LENGTH: 22
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Primer DNR CDS 5'
<400> SEQUENCE: 40
caacgcagag tggccattac gg 22
<210> SEQ ID NO 41
<211> LENGTH: 28
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Primer ODMW541
<400> SEQUENCE: 41
ctcgcggcct cgtcgcacaa tcttgaag 28
<210> SEQ ID NO 42
<211> LENGTH: 29
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Primer ODMW542
<400> SEQUENCE: 42
gagtatcaaa cttgtattcc gaggtgtag 29
<210> SEQ ID NO 43
<211> LENGTH: 26
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Primer ODMW523
<400> SEQUENCE: 43
ggttcaagtc gcaagtcgaa acctcg 26
<210> SEQ ID NO 44
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Primer AUAP
<400> SEQUENCE: 44
ggccacgcgt cgactagtac 20
<210> SEQ ID NO 45
<211> LENGTH: 25
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Primer ODMW524
<400> SEQUENCE: 45
caacttacgg aggtttcatc tcggg 25
<210> SEQ ID NO 46
<211> LENGTH: 31
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Primer YL807
<400> SEQUENCE: 46
tttccatggc tccagatgcg gacaagttga g 31
<210> SEQ ID NO 47
<211> LENGTH: 29
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Primer YL810
<400> SEQUENCE: 47
caaggcatcg ggatccttct catggtggt 29
<210> SEQ ID NO 48
<211> LENGTH: 42
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Primer YL808
<400> SEQUENCE: 48
tttgcggccg cttacaattt tcccgagtag gggttggcgc ca 42
<210> SEQ ID NO 49
<211> LENGTH: 29
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Primer YL809
<400> SEQUENCE: 49
accaccatga gaaggatccc gatgccttg 29
<210> SEQ ID NO 50
<211> LENGTH: 4112
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Plasmid pRD5S
<400> SEQUENCE: 50
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa 420
tgcatctaga tccatggctc ccgacgccga caagctgcga cagcgaaagg ctcagtccat 480
ccaggacact gccgattctc aggctaccga gctcaagatt ggcaccctga agggtctcca 540
aggcaccgag atcgtcattg atggcgacat ctacgacatc aaagacttcg atcaccctgg 600
aggcgaatcc atcatgacct ttggtggcaa cgacgttact gccacctaca agatgattca 660
tccctaccac tcgaagcatc acctggagaa gatgaaaaag gtcggtcgag tgcccgacta 720
cacctccgag tacaagttcg atactccctt cgaacgagag atcaaacagg aggtcttcaa 780
gattgtgcga agaggtcgag agtttggaac acctggctac ttctttcgag ccttctgcta 840
catcggtctc ttcttttacc tgcagtatct ctgggttacc actcctacca ctttcgccct 900
tgctatcttc tacggtgtgt ctcaggcctt cattggcctg aacgtccagc acgacgccaa 960
ccacggagct gcctccaaaa agccctggat caacaatttg ctcggcctgg gtgccgactt 1020
tatcggaggc tccaagtggc tctggatgaa ccagcactgg acccatcaca cttacaccaa 1080
ccatcacgag aaggatcccg acgccctggg tgcagagcct atgctgctct tcaacgacta 1140
tcccttgggt caccccaagc gaaccctcat tcatcacttc caagccttct actatctgtt 1200
tgtccttgct ggctactggg tgtcttcggt gttcaaccct cagatcctgg acctccagca 1260
ccgaggtgcc caggctgtcg gcatgaagat ggagaacgac tacattgcca agtctcgaaa 1320
gtacgctatc ttcctgcgac tcctgtacat ctacaccaac attgtggctc ccatccagaa 1380
ccaaggcttt tcgctcaccg tcgttgctca cattcttact atgggtgtcg cctccagcct 1440
gaccctcgct actctgttcg ccctctccca caacttcgag aacgcagatc gggatcccac 1500
ctacgaggct cgaaagggag gcgagcctgt ctgttggttc aagtcgcagg tggaaacctc 1560
ctctacttac ggtggcttca tttccggttg ccttacaggc ggactcaact ttcaggtcga 1620
gcatcacctg tttcctcgaa tgtcctctgc ctggtacccc tacatcgctc ctaccgttcg 1680
agaggtctgc aaaaagcacg gcgtcaagta cgcctactat ccctgggtgt ggcagaacct 1740
catctcgacc gtcaagtacc tgcatcagtc cggaactggc tcgaactgga agaacggtgc 1800
caatccctac tctggcaagc tgtaagcggc cgcatcggat cccgggcccg tcgactgcag 1860
aggcctgcat gcaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta 1920
tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc 1980
ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 2040
aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 2100
tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 2160
gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 2220
cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 2280
gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 2340
aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 2400
ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 2460
cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 2520
ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 2580
cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 2640
agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 2700
gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct 2760
gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 2820
tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 2880
agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 2940
agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 3000
atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 3060
cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 3120
actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 3180
aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 3240
cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 3300
ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 3360
cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 3420
ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 3480
cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 3540
ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 3600
tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 3660
ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 3720
aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 3780
gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 3840
gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 3900
ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 3960
catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 4020
atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta 4080
taaaaatagg cgtatcacga ggccctttcg tc 4112
<210> SEQ ID NO 51
<211> LENGTH: 8480
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Plasmid pZURD5S
<400> SEQUENCE: 51
catggctccc gacgccgaca agctgcgaca gcgaaaggct cagtccatcc aggacactgc 60
cgattctcag gctaccgagc tcaagattgg caccctgaag ggtctccaag gcaccgagat 120
cgtcattgat ggcgacatct acgacatcaa agacttcgat caccctggag gcgaatccat 180
catgaccttt ggtggcaacg acgttactgc cacctacaag atgattcatc cctaccactc 240
gaagcatcac ctggagaaga tgaaaaaggt cggtcgagtg cccgactaca cctccgagta 300
caagttcgat actcccttcg aacgagagat caaacaggag gtcttcaaga ttgtgcgaag 360
aggtcgagag tttggaacac ctggctactt ctttcgagcc ttctgctaca tcggtctctt 420
cttttacctg cagtatctct gggttaccac tcctaccact ttcgcccttg ctatcttcta 480
cggtgtgtct caggccttca ttggcctgaa cgtccagcac gacgccaacc acggagctgc 540
ctccaaaaag ccctggatca acaatttgct cggcctgggt gccgacttta tcggaggctc 600
caagtggctc tggatgaacc agcactggac ccatcacact tacaccaacc atcacgagaa 660
ggatcccgac gccctgggtg cagagcctat gctgctcttc aacgactatc ccttgggtca 720
ccccaagcga accctcattc atcacttcca agccttctac tatctgtttg tccttgctgg 780
ctactgggtg tcttcggtgt tcaaccctca gatcctggac ctccagcacc gaggtgccca 840
ggctgtcggc atgaagatgg agaacgacta cattgccaag tctcgaaagt acgctatctt 900
cctgcgactc ctgtacatct acaccaacat tgtggctccc atccagaacc aaggcttttc 960
gctcaccgtc gttgctcaca ttcttactat gggtgtcgcc tccagcctga ccctcgctac 1020
tctgttcgcc ctctcccaca acttcgagaa cgcagatcgg gatcccacct acgaggctcg 1080
aaagggaggc gagcctgtct gttggttcaa gtcgcaggtg gaaacctcct ctacttacgg 1140
tggcttcatt tccggttgcc ttacaggcgg actcaacttt caggtcgagc atcacctgtt 1200
tcctcgaatg tcctctgcct ggtaccccta catcgctcct accgttcgag aggtctgcaa 1260
aaagcacggc gtcaagtacg cctactatcc ctgggtgtgg cagaacctca tctcgaccgt 1320
caagtacctg catcagtccg gaactggctc gaactggaag aacggtgcca atccctactc 1380
tggcaagctg taagcggccg caagtgtgga tggggaagtg agtgcccggt tctgtgtgca 1440
caattggcaa tccaagatgg atggattcaa cacagggata tagcgagcta cgtggtggtg 1500
cgaggatata gcaacggata tttatgtttg acacttgaga atgtacgata caagcactgt 1560
ccaagtacaa tactaaacat actgtacata ctcatactcg tacccgggca acggtttcac 1620
ttgagtgcag tggctagtgc tcttactcgt acagtgtgca atactgcgta tcatagtctt 1680
tgatgtatat cgtattcatt catgttagtt gcgtacgagc cggaagcata aagtgtaaag 1740
cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt 1800
tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 1860
gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 1920
ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 1980
caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 2040
aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 2100
atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 2160
cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 2220
ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 2280
gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 2340
accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 2400
cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 2460
cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct 2520
gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 2580
aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 2640
aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 2700
actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 2760
taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 2820
gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 2880
tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 2940
ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 3000
accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 3060
agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 3120
acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 3180
tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 3240
cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 3300
tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 3360
ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 3420
gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 3480
tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 3540
ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 3600
gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 3660
cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg 3720
gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 3780
ttccgcgcac atttccccga aaagtgccac ctgacgcgcc ctgtagcggc gcattaagcg 3840
cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg 3900
ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 3960
taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 4020
aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 4080
ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 4140
tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt 4200
ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc 4260
ttacaatttc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc 4320
ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt 4380
aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgaat tgtaatacga 4440
ctcactatag ggcgaattgg gtaccgggcc ccccctcgag gtcgatggtg tcgataagct 4500
tgatatcgaa ttcatgtcac acaaaccgat cttcgcctca aggaaaccta attctacatc 4560
cgagagactg ccgagatcca gtctacactg attaattttc gggccaataa tttaaaaaaa 4620
tcgtgttata taatattata tgtattatat atatacatca tgatgatact gacagtcatg 4680
tcccattgct aaatagacag actccatctg ccgcctccaa ctgatgttct caatatttaa 4740
ggggtcatct cgcattgttt aataataaac agactccatc taccgcctcc aaatgatgtt 4800
ctcaaaatat attgtatgaa cttattttta ttacttagta ttattagaca acttacttgc 4860
tttatgaaaa acacttccta tttaggaaac aatttataat ggcagttcgt tcatttaaca 4920
atttatgtag aataaatgtt ataaatgcgt atgggaaatc ttaaatatgg atagcataaa 4980
tgatatctgc attgcctaat tcgaaatcaa cagcaacgaa aaaaatccct tgtacaacat 5040
aaatagtcat cgagaaatat caactatcaa agaacagcta ttcacacgtt actattgaga 5100
ttattattgg acgagaatca cacactcaac tgtctttctc tcttctagaa atacaggtac 5160
aagtatgtac tattctcatt gttcatactt ctagtcattt catcccacat attccttgga 5220
tttctctcca atgaatgaca ttctatcttg caaattcaac aattataata agatatacca 5280
aagtagcggt atagtggcaa tcaaaaagct tctctggtgt gcttctcgta tttattttta 5340
ttctaatgat ccattaaagg tatatattta tttcttgtta tataatcctt ttgtttatta 5400
catgggctgg atacataaag gtattttgat ttaatttttt gcttaaattc aatcccccct 5460
cgttcagtgt caactgtaat ggtaggaaat taccatactt ttgaagaagc aaaaaaaatg 5520
aaagaaaaaa aaaatcgtat ttccaggtta gacgttccgc agaatctaga atgcggtatg 5580
cggtacattg ttcttcgaac gtaaaagttg cgctccctga gatattgtac atttttgctt 5640
ttacaagtac aagtacatcg tacaactatg tactactgtt gatgcatcca caacagtttg 5700
ttttgttttt ttttgttttt tttttttcta atgattcatt accgctatgt atacctactt 5760
gtacttgtag taagccgggt tattggcgtt caattaatca tagacttatg aatctgcacg 5820
gtgtgcgctg cgagttactt ttagcttatg catgctactt gggtgtaata ttgggatctg 5880
ttcggaaatc aacggatgct caatcgattt cgacagtaat taattaagtc atacacaagt 5940
cagctttctt cgagcctcat ataagtataa gtagttcaac gtattagcac tgtacccagc 6000
atctccgtat cgagaaacac aacaacatgc cccattggac agatcatgcg gatacacagg 6060
ttgtgcagta tcatacatac tcgatcagac aggtcgtctg accatcatac aagctgaaca 6120
agcgctccat acttgcacgc tctctatata cacagttaaa ttacatatcc atagtctaac 6180
ctctaacagt taatcttctg gtaagcctcc cagccagcct tctggtatcg cttggcctcc 6240
tcaataggat ctcggttctg gccgtacaga cctcggccga caattatgat atccgttccg 6300
gtagacatga catcctcaac agttcggtac tgctgtccga gagcgtctcc cttgtcgtca 6360
agacccaccc cgggggtcag aataagccag tcctcagagt cgcccttagg tcggttctgg 6420
gcaatgaagc caaccacaaa ctcggggtcg gatcgggcaa gctcaatggt ctgcttggag 6480
tactcgccag tggccagaga gcccttgcaa gacagctcgg ccagcatgag cagacctctg 6540
gccagcttct cgttgggaga ggggactagg aactccttgt actgggagtt ctcgtagtca 6600
gagacgtcct ccttcttctg ttcagagaca gtttcctcgg caccagctcg caggccagca 6660
atgattccgg ttccgggtac accgtgggcg ttggtgatat cggaccactc ggcgattcgg 6720
tgacaccggt actggtgctt gacagtgttg ccaatatctg cgaactttct gtcctcgaac 6780
aggaagaaac cgtgcttaag agcaagttcc ttgaggggga gcacagtgcc ggcgtaggtg 6840
aagtcgtcaa tgatgtcgat atgggttttg atcatgcaca cataaggtcc gaccttatcg 6900
gcaagctcaa tgagctcctt ggtggtggta acatccagag aagcacacag gttggttttc 6960
ttggctgcca cgagcttgag cactcgagcg gcaaaggcgg acttgtggac gttagctcga 7020
gcttcgtagg agggcatttt ggtggtgaag aggagactga aataaattta gtctgcagaa 7080
ctttttatcg gaaccttatc tggggcagtg aagtatatgt tatggtaata gttacgagtt 7140
agttgaactt atagatagac tggactatac ggctatcggt ccaaattaga aagaacgtca 7200
atggctctct gggcgtcgcc tttgccgaca aaaatgtgat catgatgaaa gccagcaatg 7260
acgttgcagc tgatattgtt gtcggccaac cgcgccgaaa acgcagctgt cagacccaca 7320
gcctccaacg aagaatgtat cgtcaaagtg atccaagcac actcatagtt ggagtcgtac 7380
tccaaaggcg gcaatgacga gtcagacaga tactcgtcga ctcaggcgac gacggaattc 7440
ctgcagccca tctgcagaat tcaggagaga ccgggttggc ggcgtatttg tgtcccaaaa 7500
aacagcccca attgccccgg agaagacggc caggccgcct agatgacaaa ttcaacaact 7560
cacagctgac tttctgccat tgccactagg ggggggcctt tttatatggc caagccaagc 7620
tctccacgtc ggttgggctg cacccaacaa taaatgggta gggttgcacc aacaaaggga 7680
tgggatgggg ggtagaagat acgaggataa cggggctcaa tggcacaaat aagaacgaat 7740
actgccatta agactcgtga tccagcgact gacaccattg catcatctaa gggcctcaaa 7800
actacctcgg aactgctgcg ctgatctgga caccacagag gttccgagca ctttaggttg 7860
caccaaatgt cccaccaggt gcaggcagaa aacgctggaa cagcgtgtac agtttgtctt 7920
aacaaaaagt gagggcgctg aggtcgagca gggtggtgtg acttgttata gcctttagag 7980
ctgcgaaagc gcgtatggat ttggctcatc aggccagatt gagggtctgt ggacacatgt 8040
catgttagtg tacttcaatc gccccctgga tatagccccg acaataggcc gtggcctcat 8100
ttttttgcct tccgcacatt tccattgctc ggtacccaca ccttgcttct cctgcacttg 8160
ccaaccttaa tactggttta cattgaccaa catcttacaa gcggggggct tgtctagggt 8220
atatataaac agtggctctc ccaatcggtt gccagtctct tttttccttt ctttccccac 8280
agattcgaaa tctaaactac acatcacaca atgcctgtta ctgacgtcct taagcgaaag 8340
tccggtgtca tcgtcggcga cgatgtccga gccgtgagta tccacgacaa gatcagtgtc 8400
gagacgacgc gttttgtgta atgacacaat ccgaaagtcg ctagcaacac acactctcta 8460
cacaaactaa cccagctctc 8480
<210> SEQ ID NO 52
<211> LENGTH: 19
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Primer T7
<400> SEQUENCE: 52
ggaaacagct atgaccatg 19
<210> SEQ ID NO 53
<211> LENGTH: 459
<212> TYPE: PRT
<213> ORGANISM: Rhizopus stolonifer
<300> PUBLICATION INFORMATION:
<308> DATABASE ACCESSION NUMBER: GenBank Accession No. AAX22052
<400> SEQUENCE: 53
Met Ser Thr Leu Asp Arg Gln Ser Ile Phe Thr Ile Lys Glu Leu Glu
1 5 10 15
Ser Ile Ser Gln Arg Ile His Asp Gly Asp Glu Glu Ala Met Lys Phe
20 25 30
Ile Ile Ile Asp Lys Lys Val Tyr Asp Val Thr Glu Phe Ile Glu Asp
35 40 45
His Pro Gly Gly Ala Gln Val Leu Leu Thr His Val Gly Lys Asp Ala
50 55 60
Ser Asp Val Phe His Ala Met His Pro Glu Ser Ala Tyr Glu Val Leu
65 70 75 80
Asn Asn Tyr Phe Val Gly Asp Val Gln Glu Thr Val Val Thr Glu Lys
85 90 95
Ser Ser Ser Ala Gln Phe Ala Val Glu Met Arg Gln Leu Arg Asp Gln
100 105 110
Leu Lys Lys Glu Gly Tyr Phe His Ser Ser Lys Leu Phe Tyr Ala Tyr
115 120 125
Lys Val Leu Ser Thr Leu Ala Ile Cys Ile Ala Gly Leu Ser Leu Leu
130 135 140
Tyr Ala Tyr Gly Arg Thr Ser Thr Leu Ala Val Val Ala Ser Ala Ile
145 150 155 160
Thr Val Gly Ile Phe Trp Gln Gln Cys Gly Trp Leu Ala His Asp Phe
165 170 175
Gly His His Gln Cys Phe Glu Asp Arg Thr Trp Asn Asp Val Leu Val
180 185 190
Val Phe Leu Gly Asn Phe Cys Gln Gly Phe Ser Leu Ser Trp Trp Lys
195 200 205
Asn Lys His Asn Thr His His Ala Ser Thr Asn Val His Gly Gln Asp
210 215 220
Pro Asp Ile Asp Thr Ala Pro Val Leu Leu Trp Asp Glu Tyr Ala Ser
225 230 235 240
Ala Ala Tyr Tyr Ala Ser Leu Asp Gln Glu Pro Thr Met Val Ser Arg
245 250 255
Phe Leu Ala Glu Gln Val Leu Pro His Gln Thr Arg Tyr Phe Phe Phe
260 265 270
Ile Leu Ala Phe Ala Arg Leu Ser Trp Ala Leu Gln Ser Leu Ser Tyr
275 280 285
Ser Phe Lys Lys Glu Ser Ile Asn Lys Ser Arg Gln Leu Asn Leu Phe
290 295 300
Glu Arg Val Cys Ile Val Gly His Trp Ala Leu Phe Ala Phe Cys Ile
305 310 315 320
Tyr Ser Trp Cys Ser Asn Val Tyr His Met Val Leu Phe Phe Leu Val
325 330 335
Ser Gln Ala Thr Thr Gly Tyr Thr Leu Ala Leu Val Phe Ala Leu Asn
340 345 350
His Asn Gly Met Pro Val Ile Thr Glu Glu Lys Ala Glu Ser Met Glu
355 360 365
Phe Phe Glu Ile Gln Val Ile Thr Gly Arg Asp Val Thr Leu Ser Pro
370 375 380
Leu Gly Asp Trp Phe Met Gly Gly Leu Asn Tyr Gln Ile Glu His His
385 390 395 400
Val Phe Pro Asn Met Pro Arg His Asn Leu Pro Thr Val Lys Pro Met
405 410 415
Val Lys Ser Leu Cys Gln Lys Tyr Asp Ile Asn Tyr His Asp Thr Gly
420 425 430
Phe Leu Lys Gly Thr Leu Glu Val Leu Gln Thr Leu Asp Ile Thr Ser
435 440 445
Lys Leu Ser Leu Gln Leu Ser Lys Lys Ser Phe
450 455
<210> SEQ ID NO 54
<211> LENGTH: 695
<212> TYPE: DNA
<213> ORGANISM: Pavlova lutheri
<400> SEQUENCE: 54
agggccaagg gtgccaacca ccttccacgt gagactacac accgtaggcc gatgggcaag 60
ggtggagacg gcggcgcgca ggcggtgagc gggaccgacg cgtctctcgc tgaggtgagc 120
tccgtcgata gcaagagcgt gcacgtcgtg ctctacggca agcgcgtgga tgtcacaaag 180
ttccagggct acgacgtggc ctggtggcgc gcgcgccata acacgcacca cgtgtgcacc 240
aacgaggatg gttcggaccc ggacatcaag acggcgcccc tgctcatcta cgtgcgagag 300
aacccgtcca ttgccaagcg gctcaacttc ttccagcgct ggcagcagta ctactatgtg 360
ccgaccatgg ccatcctcga cctctactgg cgcctggagt ccatcgcgta cgtggctgtg 420
cgcctgccta agatgtggat gcaggccgcc gctcttgccg ctcactacgc gctcctgtgc 480
tgggtcttcg cagcgcatct caacctcatc cctctcatga tggttgcacg cggcttcgcg 540
acgggcatcg ttgtctttgc aacccactat ggtgaggaca tcctcgaccg cgagcacgtc 600
gagggcatga cgctcgtcga gcagaccgcc aagacctccc gtaacatcac gggcggctgg 660
ctagtgaacg tgctcacggg cttcatctcc ctgca 695
<210> SEQ ID NO 55
<211> LENGTH: 1106
<212> TYPE: DNA
<213> ORGANISM: Pavlova lutheri
<400> SEQUENCE: 55
agggccaagg gtgccaacca ccttccacgt gagactacac accgtaggcc gatgggcaag 60
ggtggagacg gcggcgcgca ggcggtgagc gggaccgacg cgtctctcgc tgaggtgagc 120
tccgtcgata gcaagagcgt gcacgtcgtg ctctacggca agcgcgtgga tgtcacaaag 180
ttccagggct acgacgtggc ctggtggcgc gcgcgccata acacgcacca cgtgtgcacc 240
aacgaggatg gttcggaccc ggacatcaag acggcgcccc tgctcatcta cgtgcgagag 300
aacccgtcca ttgccaagcg gctcaacttc ttccagcgct ggcagcagta ctactatgtg 360
ccgaccatgg ccatcctcga cctctactgg cgcctggagt ccatcgcgta cgtggctgtg 420
cgcctgccta agatgtggat gcaggccgcc gctcttgccg ctcactacgc gctcctgtgc 480
tgggtcttcg cagcgcatct caacctcatc cctctcatga tggttgcacg cggcttcgcg 540
acgggcatcg ttgtctttgc aacccactat ggtgaggaca tcctcgaccg cgagcacgtc 600
gagggcatga cgctcgtcga gcagaccgcc aagacctccc gtaacatcac gggcggctgg 660
ctagtgaacg tgctcacggg cttcatctcc ctgcagaccg agcatcacct cttccccatg 720
atgcccaccg gcaacctaat gactatccag cccgaggtac gcgacttctt caagaagcat 780
ggcctcgagt accgcgaggg caacctcttc cagtgcgtgc accagaacat caaggctctc 840
gccttcgagc acctcctcca ctgagcgtca ccactcaagc gtcctaagtg cacaggtact 900
gtcttctgac cgatggccgc gcggctccct cggctggcag tggggccaac gagtggcctc 960
gcgggatcgg gcacgatcgg gcctccatga aacttcagtg ttcagagaca agccgacaac 1020
ctccgcatcg tgagaaatct tttaaagcag tatgttccat cacgccgctt ttgcagtcaa 1080
taacattacc caaaaaaaaa aaaaaa 1106
<210> SEQ ID NO 56
<211> LENGTH: 287
<212> TYPE: PRT
<213> ORGANISM: Pavlova lutheri
<400> SEQUENCE: 56
Arg Ala Lys Gly Ala Asn His Leu Pro Arg Glu Thr Thr His Arg Arg
1 5 10 15
Pro Met Gly Lys Gly Gly Asp Gly Gly Ala Gln Ala Val Ser Gly Thr
20 25 30
Asp Ala Ser Leu Ala Glu Val Ser Ser Val Asp Ser Lys Ser Val His
35 40 45
Val Val Leu Tyr Gly Lys Arg Val Asp Val Thr Lys Phe Gln Gly Tyr
50 55 60
Asp Val Ala Trp Trp Arg Ala Arg His Asn Thr His His Val Cys Thr
65 70 75 80
Asn Glu Asp Gly Ser Asp Pro Asp Ile Lys Thr Ala Pro Leu Leu Ile
85 90 95
Tyr Val Arg Glu Asn Pro Ser Ile Ala Lys Arg Leu Asn Phe Phe Gln
100 105 110
Arg Trp Gln Gln Tyr Tyr Tyr Val Pro Thr Met Ala Ile Leu Asp Leu
115 120 125
Tyr Trp Arg Leu Glu Ser Ile Ala Tyr Val Ala Val Arg Leu Pro Lys
130 135 140
Met Trp Met Gln Ala Ala Ala Leu Ala Ala His Tyr Ala Leu Leu Cys
145 150 155 160
Trp Val Phe Ala Ala His Leu Asn Leu Ile Pro Leu Met Met Val Ala
165 170 175
Arg Gly Phe Ala Thr Gly Ile Val Val Phe Ala Thr His Tyr Gly Glu
180 185 190
Asp Ile Leu Asp Arg Glu His Val Glu Gly Met Thr Leu Val Glu Gln
195 200 205
Thr Ala Lys Thr Ser Arg Asn Ile Thr Gly Gly Trp Leu Val Asn Val
210 215 220
Leu Thr Gly Phe Ile Ser Leu Gln Thr Glu His His Leu Phe Pro Met
225 230 235 240
Met Pro Thr Gly Asn Leu Met Thr Ile Gln Pro Glu Val Arg Asp Phe
245 250 255
Phe Lys Lys His Gly Leu Glu Tyr Arg Glu Gly Asn Leu Phe Gln Cys
260 265 270
Val His Gln Asn Ile Lys Ala Leu Ala Phe Glu His Leu Leu His
275 280 285
<210> SEQ ID NO 57
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Primer SeqE
<400> SEQUENCE: 57
cgacacactc caatctttcc 20
<210> SEQ ID NO 58
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Primer SeqW
<400> SEQUENCE: 58
ggtggctgga gttagacatc 20
<210> SEQ ID NO 59
<211> LENGTH: 22
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Primer AP1
<400> SEQUENCE: 59
gtaatacgac tcactatagg gc 22
<210> SEQ ID NO 60
<211> LENGTH: 20
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Primer GSP PvDES
<400> SEQUENCE: 60
ctgcgaagac ccagcacagg 20
<210> SEQ ID NO 61
<211> LENGTH: 22
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Primer M13-28Rev
<400> SEQUENCE: 61
gtaatacgac tcactatagg gc 22
<210> SEQ ID NO 62
<211> LENGTH: 21
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Primer PavDes seq
<400> SEQUENCE: 62
ttgtggcgct caatcatctc c 21
<210> SEQ ID NO 63
<211> LENGTH: 1294
<212> TYPE: DNA
<213> ORGANISM: Pavlova lutheri
<400> SEQUENCE: 63
ctttgcgagc gcggcgcaga cgattgcggc ccgtagtgat cgcggtgcgc attgctgtgt 60
ttctagtttt gctgacgccc ggcccgataa tgacaccttc tcccgtttga aatactaata 120
agtaactata ttataatatt caaaggtggc gactatggat ctccttttct aaagttcagc 180
ggaattggga atcggagaaa tttcgagata tgtcataatc acgtgctcta tctcgaatga 240
accgcggccg gtgagcgatt actcgggaag ccaattccta ttaacgagtc aggggggatc 300
tttgaggtga gtcggccacg cagagagagc aaggaatcat cctcatccgc cgttctcgag 360
aaagagccaa gggtgccaac caccttccac gtgagactac acaccgtagg ccgatgggca 420
agggtggaga cggcggcgcg caggcggcga gcgggaccga cgcatctctc gctgaggtga 480
gctccgtcga tagcaagagc gtgcgcgtcg tgctctacgg caagcgcgtg gatgtcacaa 540
agttccagag ggcacacccg ggcgggagca aggtgttccg catcttccag gagcgcgacg 600
cgacggagca gttcgagtct taccactcgc ccaaggccat caagatgatg gagggcatgc 660
tcaagaagtc ggaggatgcg cccgcttccg tgcccctgcc ctcgcggtcc accatgggca 720
cggagttcaa ggagatgatt gagcgccaca agagggctgg tctctacgac ccttgcccgt 780
tggacgagct gttcaagctc accatcgtcc ttgcgcccat cttcgtgggc gcctatctcg 840
tgcggagcgg cgtctcgccc ctcgcgggcg cgctctccat gggctttggc ttctacctcg 900
acggctggct tgctcacrac tacctgcatc acgcagtctt caagggctcg gtcaacacgc 960
tcgtcaaggc gaacaacgcc atgggatacg ccctcggctt cctccagggc tacgacgtgg 1020
cctggtggcg cgcgcgccat aacacgcacc acgtgtgcac caacgaggat ggttcggacc 1080
cggacatcaa gacggcgccc ctgctcatct acgtgcgaga gaacccgtcc attgccaagc 1140
ggctcaactt cttccagcgc tggcagcagt actactatgt gccgaccatg gccatcctcg 1200
acctctactg gcgcctggag tccatcgcgt acgtggctgt gcgcctgcct aagatgtgga 1260
tgcaggccgc cgctcttgcc gctcactacg cgct 1294
<210> SEQ ID NO 64
<211> LENGTH: 1927
<212> TYPE: DNA
<213> ORGANISM: Pavlova lutheri
<400> SEQUENCE: 64
ctttgcgagc gcggcgcaga cgattgcggc ccgtagtgat cgcggtgcgc attgctgtgt 60
ttctagtttt gctgacgccc ggcccgataa tgacaccttc tcccgtttga aatactaata 120
agtaactata ttataatatt caaaggtggc gactatggat ctccttttct aaagttcagc 180
ggaattggga atcggagaaa tttcgagata tgtcataatc acgtgctcta tctcgaatga 240
accgcggccg gtgagcgatt actcgggaag ccaattccta ttaacgagtc aggggggatc 300
tttgaggtga gtcggccacg cagagagagc aaggaatcat cctcatccgc cgttctcgag 360
aaagagccaa gggtgccaac caccttccac gtgagactac acaccgtagg ccgatgggca 420
agggtggaga cggcggcgcg caggcggtga gcgggaccga cgcgtctctc gctgaggtga 480
gctccgtcga tagcaagagc gtgcacgtcg tgctctacgg caagcgcgtg gatgtcacaa 540
agttccagaa ggcacacccg ggcgggagca aggtgttccg catcttccag gagcgcgacg 600
cgacggagca gttcgagtct taccactcgc ccaaggccat caagatgatg gagggcatgc 660
tcaagaagtc ggaggatgcg cccgcttccg tgcccctgcc ctcgcggtcc accatgggca 720
cggagttcaa ggagatgatt gagcgccaca agagggctgg tctctacgac ccttgcccgt 780
tggacgagct gttcaagctc accatcgtcc ttgcgcccat cttcgtgggc gcctatctcg 840
tgcggagcgg cgtctcgccc ctcgcgggcg cgctctccat gggctttggc ttctacctcg 900
acggctggct tgctcacgac tacctgcatc acgcagtctt caagggctcg gtcaacacgc 960
tcgtcaaggc gaacaacgcc atgggatacg ccctcggctt cctccagggc tacgacgtgg 1020
cctggtggcg cgcgcgccat aacacgcacc acgtgtgcac caacgaggat ggttcggacc 1080
cggacatcaa gacggcgccc ctgctcatct acgtgcgaga gaacccgtcc attgccaagc 1140
ggctcaactt cttccagcgc tggcagcagt actactatgt gccgaccatg gccatcctcg 1200
acctctactg gcgcctggag tccatcgcgt acgtggctgt gcgcctgcct aagatgtgga 1260
tgcaggccgc cgctcttgcc gctcactacg cgctcctgtg ctgggtcttc gcagcgcatc 1320
tcaacctcat ccctctcatg atggttgcac gcggcttcgc gacgggcatc gttgtctttg 1380
caacccacta tggtgaggac atcctcgacc gcgagcacgt cgagggcatg acgctcgtcg 1440
agcagaccgc caagacctcc cgtaacatca cgggcggctg gctagtgaac gtgctcacgg 1500
gcttcatctc cctgcagacc gagcatcacc tcttccccat gatgcccacc ggcaacctaa 1560
tgactatcca gcccgaggta cgcgacttct tcaagaagca tggcctcgag taccgcgagg 1620
gcaacctctt ccagtgcgtg caccagaaca tcaaggctct cgccttcgag cacctcctcc 1680
actgagcgtc accactcaag cgtcctaagt gcacaggtac tgtcttctga ccgatggccg 1740
cgcggctccc tcggctggca gtggggccaa cgagtggcct cgcgggatcg ggcacgatcg 1800
ggcctccatg aaacttcagt gttcagagac aagccgacaa cctccgcatc gtgagaaatc 1860
ttttaaagca gtatgttcca tcacgccgct tttgcagtca ataacattac ccaaaaaaaa 1920
aaaaaaa 1927
<210> SEQ ID NO 65
<211> LENGTH: 459
<212> TYPE: PRT
<213> ORGANISM: Rhizopus stolonifer
<300> PUBLICATION INFORMATION:
<308> DATABASE ACCESSION NUMBER: GenBank Accession No. ABB96724
<400> SEQUENCE: 65
Met Ser Thr Leu Asp Arg Gln Ser Ile Phe Thr Ile Lys Glu Leu Glu
1 5 10 15
Ser Ile Ser Gln Arg Ile His Asp Gly Asp Glu Glu Ala Met Lys Phe
20 25 30
Ile Ile Ile Asp Lys Lys Val Tyr Asp Val Thr Glu Phe Ile Glu Asp
35 40 45
His Pro Gly Gly Ala Gln Val Leu Leu Thr His Val Gly Lys Asp Ala
50 55 60
Ser Asp Val Phe His Ala Met His Pro Glu Ser Ala Tyr Glu Val Leu
65 70 75 80
Asn Asn Tyr Phe Val Gly Asp Val Gln Glu Thr Val Val Thr Glu Lys
85 90 95
Ser Ser Ser Ala Gln Phe Ala Val Glu Met Arg Gln Leu Arg Asp Gln
100 105 110
Leu Lys Lys Glu Gly Tyr Phe His Ser Ser Lys Leu Phe Tyr Ala Tyr
115 120 125
Lys Val Leu Ser Thr Leu Ala Ile Cys Ile Ala Gly Leu Ser Pro Leu
130 135 140
Tyr Ala Tyr Gly Arg Thr Ser Thr Leu Ala Val Val Ala Ser Ala Ile
145 150 155 160
Thr Val Gly Ile Phe Trp Gln Gln Cys Gly Trp Leu Ala His Asp Phe
165 170 175
Gly His His Gln Cys Phe Glu Asp Arg Thr Trp Asn Asp Val Leu Val
180 185 190
Val Phe Leu Gly Asn Phe Cys Gln Gly Phe Ser Leu Ser Trp Trp Lys
195 200 205
Asn Lys His Asn Thr His His Ala Ser Thr Asn Val His Gly Gln Asp
210 215 220
Pro Asp Ile Asp Thr Ala Pro Val Leu Leu Trp Asp Glu Tyr Ala Ser
225 230 235 240
Ala Ala Tyr Tyr Ala Ser Leu Asp Gln Glu Pro Thr Met Val Ser Arg
245 250 255
Phe Leu Ala Glu Gln Val Leu Pro His Gln Thr Arg Tyr Phe Phe Phe
260 265 270
Ile Leu Ala Phe Ala Arg Leu Ser Trp Ala Leu Gln Ser Leu Ser Tyr
275 280 285
Ser Phe Lys Lys Glu Ser Ile Asn Lys Ser Arg Gln Leu Asn Leu Phe
290 295 300
Glu Arg Val Cys Ile Val Gly His Trp Ala Leu Ser Ala Phe Cys Ile
305 310 315 320
Tyr Ser Trp Cys Ser Asn Val Tyr His Met Val Leu Phe Phe Leu Val
325 330 335
Ser Gln Ala Thr Thr Gly Tyr Thr Leu Ala Leu Val Phe Ala Leu Asn
340 345 350
His Asn Gly Met Pro Val Ile Thr Glu Glu Lys Ala Glu Ser Met Glu
355 360 365
Phe Phe Glu Ile Gln Val Ile Thr Gly Arg Asp Val Thr Leu Ser Pro
370 375 380
Leu Gly Asp Trp Phe Met Gly Gly Leu Asn Tyr Gln Ile Glu His His
385 390 395 400
Val Phe Pro Asn Met Pro Arg His Asn Leu Pro Thr Val Lys Pro Met
405 410 415
Val Lys Ser Leu Cys Gln Lys Tyr Asp Ile Asn Tyr His Asp Thr Gly
420 425 430
Phe Leu Lys Gly Thr Leu Glu Val Leu Gln Thr Leu Asp Ile Thr Ser
435 440 445
Lys Leu Ser Leu Gln Leu Ser Lys Lys Ser Phe
450 455
<210> SEQ ID NO 66
<211> LENGTH: 427
<212> TYPE: PRT
<213> ORGANISM: Pavlova salina
<300> PUBLICATION INFORMATION:
<302> TITLE: SYNTHESIS OF LONG-CHAIN POLYUNSATURATED FATTY ACIDS BY
RECOMBINANT CELLS
<310> PATENT DOCUMENT NUMBER: WO 2005/103253
<311> PATENT FILING DATE: 2005-04-22
<312> PUBLICATION DATE: 2005-11-03
<313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(427)
<400> SEQUENCE: 66
Met Gly Arg Gly Gly Asp Ser Ser Gly Gln Ala His Pro Ala Ala Glu
1 5 10 15
Leu Ala Val Pro Ser Asp Arg Ala Glu Val Ser Asn Ala Asp Ser Lys
20 25 30
Ala Leu His Ile Val Leu Tyr Gly Lys Arg Val Asp Val Thr Lys Phe
35 40 45
Gln Arg Thr His Pro Gly Gly Ser Lys Val Phe Arg Ile Phe Gln Asp
50 55 60
Arg Asp Ala Thr Glu Gln Phe Glu Ser Tyr His Ser Lys Arg Ala Ile
65 70 75 80
Lys Met Met Glu Gly Met Leu Lys Lys Ser Glu Asp Ala Pro Ala Asp
85 90 95
Thr Pro Leu Pro Ser Gln Ser Pro Met Gly Lys Asp Phe Lys Ala Met
100 105 110
Ile Glu Arg His Val Ala Ala Gly Tyr Tyr Asp Pro Cys Pro Leu Asp
115 120 125
Glu Leu Phe Lys Leu Ser Leu Val Leu Leu Pro Thr Phe Ala Gly Met
130 135 140
Tyr Met Leu Lys Ala Gly Val Gly Ser Pro Leu Cys Gly Ala Leu Met
145 150 155 160
Val Ser Phe Gly Trp Tyr Leu Asp Gly Trp Leu Ala His Asp Tyr Leu
165 170 175
His His Ser Val Phe Lys Gly Ser Val Ala Arg Thr Val Gly Trp Asn
180 185 190
Asn Ala Ala Gly Tyr Phe Leu Gly Phe Val Gln Gly Tyr Ala Val Glu
195 200 205
Trp Trp Arg Ala Arg His Asn Thr His His Val Cys Thr Asn Glu Asp
210 215 220
Gly Ser Asp Pro Asp Ile Lys Thr Ala Pro Leu Leu Ile Tyr Val Arg
225 230 235 240
Asn Lys Pro Ser Ile Ala Lys Arg Leu Asn Ala Phe Gln Arg Tyr Gln
245 250 255
Gln Tyr Tyr Tyr Val Pro Val Met Ala Ile Leu Asp Leu Tyr Trp Arg
260 265 270
Leu Glu Ser Ile Ala Tyr Val Ala Met Arg Leu Pro Lys Met Leu Pro
275 280 285
Gln Ala Leu Ala Leu Val Ala His Tyr Ala Ile Val Ala Trp Val Phe
290 295 300
Ala Gly Asn Tyr His Leu Leu Pro Leu Val Thr Val Leu Arg Gly Phe
305 310 315 320
Gly Thr Gly Ile Thr Val Phe Ala Thr His Tyr Gly Glu Asp Ile Leu
325 330 335
Asp Ala Asp Gln Val Arg His Met Thr Leu Val Glu Gln Thr Ala Leu
340 345 350
Thr Ser Arg Asn Ile Ser Gly Gly Trp Leu Val Asn Val Leu Thr Gly
355 360 365
Phe Ile Ser Leu Gln Thr Glu His His Leu Phe Pro Met Met Pro Thr
370 375 380
Gly Asn Leu Met Thr Ile Gln Pro Glu Val Arg Ala Phe Phe Lys Lys
385 390 395 400
His Gly Leu Glu Tyr Arg Glu Gly Asn Leu Ile Glu Cys Val Arg Gln
405 410 415
Asn Ile Arg Ala Leu Ala Phe Glu His Leu Leu
420 425
<210> SEQ ID NO 67
<211> LENGTH: 1338
<212> TYPE: DNA
<213> ORGANISM: Mortierella alpina
<400> SEQUENCE: 67
atgggaacgg accaaggaaa aaccttcacc tgggaagagc tggcggccca taacaccaag 60
gacgacctac tcttggccat ccgcggcagg gtgtacgatg tcacaaagtt cttgagccgc 120
catcctggtg gagtggacac tctcctgctc ggagctggcc gagatgttac tccggtcttt 180
gagatgtatc acgcgtttgg ggctgcagat gccattatga agaagtacta tgtcggtaca 240
ctggtctcga atgagctgcc catcttcccg gagccaacgg tgttccacaa aaccatcaag 300
acgagagtcg agggctactt tacggatcgg aacattgatc ccaagaatag accagagatc 360
tggggacgat acgctcttat ctttggatcc ttgatcgctt cctactacgc gcagctcttt 420
gtgcctttcg ttgtcgaacg cacatggctt caggtggtgt ttgcaatcat catgggattt 480
gcgtgcgcac aagtcggact caaccctctt catgatgcgt ctcacttttc agtgacccac 540
aaccccactg tctggaagat tctgggagcc acgcacgact ttttcaacgg agcatcgtac 600
ctggtgtgga tgtaccaaca tatgctcggc catcacccct acaccaacat tgctggagca 660
gatcccgacg tgtcgacgtc tgagcccgat gttcgtcgta tcaagcccaa ccaaaagtgg 720
tttgtcaacc acatcaacca gcacatgttt gttcctttcc tgtacggact gctggcgttc 780
aaggtgcgca ttcaggacat caacattttg tactttgtca agaccaatga cgctattcgt 840
gtcaatccca tctcgacatg gcacactgtg atgttctggg gcggcaaggc tttctttgtc 900
tggtatcgcc tgattgttcc cctgcagtat ctgcccctgg gcaaggtgct gctcttgttc 960
acggtcgcgg acatggtgtc gtcttactgg ctggcgctga ccttccaggc gaaccacgtt 1020
gttgaggaag ttcagtggcc gttgcctgac gagaacggga tcatccaaaa ggactgggca 1080
gctatgcagg tcgagactac gcaggattac gcacacgatt cgcacctctg gaccagcatc 1140
actggcagct tgaactacca ggctgtgcac catctgttcc ccaacgtgtc gcagcaccat 1200
tatcccgata ttctggccat catcaagaac acctgcagcg agtacaaggt tccatacctt 1260
gtcaaggata cgttttggca agcatttgct tcacatttgg agcacttgcg tgttcttgga 1320
ctccgtccca aggaagag 1338
<210> SEQ ID NO 68
<211> LENGTH: 446
<212> TYPE: PRT
<213> ORGANISM: Mortierella alpina
<400> SEQUENCE: 68
Met Gly Thr Asp Gln Gly Lys Thr Phe Thr Trp Glu Glu Leu Ala Ala
1 5 10 15
His Asn Thr Lys Asp Asp Leu Leu Leu Ala Ile Arg Gly Arg Val Tyr
20 25 30
Asp Val Thr Lys Phe Leu Ser Arg His Pro Gly Gly Val Asp Thr Leu
35 40 45
Leu Leu Gly Ala Gly Arg Asp Val Thr Pro Val Phe Glu Met Tyr His
50 55 60
Ala Phe Gly Ala Ala Asp Ala Ile Met Lys Lys Tyr Tyr Val Gly Thr
65 70 75 80
Leu Val Ser Asn Glu Leu Pro Ile Phe Pro Glu Pro Thr Val Phe His
85 90 95
Lys Thr Ile Lys Thr Arg Val Glu Gly Tyr Phe Thr Asp Arg Asn Ile
100 105 110
Asp Pro Lys Asn Arg Pro Glu Ile Trp Gly Arg Tyr Ala Leu Ile Phe
115 120 125
Gly Ser Leu Ile Ala Ser Tyr Tyr Ala Gln Leu Phe Val Pro Phe Val
130 135 140
Val Glu Arg Thr Trp Leu Gln Val Val Phe Ala Ile Ile Met Gly Phe
145 150 155 160
Ala Cys Ala Gln Val Gly Leu Asn Pro Leu His Asp Ala Ser His Phe
165 170 175
Ser Val Thr His Asn Pro Thr Val Trp Lys Ile Leu Gly Ala Thr His
180 185 190
Asp Phe Phe Asn Gly Ala Ser Tyr Leu Val Trp Met Tyr Gln His Met
195 200 205
Leu Gly His His Pro Tyr Thr Asn Ile Ala Gly Ala Asp Pro Asp Val
210 215 220
Ser Thr Ser Glu Pro Asp Val Arg Arg Ile Lys Pro Asn Gln Lys Trp
225 230 235 240
Phe Val Asn His Ile Asn Gln His Met Phe Val Pro Phe Leu Tyr Gly
245 250 255
Leu Leu Ala Phe Lys Val Arg Ile Gln Asp Ile Asn Ile Leu Tyr Phe
260 265 270
Val Lys Thr Asn Asp Ala Ile Arg Val Asn Pro Ile Ser Thr Trp His
275 280 285
Thr Val Met Phe Trp Gly Gly Lys Ala Phe Phe Val Trp Tyr Arg Leu
290 295 300
Ile Val Pro Leu Gln Tyr Leu Pro Leu Gly Lys Val Leu Leu Leu Phe
305 310 315 320
Thr Val Ala Asp Met Val Ser Ser Tyr Trp Leu Ala Leu Thr Phe Gln
325 330 335
Ala Asn His Val Val Glu Glu Val Gln Trp Pro Leu Pro Asp Glu Asn
340 345 350
Gly Ile Ile Gln Lys Asp Trp Ala Ala Met Gln Val Glu Thr Thr Gln
355 360 365
Asp Tyr Ala His Asp Ser His Leu Trp Thr Ser Ile Thr Gly Ser Leu
370 375 380
Asn Tyr Gln Ala Val His His Leu Phe Pro Asn Val Ser Gln His His
385 390 395 400
Tyr Pro Asp Ile Leu Ala Ile Ile Lys Asn Thr Cys Ser Glu Tyr Lys
405 410 415
Val Pro Tyr Leu Val Lys Asp Thr Phe Trp Gln Ala Phe Ala Ser His
420 425 430
Leu Glu His Leu Arg Val Leu Gly Leu Arg Pro Lys Glu Glu
435 440 445
<210> SEQ ID NO 69
<211> LENGTH: 6473
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Plasmid pY5-22
<400> SEQUENCE: 69
ggtggagctc cagcttttgt tccctttagt gagggttaat ttcgagcttg gcgtaatcat 60
ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacgtacgag 120
ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg 180
cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 240
tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca 300
ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 360
taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 420
agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 480
cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 540
tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 600
tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 660
gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 720
acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 780
acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 840
cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 900
gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 960
gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 1020
agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 1080
ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 1140
ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 1200
atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 1260
tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 1320
gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 1380
ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 1440
caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 1500
cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 1560
cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 1620
cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 1680
agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 1740
tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 1800
agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 1860
atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 1920
ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 1980
cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 2040
caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat 2100
attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 2160
agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgcgc 2220
cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 2280
ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 2340
ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 2400
tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc 2460
cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 2520
tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga 2580
ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 2640
attttaacaa aatattaacg cttacaattt ccattcgcca ttcaggctgc gcaactgttg 2700
ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc 2760
tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac 2820
ggccagtgaa ttgtaatacg actcactata gggcgaattg ggtaccgggc cccccctcga 2880
ggtcgatggt gtcgataagc ttgatatcga attcatgtca cacaaaccga tcttcgcctc 2940
aaggaaacct aattctacat ccgagagact gccgagatcc agtctacact gattaatttt 3000
cgggccaata atttaaaaaa atcgtgttat ataatattat atgtattata tatatacatc 3060
atgatgatac tgacagtcat gtcccattgc taaatagaca gactccatct gccgcctcca 3120
actgatgttc tcaatattta aggggtcatc tcgcattgtt taataataaa cagactccat 3180
ctaccgcctc caaatgatgt tctcaaaata tattgtatga acttattttt attacttagt 3240
attattagac aacttacttg ctttatgaaa aacacttcct atttaggaaa caatttataa 3300
tggcagttcg ttcatttaac aatttatgta gaataaatgt tataaatgcg tatgggaaat 3360
cttaaatatg gatagcataa atgatatctg cattgcctaa ttcgaaatca acagcaacga 3420
aaaaaatccc ttgtacaaca taaatagtca tcgagaaata tcaactatca aagaacagct 3480
attcacacgt tactattgag attattattg gacgagaatc acacactcaa ctgtctttct 3540
ctcttctaga aatacaggta caagtatgta ctattctcat tgttcatact tctagtcatt 3600
tcatcccaca tattccttgg atttctctcc aatgaatgac attctatctt gcaaattcaa 3660
caattataat aagatatacc aaagtagcgg tatagtggca atcaaaaagc ttctctggtg 3720
tgcttctcgt atttattttt attctaatga tccattaaag gtatatattt atttcttgtt 3780
atataatcct tttgtttatt acatgggctg gatacataaa ggtattttga tttaattttt 3840
tgcttaaatt caatcccccc tcgttcagtg tcaactgtaa tggtaggaaa ttaccatact 3900
tttgaagaag caaaaaaaat gaaagaaaaa aaaaatcgta tttccaggtt agacgttccg 3960
cagaatctag aatgcggtat gcggtacatt gttcttcgaa cgtaaaagtt gcgctccctg 4020
agatattgta catttttgct tttacaagta caagtacatc gtacaactat gtactactgt 4080
tgatgcatcc acaacagttt gttttgtttt tttttgtttt ttttttttct aatgattcat 4140
taccgctatg tatacctact tgtacttgta gtaagccggg ttattggcgt tcaattaatc 4200
atagacttat gaatctgcac ggtgtgcgct gcgagttact tttagcttat gcatgctact 4260
tgggtgtaat attgggatct gttcggaaat caacggatgc tcaaccgatt tcgacagtaa 4320
ttaattaagt catacacaag tcagctttct tcgagcctca tataagtata agtagttcaa 4380
cgtattagca ctgtacccag catctccgta tcgagaaaca caacaacatg ccccattgga 4440
cagatcatgc ggatacacag gttgtgcagt atcatacata ctcgatcaga caggtcgtct 4500
gaccatcata caagctgaac aagcgctcca tacttgcacg ctctctatat acacagttaa 4560
attacatatc catagtctaa cctctaacag ttaatcttct ggtaagcctc ccagccagcc 4620
ttctggtatc gcttggcctc ctcaatagga tctcggttct ggccgtacag acctcggccg 4680
acaattatga tatccgttcc ggtagacatg acatcctcaa cagttcggta ctgctgtccg 4740
agagcgtctc ccttgtcgtc aagacccacc ccgggggtca gaataagcca gtcctcagag 4800
tcgcccttag gtcggttctg ggcaatgaag ccaaccacaa actcggggtc ggatcgggca 4860
agctcaatgg tctgcttgga gtactcgcca gtggccagag agcccttgca agacagctcg 4920
gccagcatga gcagacctct ggccagcttc tcgttgggag aggggactag gaactccttg 4980
tactgggagt tctcgtagtc agagacgtcc tccttcttct gttcagagac agtttcctcg 5040
gcaccagctc gcaggccagc aatgattccg gttccgggta caccgtgggc gttggtgata 5100
tcggaccact cggcgattcg gtgacaccgg tactggtgct tgacagtgtt gccaatatct 5160
gcgaactttc tgtcctcgaa caggaagaaa ccgtgcttaa gagcaagttc cttgaggggg 5220
agcacagtgc cggcgtaggt gaagtcgtca atgatgtcga tatgggtttt gatcatgcac 5280
acataaggtc cgaccttatc ggcaagctca atgagctcct tggtggtggt aacatccaga 5340
gaagcacaca ggttggtttt cttggctgcc acgagcttga gcactcgagc ggcaaaggcg 5400
gacttgtgga cgttagctcg agcttcgtag gagggcattt tggtggtgaa gaggagactg 5460
aaataaattt agtctgcaga actttttatc ggaaccttat ctggggcagt gaagtatatg 5520
ttatggtaat agttacgagt tagttgaact tatagataga ctggactata cggctatcgg 5580
tccaaattag aaagaacgtc aatggctctc tgggcgtcgc ctttgccgac aaaaatgtga 5640
tcatgatgaa agccagcaat gacgttgcag ctgatattgt tgtcggccaa ccgcgccgaa 5700
aacgcagctg tcagacccac agcctccaac gaagaatgta tcgtcaaagt gatccaagca 5760
cactcatagt tggagtcgta ctccaaaggc ggcaatgacg agtcagacag atactcgtcg 5820
actcaggcga cgacggaatt cctgcagccc atctgcagaa ttcaggagag accgggttgg 5880
cggcgtattt gtgtcccaaa aaacagcccc aattgcccca attgacccca aattgaccca 5940
gtagcgggcc caaccccggc gagagccccc ttcaccccac atatcaaacc tcccccggtt 6000
cccacacttg ccgttaaggg cgtagggtac tgcagtctgg aatctacgct tgttcagact 6060
ttgtactagt ttctttgtct ggccatccgg gtaacccatg ccggacgcaa aatagactac 6120
tgaaaatttt tttgctttgt ggttgggact ttagccaagg gtataaaaga ccaccgtccc 6180
cgaattacct ttcctcttct tttctctctc tccttgtcaa ctcacacccg aaatcgttaa 6240
gcatttcctt ctgagtataa gaatcattca ccatggatcc actagttcta gagcggccgc 6300
caccgcggcc cgagattccg gcctcttcgg ccgccaagcg acccgggtgg acgtctagag 6360
gtacctagca attaacagat agtttgccgg tgataattct cttaacctcc cacactcctt 6420
tgacataacg atttatgtaa cgaaactgaa atttgaccag atattgtgtc cgc 6473
<210> SEQ ID NO 70
<211> LENGTH: 6970
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Plasmid pY5-22GPD
<400> SEQUENCE: 70
tcgacgcagt aggatgtcct gcacgggtct ttttgtgggg tgtggagaaa ggggtgcttg 60
gagatggaag ccggtagaac cgggctgctt gtgcttggag atggaagccg gtagaaccgg 120
gctgcttggg gggatttggg gccgctgggc tccaaagagg ggtaggcatt tcgttggggt 180
tacgtaattg cggcatttgg gtcctgcgcg catgtcccat tggtcagaat tagtccggat 240
aggagactta tcagccaatc acagcgccgg atccacctgt aggttgggtt gggtgggagc 300
acccctccac agagtagagt caaacagcag cagcaacatg atagttgggg gtgtgcgtgt 360
taaaggaaaa aaaagaagct tgggttatat tcccgctcta tttagaggtt gcgggataga 420
cgccgacgga gggcaatggc gccatggaac cttgcggata tcgatacgcc gcggcggact 480
gcgtccgaac cagctccagc agcgtttttt ccgggccatt gagccgactg cgaccccgcc 540
aacgtgtctt ggcccacgca ctcatgtcat gttggtgttg ggaggccact ttttaagtag 600
cacaaggcac ctagctcgca gcaaggtgtc cgaaccaaag aagcggctgc agtggtgcaa 660
acggggcgga aacggcggga aaaagccacg ggggcacgaa ttgaggcacg ccctcgaatt 720
tgagacgagt cacggcccca ttcgcccgcg caatggctcg ccaacgcccg gtcttttgca 780
ccacatcagg ttaccccaag ccaaaccttt gtgttaaaaa gcttaacata ttataccgaa 840
cgtaggtttg ggcgggcttg ctccgtctgt ccaaggcaac atttatataa gggtctgcat 900
cgccggctca attgaatctt ttttcttctt ctcttctcta tattcattct tgaattaaac 960
acacatcaat ccgcggccgc caccgcggcc cgagattccg gcctcttcgg ccgccaagcg 1020
acccgggtgg acgtctagag gtacctagca attaacagat agtttgccgg tgataattct 1080
cttaacctcc cacactcctt tgacataacg atttatgtaa cgaaactgaa atttgaccag 1140
atattgtgtc cgcggtggag ctccagcttt tgttcccttt agtgagggtt aatttcgagc 1200
ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 1260
cacaacgtac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 1320
ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 1380
ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 1440
gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 1500
cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 1560
tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 1620
cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 1680
aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 1740
cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 1800
gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 1860
ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 1920
cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 1980
aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 2040
tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc 2100
ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 2160
tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 2220
ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 2280
agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 2340
atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 2400
cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 2460
ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 2520
ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 2580
agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 2640
agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 2700
gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 2760
cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 2820
gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 2880
tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 2940
tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 3000
aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 3060
cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 3120
cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 3180
aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 3240
ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 3300
tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 3360
ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc 3420
gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt 3480
ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc 3540
cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt 3600
agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt 3660
aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt 3720
gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa 3780
aaatttaacg cgaattttaa caaaatatta acgcttacaa tttccattcg ccattcaggc 3840
tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga 3900
aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac 3960
gttgtaaaac gacggccagt gaattgtaat acgactcact atagggcgaa ttgggtaccg 4020
ggccccccct cgaggtcgat ggtgtcgata agcttgatat cgaattcatg tcacacaaac 4080
cgatcttcgc ctcaaggaaa cctaattcta catccgagag actgccgaga tccagtctac 4140
actgattaat tttcgggcca ataatttaaa aaaatcgtgt tatataatat tatatgtatt 4200
atatatatac atcatgatga tactgacagt catgtcccat tgctaaatag acagactcca 4260
tctgccgcct ccaactgatg ttctcaatat ttaaggggtc atctcgcatt gtttaataat 4320
aaacagactc catctaccgc ctccaaatga tgttctcaaa atatattgta tgaacttatt 4380
tttattactt agtattatta gacaacttac ttgctttatg aaaaacactt cctatttagg 4440
aaacaattta taatggcagt tcgttcattt aacaatttat gtagaataaa tgttataaat 4500
gcgtatggga aatcttaaat atggatagca taaatgatat ctgcattgcc taattcgaaa 4560
tcaacagcaa cgaaaaaaat cccttgtaca acataaatag tcatcgagaa atatcaacta 4620
tcaaagaaca gctattcaca cgttactatt gagattatta ttggacgaga atcacacact 4680
caactgtctt tctctcttct agaaatacag gtacaagtat gtactattct cattgttcat 4740
acttctagtc atttcatccc acatattcct tggatttctc tccaatgaat gacattctat 4800
cttgcaaatt caacaattat aataagatat accaaagtag cggtatagtg gcaatcaaaa 4860
agcttctctg gtgtgcttct cgtatttatt tttattctaa tgatccatta aaggtatata 4920
tttatttctt gttatataat ccttttgttt attacatggg ctggatacat aaaggtattt 4980
tgatttaatt ttttgcttaa attcaatccc ccctcgttca gtgtcaactg taatggtagg 5040
aaattaccat acttttgaag aagcaaaaaa aatgaaagaa aaaaaaaatc gtatttccag 5100
gttagacgtt ccgcagaatc tagaatgcgg tatgcggtac attgttcttc gaacgtaaaa 5160
gttgcgctcc ctgagatatt gtacattttt gcttttacaa gtacaagtac atcgtacaac 5220
tatgtactac tgttgatgca tccacaacag tttgttttgt ttttttttgt tttttttttt 5280
tctaatgatt cattaccgct atgtatacct acttgtactt gtagtaagcc gggttattgg 5340
cgttcaatta atcatagact tatgaatctg cacggtgtgc gctgcgagtt acttttagct 5400
tatgcatgct acttgggtgt aatattggga tctgttcgga aatcaacgga tgctcaaccg 5460
atttcgacag taattaatta agtcatacac aagtcagctt tcttcgagcc tcatataagt 5520
ataagtagtt caacgtatta gcactgtacc cagcatctcc gtatcgagaa acacaacaac 5580
atgccccatt ggacagatca tgcggataca caggttgtgc agtatcatac atactcgatc 5640
agacaggtcg tctgaccatc atacaagctg aacaagcgct ccatacttgc acgctctcta 5700
tatacacagt taaattacat atccatagtc taacctctaa cagttaatct tctggtaagc 5760
ctcccagcca gccttctggt atcgcttggc ctcctcaata ggatctcggt tctggccgta 5820
cagacctcgg ccgacaatta tgatatccgt tccggtagac atgacatcct caacagttcg 5880
gtactgctgt ccgagagcgt ctcccttgtc gtcaagaccc accccggggg tcagaataag 5940
ccagtcctca gagtcgccct taggtcggtt ctgggcaatg aagccaacca caaactcggg 6000
gtcggatcgg gcaagctcaa tggtctgctt ggagtactcg ccagtggcca gagagccctt 6060
gcaagacagc tcggccagca tgagcagacc tctggccagc ttctcgttgg gagaggggac 6120
taggaactcc ttgtactggg agttctcgta gtcagagacg tcctccttct tctgttcaga 6180
gacagtttcc tcggcaccag ctcgcaggcc agcaatgatt ccggttccgg gtacaccgtg 6240
ggcgttggtg atatcggacc actcggcgat tcggtgacac cggtactggt gcttgacagt 6300
gttgccaata tctgcgaact ttctgtcctc gaacaggaag aaaccgtgct taagagcaag 6360
ttccttgagg gggagcacag tgccggcgta ggtgaagtcg tcaatgatgt cgatatgggt 6420
tttgatcatg cacacataag gtccgacctt atcggcaagc tcaatgagct ccttggtggt 6480
ggtaacatcc agagaagcac acaggttggt tttcttggct gccacgagct tgagcactcg 6540
agcggcaaag gcggacttgt ggacgttagc tcgagcttcg taggagggca ttttggtggt 6600
gaagaggaga ctgaaataaa tttagtctgc agaacttttt atcggaacct tatctggggc 6660
agtgaagtat atgttatggt aatagttacg agttagttga acttatagat agactggact 6720
atacggctat cggtccaaat tagaaagaac gtcaatggct ctctgggcgt cgcctttgcc 6780
gacaaaaatg tgatcatgat gaaagccagc aatgacgttg cagctgatat tgttgtcggc 6840
caaccgcgcc gaaaacgcag ctgtcagacc cacagcctcc aacgaagaat gtatcgtcaa 6900
agtgatccaa gcacactcat agttggagtc gtactccaaa ggcggcaatg acgagtcaga 6960
cagatactcg 6970
<210> SEQ ID NO 71
<211> LENGTH: 968
<212> TYPE: DNA
<213> ORGANISM: Yarrowia lipolytica
<300> PUBLICATION INFORMATION:
<302> TITLE: GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE AND
PHOSPHOGLYCERATE
MUTASE PROMOTERS FOR GENE EXPRESSION IN OLEAGINOUS YEAST
<310> PATENT DOCUMENT NUMBER: US-2005-0014270-A1
<311> PATENT FILING DATE: 2004-06-16
<312> PUBLICATION DATE: 2005-01-20
<313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(968)
<400> SEQUENCE: 71
tcgacgcagt aggatgtcct gcacgggtct ttttgtgggg tgtggagaaa ggggtgcttg 60
gagatggaag ccggtagaac cgggctgctt gtgcttggag atggaagccg gtagaaccgg 120
gctgcttggg gggatttggg gccgctgggc tccaaagagg ggtaggcatt tcgttggggt 180
tacgtaattg cggcatttgg gtcctgcgcg catgtcccat tggtcagaat tagtccggat 240
aggagactta tcagccaatc acagcgccgg atccacctgt aggttgggtt gggtgggagc 300
acccctccac agagtagagt caaacagcag cagcaacatg atagttgggg gtgtgcgtgt 360
taaaggaaaa aaaagaagct tgggttatat tcccgctcta tttagaggtt gcgggataga 420
cgccgacgga gggcaatggc gccatggaac cttgcggata tcgatacgcc gcggcggact 480
gcgtccgaac cagctccagc agcgtttttt ccgggccatt gagccgactg cgaccccgcc 540
aacgtgtctt ggcccacgca ctcatgtcat gttggtgttg ggaggccact ttttaagtag 600
cacaaggcac ctagctcgca gcaaggtgtc cgaaccaaag aagcggctgc agtggtgcaa 660
acggggcgga aacggcggga aaaagccacg ggggcacgaa ttgaggcacg ccctcgaatt 720
tgagacgagt cacggcccca ttcgcccgcg caatggctcg ccaacgcccg gtcttttgca 780
ccacatcagg ttaccccaag ccaaaccttt gtgttaaaaa gcttaacata ttataccgaa 840
cgtaggtttg ggcgggcttg ctccgtctgt ccaaggcaac atttatataa gggtctgcat 900
cgccggctca attgaatctt ttttcttctt ctcttctcta tattcattct tgaattaaac 960
acacatca 968
<210> SEQ ID NO 72
<211> LENGTH: 8630
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Plasmid pYZDE2-S
<400> SEQUENCE: 72
ggtggagctc cagcttttgt tccctttagt gagggttaat ttcgagcttg gcgtaatcat 60
ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacgtacgag 120
ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg 180
cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 240
tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca 300
ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 360
taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 420
agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 480
cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 540
tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 600
tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 660
gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 720
acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 780
acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 840
cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 900
gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 960
gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 1020
agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 1080
ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 1140
ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 1200
atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 1260
tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 1320
gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 1380
ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 1440
caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 1500
cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 1560
cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 1620
cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 1680
agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 1740
tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 1800
agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 1860
atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 1920
ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 1980
cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 2040
caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat 2100
attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 2160
agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgcgc 2220
cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 2280
ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 2340
ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 2400
tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc 2460
cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 2520
tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga 2580
ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 2640
attttaacaa aatattaacg cttacaattt ccattcgcca ttcaggctgc gcaactgttg 2700
ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc 2760
tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac 2820
ggccagtgaa ttgtaatacg actcactata gggcgaattg ggtaccgggc cccccctcga 2880
ggtcgatggt gtcgataagc ttgatatcga attcatgtca cacaaaccga tcttcgcctc 2940
aaggaaacct aattctacat ccgagagact gccgagatcc agtctacact gattaatttt 3000
cgggccaata atttaaaaaa atcgtgttat ataatattat atgtattata tatatacatc 3060
atgatgatac tgacagtcat gtcccattgc taaatagaca gactccatct gccgcctcca 3120
actgatgttc tcaatattta aggggtcatc tcgcattgtt taataataaa cagactccat 3180
ctaccgcctc caaatgatgt tctcaaaata tattgtatga acttattttt attacttagt 3240
attattagac aacttacttg ctttatgaaa aacacttcct atttaggaaa caatttataa 3300
tggcagttcg ttcatttaac aatttatgta gaataaatgt tataaatgcg tatgggaaat 3360
cttaaatatg gatagcataa atgatatctg cattgcctaa ttcgaaatca acagcaacga 3420
aaaaaatccc ttgtacaaca taaatagtca tcgagaaata tcaactatca aagaacagct 3480
attcacacgt tactattgag attattattg gacgagaatc acacactcaa ctgtctttct 3540
ctcttctaga aatacaggta caagtatgta ctattctcat tgttcatact tctagtcatt 3600
tcatcccaca tattccttgg atttctctcc aatgaatgac attctatctt gcaaattcaa 3660
caattataat aagatatacc aaagtagcgg tatagtggca atcaaaaagc ttctctggtg 3720
tgcttctcgt atttattttt attctaatga tccattaaag gtatatattt atttcttgtt 3780
atataatcct tttgtttatt acatgggctg gatacataaa ggtattttga tttaattttt 3840
tgcttaaatt caatcccccc tcgttcagtg tcaactgtaa tggtaggaaa ttaccatact 3900
tttgaagaag caaaaaaaat gaaagaaaaa aaaaatcgta tttccaggtt agacgttccg 3960
cagaatctag aatgcggtat gcggtacatt gttcttcgaa cgtaaaagtt gcgctccctg 4020
agatattgta catttttgct tttacaagta caagtacatc gtacaactat gtactactgt 4080
tgatgcatcc acaacagttt gttttgtttt tttttgtttt ttttttttct aatgattcat 4140
taccgctatg tatacctact tgtacttgta gtaagccggg ttattggcgt tcaattaatc 4200
atagacttat gaatctgcac ggtgtgcgct gcgagttact tttagcttat gcatgctact 4260
tgggtgtaat attgggatct gttcggaaat caacggatgc tcaaccgatt tcgacagtaa 4320
taatttgaat cgaatcggag cctaaaatga acccgagtat atctcataaa attctcggtg 4380
agaggtctgt gactgtcagt acaaggtgcc ttcattatgc cctcaacctt accatacctc 4440
actgaatgta gtgtacctct aaaaatgaaa tacagtgcca aaagccaagg cactgagctc 4500
gtctaacgga cttgatatac aaccaattaa aacaaatgaa aagaaataca gttctttgta 4560
tcatttgtaa caattaccct gtacaaacta aggtattgaa atcccacaat attcccaaag 4620
tccacccctt tccaaattgt catgcctaca actcatatac caagcactaa cctaccaaac 4680
accactaaaa ccccacaaaa tatatcttac cgaatataca gtaacaagct accaccacac 4740
tcgttgggtg cagtcgccag cttaaagata tctatccaca tcagccacaa ctcccttcct 4800
ttaataaacc gactacaccc ttggctattg aggttatgag tgaatatact gtagacaaga 4860
cactttcaag aagactgttt ccaaaacgta ccactgtcct ccactacaaa cacacccaat 4920
ctgcttcttc tagtcaaggt tgctacaccg gtaaattata aatcatcatt tcattagcag 4980
ggcagggccc tttttataga gtcttataca ctagcggacc ctgccggtag accaacccgc 5040
aggcgcgtca gtttgctcct tccatcaatg cgtcgtagaa acgacttact ccttcttgag 5100
cagctccttg accttgttgg caacaagtct ccgacctcgg aggtggagga agagcctccg 5160
atatcggcgg tagtgatacc agcctcgacg gactccttga cggcagcctc aacagcgtca 5220
ccggcgggct tcatgttaag agagaacttg agcatcatgg cggcagacag aatggtggca 5280
atggggttga ccttctgctt gccgagatcg ggggcagatc cgtgacaggg ctcgtacaga 5340
ccgaacgcct cgttggtgtc gggcagagaa gccagagagg cggagggcag cagacccaga 5400
gaaccgggga tgacggaggc ctcgtcggag atgatatcgc caaacatgtt ggtggtgatg 5460
atgataccat tcatcttgga gggctgcttg atgaggatca tggcggccga gtcgatcagc 5520
tggtggttga gctcgagctg ggggaattcg tccttgagga ctcgagtgac agtctttcgc 5580
caaagtcgag aggaggccag cacgttggcc ttgtcaagag accacacggg aagagggggg 5640
ttgtgctgaa gggccaggaa ggcggccatt cgggcaattc gctcaacctc aggaacggag 5700
taggtctcgg tgtcggaagc gacgccagat ccgtcatcct cctttcgctc tccaaagtag 5760
atacctccga cgagctctcg gacaatgatg aagtcggtgc cctcaacgtt tcggatgggg 5820
gagagatcgg cgagcttggg cgacagcagc tggcagggtc gcaggttggc gtacaggttc 5880
aggtcctttc gcagcttgag gagaccctgc tcgggtcgca cgtcggttcg tccgtcggga 5940
gtggtccata cggtgttggc agcgcctccg acagcaccga gcataataga gtcagccttt 6000
cggcagatgt cgagagtagc gtcggtgatg ggctcgccct ccttctcaat ggcagctcct 6060
ccaatgagtc ggtcctcaaa cacaaactcg gtgccggagg cctcagcaac agacttgagc 6120
accttgacgg cctcggcaat cacctcgggg ccacagaagt cgccgccgag aagaacaatc 6180
ttcttggagt cagtcttggt cttcttagtt tcgggttcca ttgtggatgt gtgtggttgt 6240
atgtgtgatg tggtgtgtgg agtgaaaatc tgtggctggc aaacgctctt gtatatatac 6300
gcacttttgc ccgtgctatg tggaagacta aacctccgaa gattgtgact caggtagtgc 6360
ggtatcggct agggacccaa accttgtcga tgccgatagc gctatcgaac gtaccccagc 6420
cggccgggag tatgtcggag gggacatacg agatcgtcaa gggtttgtgg ccaactggta 6480
aatttaaatg atgtcgacgc agtaggatgt cctgcacggg tctttttgtg gggtgtggag 6540
aaaggggtgc ttggagatgg aagccggtag aaccgggctg cttgtgcttg gagatggaag 6600
ccggtagaac cgggctgctt ggggggattt ggggccgctg ggctccaaag aggggtaggc 6660
atttcgttgg ggttacgtaa ttgcggcatt tgggtcctgc gcgcatgtcc cattggtcag 6720
aattagtccg gataggagac ttatcagcca atcacagcgc cggatccacc tgtaggttgg 6780
gttgggtggg agcacccctc cacagagtag agtcaaacag cagcagcaac atgatagttg 6840
ggggtgtgcg tgttaaagga aaaaaaagaa gcttgggtta tattcccgct ctatttagag 6900
gttgcgggat agacgccgac ggagggcaat ggcgccatgg aaccttgcgg atatcgatac 6960
gccgcggcgg actgcgtccg aaccagctcc agcagcgttt tttccgggcc attgagccga 7020
ctgcgacccc gccaacgtgt cttggcccac gcactcatgt catgttggtg ttgggaggcc 7080
actttttaag tagcacaagg cacctagctc gcagcaaggt gtccgaacca aagaagcggc 7140
tgcagtggtg caaacggggc ggaaacggcg ggaaaaagcc acgggggcac gaattgaggc 7200
acgccctcga atttgagacg agtcacggcc ccattcgccc gcgcaatggc tcgccaacgc 7260
ccggtctttt gcaccacatc aggttacccc aagccaaacc tttgtgttaa aaagcttaac 7320
atattatacc gaacgtaggt ttgggcgggc ttgctccgtc tgtccaaggc aacatttata 7380
taagggtctg catcgccggc tcaattgaat cttttttctt cttctcttct ctatattcat 7440
tcttgaatta aacacacatc aatccatggc aaacagcagc gtgtgggatg atgtggtggg 7500
ccgcgtggag accggcgtgg accagtggat ggatggcgcc aagccgtacg cactcaccga 7560
tgggctcccg atgatggacg tgtccaccat gctggcattc gaggtgggat acatggccat 7620
gctgctcttc ggcatcccga tcatgaagca gatggagaag ccttttgagc tcaagaccat 7680
caagctcttg cacaacttgt ttctcttcgg actttccttg tacatgtgcg tggagaccat 7740
ccgccaggct atcctcggag gctacaaagt gtttggaaac gacatggaga agggcaacga 7800
gtctcatgct cagggcatgt ctcgcatcgt gtacgtgttc tacgtgtcca aggcatacga 7860
gttcttggat accgccatca tgatcctttg caagaagttc aaccaggttt ccttcttgca 7920
tgtgtaccac catgccactc atttttgcca tctggtgggc tatccgccaa gtacgctcca 7980
ggaggtgatg cgtacttttt cagtgatcct caactctttc gtgcacaccg tcatgtacgg 8040
catactactt cttctcctcc caagggttcg ggttcgtgaa gccaatcaag ccgtacatca 8100
ccacccttca gatgacccag ttcatggcaa tgcttgtgca gtccttgtac gactacctct 8160
tcccatgcga ctacccacag gctcttgtgc agctccttgg agtgtacatg atcaccttgc 8220
ttgccctctt cggcaacttt tttgtgcaga gctatcttaa aaagccaaaa aagagcaaga 8280
ccaactaaaa ctgcctgcat gatatgccgc tcgccggcgt tcgaattgac tcagaaagcg 8340
agttaaggcg acacgcaaac tctatatttt ttcaaacgtg ttgccgtcac tcattcgcca 8400
tctgtttact acgtgtctgt tcaatgagca tgttcttgaa tctaaagaat ctcgaatgtt 8460
ttttaaaaaa agaattcgat atcaagctta cgcgtcgacc cgggtggacg tctagaggta 8520
cctagcaatt aacagatagt ttgccggtga taattctctt aacctcccac actcctttga 8580
cataacgatt tatgtaacga aactgaaatt tgaccagata ttgtgtccgc 8630
<210> SEQ ID NO 73
<211> LENGTH: 18
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Primer GPDsense
<400> SEQUENCE: 73
atacgagatc gtcaaggg 18
<210> SEQ ID NO 74
<211> LENGTH: 26
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Primer GPDantisense
<400> SEQUENCE: 74
gcggccgcgg attgatgtgt gtttaa 26
<210> SEQ ID NO 75
<211> LENGTH: 6339
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Plasmid pKR136
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (4078)..(4078)
<223> OTHER INFORMATION: n is a, c, g, or t
<400> SEQUENCE: 75
ggccgcgaca caagtgtgag agtactaaat aaatgctttg gttgtacgaa atcattacac 60
taaataaaat aatcaaagct tatatatgcc ttccgctaag gccgaatgca aagaaattgg 120
ttctttctcg ttatcttttg ccacttttac tagtacgtat taattactac ttaatcatct 180
ttgtttacgg ctcattatat ccgtacgtcg agtcgacctg caggcatgca agcttggcgt 240
aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca 300
tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat 360
taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt 420
aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct 480
cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 540
aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 600
aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 660
tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 720
caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 780
cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 840
ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 900
gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 960
agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 1020
gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 1080
acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 1140
gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 1200
gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 1260
cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 1320
caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 1380
gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 1440
cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 1500
cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 1560
caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 1620
gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 1680
gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 1740
cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 1800
catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 1860
gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 1920
ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 1980
gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 2040
cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 2100
tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 2160
gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 2220
atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 2280
ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 2340
gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 2400
acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc 2460
cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg 2520
agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt 2580
cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac 2640
tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca 2700
tcaggcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct 2760
cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa 2820
cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattc gagctcggta 2880
cccggggatc ctctagacgt acgtcctcga agagaagggt taataacaca ttttttaaca 2940
tttttaacac aaattttagt tatttaaaaa tttattaaaa aatttaaaat aagaagagga 3000
actctttaaa taaatctaac ttacaaaatt tatgattttt aataagtttt caccaataaa 3060
aaatgtcata aaaatatgtt aaaaagtata ttatcaatat tctctttatg ataaataaaa 3120
agaaaaaaaa aataaaagtt aagtgaaaat gagattgaag tgactttagg tgtgtataaa 3180
tatatcaacc ccgccaacaa tttatttaat ccaaatatat tgaagtatat tattccatag 3240
cctttattta tttatatatt tattatataa aagctttatt tgttctaggt tgttcatgaa 3300
atattttttt ggttttatct ccgttgtaag aaaatcatgt gctttgtgtc gccactcact 3360
attgcagctt tttcatgcat tggtcagatt gacggttgat tgtatttttg ttttttatgg 3420
ttttgtgtta tgacttaagt cttcatctct ttatctcttc atcaggtttg atggttacct 3480
aatatggtcc atgggtacat gcatggttaa attaggtggc caactttgtt gtgaacgata 3540
gaattttttt tatattaagt aaactatttt tatattatga aataataata aaaaaaatat 3600
tttatcatta ttaacaaaat catattagtt aatttgttaa ctctataata aaagaaatac 3660
tgtaacattc acattacatg gtaacatctt tccacccttt catttgtttt ttgtttgatg 3720
actttttttc ttgtttaaat ttatttccct tcttttaaat ttggaataca ttatcatcat 3780
atataaacta aaatactaaa aacaggatta cacaaatgat aaataataac acaaatattt 3840
ataaatctag ctgcaatata tttaaactag ctatatcgat attgtaaaat aaaactagct 3900
gcattgatac tgataaaaaa atatcatgtg ctttctggac tgatgatgca gtatactttt 3960
gacattgcct ttattttatt tttcagaaaa gctttcttag ttctgggttc ttcattattt 4020
gtttcccatc tccattgtga attgaatcat ttgcttcgtg tcacaaatac aatttagnta 4080
ggtacatgca ttggtcagat tcacggttta ttatgtcatg acttaagttc atggtagtac 4140
attacctgcc acgcatgcat tatattggtt agatttgata ggcaaatttg gttgtcaaca 4200
atataaatat aaataatgtt tttatattac gaaataacag tgatcaaaac aaacagtttt 4260
atctttatta acaagatttt gtttttgttt gatgacgttt tttaatgttt acgctttccc 4320
ccttcttttg aatttagaac actttatcat cataaaatca aatactaaaa aaattacata 4380
tttcataaat aataacacaa atatttttaa aaaatctgaa ataataatga acaatattac 4440
atattatcac gaaaattcat taataaaaat attatataaa taaaatgtaa tagtagttat 4500
atgtaggaaa aaagtactgc acgcataata tatacaaaaa gattaaaatg aactattata 4560
aataataaca ctaaattaat ggtgaatcat atcaaaataa tgaaaaagta aataaaattt 4620
gtaattaact tctatatgta ttacacacac aaataataaa taatagtaaa aaaaattatg 4680
ataaatattt accatctcat aagatattta aaataatgat aaaaatatag attatttttt 4740
atgcaactag ctagccaaaa agagaacacg ggtatatata aaaagagtac ctttaaattc 4800
tactgtactt cctttattcc tgacgttttt atatcaagtg gacatacgtg aagattttaa 4860
ttatcagtct aaatatttca ttagcactta atacttttct gttttattcc tatcctataa 4920
gtagtcccga ttctcccaac attgcttatt cacacaacta actaagaaag tcttccatag 4980
ccccccaagc ggccgcatgg gaacggacca aggaaaaacc ttcacctggg aagagctggc 5040
ggcccataac accaaggacg acctactctt ggccatccgc ggcagggtgt acgatgtcac 5100
aaagttcttg agccgccatc ctggtggagt ggacactctc ctgctcggag ctggccgaga 5160
tgttactccg gtctttgaga tgtatcacgc gtttggggct gcagatgcca ttatgaagaa 5220
gtactatgtc ggtacactgg tctcgaatga gctgcccatc ttcccggagc caacggtgtt 5280
ccacaaaacc atcaagacga gagtcgaggg ctactttacg gatcggaaca ttgatcccaa 5340
gaatagacca gagatctggg gacgatacgc tcttatcttt ggatccttga tcgcttccta 5400
ctacgcgcag ctctttgtgc ctttcgttgt cgaacgcaca tggcttcagg tggtgtttgc 5460
aatcatcatg ggatttgcgt gcgcacaagt cggactcaac cctcttcatg atgcgtctca 5520
cttttcagtg acccacaacc ccactgtctg gaagattctg ggagccacgc acgacttttt 5580
caacggagca tcgtacctgg tgtggatgta ccaacatatg ctcggccatc acccctacac 5640
caacattgct ggagcagatc ccgacgtgtc gacgtctgag cccgatgttc gtcgtatcaa 5700
gcccaaccaa aagtggtttg tcaaccacat caaccagcac atgtttgttc ctttcctgta 5760
cggactgctg gcgttcaagg tgcgcattca ggacatcaac attttgtact ttgtcaagac 5820
caatgacgct attcgtgtca atcccatctc gacatggcac actgtgatgt tctggggcgg 5880
caaggctttc tttgtctggt atcgcctgat tgttcccctg cagtatctgc ccctgggcaa 5940
ggtgctgctc ttgttcacgg tcgcggacat ggtgtcgtct tactggctgg cgctgacctt 6000
ccaggcgaac cacgttgttg aggaagttca gtggccgttg cctgacgaga acgggatcat 6060
ccaaaaggac tgggcagcta tgcaggtcga gactacgcag gattacgcac acgattcgca 6120
cctctggacc agcatcactg gcagcttgaa ctaccaggct gtgcaccatc tgttccccaa 6180
cgtgtcgcag caccattatc ccgatattct ggccatcatc aagaacacct gcagcgagta 6240
caaggttcca taccttgtca aggatacgtt ttggcaagca tttgcttcac atttggagca 6300
cttgcgtgtt cttggactcc gtcccaagga agagtaggc 6339
<210> SEQ ID NO 76
<211> LENGTH: 8319
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Plasmid pY98
<400> SEQUENCE: 76
ggccgccacc gcggcccgag attccggcct cttcggccgc caagcgaccc gggtggacgt 60
ctagaggtac ctagcaatta acagatagtt tgccggtgat aattctctta acctcccaca 120
ctcctttgac ataacgattt atgtaacgaa actgaaattt gaccagatat tgtgtccgcg 180
gtggagctcc agcttttgtt ccctttagtg agggttaatt tcgagcttgg cgtaatcatg 240
gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acgtacgagc 300
cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc 360
gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat 420
cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac 480
tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 540
aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca 600
gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 660
ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 720
ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 780
gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag 840
ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 900
cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 960
cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 1020
gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 1080
aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 1140
tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 1200
gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc 1260
tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag 1320
gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata 1380
tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat 1440
ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg 1500
ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc 1560
tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc 1620
aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc 1680
gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc 1740
gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc 1800
ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa 1860
gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat 1920
gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata 1980
gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca 2040
tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag 2100
gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc 2160
agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc 2220
aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata 2280
ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta 2340
gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgcgcc 2400
ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact 2460
tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc 2520
cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt 2580
acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc 2640
ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt 2700
gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat 2760
tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa 2820
ttttaacaaa atattaacgc ttacaatttc cattcgccat tcaggctgcg caactgttgg 2880
gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct 2940
gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg 3000
gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtaccgggcc ccccctcgag 3060
gtcgatggtg tcgataagct tgatatcgaa ttcatgtcac acaaaccgat cttcgcctca 3120
aggaaaccta attctacatc cgagagactg ccgagatcca gtctacactg attaattttc 3180
gggccaataa tttaaaaaaa tcgtgttata taatattata tgtattatat atatacatca 3240
tgatgatact gacagtcatg tcccattgct aaatagacag actccatctg ccgcctccaa 3300
ctgatgttct caatatttaa ggggtcatct cgcattgttt aataataaac agactccatc 3360
taccgcctcc aaatgatgtt ctcaaaatat attgtatgaa cttattttta ttacttagta 3420
ttattagaca acttacttgc tttatgaaaa acacttccta tttaggaaac aatttataat 3480
ggcagttcgt tcatttaaca atttatgtag aataaatgtt ataaatgcgt atgggaaatc 3540
ttaaatatgg atagcataaa tgatatctgc attgcctaat tcgaaatcaa cagcaacgaa 3600
aaaaatccct tgtacaacat aaatagtcat cgagaaatat caactatcaa agaacagcta 3660
ttcacacgtt actattgaga ttattattgg acgagaatca cacactcaac tgtctttctc 3720
tcttctagaa atacaggtac aagtatgtac tattctcatt gttcatactt ctagtcattt 3780
catcccacat attccttgga tttctctcca atgaatgaca ttctatcttg caaattcaac 3840
aattataata agatatacca aagtagcggt atagtggcaa tcaaaaagct tctctggtgt 3900
gcttctcgta tttattttta ttctaatgat ccattaaagg tatatattta tttcttgtta 3960
tataatcctt ttgtttatta catgggctgg atacataaag gtattttgat ttaatttttt 4020
gcttaaattc aatcccccct cgttcagtgt caactgtaat ggtaggaaat taccatactt 4080
ttgaagaagc aaaaaaaatg aaagaaaaaa aaaatcgtat ttccaggtta gacgttccgc 4140
agaatctaga atgcggtatg cggtacattg ttcttcgaac gtaaaagttg cgctccctga 4200
gatattgtac atttttgctt ttacaagtac aagtacatcg tacaactatg tactactgtt 4260
gatgcatcca caacagtttg ttttgttttt ttttgttttt tttttttcta atgattcatt 4320
accgctatgt atacctactt gtacttgtag taagccgggt tattggcgtt caattaatca 4380
tagacttatg aatctgcacg gtgtgcgctg cgagttactt ttagcttatg catgctactt 4440
gggtgtaata ttgggatctg ttcggaaatc aacggatgct caaccgattt cgacagtaat 4500
taattaagtc atacacaagt cagctttctt cgagcctcat ataagtataa gtagttcaac 4560
gtattagcac tgtacccagc atctccgtat cgagaaacac aacaacatgc cccattggac 4620
agatcatgcg gatacacagg ttgtgcagta tcatacatac tcgatcagac aggtcgtctg 4680
accatcatac aagctgaaca agcgctccat acttgcacgc tctctatata cacagttaaa 4740
ttacatatcc atagtctaac ctctaacagt taatcttctg gtaagcctcc cagccagcct 4800
tctggtatcg cttggcctcc tcaataggat ctcggttctg gccgtacaga cctcggccga 4860
caattatgat atccgttccg gtagacatga catcctcaac agttcggtac tgctgtccga 4920
gagcgtctcc cttgtcgtca agacccaccc cgggggtcag aataagccag tcctcagagt 4980
cgcccttagg tcggttctgg gcaatgaagc caaccacaaa ctcggggtcg gatcgggcaa 5040
gctcaatggt ctgcttggag tactcgccag tggccagaga gcccttgcaa gacagctcgg 5100
ccagcatgag cagacctctg gccagcttct cgttgggaga ggggactagg aactccttgt 5160
actgggagtt ctcgtagtca gagacgtcct ccttcttctg ttcagagaca gtttcctcgg 5220
caccagctcg caggccagca atgattccgg ttccgggtac accgtgggcg ttggtgatat 5280
cggaccactc ggcgattcgg tgacaccggt actggtgctt gacagtgttg ccaatatctg 5340
cgaactttct gtcctcgaac aggaagaaac cgtgcttaag agcaagttcc ttgaggggga 5400
gcacagtgcc ggcgtaggtg aagtcgtcaa tgatgtcgat atgggttttg atcatgcaca 5460
cataaggtcc gaccttatcg gcaagctcaa tgagctcctt ggtggtggta acatccagag 5520
aagcacacag gttggttttc ttggctgcca cgagcttgag cactcgagcg gcaaaggcgg 5580
acttgtggac gttagctcga gcttcgtagg agggcatttt ggtggtgaag aggagactga 5640
aataaattta gtctgcagaa ctttttatcg gaaccttatc tggggcagtg aagtatatgt 5700
tatggtaata gttacgagtt agttgaactt atagatagac tggactatac ggctatcggt 5760
ccaaattaga aagaacgtca atggctctct gggcgtcgcc tttgccgaca aaaatgtgat 5820
catgatgaaa gccagcaatg acgttgcagc tgatattgtt gtcggccaac cgcgccgaaa 5880
acgcagctgt cagacccaca gcctccaacg aagaatgtat cgtcaaagtg atccaagcac 5940
actcatagtt ggagtcgtac tccaaaggcg gcaatgacga gtcagacaga tactcgtcga 6000
cgcagtagga tgtcctgcac gggtcttttt gtggggtgtg gagaaagggg tgcttggaga 6060
tggaagccgg tagaaccggg ctgcttgtgc ttggagatgg aagccggtag aaccgggctg 6120
cttgggggga tttggggccg ctgggctcca aagaggggta ggcatttcgt tggggttacg 6180
taattgcggc atttgggtcc tgcgcgcatg tcccattggt cagaattagt ccggatagga 6240
gacttatcag ccaatcacag cgccggatcc acctgtaggt tgggttgggt gggagcaccc 6300
ctccacagag tagagtcaaa cagcagcagc aacatgatag ttgggggtgt gcgtgttaaa 6360
ggaaaaaaaa gaagcttggg ttatattccc gctctattta gaggttgcgg gatagacgcc 6420
gacggagggc aatggcgcca tggaaccttg cggatatcga tacgccgcgg cggactgcgt 6480
ccgaaccagc tccagcagcg ttttttccgg gccattgagc cgactgcgac cccgccaacg 6540
tgtcttggcc cacgcactca tgtcatgttg gtgttgggag gccacttttt aagtagcaca 6600
aggcacctag ctcgcagcaa ggtgtccgaa ccaaagaagc ggctgcagtg gtgcaaacgg 6660
ggcggaaacg gcgggaaaaa gccacggggg cacgaattga ggcacgccct cgaatttgag 6720
acgagtcacg gccccattcg cccgcgcaat ggctcgccaa cgcccggtct tttgcaccac 6780
atcaggttac cccaagccaa acctttgtgt taaaaagctt aacatattat accgaacgta 6840
ggtttgggcg ggcttgctcc gtctgtccaa ggcaacattt atataagggt ctgcatcgcc 6900
ggctcaattg aatctttttt cttcttctct tctctatatt cattcttgaa ttaaacacac 6960
atcaatccgc ggccgcatgg gaacggacca aggaaaaacc ttcacctggg aagagctggc 7020
ggcccataac accaaggacg acctactctt ggccatccgc ggcagggtgt acgatgtcac 7080
aaagttcttg agccgccatc ctggtggagt ggacactctc ctgctcggag ctggccgaga 7140
tgttactccg gtctttgaga tgtatcacgc gtttggggct gcagatgcca ttatgaagaa 7200
gtactatgtc ggtacactgg tctcgaatga gctgcccatc ttcccggagc caacggtgtt 7260
ccacaaaacc atcaagacga gagtcgaggg ctactttacg gatcggaaca ttgatcccaa 7320
gaatagacca gagatctggg gacgatacgc tcttatcttt ggatccttga tcgcttccta 7380
ctacgcgcag ctctttgtgc ctttcgttgt cgaacgcaca tggcttcagg tggtgtttgc 7440
aatcatcatg ggatttgcgt gcgcacaagt cggactcaac cctcttcatg atgcgtctca 7500
cttttcagtg acccacaacc ccactgtctg gaagattctg ggagccacgc acgacttttt 7560
caacggagca tcgtacctgg tgtggatgta ccaacatatg ctcggccatc acccctacac 7620
caacattgct ggagcagatc ccgacgtgtc gacgtctgag cccgatgttc gtcgtatcaa 7680
gcccaaccaa aagtggtttg tcaaccacat caaccagcac atgtttgttc ctttcctgta 7740
cggactgctg gcgttcaagg tgcgcattca ggacatcaac attttgtact ttgtcaagac 7800
caatgacgct attcgtgtca atcccatctc gacatggcac actgtgatgt tctggggcgg 7860
caaggctttc tttgtctggt atcgcctgat tgttcccctg cagtatctgc ccctgggcaa 7920
ggtgctgctc ttgttcacgg tcgcggacat ggtgtcgtct tactggctgg cgctgacctt 7980
ccaggcgaac cacgttgttg aggaagttca gtggccgttg cctgacgaga acgggatcat 8040
ccaaaaggac tgggcagcta tgcaggtcga gactacgcag gattacgcac acgattcgca 8100
cctctggacc agcatcactg gcagcttgaa ctaccaggct gtgcaccatc tgttccccaa 8160
cgtgtcgcag caccattatc ccgatattct ggccatcatc aagaacacct gcagcgagta 8220
caaggttcca taccttgtca aggatacgtt ttggcaagca tttgcttcac atttggagca 8280
cttgcgtgtt cttggactcc gtcccaagga agagtaggc 8319
<210> SEQ ID NO 77
<211> LENGTH: 774
<212> TYPE: DNA
<213> ORGANISM: Euglena gracilis
<400> SEQUENCE: 77
atggaggtgg tgaatgaaat agtctcaatt gggcaggaag ttttacccaa agttgattat 60
gcccaactct ggagtgatgc cagtcactgt gaggtgcttt acttgtccat cgcatttgtc 120
atcttgaagt tcactcttgg cccccttggt ccaaaaggtc agtctcgtat gaagtttgtt 180
ttcaccaatt acaaccttct catgtccatt tattcgttgg gatcattcct ctcaatggca 240
tatgccatgt acaccatcgg tgttatgtct gacaactgcg agaaggcttt tgacaacaac 300
gtcttcagga tcaccacgca gttgttctat ttgagcaagt tcctggagta tattgactcc 360
ttctatttgc cactgatggg caagcctctg acctggttgc aattcttcca tcatttgggg 420
gcaccgatgg atatgtggct gttctataat taccgaaatg aagctgtttg gatttttgtg 480
ctgttgaatg gtttcatcca ctggatcatg tacggttatt attggaccag attgatcaag 540
ctgaagttcc ccatgccaaa atccctgatt acatcaatgc agatcattca attcaatgtt 600
ggtttctaca ttgtctggaa gtacaggaac attccctgtt atcgccaaga tgggatgagg 660
atgtttggct ggttcttcaa ttacttttat gttggcacag tcttgtgttt gttcttgaat 720
ttctatgtgc aaacgtatat cgtcaggaag cacaagggag ccaaaaagat tcag 774
<210> SEQ ID NO 78
<211> LENGTH: 1263
<212> TYPE: DNA
<213> ORGANISM: Euglena gracilis
<400> SEQUENCE: 78
atgaagtcaa agcgccaagc gcttcccctt acaattgatg gaacaacata tgatgtgtct 60
gcctgggtca atttccaccc tggtggtgcg gaaattatag agaattacca aggaagggat 120
gccactgatg ccttcatggt tatgcactct caagaagcct tcgacaagct caagcgcatg 180
cccaaaatca atcccagttc tgagttgcca ccccaggctg cagtgaatga agctcaagag 240
gatttccgga agctccgaga agagttgatc gcaactggca tgtttgatgc ctcccccctc 300
tggtactcat acaaaatcag caccacactg ggccttggag tgctgggtta tttcctgatg 360
gttcagtatc agatgtattt cattggggca gtgttgcttg ggatgcacta tcaacagatg 420
ggctggcttt ctcatgacat ttgccaccac cagactttca agaaccggaa ctggaacaac 480
ctcgtgggac tggtatttgg caatggtctg caaggttttt ccgtgacatg gtggaaggac 540
agacacaatg cacatcattc ggcaaccaat gttcaagggc acgaccctga tattgacaac 600
ctccccctct tagcctggtc tgaggatgac gtcacacggg cgtcaccgat ttcccgcaag 660
ctcattcagt tccagcagta ctatttcttg gtcatctgta tcttgttgcg gttcatttgg 720
tgtttccaga gcgtgttgac cgtgcgcagt ttgaaggaca gagataacca attctatcgc 780
tctcagtata agaaggaggc cattggcctc gccctgcact ggaccttgaa gaccctgttc 840
cacttattct ttatgcccag catcctcaca tcgctgttgg tgtttttcgt ttcggagctg 900
gttggcggct tcggcattgc gatcgtggtg ttcatgaacc actacccact ggagaagatc 960
ggggactcag tctgggatgg ccatggattc tcggttggcc agatccatga gaccatgaac 1020
attcggcgag ggattatcac agattggttt ttcggaggct tgaattacca gattgagcac 1080
catttgtggc cgaccctccc tcgccacaac ctgacagcgg ttagctacca ggtggaacag 1140
ctgtgccaga agcacaacct gccgtatcgg aacccgctgc cccatgaagg gttggtcatc 1200
ctgctgcgct atctggcggt gttcgcccgg atggcggaga agcaacccgc ggggaaggct 1260
cta 1263
<210> SEQ ID NO 79
<211> LENGTH: 30
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Primer oEugEL1-1
<400> SEQUENCE: 79
agcggccgca ccatggaggt ggtgaatgaa 30
<210> SEQ ID NO 80
<211> LENGTH: 30
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Primer oEugEL1-2
<400> SEQUENCE: 80
tgcggccgct cactgaatct ttttggctcc 30
<210> SEQ ID NO 81
<211> LENGTH: 4311
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Plasmid pKR906
<400> SEQUENCE: 81
agcggccgca ccatggaggt ggtgaatgaa atagtctcaa ttgggcagga agttttaccc 60
aaagttgatt atgcccaact ctggagtgat gccagtcact gtgaggtgct ttacttgtcc 120
atcgcatttg tcatcttgaa gttcactctt ggcccccttg gtccaaaagg tcagtctcgt 180
atgaagtttg ttttcaccaa ttacaacctt ctcatgtcca tttattcgtt gggatcattc 240
ctctcaatgg catatgccat gtacaccatc ggtgttatgt ctgacaactg cgagaaggct 300
tttgacaaca acgtcttcag gatcaccacg cagttgttct atttgagcaa gttcctggag 360
tatattgact ccttctattt gccactgatg ggcaagcctc tgacctggtt gcaattcttc 420
catcatttgg gggcaccgat ggatatgtgg ctgttctata attaccgaaa tgaagctgtt 480
tggatttttg tgctgttgaa tggtttcatc cactggatca tgtacggtta ttattggacc 540
agattgatca agctgaagtt ccccatgcca aaatccctga ttacatcaat gcagatcatt 600
caattcaatg ttggtttcta cattgtctgg aagtacagga acattccctg ttatcgccaa 660
gatgggatga ggatgtttgg ctggttcttc aattactttt atgttggcac agtcttgtgt 720
ttgttcttga atttctatgt gcaaacgtat atcgtcagga agcacaaggg agccaaaaag 780
attcagtgag cggccgcacc tgaattccag cacactggcg gccgttacta gtggatccga 840
gctcggtacc aagcttgatg catagcttga gtattctaac gcgtcaccta aatagcttgg 900
cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca 960
acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca 1020
cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 1080
attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt 1140
cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 1200
caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 1260
caaaaggcca gcaaaagccc aggaaccgta aaaaggccgc gttgctggcg tttttccata 1320
ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 1380
cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 1440
ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 1500
tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 1560
gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 1620
ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 1680
ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 1740
gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 1800
aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 1860
tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 1920
ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 1980
tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt agcacgtgtc 2040
agtcctgctc ctcggccacg aagtgcacgc agttgccggc cgggtcgcgc agggcgaact 2100
cccgccccca cggctgctcg ccgatctcgg tcatggccgg cccggaggcg tcccggaagt 2160
tcgtggacac gacctccgac cactcggcgt acagctcgtc caggccgcgc acccacaccc 2220
aggccagggt gttgtccggc accacctggt cctggaccgc gctgatgaac agggtcacgt 2280
cgtcccggac cacaccggcg aagtcgtcct ccacgaagtc ccgggagaac ccgagccggt 2340
cggtccagaa ctcgaccgct ccggcgacgt cgcgcgcggt gagcaccgga acggcactgg 2400
tcaacttggc catggtggcc ctcctcacgt gctattattg aagcatttat cagggttatt 2460
gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 2520
gcacatttcc ccgaaaagtg ccacctgtat gcggtgtgaa ataccgcaca gatgcgtaag 2580
gagaaaatac cgcatcagga aattgtaagc gttaataatt cagaagaact cgtcaagaag 2640
gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg 2700
gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg ctatgtcctg 2760
atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc ggccattttc 2820
caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct cgccgtcggg 2880
catgctcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat gctcttcgtc 2940
cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct cgatgcgatg 3000
tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc 3060
atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga gatcctgccc 3120
cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt cgagcacagc 3180
tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt cttgcagttc 3240
attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct gcgctgacag 3300
ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat agccgaatag 3360
cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa 3420
cgatcctcat cctgtctctt gatcagagct tgatcccctg cgccatcaga tccttggcgg 3480
cgagaaagcc atccagttta ctttgcaggg cttcccaacc ttaccagagg gcgccccagc 3540
tggcaattcc ggttcgcttg ctgtccataa aaccgcccag tctagctatc gccatgtaag 3600
cccactgcaa gctacctgct ttctctttgc gcttgcgttt tcccttgtcc agatagccca 3660
gtagctgaca ttcatccggg gtcagcaccg tttctgcgga ctggctttct acgtgaaaag 3720
gatctaggtg aagatccttt ttgataatct catgcctgac atttatattc cccagaacat 3780
caggttaatg gcgtttttga tgtcattttc gcggtggctg agatcagcca cttcttcccc 3840
gataacggag accggcacac tggccatatc ggtggtcatc atgcgccagc tttcatcccc 3900
gatatgcacc accgggtaaa gttcacggga gactttatct gacagcagac gtgcactggc 3960
cagggggatc accatccgtc gccccggcgt gtcaataata tcactctgta catccacaaa 4020
cagacgataa cggctctctc ttttataggt gtaaacctta aactgccgta cgtataggct 4080
gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 4140
agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg 4200
ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tgggccctct 4260
agatgcatgc tcgagcggcc gccagtgtga tggatatctg cagaattcag g 4311
<210> SEQ ID NO 82
<211> LENGTH: 7085
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Plasmid pKR72
<400> SEQUENCE: 82
gtacggatcc gtcgacggcg cgcccgatca tccggatata gttcctcctt tcagcaaaaa 60
acccctcaag acccgtttag aggccccaag gggttatgct agttattgct cagcggtggc 120
agcagccaac tcagcttcct ttcgggcttt gttagcagcc ggatcgatcc aagctgtacc 180
tcactattcc tttgccctcg gacgagtgct ggggcgtcgg tttccactat cggcgagtac 240
ttctacacag ccatcggtcc agacggccgc gcttctgcgg gcgatttgtg tacgcccgac 300
agtcccggct ccggatcgga cgattgcgtc gcatcgaccc tgcgcccaag ctgcatcatc 360
gaaattgccg tcaaccaagc tctgatagag ttggtcaaga ccaatgcgga gcatatacgc 420
ccggagccgc ggcgatcctg caagctccgg atgcctccgc tcgaagtagc gcgtctgctg 480
ctccatacaa gccaaccacg gcctccagaa gaagatgttg gcgacctcgt attgggaatc 540
cccgaacatc gcctcgctcc agtcaatgac cgctgttatg cggccattgt ccgtcaggac 600
attgttggag ccgaaatccg cgtgcacgag gtgccggact tcggggcagt cctcggccca 660
aagcatcagc tcatcgagag cctgcgcgac ggacgcactg acggtgtcgt ccatcacagt 720
ttgccagtga tacacatggg gatcagcaat cgcgcatatg aaatcacgcc atgtagtgta 780
ttgaccgatt ccttgcggtc cgaatgggcc gaacccgctc gtctggctaa gatcggccgc 840
agcgatcgca tccatagcct ccgcgaccgg ctgcagaaca gcgggcagtt cggtttcagg 900
caggtcttgc aacgtgacac cctgtgcacg gcgggagatg caataggtca ggctctcgct 960
gaattcccca atgtcaagca cttccggaat cgggagcgcg gccgatgcaa agtgccgata 1020
aacataacga tctttgtaga aaccatcggc gcagctattt acccgcagga catatccacg 1080
ccctcctaca tcgaagctga aagcacgaga ttcttcgccc tccgagagct gcatcaggtc 1140
ggagacgctg tcgaactttt cgatcagaaa cttctcgaca gacgtcgcgg tgagttcagg 1200
cttttccatg ggtatatctc cttcttaaag ttaaacaaaa ttatttctag agggaaaccg 1260
ttgtggtctc cctatagtga gtcgtattaa tttcgcggga tcgagatcga tccaattcca 1320
atcccacaaa aatctgagct taacagcaca gttgctcctc tcagagcaga atcgggtatt 1380
caacaccctc atatcaacta ctacgttgtg tataacggtc cacatgccgg tatatacgat 1440
gactggggtt gtacaaaggc ggcaacaaac ggcgttcccg gagttgcaca caagaaattt 1500
gccactatta cagaggcaag agcagcagct gacgcgtaca caacaagtca gcaaacagac 1560
aggttgaact tcatccccaa aggagaagct caactcaagc ccaagagctt tgctaaggcc 1620
ctaacaagcc caccaaagca aaaagcccac tggctcacgc taggaaccaa aaggcccagc 1680
agtgatccag ccccaaaaga gatctccttt gccccggaga ttacaatgga cgatttcctc 1740
tatctttacg atctaggaag gaagttcgaa ggtgaaggtg acgacactat gttcaccact 1800
gataatgaga aggttagcct cttcaatttc agaaagaatg ctgacccaca gatggttaga 1860
gaggcctacg cagcaggtct catcaagacg atctacccga gtaacaatct ccaggagatc 1920
aaataccttc ccaagaaggt taaagatgca gtcaaaagat tcaggactaa ttgcatcaag 1980
aacacagaga aagacatatt tctcaagatc agaagtacta ttccagtatg gacgattcaa 2040
ggcttgcttc ataaaccaag gcaagtaata gagattggag tctctaaaaa ggtagttcct 2100
actgaatcta aggccatgca tggagtctaa gattcaaatc gaggatctaa cagaactcgc 2160
cgtgaagact ggcgaacagt tcatacagag tcttttacga ctcaatgaca agaagaaaat 2220
cttcgtcaac atggtggagc acgacactct ggtctactcc aaaaatgtca aagatacagt 2280
ctcagaagac caaagggcta ttgagacttt tcaacaaagg ataatttcgg gaaacctcct 2340
cggattccat tgcccagcta tctgtcactt catcgaaagg acagtagaaa aggaaggtgg 2400
ctcctacaaa tgccatcatt gcgataaagg aaaggctatc attcaagatg cctctgccga 2460
cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag aagacgttcc 2520
aaccacgtct tcaaagcaag tggattgatg tgacatctcc actgacgtaa gggatgacgc 2580
acaatcccac tatccttcgc aagacccttc ctctatataa ggaagttcat ttcatttgga 2640
gaggacacgc tcgagctcat ttctctatta cttcagccat aacaaaagaa ctcttttctc 2700
ttcttattaa accatgaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat 2760
cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc 2820
tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg 2880
tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga 2940
agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca 3000
gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc 3060
ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt 3120
cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga 3180
tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca 3240
ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca 3300
cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga 3360
ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag 3420
gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct 3480
tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca 3540
gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat 3600
cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt 3660
ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg 3720
tccgagggca aaggaatagt gaggtaccta aagaaggagt gcgtcgaagc agatcgttca 3780
aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc 3840
atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta 3900
tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa 3960
aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta 4020
gatcgatgtc gaatcgatca acctgcatta atgaatcggc caacgcgcgg ggagaggcgg 4080
tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 4140
gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 4200
ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4260
ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 4320
acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 4380
tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4440
ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc 4500
ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4560
ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4620
actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 4680
gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 4740
tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4800
caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 4860
atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 4920
acgttaaggg attttggtca tgacattaac ctataaaaat aggcgtatca cgaggccctt 4980
tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac 5040
ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc 5100
gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag 5160
agtgcaccat atggacatat tgtcgttaga acgcggctac aattaataca taaccttatg 5220
tatcatacac atacgattta ggtgacacta tagaacggcg cgccaagctt gttgaaacat 5280
ccctgaagtg tctcatttta ttttatttat tctttgctga taaaaaaata aaataaaaga 5340
agctaagcac acggtcaacc attgctctac tgctaaaagg gttatgtgta gtgttttact 5400
gcataaatta tgcagcaaac aagacaactc aaattaaaaa atttcctttg cttgtttttt 5460
tgttgtctct gacttgactt tcttgtggaa gttggttgta taaggattgg gacaccattg 5520
tccttcttaa tttaatttta ttctttgctg ataaaaaaaa aaatttcata tagtgttaaa 5580
taataatttg ttaaataacc aaaaagtcaa atatgtttac tctcgtttaa ataattgaga 5640
ttcgtccagc aaggctaaac gattgtatag atttatgaca atatttactt ttttatagat 5700
aaatgttata ttataataaa tttatataca tatattatat gttatttatt attattttaa 5760
atccttcaat attttatcaa accaactcat aatttttttt ttatctgtaa gaagcaataa 5820
aattaaatag acccacttta aggatgatcc aacctttata cagagtaaga gagttcaaat 5880
agtacccttt catatacata tcaactaaaa tattagaaat atcatggatc aaaccttata 5940
aagacattaa ataagtggat aagtataata tataaatggg tagtatataa tatataaatg 6000
gatacaaact tctctcttta taattgttat gtctccttaa catcctaata taatacataa 6060
gtgggtaata tataatatat aaatggagac aaacttcttc cattataatt gttatgtctt 6120
cttaacactt atgtctcgtt cacaatgcta aggttagaat tgtttagaaa gtcttatagt 6180
acacatttgt ttttgtacta tttgaagcat tccataagcc gtcacgattc agatgattta 6240
taataataag aggaaattta tcatagaaca ataaggtgca tagatagagt gttaatatat 6300
cataacatcc tttgtttatt catagaagaa gtgagatgga gctcagttat tatactgtta 6360
catggtcgga tacaatattc catgctctcc atgagctctt acacctacat gcattttagt 6420
tcatacttgc ggccgcagta tatcttaaat tctttaatac ggtgtactag gatattgaac 6480
tggttcttga tgatgaaaac ctgggccgag attgcagcta tttatagtca taggtcttgt 6540
taacatgcat ggacatttgg ccacggggtg gcatgcagtt tgacgggtgt tgaaataaac 6600
aaaaatgagg tggcggaaga gaatacgagt ttgaggttgg gttagaaaca acaaatgtga 6660
gggctcatga tgggttgagt tggtgaatgt tttgggctgc tcgattgaca cctttgtgag 6720
tacgtgttgt tgtgcatggc ttttggggtc cagttttttt ttcttgacgc ggcgatcctg 6780
atcagctagt ggataagtga tgtccactgt gtgtgattgc gtttttgttt gaattttatg 6840
aacttagaca ttgctatgca aaggatactc tcattgtgtt ttgtcttctt ttgttccttg 6900
gctttttctt atgatccaag agactagtca gtgttgtggc attcgagact accaagatta 6960
attatgatgg gggaaggata agtaactgat tagtacggac tgttaccaaa ttaattaata 7020
agcggcaaat gaagggcatg gatcaaaagc ttggatctcc tgcaggatct ggccggccgg 7080
atctc 7085
<210> SEQ ID NO 83
<211> LENGTH: 2540
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Plasmid pKS102
<400> SEQUENCE: 83
cgatcatccg gatatagttc ctcctttcag caaaaaaccc ctcaagaccc gtttagaggc 60
cccaaggggt tatgctagtt attgctcagc ggtggcagca gccaactcag cttcctttcg 120
ggctttgtta gcagccggat cgatccaagc tgtacctcac tattcctttg ccctcggacg 180
agtgctgggg cgtcggtttc cactatcggc gagtacttct acacagccat cggtccagac 240
ggccgcgctt ctgcgggcga tttgtgtacg cccgacagtc ccggctccgg atcggacgat 300
tgcgtcgcat cgaccctgcg cccaagctgc atcatcgaaa ttgccgtcaa ccaagctctg 360
atagagttgg tcaagaccaa tgcggagcat atacgcccgg agccgcggcg atcctgcaag 420
ctccggatgc ctccgctcga agtagcgcgt ctgctgctcc atacaagcca accacggcct 480
ccagaagaag atgttggcga cctcgtattg ggaatccccg aacatcgcct cgctccagtc 540
aatgaccgct gttatgcggc cattgtccgt caggacattg ttggagccga aatccgcgtg 600
cacgaggtgc cggacttcgg ggcagtcctc ggcccaaagc atcagctcat cgagagcctg 660
cgcgacggac gcactgacgg tgtcgtccat cacagtttgc cagtgataca catggggatc 720
agcaatcgcg catatgaaat cacgccatgt agtgtattga ccgattcctt gcggtccgaa 780
tgggccgaac ccgctcgtct ggctaagatc ggccgcagcg atcgcatcca tagcctccgc 840
gaccggctgc agaacagcgg gcagttcggt ttcaggcagg tcttgcaacg tgacaccctg 900
tgcacggcgg gagatgcaat aggtcaggct ctcgctgaat tccccaatgt caagcacttc 960
cggaatcggg agcgcggccg atgcaaagtg ccgataaaca taacgatctt tgtagaaacc 1020
atcggcgcag ctatttaccc gcaggacata tccacgccct cctacatcga agctgaaagc 1080
acgagattct tcgccctccg agagctgcat caggtcggag acgctgtcga acttttcgat 1140
cagaaacttc tcgacagacg tcgcggtgag ttcaggcttt tccatgggta tatctccttc 1200
ttaaagttaa acaaaattat ttctagaggg aaaccgttgt ggtctcccta tagtgagtcg 1260
tattaatttc gcgggatcga gatctgatca acctgcatta atgaatcggc caacgcgcgg 1320
ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 1380
cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 1440
cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 1500
accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 1560
acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 1620
cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 1680
acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt 1740
atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 1800
agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 1860
acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 1920
gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg 1980
gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 2040
gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 2100
gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 2160
acgaaaactc acgttaaggg attttggtca tgacattaac ctataaaaat aggcgtatca 2220
cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc 2280
tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg 2340
gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga 2400
ttgtactgag agtgcaccat atggacatat tgtcgttaga acgcggctac aattaataca 2460
taaccttatg tatcatacac atacgattta ggtgacacta tagaacggcg cgccaagctt 2520
ggatccgtcg acggcgcgcc 2540
<210> SEQ ID NO 84
<211> LENGTH: 4359
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Plasmid pKR197
<400> SEQUENCE: 84
cgcgcccgat catccggata tagttcctcc tttcagcaaa aaacccctca agacccgttt 60
agaggcccca aggggttatg ctagttattg ctcagcggtg gcagcagcca actcagcttc 120
ctttcgggct ttgttagcag ccggatcgat ccaagctgta cctcactatt cctttgccct 180
cggacgagtg ctggggcgtc ggtttccact atcggcgagt acttctacac agccatcggt 240
ccagacggcc gcgcttctgc gggcgatttg tgtacgcccg acagtcccgg ctccggatcg 300
gacgattgcg tcgcatcgac cctgcgccca agctgcatca tcgaaattgc cgtcaaccaa 360
gctctgatag agttggtcaa gaccaatgcg gagcatatac gcccggagcc gcggcgatcc 420
tgcaagctcc ggatgcctcc gctcgaagta gcgcgtctgc tgctccatac aagccaacca 480
cggcctccag aagaagatgt tggcgacctc gtattgggaa tccccgaaca tcgcctcgct 540
ccagtcaatg accgctgtta tgcggccatt gtccgtcagg acattgttgg agccgaaatc 600
cgcgtgcacg aggtgccgga cttcggggca gtcctcggcc caaagcatca gctcatcgag 660
agcctgcgcg acggacgcac tgacggtgtc gtccatcaca gtttgccagt gatacacatg 720
gggatcagca atcgcgcata tgaaatcacg ccatgtagtg tattgaccga ttccttgcgg 780
tccgaatggg ccgaacccgc tcgtctggct aagatcggcc gcagcgatcg catccatagc 840
ctccgcgacc ggctgcagaa cagcgggcag ttcggtttca ggcaggtctt gcaacgtgac 900
accctgtgca cggcgggaga tgcaataggt caggctctcg ctgaattccc caatgtcaag 960
cacttccgga atcgggagcg cggccgatgc aaagtgccga taaacataac gatctttgta 1020
gaaaccatcg gcgcagctat ttacccgcag gacatatcca cgccctccta catcgaagct 1080
gaaagcacga gattcttcgc cctccgagag ctgcatcagg tcggagacgc tgtcgaactt 1140
ttcgatcaga aacttctcga cagacgtcgc ggtgagttca ggcttttcca tgggtatatc 1200
tccttcttaa agttaaacaa aattatttct agagggaaac cgttgtggtc tccctatagt 1260
gagtcgtatt aatttcgcgg gatcgagatc tgatcaacct gcattaatga atcggccaac 1320
gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 1380
tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 1440
tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 1500
ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 1560
agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 1620
accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 1680
ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct 1740
gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 1800
ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 1860
gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 1920
taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag 1980
tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 2040
gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 2100
cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 2160
agtggaacga aaactcacgt taagggattt tggtcatgac attaacctat aaaaataggc 2220
gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca 2280
tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc 2340
gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag 2400
agcagattgt actgagagtg caccatatgg acatattgtc gttagaacgc ggctacaatt 2460
aatacataac cttatgtatc atacacatac gatttaggtg acactataga acggcgcgcc 2520
aagcttgttg aaacatccct gaagtgtctc attttatttt atttattctt tgctgataaa 2580
aaaataaaat aaaagaagct aagcacacgg tcaaccattg ctctactgct aaaagggtta 2640
tgtgtagtgt tttactgcat aaattatgca gcaaacaaga caactcaaat taaaaaattt 2700
cctttgcttg tttttttgtt gtctctgact tgactttctt gtggaagttg gttgtataag 2760
gattgggaca ccattgtcct tcttaattta attttattct ttgctgataa aaaaaaaaat 2820
ttcatatagt gttaaataat aatttgttaa ataaccaaaa agtcaaatat gtttactctc 2880
gtttaaataa ttgagattcg tccagcaagg ctaaacgatt gtatagattt atgacaatat 2940
ttactttttt atagataaat gttatattat aataaattta tatacatata ttatatgtta 3000
tttattatta ttttaaatcc ttcaatattt tatcaaacca actcataatt ttttttttat 3060
ctgtaagaag caataaaatt aaatagaccc actttaagga tgatccaacc tttatacaga 3120
gtaagagagt tcaaatagta ccctttcata tacatatcaa ctaaaatatt agaaatatca 3180
tggatcaaac cttataaaga cattaaataa gtggataagt ataatatata aatgggtagt 3240
atataatata taaatggata caaacttctc tctttataat tgttatgtct ccttaacatc 3300
ctaatataat acataagtgg gtaatatata atatataaat ggagacaaac ttcttccatt 3360
ataattgtta tgtcttctta acacttatgt ctcgttcaca atgctaaggt tagaattgtt 3420
tagaaagtct tatagtacac atttgttttt gtactatttg aagcattcca taagccgtca 3480
cgattcagat gatttataat aataagagga aatttatcat agaacaataa ggtgcataga 3540
tagagtgtta atatatcata acatcctttg tttattcata gaagaagtga gatggagctc 3600
agttattata ctgttacatg gtcggataca atattccatg ctctccatga gctcttacac 3660
ctacatgcat tttagttcat acttgcggcc gcagtatatc ttaaattctt taatacggtg 3720
tactaggata ttgaactggt tcttgatgat gaaaacctgg gccgagattg cagctattta 3780
tagtcatagg tcttgttaac atgcatggac atttggccac ggggtggcat gcagtttgac 3840
gggtgttgaa ataaacaaaa atgaggtggc ggaagagaat acgagtttga ggttgggtta 3900
gaaacaacaa atgtgagggc tcatgatggg ttgagttggt gaatgttttg ggctgctcga 3960
ttgacacctt tgtgagtacg tgttgttgtg catggctttt ggggtccagt ttttttttct 4020
tgacgcggcg atcctgatca gctagtggat aagtgatgtc cactgtgtgt gattgcgttt 4080
ttgtttgaat tttatgaact tagacattgc tatgcaaagg atactctcat tgtgttttgt 4140
cttcttttgt tccttggctt tttcttatga tccaagagac tagtcagtgt tgtggcattc 4200
gagactacca agattaatta tgatggggga aggataagta actgattagt acggactgtt 4260
accaaattaa ttaataagcg gcaaatgaag ggcatggatc aaaagcttgg atctcctgca 4320
ggatctggcc ggccggatct cgtacggatc cgtcgacgg 4359
<210> SEQ ID NO 85
<211> LENGTH: 5147
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Plasmid pKR911
<400> SEQUENCE: 85
ggccgcaagt atgaactaaa atgcatgtag gtgtaagagc tcatggagag catggaatat 60
tgtatccgac catgtaacag tataataact gagctccatc tcacttcttc tatgaataaa 120
caaaggatgt tatgatatat taacactcta tctatgcacc ttattgttct atgataaatt 180
tcctcttatt attataaatc atctgaatcg tgacggctta tggaatgctt caaatagtac 240
aaaaacaaat gtgtactata agactttcta aacaattcta accttagcat tgtgaacgag 300
acataagtgt taagaagaca taacaattat aatggaagaa gtttgtctcc atttatatat 360
tatatattac ccacttatgt attatattag gatgttaagg agacataaca attataaaga 420
gagaagtttg tatccattta tatattatat actacccatt tatatattat acttatccac 480
ttatttaatg tctttataag gtttgatcca tgatatttct aatattttag ttgatatgta 540
tatgaaaggg tactatttga actctcttac tctgtataaa ggttggatca tccttaaagt 600
gggtctattt aattttattg cttcttacag ataaaaaaaa aattatgagt tggtttgata 660
aaatattgaa ggatttaaaa taataataaa taacatataa tatatgtata taaatttatt 720
ataatataac atttatctat aaaaaagtaa atattgtcat aaatctatac aatcgtttag 780
ccttgctgga cgaatctcaa ttatttaaac gagagtaaac atatttgact ttttggttat 840
ttaacaaatt attatttaac actatatgaa attttttttt ttatcagcaa agaataaaat 900
taaattaaga aggacaatgg tgtcccaatc cttatacaac caacttccac aagaaagtca 960
agtcagagac aacaaaaaaa caagcaaagg aaatttttta atttgagttg tcttgtttgc 1020
tgcataattt atgcagtaaa acactacaca taaccctttt agcagtagag caatggttga 1080
ccgtgtgctt agcttctttt attttatttt tttatcagca aagaataaat aaaataaaat 1140
gagacacttc agggatgttt caacaagctt ggcgcgccgt tctatagtgt cacctaaatc 1200
gtatgtgtat gatacataag gttatgtatt aattgtagcc gcgttctaac gacaatatgt 1260
ccatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac 1320
acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca 1380
gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga 1440
aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgacca 1500
aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag 1560
gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac 1620
cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa 1680
ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc 1740
accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag 1800
tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac 1860
cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc 1920
gaacgaccta caccgaactg agatacctac agcgtgagca ttgagaaagc gccacgcttc 1980
ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca 2040
cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc 2100
tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg 2160
ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct 2220
ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata 2280
ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc 2340
gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc aggttgatca 2400
gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct 2460
agaaataatt ttgtttaact ttaagaagga gatataccca tggaaaagcc tgaactcacc 2520
gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag 2580
ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc 2640
ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt 2700
gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg 2760
acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa 2820
ctgcccgctg ttctgcagcc ggtcgcggag gctatggatg cgatcgctgc ggccgatctt 2880
agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg 2940
cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac 3000
gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac 3060
tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac 3120
aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac 3180
gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc 3240
tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc 3300
cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct 3360
tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca 3420
caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat 3480
agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagtgagg tacagcttgg 3540
atcgatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag 3600
caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa 3660
ggaggaacta tatccggatg atcgggcgcg ccgtcgacgg atccgtacga gatccggccg 3720
gccagatcct gcaggagatc caagcttttg atccatgccc ttcatttgcc gcttattaat 3780
taatttggta acagtccgta ctaatcagtt acttatcctt cccccatcat aattaatctt 3840
ggtagtctcg aatgccacaa cactgactag tctcttggat cataagaaaa agccaaggaa 3900
caaaagaaga caaaacacaa tgagagtatc ctttgcatag caatgtctaa gttcataaaa 3960
ttcaaacaaa aacgcaatca cacacagtgg acatcactta tccactagct gatcaggatc 4020
gccgcgtcaa gaaaaaaaaa ctggacccca aaagccatgc acaacaacac gtactcacaa 4080
aggtgtcaat cgagcagccc aaaacattca ccaactcaac ccatcatgag ccctcacatt 4140
tgttgtttct aacccaacct caaactcgta ttctcttccg ccacctcatt tttgtttatt 4200
tcaacacccg tcaaactgca tgccaccccg tggccaaatg tccatgcatg ttaacaagac 4260
ctatgactat aaatagctgc aatctcggcc caggttttca tcatcaagaa ccagttcaat 4320
atcctagtac accgtattaa agaatttaag atatactgcg gccgcaccat ggaggtggtg 4380
aatgaaatag tctcaattgg gcaggaagtt ttacccaaag ttgattatgc ccaactctgg 4440
agtgatgcca gtcactgtga ggtgctttac ttgtccatcg catttgtcat cttgaagttc 4500
actcttggcc cccttggtcc aaaaggtcag tctcgtatga agtttgtttt caccaattac 4560
aaccttctca tgtccattta ttcgttggga tcattcctct caatggcata tgccatgtac 4620
accatcggtg ttatgtctga caactgcgag aaggcttttg acaacaacgt cttcaggatc 4680
accacgcagt tgttctattt gagcaagttc ctggagtata ttgactcctt ctatttgcca 4740
ctgatgggca agcctctgac ctggttgcaa ttcttccatc atttgggggc accgatggat 4800
atgtggctgt tctataatta ccgaaatgaa gctgtttgga tttttgtgct gttgaatggt 4860
ttcatccact ggatcatgta cggttattat tggaccagat tgatcaagct gaagttcccc 4920
atgccaaaat ccctgattac atcaatgcag atcattcaat tcaatgttgg tttctacatt 4980
gtctggaagt acaggaacat tccctgttat cgccaagatg ggatgaggat gtttggctgg 5040
ttcttcaatt acttttatgt tggcacagtc ttgtgtttgt tcttgaattt ctatgtgcaa 5100
acgtatatcg tcaggaagca caagggagcc aaaaagattc agtgagc 5147
<210> SEQ ID NO 86
<211> LENGTH: 6559
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Plasmid pKR680
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (4340)..(4340)
<223> OTHER INFORMATION: n is a, c, g, or t
<400> SEQUENCE: 86
ggccgcgaca caagtgtgag agtactaaat aaatgctttg gttgtacgaa atcattacac 60
taaataaaat aatcaaagct tatatatgcc ttccgctaag gccgaatgca aagaaattgg 120
ttctttctcg ttatcttttg ccacttttac tagtacgtat taattactac ttaatcatct 180
ttgtttacgg ctcattatat ccggtctaga ggatccaagg ccgcgaagtt aaaagcaatg 240
ttgtcacttg tcgtactaac acatgatgtg atagtttatg ctagctagct ataacataag 300
ctgtctctga gtgtgttgta tattaataaa gatcatcact ggtgaatggt gatcgtgtac 360
gtaccctact tagtaggcaa tggaagcact tagagtgtgc tttgtgcatg gccttgcctc 420
tgttttgaga cttttgtaat gttttcgagt ttaaatcttt gcctttgcgt acgtgggcgg 480
atcccccggg ctgcaggaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc 540
ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata 600
gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc 660
gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca 720
ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac 780
ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga 840
ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac 900
gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt 960
agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct 1020
aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat 1080
attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg 1140
cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg 1200
aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc 1260
ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat 1320
gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact 1380
attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca 1440
tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact 1500
tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg 1560
atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg 1620
agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg 1680
aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg 1740
caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag 1800
ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc 1860
gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga 1920
tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat 1980
atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc 2040
tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 2100
accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 2160
gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 2220
caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc 2280
tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 2340
ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 2400
tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 2460
gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 2520
tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca 2580
gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 2640
gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 2700
ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 2760
ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta 2820
ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag 2880
tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga 2940
ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg 3000
caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg 3060
ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc 3120
atgattacgc caagcttgca tgcctgcagg tcgactcgac gtacgtcctc gaagagaagg 3180
gttaataaca cattttttaa catttttaac acaaatttta gttatttaaa aatttattaa 3240
aaaatttaaa ataagaagag gaactcttta aataaatcta acttacaaaa tttatgattt 3300
ttaataagtt ttcaccaata aaaaatgtca taaaaatatg ttaaaaagta tattatcaat 3360
attctcttta tgataaataa aaagaaaaaa aaaataaaag ttaagtgaaa atgagattga 3420
agtgacttta ggtgtgtata aatatatcaa ccccgccaac aatttattta atccaaatat 3480
attgaagtat attattccat agcctttatt tatttatata tttattatat aaaagcttta 3540
tttgttctag gttgttcatg aaatattttt ttggttttat ctccgttgta agaaaatcat 3600
gtgctttgtg tcgccactca ctattgcagc tttttcatgc attggtcaga ttgacggttg 3660
attgtatttt tgttttttat ggttttgtgt tatgacttaa gtcttcatct ctttatctct 3720
tcatcaggtt tgatggttac ctaatatggt ccatgggtac atgcatggtt aaattaggtg 3780
gccaactttg ttgtgaacga tagaattttt tttatattaa gtaaactatt tttatattat 3840
gaaataataa taaaaaaaat attttatcat tattaacaaa atcatattag ttaatttgtt 3900
aactctataa taaaagaaat actgtaacat tcacattaca tggtaacatc tttccaccct 3960
ttcatttgtt ttttgtttga tgactttttt tcttgtttaa atttatttcc cttcttttaa 4020
atttggaata cattatcatc atatataaac taaaatacta aaaacaggat tacacaaatg 4080
ataaataata acacaaatat ttataaatct agctgcaata tatttaaact agctatatcg 4140
atattgtaaa ataaaactag ctgcattgat actgataaaa aaatatcatg tgctttctgg 4200
actgatgatg cagtatactt ttgacattgc ctttatttta tttttcagaa aagctttctt 4260
agttctgggt tcttcattat ttgtttccca tctccattgt gaattgaatc atttgcttcg 4320
tgtcacaaat acaatttagn taggtacatg cattggtcag attcacggtt tattatgtca 4380
tgacttaagt tcatggtagt acattacctg ccacgcatgc attatattgg ttagatttga 4440
taggcaaatt tggttgtcaa caatataaat ataaataatg tttttatatt acgaaataac 4500
agtgatcaaa acaaacagtt ttatctttat taacaagatt ttgtttttgt ttgatgacgt 4560
tttttaatgt ttacgctttc ccccttcttt tgaatttaga acactttatc atcataaaat 4620
caaatactaa aaaaattaca tatttcataa ataataacac aaatattttt aaaaaatctg 4680
aaataataat gaacaatatt acatattatc acgaaaattc attaataaaa atattatata 4740
aataaaatgt aatagtagtt atatgtagga aaaaagtact gcacgcataa tatatacaaa 4800
aagattaaaa tgaactatta taaataataa cactaaatta atggtgaatc atatcaaaat 4860
aatgaaaaag taaataaaat ttgtaattaa cttctatatg tattacacac acaaataata 4920
aataatagta aaaaaaatta tgataaatat ttaccatctc ataagatatt taaaataatg 4980
ataaaaatat agattatttt ttatgcaact agctagccaa aaagagaaca cgggtatata 5040
taaaaagagt acctttaaat tctactgtac ttcctttatt cctgacgttt ttatatcaag 5100
tggacatacg tgaagatttt aattatcagt ctaaatattt cattagcact taatactttt 5160
ctgttttatt cctatcctat aagtagtccc gattctccca acattgctta ttcacacaac 5220
taactaagaa agtcttccat agccccccaa gcggccgcgg gaattcgatt gaaatgaagt 5280
caaagcgcca agcgcttccc cttacaattg atggaacaac atatgatgtg tctgcctggg 5340
tcaatttcca ccctggtggt gcggaaatta tagagaatta ccaaggaagg gatgccactg 5400
atgccttcat ggttatgcac tctcaagaag ccttcgacaa gctcaagcgc atgcccaaaa 5460
tcaatcccag ttctgagttg ccaccccagg ctgcagtgaa tgaagctcaa gaggatttcc 5520
ggaagctccg agaagagttg atcgcaactg gcatgtttga tgcctccccc ctctggtact 5580
catacaaaat cagcaccaca ctgggccttg gagtgctggg ttatttcctg atggttcagt 5640
atcagatgta tttcattggg gcagtgttgc ttgggatgca ctatcaacag atgggctggc 5700
tttctcatga catttgccac caccagactt tcaagaaccg gaactggaac aacctcgtgg 5760
gactggtatt tggcaatggt ctgcaaggtt tttccgtgac atggtggaag gacagacaca 5820
atgcacatca ttcggcaacc aatgttcaag ggcacgaccc tgatattgac aacctccccc 5880
tcttagcctg gtctgaggat gacgtcacac gggcgtcacc gatttcccgc aagctcattc 5940
agttccagca gtactatttc ttggtcatct gtatcttgtt gcggttcatt tggtgtttcc 6000
agagcgtgtt gaccgtgcgc agtttgaagg acagagataa ccaattctat cgctctcagt 6060
ataagaagga ggccattggc ctcgccctgc actggacctt gaagaccctg ttccacttat 6120
tctttatgcc cagcatcctc acatcgctgt tggtgttttt cgtttcggag ctggttggcg 6180
gcttcggcat tgcgatcgtg gtgttcatga accactaccc actggagaag atcggggact 6240
cagtctggga tggccatgga ttctcggttg gccagatcca tgagaccatg aacattcggc 6300
gagggattat cacagattgg tttttcggag gcttgaatta ccagattgag caccatttgt 6360
ggccgaccct ccctcgccac aacctgacag cggttagcta ccaggtggaa cagctgtgcc 6420
agaagcacaa cctgccgtat cggaacccgc tgccccatga agggttggtc atcctgctgc 6480
gctatctggc ggtgttcgcc cggatggcgg agaagcaacc cgcggggaag gctctataag 6540
gaatcactag tgaattcgc 6559
<210> SEQ ID NO 87
<211> LENGTH: 9014
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Plasmid pKR913
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (7839)..(7839)
<223> OTHER INFORMATION: n is a, c, g, or t
<400> SEQUENCE: 87
gtacgagatc cggccggcca gatcctgcag gagatccaag cttttgatcc atgcccttca 60
tttgccgctt attaattaat ttggtaacag tccgtactaa tcagttactt atccttcccc 120
catcataatt aatcttggta gtctcgaatg ccacaacact gactagtctc ttggatcata 180
agaaaaagcc aaggaacaaa agaagacaaa acacaatgag agtatccttt gcatagcaat 240
gtctaagttc ataaaattca aacaaaaacg caatcacaca cagtggacat cacttatcca 300
ctagctgatc aggatcgccg cgtcaagaaa aaaaaactgg accccaaaag ccatgcacaa 360
caacacgtac tcacaaaggt gtcaatcgag cagcccaaaa cattcaccaa ctcaacccat 420
catgagccct cacatttgtt gtttctaacc caacctcaaa ctcgtattct cttccgccac 480
ctcatttttg tttatttcaa cacccgtcaa actgcatgcc accccgtggc caaatgtcca 540
tgcatgttaa caagacctat gactataaat agctgcaatc tcggcccagg ttttcatcat 600
caagaaccag ttcaatatcc tagtacaccg tattaaagaa tttaagatat actgcggccg 660
caccatggag gtggtgaatg aaatagtctc aattgggcag gaagttttac ccaaagttga 720
ttatgcccaa ctctggagtg atgccagtca ctgtgaggtg ctttacttgt ccatcgcatt 780
tgtcatcttg aagttcactc ttggccccct tggtccaaaa ggtcagtctc gtatgaagtt 840
tgttttcacc aattacaacc ttctcatgtc catttattcg ttgggatcat tcctctcaat 900
ggcatatgcc atgtacacca tcggtgttat gtctgacaac tgcgagaagg cttttgacaa 960
caacgtcttc aggatcacca cgcagttgtt ctatttgagc aagttcctgg agtatattga 1020
ctccttctat ttgccactga tgggcaagcc tctgacctgg ttgcaattct tccatcattt 1080
gggggcaccg atggatatgt ggctgttcta taattaccga aatgaagctg tttggatttt 1140
tgtgctgttg aatggtttca tccactggat catgtacggt tattattgga ccagattgat 1200
caagctgaag ttccccatgc caaaatccct gattacatca atgcagatca ttcaattcaa 1260
tgttggtttc tacattgtct ggaagtacag gaacattccc tgttatcgcc aagatgggat 1320
gaggatgttt ggctggttct tcaattactt ttatgttggc acagtcttgt gtttgttctt 1380
gaatttctat gtgcaaacgt atatcgtcag gaagcacaag ggagccaaaa agattcagtg 1440
agcggccgca agtatgaact aaaatgcatg taggtgtaag agctcatgga gagcatggaa 1500
tattgtatcc gaccatgtaa cagtataata actgagctcc atctcacttc ttctatgaat 1560
aaacaaagga tgttatgata tattaacact ctatctatgc accttattgt tctatgataa 1620
atttcctctt attattataa atcatctgaa tcgtgacggc ttatggaatg cttcaaatag 1680
tacaaaaaca aatgtgtact ataagacttt ctaaacaatt ctaaccttag cattgtgaac 1740
gagacataag tgttaagaag acataacaat tataatggaa gaagtttgtc tccatttata 1800
tattatatat tacccactta tgtattatat taggatgtta aggagacata acaattataa 1860
agagagaagt ttgtatccat ttatatatta tatactaccc atttatatat tatacttatc 1920
cacttattta atgtctttat aaggtttgat ccatgatatt tctaatattt tagttgatat 1980
gtatatgaaa gggtactatt tgaactctct tactctgtat aaaggttgga tcatccttaa 2040
agtgggtcta tttaatttta ttgcttctta cagataaaaa aaaaattatg agttggtttg 2100
ataaaatatt gaaggattta aaataataat aaataacata taatatatgt atataaattt 2160
attataatat aacatttatc tataaaaaag taaatattgt cataaatcta tacaatcgtt 2220
tagccttgct ggacgaatct caattattta aacgagagta aacatatttg actttttggt 2280
tatttaacaa attattattt aacactatat gaaatttttt tttttatcag caaagaataa 2340
aattaaatta agaaggacaa tggtgtccca atccttatac aaccaacttc cacaagaaag 2400
tcaagtcaga gacaacaaaa aaacaagcaa aggaaatttt ttaatttgag ttgtcttgtt 2460
tgctgcataa tttatgcagt aaaacactac acataaccct tttagcagta gagcaatggt 2520
tgaccgtgtg cttagcttct tttattttat ttttttatca gcaaagaata aataaaataa 2580
aatgagacac ttcagggatg tttcaacaag cttggcgcgc cgttctatag tgtcacctaa 2640
atcgtatgtg tatgatacat aaggttatgt attaattgta gccgcgttct aacgacaata 2700
tgtccatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc 2760
gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 2820
acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 2880
cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga 2940
ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 3000
aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 3060
caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 3120
taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag 3180
gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 3240
cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 3300
taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 3360
agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gcattgagaa agcgccacgc 3420
ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 3480
gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 3540
acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 3600
acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 3660
tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg 3720
ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag 3780
agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcaggttga 3840
tcagatctcg atcccgcgaa attaatacga ctcactatag ggagaccaca acggtttccc 3900
tctagaaata attttgttta actttaagaa ggagatatac ccatggaaaa gcctgaactc 3960
accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg 4020
cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat 4080
gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac 4140
tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc 4200
ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc 4260
gaactgcccg ctgttctgca gccggtcgcg gaggctatgg atgcgatcgc tgcggccgat 4320
cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca 4380
tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg 4440
gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag 4500
gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg 4560
gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa 4620
tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg 4680
cgctacttcg agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg 4740
ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca 4800
gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt 4860
acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc 4920
gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaatagtg aggtacagct 4980
tggatcgatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct 5040
gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg 5100
aaaggaggaa ctatatccgg atgatcgggc gcgccgtcga cggatccgta cgcaaaggca 5160
aagatttaaa ctcgaaaaca ttacaaaagt ctcaaaacag aggcaaggcc atgcacaaag 5220
cacactctaa gtgcttccat tgcctactaa gtagggtacg tacacgatca ccattcacca 5280
gtgatgatct ttattaatat acaacacact cagagacagc ttatgttata gctagctagc 5340
ataaactatc acatcatgtg ttagtacgac aagtgacaac attgctttta acttcgcggc 5400
cttggatcct ctagaccgga tataatgagc cgtaaacaaa gatgattaag tagtaattaa 5460
tacgtactag taaaagtggc aaaagataac gagaaagaac caatttcttt gcattcggcc 5520
ttagcggaag gcatatataa gctttgatta ttttatttag tgtaatgatt tcgtacaacc 5580
aaagcattta tttagtactc tcacacttgt gtcgcggccg cgaattcact agtgattcct 5640
tatagagcct tccccgcggg ttgcttctcc gccatccggg cgaacaccgc cagatagcgc 5700
agcaggatga ccaacccttc atggggcagc gggttccgat acggcaggtt gtgcttctgg 5760
cacagctgtt ccacctggta gctaaccgct gtcaggttgt ggcgagggag ggtcggccac 5820
aaatggtgct caatctggta attcaagcct ccgaaaaacc aatctgtgat aatccctcgc 5880
cgaatgttca tggtctcatg gatctggcca accgagaatc catggccatc ccagactgag 5940
tccccgatct tctccagtgg gtagtggttc atgaacacca cgatcgcaat gccgaagccg 6000
ccaaccagct ccgaaacgaa aaacaccaac agcgatgtga ggatgctggg cataaagaat 6060
aagtggaaca gggtcttcaa ggtccagtgc agggcgaggc caatggcctc cttcttatac 6120
tgagagcgat agaattggtt atctctgtcc ttcaaactgc gcacggtcaa cacgctctgg 6180
aaacaccaaa tgaaccgcaa caagatacag atgaccaaga aatagtactg ctggaactga 6240
atgagcttgc gggaaatcgg tgacgcccgt gtgacgtcat cctcagacca ggctaagagg 6300
gggaggttgt caatatcagg gtcgtgccct tgaacattgg ttgccgaatg atgtgcattg 6360
tgtctgtcct tccaccatgt cacggaaaaa ccttgcagac cattgccaaa taccagtccc 6420
acgaggttgt tccagttccg gttcttgaaa gtctggtggt ggcaaatgtc atgagaaagc 6480
cagcccatct gttgatagtg catcccaagc aacactgccc caatgaaata catctgatac 6540
tgaaccatca ggaaataacc cagcactcca aggcccagtg tggtgctgat tttgtatgag 6600
taccagaggg gggaggcatc aaacatgcca gttgcgatca actcttctcg gagcttccgg 6660
aaatcctctt gagcttcatt cactgcagcc tggggtggca actcagaact gggattgatt 6720
ttgggcatgc gcttgagctt gtcgaaggct tcttgagagt gcataaccat gaaggcatca 6780
gtggcatccc ttccttggta attctctata atttccgcac caccagggtg gaaattgacc 6840
caggcagaca catcatatgt tgttccatca attgtaaggg gaagcgcttg gcgctttgac 6900
ttcatttcaa tcgaattccc gcggccgctt ggggggctat ggaagacttt cttagttagt 6960
tgtgtgaata agcaatgttg ggagaatcgg gactacttat aggataggaa taaaacagaa 7020
aagtattaag tgctaatgaa atatttagac tgataattaa aatcttcacg tatgtccact 7080
tgatataaaa acgtcaggaa taaaggaagt acagtagaat ttaaaggtac tctttttata 7140
tatacccgtg ttctcttttt ggctagctag ttgcataaaa aataatctat atttttatca 7200
ttattttaaa tatcttatga gatggtaaat atttatcata atttttttta ctattattta 7260
ttatttgtgt gtgtaataca tatagaagtt aattacaaat tttatttact ttttcattat 7320
tttgatatga ttcaccatta atttagtgtt attatttata atagttcatt ttaatctttt 7380
tgtatatatt atgcgtgcag tacttttttc ctacatataa ctactattac attttattta 7440
tataatattt ttattaatga attttcgtga taatatgtaa tattgttcat tattatttca 7500
gattttttaa aaatatttgt gttattattt atgaaatatg taattttttt agtatttgat 7560
tttatgatga taaagtgttc taaattcaaa agaaggggga aagcgtaaac attaaaaaac 7620
gtcatcaaac aaaaacaaaa tcttgttaat aaagataaaa ctgtttgttt tgatcactgt 7680
tatttcgtaa tataaaaaca ttatttatat ttatattgtt gacaaccaaa tttgcctatc 7740
aaatctaacc aatataatgc atgcgtggca ggtaatgtac taccatgaac ttaagtcatg 7800
acataataaa ccgtgaatct gaccaatgca tgtacctanc taaattgtat ttgtgacacg 7860
aagcaaatga ttcaattcac aatggagatg ggaaacaaat aatgaagaac ccagaactaa 7920
gaaagctttt ctgaaaaata aaataaaggc aatgtcaaaa gtatactgca tcatcagtcc 7980
agaaagcaca tgatattttt ttatcagtat caatgcagct agttttattt tacaatatcg 8040
atatagctag tttaaatata ttgcagctag atttataaat atttgtgtta ttatttatca 8100
tttgtgtaat cctgttttta gtattttagt ttatatatga tgataatgta ttccaaattt 8160
aaaagaaggg aaataaattt aaacaagaaa aaaagtcatc aaacaaaaaa caaatgaaag 8220
ggtggaaaga tgttaccatg taatgtgaat gttacagtat ttcttttatt atagagttaa 8280
caaattaact aatatgattt tgttaataat gataaaatat tttttttatt attatttcat 8340
aatataaaaa tagtttactt aatataaaaa aaattctatc gttcacaaca aagttggcca 8400
cctaatttaa ccatgcatgt acccatggac catattaggt aaccatcaaa cctgatgaag 8460
agataaagag atgaagactt aagtcataac acaaaaccat aaaaaacaaa aatacaatca 8520
accgtcaatc tgaccaatgc atgaaaaagc tgcaatagtg agtggcgaca caaagcacat 8580
gattttctta caacggagat aaaaccaaaa aaatatttca tgaacaacct agaacaaata 8640
aagcttttat ataataaata tataaataaa taaaggctat ggaataatat acttcaatat 8700
atttggatta aataaattgt tggcggggtt gatatattta tacacaccta aagtcacttc 8760
aatctcattt tcacttaact tttatttttt ttttcttttt atttatcata aagagaatat 8820
tgataatata ctttttaaca tatttttatg acatttttta ttggtgaaaa cttattaaaa 8880
atcataaatt ttgtaagtta gatttattta aagagttcct cttcttattt taaatttttt 8940
aataaatttt taaataacta aaatttgtgt taaaaatgtt aaaaaatgtg ttattaaccc 9000
ttctcttcga ggac 9014
<210> SEQ ID NO 88
<211> LENGTH: 5561
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Plasmid pKR767
<400> SEQUENCE: 88
catggtcaat caatgagacg ccaacttctt aatctattga gacctgcagg tctagaaggg 60
cggatccccg ggtaccgagc tcgaattcac tggccgtcgt tttacaacgt cgtgactggg 120
aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc 180
gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg 240
aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat 300
ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc 360
caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag 420
ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg 480
cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg 540
tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 600
ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 660
aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 720
tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 780
atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 840
agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 900
tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 960
tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 1020
atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 1080
ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 1140
tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 1200
acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 1260
ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 1320
aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 1380
ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 1440
cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 1500
gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 1560
actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 1620
agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 1680
cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 1740
tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 1800
agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 1860
tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 1920
acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 1980
ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 2040
gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 2100
gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 2160
gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 2220
tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 2280
caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 2340
tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc 2400
gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg 2460
agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt 2520
ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc 2580
gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc 2640
ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct 2700
atgaccatga ttacgccaag cttgcatgcc tgcaggctag cctaagtacg tactcaaaat 2760
gccaacaaat aaaaaaaaag ttgctttaat aatgccaaaa caaattaata aaacacttac 2820
aacaccggat tttttttaat taaaatgtgc catttaggat aaatagttaa tatttttaat 2880
aattatttaa aaagccgtat ctactaaaat gatttttatt tggttgaaaa tattaatatg 2940
tttaaatcaa cacaatctat caaaattaaa ctaaaaaaaa aataagtgta cgtggttaac 3000
attagtacag taatataaga ggaaaatgag aaattaagaa attgaaagcg agtctaattt 3060
ttaaattatg aacctgcata tataaaagga aagaaagaat ccaggaagaa aagaaatgaa 3120
accatgcatg gtcccctcgt catcacgagt ttctgccatt tgcaatagaa acactgaaac 3180
acctttctct ttgtcactta attgagatgc cgaagccacc tcacaccatg aacttcatga 3240
ggtgtagcac ccaaggcttc catagccatg catactgaag aatgtctcaa gctcagcacc 3300
ctacttctgt gacgtgtccc tcattcacct tcctctcttc cctataaata accacgcctc 3360
aggttctccg cttcacaact caaacattct ctccattggt ccttaaacac tcatcagtca 3420
tcaccgcggc cgcatgggaa cggaccaagg aaaaaccttc acctgggaag agctggcggc 3480
ccataacacc aaggacgacc tactcttggc catccgcggc agggtgtacg atgtcacaaa 3540
gttcttgagc cgccatcctg gtggagtgga cactctcctg ctcggagctg gccgagatgt 3600
tactccggtc tttgagatgt atcacgcgtt tggggctgca gatgccatta tgaagaagta 3660
ctatgtcggt acactggtct cgaatgagct gcccatcttc ccggagccaa cggtgttcca 3720
caaaaccatc aagacgagag tcgagggcta ctttacggat cggaacattg atcccaagaa 3780
tagaccagag atctggggac gatacgctct tatctttgga tccttgatcg cttcctacta 3840
cgcgcagctc tttgtgcctt tcgttgtcga acgcacatgg cttcaggtgg tgtttgcaat 3900
catcatggga tttgcgtgcg cacaagtcgg actcaaccct cttcatgatg cgtctcactt 3960
ttcagtgacc cacaacccca ctgtctggaa gattctggga gccacgcacg actttttcaa 4020
cggagcatcg tacctggtgt ggatgtacca acatatgctc ggccatcacc cctacaccaa 4080
cattgctgga gcagatcccg acgtgtcgac gtctgagccc gatgttcgtc gtatcaagcc 4140
caaccaaaag tggtttgtca accacatcaa ccagcacatg tttgttcctt tcctgtacgg 4200
actgctggcg ttcaaggtgc gcattcagga catcaacatt ttgtactttg tcaagaccaa 4260
tgacgctatt cgtgtcaatc ccatctcgac atggcacact gtgatgttct ggggcggcaa 4320
ggctttcttt gtctggtatc gcctgattgt tcccctgcag tatctgcccc tgggcaaggt 4380
gctgctcttg ttcacggtcg cggacatggt gtcgtcttac tggctggcgc tgaccttcca 4440
ggcgaaccac gttgttgagg aagttcagtg gccgttgcct gacgagaacg ggatcatcca 4500
aaaggactgg gcagctatgc aggtcgagac tacgcaggat tacgcacacg attcgcacct 4560
ctggaccagc atcactggca gcttgaacta ccaggctgtg caccatctgt tccccaacgt 4620
gtcgcagcac cattatcccg atattctggc catcatcaag aacacctgca gcgagtacaa 4680
ggttccatac cttgtcaagg atacgttttg gcaagcattt gcttcacatt tggagcactt 4740
gcgtgttctt ggactccgtc ccaaggaaga gtaggcggcc gcatttcgca ccaaatcaat 4800
gaaagtaata atgaaaagtc tgaataagaa tacttaggct tagatgcctt tgttacttgt 4860
gtaaaataac ttgagtcatg tacctttggc ggaaacagaa taaataaaag gtgaaattcc 4920
aatgctctat gtataagtta gtaatactta atgtgttcta cggttgtttc aatatcatca 4980
aactctaatt gaaactttag aaccacaaat ctcaatcttt tcttaatgaa atgaaaaatc 5040
ttaattgtac catgtttatg ttaaacacct tacaattggt tggagaggag gaccaaccga 5100
tgggacaaca ttgggagaaa gagattcaat ggagatttgg ataggagaac aacattcttt 5160
ttcacttcaa tacaagatga gtgcaacact aaggatatgt atgagacttt cagaagctac 5220
gacaacatag atgagtgagg tggtgattcc tagcaagaaa gacattagag gaagccaaaa 5280
tcgaacaagg aagacatcaa gggcaagaga caggaccatc catctcagga aaaggagctt 5340
tgggatagtc cgagaagttg tacaagaaat tttttggagg gtgagtgatg cattgctggt 5400
gactttaact caatcaaaat tgagaaagaa agaaaaggga gggggctcac atgtgaatag 5460
aagggaaacg ggagaatttt acagttttga tctaatgggc atcccagcta gtggtaacat 5520
attcaccatg tttaaccttc acgtacgtct agaggatccc c 5561
<210> SEQ ID NO 89
<211> LENGTH: 11889
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Plasmid pKR916
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (7810)..(7810)
<223> OTHER INFORMATION: n is a, c, g, or t
<400> SEQUENCE: 89
ggagatccaa gcttttgatc catgcccttc atttgccgct tattaattaa tttggtaaca 60
gtccgtacta atcagttact tatccttccc ccatcataat taatcttggt agtctcgaat 120
gccacaacac tgactagtct cttggatcat aagaaaaagc caaggaacaa aagaagacaa 180
aacacaatga gagtatcctt tgcatagcaa tgtctaagtt cataaaattc aaacaaaaac 240
gcaatcacac acagtggaca tcacttatcc actagctgat caggatcgcc gcgtcaagaa 300
aaaaaaactg gaccccaaaa gccatgcaca acaacacgta ctcacaaagg tgtcaatcga 360
gcagcccaaa acattcacca actcaaccca tcatgagccc tcacatttgt tgtttctaac 420
ccaacctcaa actcgtattc tcttccgcca cctcattttt gtttatttca acacccgtca 480
aactgcatgc caccccgtgg ccaaatgtcc atgcatgtta acaagaccta tgactataaa 540
tagctgcaat ctcggcccag gttttcatca tcaagaacca gttcaatatc ctagtacacc 600
gtattaaaga atttaagata tactgcggcc gcaccatgga ggtggtgaat gaaatagtct 660
caattgggca ggaagtttta cccaaagttg attatgccca actctggagt gatgccagtc 720
actgtgaggt gctttacttg tccatcgcat ttgtcatctt gaagttcact cttggccccc 780
ttggtccaaa aggtcagtct cgtatgaagt ttgttttcac caattacaac cttctcatgt 840
ccatttattc gttgggatca ttcctctcaa tggcatatgc catgtacacc atcggtgtta 900
tgtctgacaa ctgcgagaag gcttttgaca acaacgtctt caggatcacc acgcagttgt 960
tctatttgag caagttcctg gagtatattg actccttcta tttgccactg atgggcaagc 1020
ctctgacctg gttgcaattc ttccatcatt tgggggcacc gatggatatg tggctgttct 1080
ataattaccg aaatgaagct gtttggattt ttgtgctgtt gaatggtttc atccactgga 1140
tcatgtacgg ttattattgg accagattga tcaagctgaa gttccccatg ccaaaatccc 1200
tgattacatc aatgcagatc attcaattca atgttggttt ctacattgtc tggaagtaca 1260
ggaacattcc ctgttatcgc caagatggga tgaggatgtt tggctggttc ttcaattact 1320
tttatgttgg cacagtcttg tgtttgttct tgaatttcta tgtgcaaacg tatatcgtca 1380
ggaagcacaa gggagccaaa aagattcagt gagcggccgc aagtatgaac taaaatgcat 1440
gtaggtgtaa gagctcatgg agagcatgga atattgtatc cgaccatgta acagtataat 1500
aactgagctc catctcactt cttctatgaa taaacaaagg atgttatgat atattaacac 1560
tctatctatg caccttattg ttctatgata aatttcctct tattattata aatcatctga 1620
atcgtgacgg cttatggaat gcttcaaata gtacaaaaac aaatgtgtac tataagactt 1680
tctaaacaat tctaacctta gcattgtgaa cgagacataa gtgttaagaa gacataacaa 1740
ttataatgga agaagtttgt ctccatttat atattatata ttacccactt atgtattata 1800
ttaggatgtt aaggagacat aacaattata aagagagaag tttgtatcca tttatatatt 1860
atatactacc catttatata ttatacttat ccacttattt aatgtcttta taaggtttga 1920
tccatgatat ttctaatatt ttagttgata tgtatatgaa agggtactat ttgaactctc 1980
ttactctgta taaaggttgg atcatcctta aagtgggtct atttaatttt attgcttctt 2040
acagataaaa aaaaaattat gagttggttt gataaaatat tgaaggattt aaaataataa 2100
taaataacat ataatatatg tatataaatt tattataata taacatttat ctataaaaaa 2160
gtaaatattg tcataaatct atacaatcgt ttagccttgc tggacgaatc tcaattattt 2220
aaacgagagt aaacatattt gactttttgg ttatttaaca aattattatt taacactata 2280
tgaaattttt ttttttatca gcaaagaata aaattaaatt aagaaggaca atggtgtccc 2340
aatccttata caaccaactt ccacaagaaa gtcaagtcag agacaacaaa aaaacaagca 2400
aaggaaattt tttaatttga gttgtcttgt ttgctgcata atttatgcag taaaacacta 2460
cacataaccc ttttagcagt agagcaatgg ttgaccgtgt gcttagcttc ttttatttta 2520
tttttttatc agcaaagaat aaataaaata aaatgagaca cttcagggat gtttcaacaa 2580
gcttggcgcg ccgttctata gtgtcaccta aatcgtatgt gtatgataca taaggttatg 2640
tattaattgt agccgcgttc taacgacaat atgtccatat ggtgcactct cagtacaatc 2700
tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc 2760
tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc 2820
tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg 2880
atacgcctat ttttataggt taatgtcatg accaaaatcc cttaacgtga gttttcgttc 2940
cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 3000
cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 3060
gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 3120
aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 3180
cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 3240
tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 3300
acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 3360
ctacagcgtg agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 3420
ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 3480
tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 3540
tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 3600
ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg 3660
gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag 3720
cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc 3780
gcgcgttggc cgattcatta atgcaggttg atcagatctc gatcccgcga aattaatacg 3840
actcactata gggagaccac aacggtttcc ctctagaaat aattttgttt aactttaaga 3900
aggagatata cccatggaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat 3960
cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc 4020
tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg 4080
tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga 4140
agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca 4200
gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc 4260
ggaggctatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt 4320
cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga 4380
tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca 4440
ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca 4500
cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga 4560
ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag 4620
gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct 4680
tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca 4740
gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat 4800
cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt 4860
ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg 4920
tccgagggca aaggaatagt gaggtacagc ttggatcgat ccggctgcta acaaagcccg 4980
aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc 5040
ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gatgatcggg 5100
cgcgccgtcg acggatccgt acgcaaaggc aaagatttaa actcgaaaac attacaaaag 5160
tctcaaaaca gaggcaaggc catgcacaaa gcacactcta agtgcttcca ttgcctacta 5220
agtagggtac gtacacgatc accattcacc agtgatgatc tttattaata tacaacacac 5280
tcagagacag cttatgttat agctagctag cataaactat cacatcatgt gttagtacga 5340
caagtgacaa cattgctttt aacttcgcgg ccttggatcc tctagaccgg atataatgag 5400
ccgtaaacaa agatgattaa gtagtaatta atacgtacta gtaaaagtgg caaaagataa 5460
cgagaaagaa ccaatttctt tgcattcggc cttagcggaa ggcatatata agctttgatt 5520
attttattta gtgtaatgat ttcgtacaac caaagcattt atttagtact ctcacacttg 5580
tgtcgcggcc gcgaattcac tagtgattcc ttatagagcc ttccccgcgg gttgcttctc 5640
cgccatccgg gcgaacaccg ccagatagcg cagcaggatg accaaccctt catggggcag 5700
cgggttccga tacggcaggt tgtgcttctg gcacagctgt tccacctggt agctaaccgc 5760
tgtcaggttg tggcgaggga gggtcggcca caaatggtgc tcaatctggt aattcaagcc 5820
tccgaaaaac caatctgtga taatccctcg ccgaatgttc atggtctcat ggatctggcc 5880
aaccgagaat ccatggccat cccagactga gtccccgatc ttctccagtg ggtagtggtt 5940
catgaacacc acgatcgcaa tgccgaagcc gccaaccagc tccgaaacga aaaacaccaa 6000
cagcgatgtg aggatgctgg gcataaagaa taagtggaac agggtcttca aggtccagtg 6060
cagggcgagg ccaatggcct ccttcttata ctgagagcga tagaattggt tatctctgtc 6120
cttcaaactg cgcacggtca acacgctctg gaaacaccaa atgaaccgca acaagataca 6180
gatgaccaag aaatagtact gctggaactg aatgagcttg cgggaaatcg gtgacgcccg 6240
tgtgacgtca tcctcagacc aggctaagag ggggaggttg tcaatatcag ggtcgtgccc 6300
ttgaacattg gttgccgaat gatgtgcatt gtgtctgtcc ttccaccatg tcacggaaaa 6360
accttgcaga ccattgccaa ataccagtcc cacgaggttg ttccagttcc ggttcttgaa 6420
agtctggtgg tggcaaatgt catgagaaag ccagcccatc tgttgatagt gcatcccaag 6480
caacactgcc ccaatgaaat acatctgata ctgaaccatc aggaaataac ccagcactcc 6540
aaggcccagt gtggtgctga ttttgtatga gtaccagagg ggggaggcat caaacatgcc 6600
agttgcgatc aactcttctc ggagcttccg gaaatcctct tgagcttcat tcactgcagc 6660
ctggggtggc aactcagaac tgggattgat tttgggcatg cgcttgagct tgtcgaaggc 6720
ttcttgagag tgcataacca tgaaggcatc agtggcatcc cttccttggt aattctctat 6780
aatttccgca ccaccagggt ggaaattgac ccaggcagac acatcatatg ttgttccatc 6840
aattgtaagg ggaagcgctt ggcgctttga cttcatttca atcgaattcc cgcggccgct 6900
tggggggcta tggaagactt tcttagttag ttgtgtgaat aagcaatgtt gggagaatcg 6960
ggactactta taggatagga ataaaacaga aaagtattaa gtgctaatga aatatttaga 7020
ctgataatta aaatcttcac gtatgtccac ttgatataaa aacgtcagga ataaaggaag 7080
tacagtagaa tttaaaggta ctctttttat atatacccgt gttctctttt tggctagcta 7140
gttgcataaa aaataatcta tatttttatc attattttaa atatcttatg agatggtaaa 7200
tatttatcat aatttttttt actattattt attatttgtg tgtgtaatac atatagaagt 7260
taattacaaa ttttatttac tttttcatta ttttgatatg attcaccatt aatttagtgt 7320
tattatttat aatagttcat tttaatcttt ttgtatatat tatgcgtgca gtactttttt 7380
cctacatata actactatta cattttattt atataatatt tttattaatg aattttcgtg 7440
ataatatgta atattgttca ttattatttc agatttttta aaaatatttg tgttattatt 7500
tatgaaatat gtaatttttt tagtatttga ttttatgatg ataaagtgtt ctaaattcaa 7560
aagaaggggg aaagcgtaaa cattaaaaaa cgtcatcaaa caaaaacaaa atcttgttaa 7620
taaagataaa actgtttgtt ttgatcactg ttatttcgta atataaaaac attatttata 7680
tttatattgt tgacaaccaa atttgcctat caaatctaac caatataatg catgcgtggc 7740
aggtaatgta ctaccatgaa cttaagtcat gacataataa accgtgaatc tgaccaatgc 7800
atgtacctan ctaaattgta tttgtgacac gaagcaaatg attcaattca caatggagat 7860
gggaaacaaa taatgaagaa cccagaacta agaaagcttt tctgaaaaat aaaataaagg 7920
caatgtcaaa agtatactgc atcatcagtc cagaaagcac atgatatttt tttatcagta 7980
tcaatgcagc tagttttatt ttacaatatc gatatagcta gtttaaatat attgcagcta 8040
gatttataaa tatttgtgtt attatttatc atttgtgtaa tcctgttttt agtattttag 8100
tttatatatg atgataatgt attccaaatt taaaagaagg gaaataaatt taaacaagaa 8160
aaaaagtcat caaacaaaaa acaaatgaaa gggtggaaag atgttaccat gtaatgtgaa 8220
tgttacagta tttcttttat tatagagtta acaaattaac taatatgatt ttgttaataa 8280
tgataaaata ttttttttat tattatttca taatataaaa atagtttact taatataaaa 8340
aaaattctat cgttcacaac aaagttggcc acctaattta accatgcatg tacccatgga 8400
ccatattagg taaccatcaa acctgatgaa gagataaaga gatgaagact taagtcataa 8460
cacaaaacca taaaaaacaa aaatacaatc aaccgtcaat ctgaccaatg catgaaaaag 8520
ctgcaatagt gagtggcgac acaaagcaca tgattttctt acaacggaga taaaaccaaa 8580
aaaatatttc atgaacaacc tagaacaaat aaagctttta tataataaat atataaataa 8640
ataaaggcta tggaataata tacttcaata tatttggatt aaataaattg ttggcggggt 8700
tgatatattt atacacacct aaagtcactt caatctcatt ttcacttaac ttttattttt 8760
tttttctttt tatttatcat aaagagaata ttgataatat actttttaac atatttttat 8820
gacatttttt attggtgaaa acttattaaa aatcataaat tttgtaagtt agatttattt 8880
aaagagttcc tcttcttatt ttaaattttt taataaattt ttaaataact aaaatttgtg 8940
ttaaaaatgt taaaaaatgt gttattaacc cttctcttcg aggacgtacg agatccggcc 9000
ggccagatcc tgcaggtctc aatagattaa gaagttggcg tctcattgat tgaccatggg 9060
ggatcctcta gacgtacgtg aaggttaaac atggtgaata tgttaccact agctgggatg 9120
cccattagat caaaactgta aaattctccc gtttcccttc tattcacatg tgagccccct 9180
cccttttctt tctttctcaa ttttgattga gttaaagtca ccagcaatgc atcactcacc 9240
ctccaaaaaa tttcttgtac aacttctcgg actatcccaa agctcctttt cctgagatgg 9300
atggtcctgt ctcttgccct tgatgtcttc cttgttcgat tttggcttcc tctaatgtct 9360
ttcttgctag gaatcaccac ctcactcatc tatgttgtcg tagcttctga aagtctcata 9420
catatcctta gtgttgcact catcttgtat tgaagtgaaa aagaatgttg ttctcctatc 9480
caaatctcca ttgaatctct ttctcccaat gttgtcccat cggttggtcc tcctctccaa 9540
ccaattgtaa ggtgtttaac ataaacatgg tacaattaag atttttcatt tcattaagaa 9600
aagattgaga tttgtggttc taaagtttca attagagttt gatgatattg aaacaaccgt 9660
agaacacatt aagtattact aacttataca tagagcattg gaatttcacc ttttatttat 9720
tctgtttccg ccaaaggtac atgactcaag ttattttaca caagtaacaa aggcatctaa 9780
gcctaagtat tcttattcag acttttcatt attactttca ttgatttggt gcgaaatgcg 9840
gccgcctact cttccttggg acggagtcca agaacacgca agtgctccaa atgtgaagca 9900
aatgcttgcc aaaacgtatc cttgacaagg tatggaacct tgtactcgct gcaggtgttc 9960
ttgatgatgg ccagaatatc gggataatgg tgctgcgaca cgttggggaa cagatggtgc 10020
acagcctggt agttcaagct gccagtgatg ctggtccaga ggtgcgaatc gtgtgcgtaa 10080
tcctgcgtag tctcgacctg catagctgcc cagtcctttt ggatgatccc gttctcgtca 10140
ggcaacggcc actgaacttc ctcaacaacg tggttcgcct ggaaggtcag cgccagccag 10200
taagacgaca ccatgtccgc gaccgtgaac aagagcagca ccttgcccag gggcagatac 10260
tgcaggggaa caatcaggcg ataccagaca aagaaagcct tgccgcccca gaacatcaca 10320
gtgtgccatg tcgagatggg attgacacga atagcgtcat tggtcttgac aaagtacaaa 10380
atgttgatgt cctgaatgcg caccttgaac gccagcagtc cgtacaggaa aggaacaaac 10440
atgtgctggt tgatgtggtt gacaaaccac ttttggttgg gcttgatacg acgaacatcg 10500
ggctcagacg tcgacacgtc gggatctgct ccagcaatgt tggtgtaggg gtgatggccg 10560
agcatatgtt ggtacatcca caccaggtac gatgctccgt tgaaaaagtc gtgcgtggct 10620
cccagaatct tccagacagt ggggttgtgg gtcactgaaa agtgagacgc atcatgaaga 10680
gggttgagtc cgacttgtgc gcacgcaaat cccatgatga ttgcaaacac cacctgaagc 10740
catgtgcgtt cgacaacgaa aggcacaaag agctgcgcgt agtaggaagc gatcaaggat 10800
ccaaagataa gagcgtatcg tccccagatc tctggtctat tcttgggatc aatgttccga 10860
tccgtaaagt agccctcgac tctcgtcttg atggttttgt ggaacaccgt tggctccggg 10920
aagatgggca gctcattcga gaccagtgta ccgacatagt acttcttcat aatggcatct 10980
gcagccccaa acgcgtgata catctcaaag accggagtaa catctcggcc agctccgagc 11040
aggagagtgt ccactccacc aggatggcgg ctcaagaact ttgtgacatc gtacaccctg 11100
ccgcggatgg ccaagagtag gtcgtccttg gtgttatggg ccgccagctc ttcccaggtg 11160
aaggtttttc cttggtccgt tcccatgcgg ccgcggtgat gactgatgag tgtttaagga 11220
ccaatggaga gaatgtttga gttgtgaagc ggagaacctg aggcgtggtt atttataggg 11280
aagagaggaa ggtgaatgag ggacacgtca cagaagtagg gtgctgagct tgagacattc 11340
ttcagtatgc atggctatgg aagccttggg tgctacacct catgaagttc atggtgtgag 11400
gtggcttcgg catctcaatt aagtgacaaa gagaaaggtg tttcagtgtt tctattgcaa 11460
atggcagaaa ctcgtgatga cgaggggacc atgcatggtt tcatttcttt tcttcctgga 11520
ttctttcttt ccttttatat atgcaggttc ataatttaaa aattagactc gctttcaatt 11580
tcttaatttc tcattttcct cttatattac tgtactaatg ttaaccacgt acacttattt 11640
tttttttagt ttaattttga tagattgtgt tgatttaaac atattaatat tttcaaccaa 11700
ataaaaatca ttttagtaga tacggctttt taaataatta ttaaaaatat taactattta 11760
tcctaaatgg cacattttaa ttaaaaaaaa tccggtgttg taagtgtttt attaatttgt 11820
tttggcatta ttaaagcaac ttttttttta tttgttggca ttttgagtac gtacttaggc 11880
tagcctgca 11889
<210> SEQ ID NO 90
<211> LENGTH: 5661
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Plasmid pKR974
<400> SEQUENCE: 90
gtacgtctag aggatccccc atggtcaatc aatgagacgc caacttctta atctattgag 60
acctgcaggt ctagaagggc ggatccccgg gtaccgagct cgaattcact ggccgtcgtt 120
ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat 180
ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag 240
ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc 300
ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta 360
agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg 420
gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca 480
ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt 540
aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc 600
ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa 660
taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc 720
cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa 780
acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa 840
ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg 900
atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa 960
gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc 1020
acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc 1080
atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta 1140
accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag 1200
ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca 1260
acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata 1320
gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc 1380
tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca 1440
ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca 1500
actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg 1560
taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 1620
tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 1680
gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 1740
cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 1800
gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 1860
gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac 1920
tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 1980
ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 2040
cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 2100
gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 2160
gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 2220
gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 2280
cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 2340
tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc 2400
cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 2460
cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa 2520
ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 2580
tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 2640
caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 2700
tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcaggctagc 2760
ctaagtacgt actcaaaatg ccaacaaata aaaaaaaagt tgctttaata atgccaaaac 2820
aaattaataa aacacttaca acaccggatt ttttttaatt aaaatgtgcc atttaggata 2880
aatagttaat atttttaata attatttaaa aagccgtatc tactaaaatg atttttattt 2940
ggttgaaaat attaatatgt ttaaatcaac acaatctatc aaaattaaac taaaaaaaaa 3000
ataagtgtac gtggttaaca ttagtacagt aatataagag gaaaatgaga aattaagaaa 3060
ttgaaagcga gtctaatttt taaattatga acctgcatat ataaaaggaa agaaagaatc 3120
caggaagaaa agaaatgaaa ccatgcatgg tcccctcgtc atcacgagtt tctgccattt 3180
gcaatagaaa cactgaaaca cctttctctt tgtcacttaa ttgagatgcc gaagccacct 3240
cacaccatga acttcatgag gtgtagcacc caaggcttcc atagccatgc atactgaaga 3300
atgtctcaag ctcagcaccc tacttctgtg acgtgtccct cattcacctt cctctcttcc 3360
ctataaataa ccacgcctca ggttctccgc ttcacaactc aaacattctc tccattggtc 3420
cttaaacact catcagtcat caccgcggcc gccaattcat ggccccgcag acggagctcc 3480
gccagcgcca cgccgccgtc gccgagacgc cggtggccgg caagaaggcc tttacatggc 3540
aggaggtcgc gcagcacaac acggcggcct cggcctggat cattatccgc ggcaaggtct 3600
acgacgtgac cgagtgggcc aacaagcacc ccggcggccg cgagatggtg ctgctgcacg 3660
ccggtcgcga ggccaccgac acgttcgact cgtaccaccc gttcagcgac aaggccgagt 3720
cgatcttgaa caagtatgag attggcacgt tcacgggccc gtccgagttt ccgaccttca 3780
agccggacac gggcttctac aaggagtgcc gcaagcgcgt tggcgagtac ttcaagaaga 3840
acaacctcca tccgcaggac ggcttcccgg gcctctggcg catgatggtc gtgtttgcgg 3900
tcgccggcct cgccttgtac ggcatgcact tttcgactat ctttgcgctg cagctcgcgg 3960
ccgcggcgct ctttggcgtc tgccaggcgc tgccgctgct ccacgtcatg cacgactcgt 4020
cgcacgcgtc gtacaccaac atgccgttct tccattacgt cgtcggccgc tttgccatgg 4080
actggtttgc cggcggctcg atggtgtcat ggctcaacca gcacgtcgtg ggccaccaca 4140
tctacacgaa cgtcgcgggc tcggacccgg atcttccggt caacatggac ggcgacatcc 4200
gccgcatcgt gaaccgccag gtgttccagc ccatgtacgc attccagcac atctaccttc 4260
cgccgctcta tggcgtgctt ggcctcaagt tccgcatcca ggacttcacc gacacgttcg 4320
gctcgcacac gaacggcccg atccgcgtca acccgcacgc gctctcgacg tggatggcca 4380
tgatcagctc caagtcgttc tgggccttct accgcgtgta ccttccgctt gccgtgctcc 4440
agatgcccat caagacgtac cttgcgatct tcttcctcgc cgagtttgtc acgggctggt 4500
acctcgcgtt caacttccaa gtaagccatg tctcgaccga gtgcggctac ccatgcggcg 4560
acgaggccaa gatggcgctc caggacgagt gggcagtctc gcaggtcaag acgtcggtcg 4620
actacgccca tggctcgtgg atgacgacgt tccttgccgg cgcgctcaac taccaggtcg 4680
tgcaccactt gttccccagc gtgtcgcagt accactaccc ggcgatcgcg cccatcatcg 4740
tcgacgtctg caaggagtac aacatcaagt acgccatctt gccggacttt acggcggcgt 4800
tcgttgccca cttgaagcac ctccgcaaca tgggccagca gggcatcgcc gccacgatcc 4860
acatgggcta actcgagctc agctagatcg cggccgcatt tcgcaccaaa tcaatgaaag 4920
taataatgaa aagtctgaat aagaatactt aggcttagat gcctttgtta cttgtgtaaa 4980
ataacttgag tcatgtacct ttggcggaaa cagaataaat aaaaggtgaa attccaatgc 5040
tctatgtata agttagtaat acttaatgtg ttctacggtt gtttcaatat catcaaactc 5100
taattgaaac tttagaacca caaatctcaa tcttttctta atgaaatgaa aaatcttaat 5160
tgtaccatgt ttatgttaaa caccttacaa ttaattggtt ggagaggagg accaaccgat 5220
gggacaacat tgggagaaag agattcaatg gagatttgga taggagaaca acattctttt 5280
tcacttcaat acaagatgag tgcaacacta aggatatgta tgagactttc agaagctacg 5340
acaacataga tgagtgaggt ggtgattcct agcaagaaag acattagagg aagccaaaat 5400
cgaacaagga agacatcaag ggcaagagac aggaccatcc atctcaggaa aaggagcttt 5460
gggatagtcc gagaagttgt acaagaaatt ttttggaggg tgagtgatgc attgctggtg 5520
actttaactc aatcaaaatt gagaaagaaa gaaaagggag ggggctcaca tgtgaataga 5580
agggaaacgg gagaatttta cagttttgat ctaatgggca tcccagctag tggtaacata 5640
ttcaccatgt ttaaccttca c 5661
<210> SEQ ID NO 91
<211> LENGTH: 1413
<212> TYPE: DNA
<213> ORGANISM: Saprolegnia diclina
<400> SEQUENCE: 91
atggccccgc agacggagct ccgccagcgc cacgccgccg tcgccgagac gccggtggcc 60
ggcaagaagg cctttacatg gcaggaggtc gcgcagcaca acacggcggc ctcggcctgg 120
atcattatcc gcggcaaggt ctacgacgtg accgagtggg ccaacaagca ccccggcggc 180
cgcgagatgg tgctgctgca cgccggtcgc gaggccaccg acacgttcga ctcgtaccac 240
ccgttcagcg acaaggccga gtcgatcttg aacaagtatg agattggcac gttcacgggc 300
ccgtccgagt ttccgacctt caagccggac acgggcttct acaaggagtg ccgcaagcgc 360
gttggcgagt acttcaagaa gaacaacctc catccgcagg acggcttccc gggcctctgg 420
cgcatgatgg tcgtgtttgc ggtcgccggc ctcgccttgt acggcatgca cttttcgact 480
atctttgcgc tgcagctcgc ggccgcggcg ctctttggcg tctgccaggc gctgccgctg 540
ctccacgtca tgcacgactc gtcgcacgcg tcgtacacca acatgccgtt cttccattac 600
gtcgtcggcc gctttgccat ggactggttt gccggcggct cgatggtgtc atggctcaac 660
cagcacgtcg tgggccacca catctacacg aacgtcgcgg gctcggaccc ggatcttccg 720
gtcaacatgg acggcgacat ccgccgcatc gtgaaccgcc aggtgttcca gcccatgtac 780
gcattccagc acatctacct tccgccgctc tatggcgtgc ttggcctcaa gttccgcatc 840
caggacttca ccgacacgtt cggctcgcac acgaacggcc cgatccgcgt caacccgcac 900
gcgctctcga cgtggatggc catgatcagc tccaagtcgt tctgggcctt ctaccgcgtg 960
taccttccgc ttgccgtgct ccagatgccc atcaagacgt accttgcgat cttcttcctc 1020
gccgagtttg tcacgggctg gtacctcgcg ttcaacttcc aagtaagcca tgtctcgacc 1080
gagtgcggct acccatgcgg cgacgaggcc aagatggcgc tccaggacga gtgggcagtc 1140
tcgcaggtca agacgtcggt cgactacgcc catggctcgt ggatgacgac gttccttgcc 1200
ggcgcgctca actaccaggt cgtgcaccac ttgttcccca gcgtgtcgca gtaccactac 1260
ccggcgatcg cgcccatcat cgtcgacgtc tgcaaggagt acaacatcaa gtacgccatc 1320
ttgccggact ttacggcggc gttcgttgcc cacttgaagc acctccgcaa catgggccag 1380
cagggcatcg ccgccacgat ccacatgggc taa 1413
<210> SEQ ID NO 92
<211> LENGTH: 5621
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Plasmid pKR1033
<400> SEQUENCE: 92
ggccgcattt cgcaccaaat caatgaaagt aataatgaaa agtctgaata agaatactta 60
ggcttagatg cctttgttac ttgtgtaaaa taacttgagt catgtacctt tggcggaaac 120
agaataaata aaaggtgaaa ttccaatgct ctatgtataa gttagtaata cttaatgtgt 180
tctacggttg tttcaatatc atcaaactct aattgaaact ttagaaccac aaatctcaat 240
cttttcttaa tgaaatgaaa aatcttaatt gtaccatgtt tatgttaaac accttacaat 300
taattggttg gagaggagga ccaaccgatg ggacaacatt gggagaaaga gattcaatgg 360
agatttggat aggagaacaa cattcttttt cacttcaata caagatgagt gcaacactaa 420
ggatatgtat gagactttca gaagctacga caacatagat gagtgaggtg gtgattccta 480
gcaagaaaga cattagagga agccaaaatc gaacaaggaa gacatcaagg gcaagagaca 540
ggaccatcca tctcaggaaa aggagctttg ggatagtccg agaagttgta caagaaattt 600
tttggagggt gagtgatgca ttgctggtga ctttaactca atcaaaattg agaaagaaag 660
aaaagggagg gggctcacat gtgaatagaa gggaaacggg agaattttac agttttgatc 720
taatgggcat cccagctagt ggtaacatat tcaccatgtt taaccttcac gtacgtctag 780
aggatccccc atggtcaatc aatgagacgc caacttctta atctattgag acctgcaggt 840
ctagaagggc ggatccccgg gtaccgagct cgaattcact ggccgtcgtt ttacaacgtc 900
gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg 960
ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc 1020
tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 1080
accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc 1140
gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 1200
acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 1260
cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga 1320
taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta 1380
tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat 1440
aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc 1500
ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga 1560
aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca 1620
acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt 1680
ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg 1740
gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc 1800
atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata 1860
acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt 1920
tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag 1980
ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca 2040
aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg 2100
aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg 2160
ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag 2220
atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg 2280
aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag 2340
accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga 2400
tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt 2460
tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc 2520
tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc 2580
cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac 2640
caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac 2700
cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt 2760
cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct 2820
gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat 2880
acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt 2940
atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg 3000
cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt 3060
gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt 3120
tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg 3180
tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg 3240
agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc 3300
ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg 3360
gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac 3420
actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag 3480
gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcaggctagc ctaagtacgt 3540
actcaaaatg ccaacaaata aaaaaaaagt tgctttaata atgccaaaac aaattaataa 3600
aacacttaca acaccggatt ttttttaatt aaaatgtgcc atttaggata aatagttaat 3660
atttttaata attatttaaa aagccgtatc tactaaaatg atttttattt ggttgaaaat 3720
attaatatgt ttaaatcaac acaatctatc aaaattaaac taaaaaaaaa ataagtgtac 3780
gtggttaaca ttagtacagt aatataagag gaaaatgaga aattaagaaa ttgaaagcga 3840
gtctaatttt taaattatga acctgcatat ataaaaggaa agaaagaatc caggaagaaa 3900
agaaatgaaa ccatgcatgg tcccctcgtc atcacgagtt tctgccattt gcaatagaaa 3960
cactgaaaca cctttctctt tgtcacttaa ttgagatgcc gaagccacct cacaccatga 4020
acttcatgag gtgtagcacc caaggcttcc atagccatgc atactgaaga atgtctcaag 4080
ctcagcaccc tacttctgtg acgtgtccct cattcacctt cctctcttcc ctataaataa 4140
ccacgcctca ggttctccgc ttcacaactc aaacattctc tccattggtc cttaaacact 4200
catcagtcat caccgcggcc gcaaaccatg gctccagatg cggacaagtt gagacagcgc 4260
aaggcgcaat cgattcaaga cacggctgat tcgcaagcta ccgaactcaa gattggcacc 4320
ctgaagggct tgcaggggac agaaatcgtc attgatggag acatttacga tataaaagac 4380
tttgatcacc ccggtggtga atccatcatg acttttgggg gaaacgatgt caccgccacg 4440
tacaagatga tccaccccta ccactctaag caccatttgg agaagatgaa gaaagtggga 4500
cgagttccgg actacacctc ggaatacaag tttgatactc cctttgagcg tgaaatcaag 4560
caagaggtct tcaagattgt gcgacgaggc cgcgagtttg gaacacctgg atacttcttc 4620
cgggctttct gctacattgg acttttcttt tacttgcagt atttgtgggt cacgactccc 4680
actacctttg ccttggcgat cttctatggt gtttcgcaag ctttcattgg tttgaacgta 4740
caacatgatg ccaaccacgg agctgcctcc aagaagcctt ggatcaataa cttgctagga 4800
ttgggggctg actttatcgg aggttccaaa tggttgtgga tgaaccagca ctggacgcac 4860
cacacataca ccaaccacca tgagaaggat cccgatgcct tgggcgctga accaatgttg 4920
ttgttcaatg attatccctt gggtcaccca aagcgtactt tgattcacca cttccaggcc 4980
ttctattacc ttttcgtctt ggccggatac tgggtctctt cggtcttcaa ccctcaaatt 5040
ttggacttgc aacaccgcgg tgctcaagcg gttggaatga aaatggagaa cgattacatt 5100
gccaaaagcc gaaagtatgc catcttcttg cgtctcttgt atatttacac caacattgtc 5160
gctccgatcc aaaaccaagg cttctcgttg accgtggtcg cccacatttt gaccatgggc 5220
gtcgcttcca gtttgacttt ggcgactctt tttgccttgt cgcacaattt tgaaaacgcg 5280
gatcgcgatc ccacttacga ggcccgcaag ggaggagagc ctgtttgttg gttcaagtcg 5340
caagtcgaaa cctcgtcaac ttacggaggt ttcatctcgg gttgcttgac gggcggactc 5400
aacttccaag tggaacacca cttgttccct cgtatgagtt cggcctggta cccctacatt 5460
gcccctactg ttcgagaggt ttgcaaaaag cacggagtca agtacgcata ctatccctgg 5520
gtctggcaaa acttgatttc aactgtcaag tatctgcatc aaagcggaac tggatccaac 5580
tggaagaatg gcgccaaccc ctactcggga aaattgtaag c 5621
<210> SEQ ID NO 93
<211> LENGTH: 11949
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Plasmid pKR1038
<220> FEATURE:
<221> NAME/KEY: misc_feature
<222> LOCATION: (7810)..(7810)
<223> OTHER INFORMATION: n is a, c, g, or t
<400> SEQUENCE: 93
ggagatccaa gcttttgatc catgcccttc atttgccgct tattaattaa tttggtaaca 60
gtccgtacta atcagttact tatccttccc ccatcataat taatcttggt agtctcgaat 120
gccacaacac tgactagtct cttggatcat aagaaaaagc caaggaacaa aagaagacaa 180
aacacaatga gagtatcctt tgcatagcaa tgtctaagtt cataaaattc aaacaaaaac 240
gcaatcacac acagtggaca tcacttatcc actagctgat caggatcgcc gcgtcaagaa 300
aaaaaaactg gaccccaaaa gccatgcaca acaacacgta ctcacaaagg tgtcaatcga 360
gcagcccaaa acattcacca actcaaccca tcatgagccc tcacatttgt tgtttctaac 420
ccaacctcaa actcgtattc tcttccgcca cctcattttt gtttatttca acacccgtca 480
aactgcatgc caccccgtgg ccaaatgtcc atgcatgtta acaagaccta tgactataaa 540
tagctgcaat ctcggcccag gttttcatca tcaagaacca gttcaatatc ctagtacacc 600
gtattaaaga atttaagata tactgcggcc gcaccatgga ggtggtgaat gaaatagtct 660
caattgggca ggaagtttta cccaaagttg attatgccca actctggagt gatgccagtc 720
actgtgaggt gctttacttg tccatcgcat ttgtcatctt gaagttcact cttggccccc 780
ttggtccaaa aggtcagtct cgtatgaagt ttgttttcac caattacaac cttctcatgt 840
ccatttattc gttgggatca ttcctctcaa tggcatatgc catgtacacc atcggtgtta 900
tgtctgacaa ctgcgagaag gcttttgaca acaacgtctt caggatcacc acgcagttgt 960
tctatttgag caagttcctg gagtatattg actccttcta tttgccactg atgggcaagc 1020
ctctgacctg gttgcaattc ttccatcatt tgggggcacc gatggatatg tggctgttct 1080
ataattaccg aaatgaagct gtttggattt ttgtgctgtt gaatggtttc atccactgga 1140
tcatgtacgg ttattattgg accagattga tcaagctgaa gttccccatg ccaaaatccc 1200
tgattacatc aatgcagatc attcaattca atgttggttt ctacattgtc tggaagtaca 1260
ggaacattcc ctgttatcgc caagatggga tgaggatgtt tggctggttc ttcaattact 1320
tttatgttgg cacagtcttg tgtttgttct tgaatttcta tgtgcaaacg tatatcgtca 1380
ggaagcacaa gggagccaaa aagattcagt gagcggccgc aagtatgaac taaaatgcat 1440
gtaggtgtaa gagctcatgg agagcatgga atattgtatc cgaccatgta acagtataat 1500
aactgagctc catctcactt cttctatgaa taaacaaagg atgttatgat atattaacac 1560
tctatctatg caccttattg ttctatgata aatttcctct tattattata aatcatctga 1620
atcgtgacgg cttatggaat gcttcaaata gtacaaaaac aaatgtgtac tataagactt 1680
tctaaacaat tctaacctta gcattgtgaa cgagacataa gtgttaagaa gacataacaa 1740
ttataatgga agaagtttgt ctccatttat atattatata ttacccactt atgtattata 1800
ttaggatgtt aaggagacat aacaattata aagagagaag tttgtatcca tttatatatt 1860
atatactacc catttatata ttatacttat ccacttattt aatgtcttta taaggtttga 1920
tccatgatat ttctaatatt ttagttgata tgtatatgaa agggtactat ttgaactctc 1980
ttactctgta taaaggttgg atcatcctta aagtgggtct atttaatttt attgcttctt 2040
acagataaaa aaaaaattat gagttggttt gataaaatat tgaaggattt aaaataataa 2100
taaataacat ataatatatg tatataaatt tattataata taacatttat ctataaaaaa 2160
gtaaatattg tcataaatct atacaatcgt ttagccttgc tggacgaatc tcaattattt 2220
aaacgagagt aaacatattt gactttttgg ttatttaaca aattattatt taacactata 2280
tgaaattttt ttttttatca gcaaagaata aaattaaatt aagaaggaca atggtgtccc 2340
aatccttata caaccaactt ccacaagaaa gtcaagtcag agacaacaaa aaaacaagca 2400
aaggaaattt tttaatttga gttgtcttgt ttgctgcata atttatgcag taaaacacta 2460
cacataaccc ttttagcagt agagcaatgg ttgaccgtgt gcttagcttc ttttatttta 2520
tttttttatc agcaaagaat aaataaaata aaatgagaca cttcagggat gtttcaacaa 2580
gcttggcgcg ccgttctata gtgtcaccta aatcgtatgt gtatgataca taaggttatg 2640
tattaattgt agccgcgttc taacgacaat atgtccatat ggtgcactct cagtacaatc 2700
tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc 2760
tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc 2820
tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg 2880
atacgcctat ttttataggt taatgtcatg accaaaatcc cttaacgtga gttttcgttc 2940
cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 3000
cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 3060
gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 3120
aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 3180
cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 3240
tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 3300
acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 3360
ctacagcgtg agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 3420
ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 3480
tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 3540
tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 3600
ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg 3660
gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag 3720
cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc 3780
gcgcgttggc cgattcatta atgcaggttg atcagatctc gatcccgcga aattaatacg 3840
actcactata gggagaccac aacggtttcc ctctagaaat aattttgttt aactttaaga 3900
aggagatata cccatggaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat 3960
cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc 4020
tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg 4080
tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga 4140
agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca 4200
gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc 4260
ggaggctatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt 4320
cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga 4380
tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca 4440
ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca 4500
cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga 4560
ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag 4620
gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct 4680
tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca 4740
gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat 4800
cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt 4860
ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg 4920
tccgagggca aaggaatagt gaggtacagc ttggatcgat ccggctgcta acaaagcccg 4980
aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc 5040
ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gatgatcggg 5100
cgcgccgtcg acggatccgt acgcaaaggc aaagatttaa actcgaaaac attacaaaag 5160
tctcaaaaca gaggcaaggc catgcacaaa gcacactcta agtgcttcca ttgcctacta 5220
agtagggtac gtacacgatc accattcacc agtgatgatc tttattaata tacaacacac 5280
tcagagacag cttatgttat agctagctag cataaactat cacatcatgt gttagtacga 5340
caagtgacaa cattgctttt aacttcgcgg ccttggatcc tctagaccgg atataatgag 5400
ccgtaaacaa agatgattaa gtagtaatta atacgtacta gtaaaagtgg caaaagataa 5460
cgagaaagaa ccaatttctt tgcattcggc cttagcggaa ggcatatata agctttgatt 5520
attttattta gtgtaatgat ttcgtacaac caaagcattt atttagtact ctcacacttg 5580
tgtcgcggcc gcgaattcac tagtgattcc ttatagagcc ttccccgcgg gttgcttctc 5640
cgccatccgg gcgaacaccg ccagatagcg cagcaggatg accaaccctt catggggcag 5700
cgggttccga tacggcaggt tgtgcttctg gcacagctgt tccacctggt agctaaccgc 5760
tgtcaggttg tggcgaggga gggtcggcca caaatggtgc tcaatctggt aattcaagcc 5820
tccgaaaaac caatctgtga taatccctcg ccgaatgttc atggtctcat ggatctggcc 5880
aaccgagaat ccatggccat cccagactga gtccccgatc ttctccagtg ggtagtggtt 5940
catgaacacc acgatcgcaa tgccgaagcc gccaaccagc tccgaaacga aaaacaccaa 6000
cagcgatgtg aggatgctgg gcataaagaa taagtggaac agggtcttca aggtccagtg 6060
cagggcgagg ccaatggcct ccttcttata ctgagagcga tagaattggt tatctctgtc 6120
cttcaaactg cgcacggtca acacgctctg gaaacaccaa atgaaccgca acaagataca 6180
gatgaccaag aaatagtact gctggaactg aatgagcttg cgggaaatcg gtgacgcccg 6240
tgtgacgtca tcctcagacc aggctaagag ggggaggttg tcaatatcag ggtcgtgccc 6300
ttgaacattg gttgccgaat gatgtgcatt gtgtctgtcc ttccaccatg tcacggaaaa 6360
accttgcaga ccattgccaa ataccagtcc cacgaggttg ttccagttcc ggttcttgaa 6420
agtctggtgg tggcaaatgt catgagaaag ccagcccatc tgttgatagt gcatcccaag 6480
caacactgcc ccaatgaaat acatctgata ctgaaccatc aggaaataac ccagcactcc 6540
aaggcccagt gtggtgctga ttttgtatga gtaccagagg ggggaggcat caaacatgcc 6600
agttgcgatc aactcttctc ggagcttccg gaaatcctct tgagcttcat tcactgcagc 6660
ctggggtggc aactcagaac tgggattgat tttgggcatg cgcttgagct tgtcgaaggc 6720
ttcttgagag tgcataacca tgaaggcatc agtggcatcc cttccttggt aattctctat 6780
aatttccgca ccaccagggt ggaaattgac ccaggcagac acatcatatg ttgttccatc 6840
aattgtaagg ggaagcgctt ggcgctttga cttcatttca atcgaattcc cgcggccgct 6900
tggggggcta tggaagactt tcttagttag ttgtgtgaat aagcaatgtt gggagaatcg 6960
ggactactta taggatagga ataaaacaga aaagtattaa gtgctaatga aatatttaga 7020
ctgataatta aaatcttcac gtatgtccac ttgatataaa aacgtcagga ataaaggaag 7080
tacagtagaa tttaaaggta ctctttttat atatacccgt gttctctttt tggctagcta 7140
gttgcataaa aaataatcta tatttttatc attattttaa atatcttatg agatggtaaa 7200
tatttatcat aatttttttt actattattt attatttgtg tgtgtaatac atatagaagt 7260
taattacaaa ttttatttac tttttcatta ttttgatatg attcaccatt aatttagtgt 7320
tattatttat aatagttcat tttaatcttt ttgtatatat tatgcgtgca gtactttttt 7380
cctacatata actactatta cattttattt atataatatt tttattaatg aattttcgtg 7440
ataatatgta atattgttca ttattatttc agatttttta aaaatatttg tgttattatt 7500
tatgaaatat gtaatttttt tagtatttga ttttatgatg ataaagtgtt ctaaattcaa 7560
aagaaggggg aaagcgtaaa cattaaaaaa cgtcatcaaa caaaaacaaa atcttgttaa 7620
taaagataaa actgtttgtt ttgatcactg ttatttcgta atataaaaac attatttata 7680
tttatattgt tgacaaccaa atttgcctat caaatctaac caatataatg catgcgtggc 7740
aggtaatgta ctaccatgaa cttaagtcat gacataataa accgtgaatc tgaccaatgc 7800
atgtacctan ctaaattgta tttgtgacac gaagcaaatg attcaattca caatggagat 7860
gggaaacaaa taatgaagaa cccagaacta agaaagcttt tctgaaaaat aaaataaagg 7920
caatgtcaaa agtatactgc atcatcagtc cagaaagcac atgatatttt tttatcagta 7980
tcaatgcagc tagttttatt ttacaatatc gatatagcta gtttaaatat attgcagcta 8040
gatttataaa tatttgtgtt attatttatc atttgtgtaa tcctgttttt agtattttag 8100
tttatatatg atgataatgt attccaaatt taaaagaagg gaaataaatt taaacaagaa 8160
aaaaagtcat caaacaaaaa acaaatgaaa gggtggaaag atgttaccat gtaatgtgaa 8220
tgttacagta tttcttttat tatagagtta acaaattaac taatatgatt ttgttaataa 8280
tgataaaata ttttttttat tattatttca taatataaaa atagtttact taatataaaa 8340
aaaattctat cgttcacaac aaagttggcc acctaattta accatgcatg tacccatgga 8400
ccatattagg taaccatcaa acctgatgaa gagataaaga gatgaagact taagtcataa 8460
cacaaaacca taaaaaacaa aaatacaatc aaccgtcaat ctgaccaatg catgaaaaag 8520
ctgcaatagt gagtggcgac acaaagcaca tgattttctt acaacggaga taaaaccaaa 8580
aaaatatttc atgaacaacc tagaacaaat aaagctttta tataataaat atataaataa 8640
ataaaggcta tggaataata tacttcaata tatttggatt aaataaattg ttggcggggt 8700
tgatatattt atacacacct aaagtcactt caatctcatt ttcacttaac ttttattttt 8760
tttttctttt tatttatcat aaagagaata ttgataatat actttttaac atatttttat 8820
gacatttttt attggtgaaa acttattaaa aatcataaat tttgtaagtt agatttattt 8880
aaagagttcc tcttcttatt ttaaattttt taataaattt ttaaataact aaaatttgtg 8940
ttaaaaatgt taaaaaatgt gttattaacc cttctcttcg aggacgtacg agatccggcc 9000
ggccagatcc tgcaggtctc aatagattaa gaagttggcg tctcattgat tgaccatggg 9060
ggatcctcta gacgtacgtg aaggttaaac atggtgaata tgttaccact agctgggatg 9120
cccattagat caaaactgta aaattctccc gtttcccttc tattcacatg tgagccccct 9180
cccttttctt tctttctcaa ttttgattga gttaaagtca ccagcaatgc atcactcacc 9240
ctccaaaaaa tttcttgtac aacttctcgg actatcccaa agctcctttt cctgagatgg 9300
atggtcctgt ctcttgccct tgatgtcttc cttgttcgat tttggcttcc tctaatgtct 9360
ttcttgctag gaatcaccac ctcactcatc tatgttgtcg tagcttctga aagtctcata 9420
catatcctta gtgttgcact catcttgtat tgaagtgaaa aagaatgttg ttctcctatc 9480
caaatctcca ttgaatctct ttctcccaat gttgtcccat cggttggtcc tcctctccaa 9540
ccaattaatt gtaaggtgtt taacataaac atggtacaat taagattttt catttcatta 9600
agaaaagatt gagatttgtg gttctaaagt ttcaattaga gtttgatgat attgaaacaa 9660
ccgtagaaca cattaagtat tactaactta tacatagagc attggaattt caccttttat 9720
ttattctgtt tccgccaaag gtacatgact caagttattt tacacaagta acaaaggcat 9780
ctaagcctaa gtattcttat tcagactttt cattattact ttcattgatt tggtgcgaaa 9840
tgcggccgct tacaattttc ccgagtaggg gttggcgcca ttcttccagt tggatccagt 9900
tccgctttga tgcagatact tgacagttga aatcaagttt tgccagaccc agggatagta 9960
tgcgtacttg actccgtgct ttttgcaaac ctctcgaaca gtaggggcaa tgtaggggta 10020
ccaggccgaa ctcatacgag ggaacaagtg gtgttccact tggaagttga gtccgcccgt 10080
caagcaaccc gagatgaaac ctccgtaagt tgacgaggtt tcgacttgcg acttgaacca 10140
acaaacaggc tctcctccct tgcgggcctc gtaagtggga tcgcgatccg cgttttcaaa 10200
attgtgcgac aaggcaaaaa gagtcgccaa agtcaaactg gaagcgacgc ccatggtcaa 10260
aatgtgggcg accacggtca acgagaagcc ttggttttgg atcggagcga caatgttggt 10320
gtaaatatac aagagacgca agaagatggc atactttcgg cttttggcaa tgtaatcgtt 10380
ctccattttc attccaaccg cttgagcacc gcggtgttgc aagtccaaaa tttgagggtt 10440
gaagaccgaa gagacccagt atccggccaa gacgaaaagg taatagaagg cctggaagtg 10500
gtgaatcaaa gtacgctttg ggtgacccaa gggataatca ttgaacaaca acattggttc 10560
agcgcccaag gcatcgggat ccttctcatg gtggttggtg tatgtgtggt gcgtccagtg 10620
ctggttcatc cacaaccatt tggaacctcc gataaagtca gcccccaatc ctagcaagtt 10680
attgatccaa ggcttcttgg aggcagctcc gtggttggca tcatgttgta cgttcaaacc 10740
aatgaaagct tgcgaaacac catagaagat cgccaaggca aaggtagtgg gagtcgtgac 10800
ccacaaatac tgcaagtaaa agaaaagtcc aatgtagcag aaagcccgga agaagtatcc 10860
aggtgttcca aactcgcggc ctcgtcgcac aatcttgaag acctcttgct tgatttcacg 10920
ctcaaaggga gtatcaaact tgtattccga ggtgtagtcc ggaactcgtc ccactttctt 10980
catcttctcc aaatggtgct tagagtggta ggggtggatc atcttgtacg tggcggtgac 11040
atcgtttccc ccaaaagtca tgatggattc accaccgggg tgatcaaagt cttttatatc 11100
gtaaatgtct ccatcaatga cgatttctgt cccctgcaag cccttcaggg tgccaatctt 11160
gagttcggta gcttgcgaat cagccgtgtc ttgaatcgat tgcgccttgc gctgtctcaa 11220
cttgtccgca tctggagcca tggtttgcgg ccgcggtgat gactgatgag tgtttaagga 11280
ccaatggaga gaatgtttga gttgtgaagc ggagaacctg aggcgtggtt atttataggg 11340
aagagaggaa ggtgaatgag ggacacgtca cagaagtagg gtgctgagct tgagacattc 11400
ttcagtatgc atggctatgg aagccttggg tgctacacct catgaagttc atggtgtgag 11460
gtggcttcgg catctcaatt aagtgacaaa gagaaaggtg tttcagtgtt tctattgcaa 11520
atggcagaaa ctcgtgatga cgaggggacc atgcatggtt tcatttcttt tcttcctgga 11580
ttctttcttt ccttttatat atgcaggttc ataatttaaa aattagactc gctttcaatt 11640
tcttaatttc tcattttcct cttatattac tgtactaatg ttaaccacgt acacttattt 11700
tttttttagt ttaattttga tagattgtgt tgatttaaac atattaatat tttcaaccaa 11760
ataaaaatca ttttagtaga tacggctttt taaataatta ttaaaaatat taactattta 11820
tcctaaatgg cacattttaa ttaaaaaaaa tccggtgttg taagtgtttt attaatttgt 11880
tttggcatta ttaaagcaac ttttttttta tttgttggca ttttgagtac gtacttaggc 11940
tagcctgca 11949
<210> SEQ ID NO 94
<211> LENGTH: 8671
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Plasmid pKR328
<400> SEQUENCE: 94
ggatctggcc ggccggatct cgtacggatc cgtcgacggc gcgcccgatc atccggatat 60
agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa ggggttatgc 120
tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt tgttagcagc 180
cggatcgatc caagctgtac ctcactattc ctttgccctc ggacgagtgc tggggcgtcg 240
gtttccacta tcggcgagta cttctacaca gccatcggtc cagacggccg cgcttctgcg 300
ggcgatttgt gtacgcccga cagtcccggc tccggatcgg acgattgcgt cgcatcgacc 360
ctgcgcccaa gctgcatcat cgaaattgcc gtcaaccaag ctctgataga gttggtcaag 420
accaatgcgg agcatatacg cccggagccg cggcgatcct gcaagctccg gatgcctccg 480
ctcgaagtag cgcgtctgct gctccataca agccaaccac ggcctccaga agaagatgtt 540
ggcgacctcg tattgggaat ccccgaacat cgcctcgctc cagtcaatga ccgctgttat 600
gcggccattg tccgtcagga cattgttgga gccgaaatcc gcgtgcacga ggtgccggac 660
ttcggggcag tcctcggccc aaagcatcag ctcatcgaga gcctgcgcga cggacgcact 720
gacggtgtcg tccatcacag tttgccagtg atacacatgg ggatcagcaa tcgcgcatat 780
gaaatcacgc catgtagtgt attgaccgat tccttgcggt ccgaatgggc cgaacccgct 840
cgtctggcta agatcggccg cagcgatcgc atccatagcc tccgcgaccg gctgcagaac 900
agcgggcagt tcggtttcag gcaggtcttg caacgtgaca ccctgtgcac ggcgggagat 960
gcaataggtc aggctctcgc tgaattcccc aatgtcaagc acttccggaa tcgggagcgc 1020
ggccgatgca aagtgccgat aaacataacg atctttgtag aaaccatcgg cgcagctatt 1080
tacccgcagg acatatccac gccctcctac atcgaagctg aaagcacgag attcttcgcc 1140
ctccgagagc tgcatcaggt cggagacgct gtcgaacttt tcgatcagaa acttctcgac 1200
agacgtcgcg gtgagttcag gcttttccat gggtatatct ccttcttaaa gttaaacaaa 1260
attatttcta gagggaaacc gttgtggtct ccctatagtg agtcgtatta atttcgcggg 1320
atcgagatcg atccaattcc aatcccacaa aaatctgagc ttaacagcac agttgctcct 1380
ctcagagcag aatcgggtat tcaacaccct catatcaact actacgttgt gtataacggt 1440
ccacatgccg gtatatacga tgactggggt tgtacaaagg cggcaacaaa cggcgttccc 1500
ggagttgcac acaagaaatt tgccactatt acagaggcaa gagcagcagc tgacgcgtac 1560
acaacaagtc agcaaacaga caggttgaac ttcatcccca aaggagaagc tcaactcaag 1620
cccaagagct ttgctaaggc cctaacaagc ccaccaaagc aaaaagccca ctggctcacg 1680
ctaggaacca aaaggcccag cagtgatcca gccccaaaag agatctcctt tgccccggag 1740
attacaatgg acgatttcct ctatctttac gatctaggaa ggaagttcga aggtgaaggt 1800
gacgacacta tgttcaccac tgataatgag aaggttagcc tcttcaattt cagaaagaat 1860
gctgacccac agatggttag agaggcctac gcagcaggtc tcatcaagac gatctacccg 1920
agtaacaatc tccaggagat caaatacctt cccaagaagg ttaaagatgc agtcaaaaga 1980
ttcaggacta attgcatcaa gaacacagag aaagacatat ttctcaagat cagaagtact 2040
attccagtat ggacgattca aggcttgctt cataaaccaa ggcaagtaat agagattgga 2100
gtctctaaaa aggtagttcc tactgaatct aaggccatgc atggagtcta agattcaaat 2160
cgaggatcta acagaactcg ccgtgaagac tggcgaacag ttcatacaga gtcttttacg 2220
actcaatgac aagaagaaaa tcttcgtcaa catggtggag cacgacactc tggtctactc 2280
caaaaatgtc aaagatacag tctcagaaga ccaaagggct attgagactt ttcaacaaag 2340
gataatttcg ggaaacctcc tcggattcca ttgcccagct atctgtcact tcatcgaaag 2400
gacagtagaa aaggaaggtg gctcctacaa atgccatcat tgcgataaag gaaaggctat 2460
cattcaagat gcctctgccg acagtggtcc caaagatgga cccccaccca cgaggagcat 2520
cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa gtggattgat gtgacatctc 2580
cactgacgta agggatgacg cacaatccca ctatccttcg caagaccctt cctctatata 2640
aggaagttca tttcatttgg agaggacacg ctcgagctca tttctctatt acttcagcca 2700
taacaaaaga actcttttct cttcttatta aaccatgaaa aagcctgaac tcaccgcgac 2760
gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc 2820
ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg 2880
ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc 2940
ggccgcgctc ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta 3000
ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc 3060
cgctgttctg cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca 3120
gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga 3180
tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac 3240
cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc 3300
cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg 3360
ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt 3420
cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt 3480
cgagcggagg catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat 3540
tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc 3600
gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat 3660
cgcccgcaga agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg 3720
aaaccgacgc cccagcactc gtccgagggc aaaggaatag tgaggtacct aaagaaggag 3780
tgcgtcgaag cagatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgtt 3840
gccggtcttg cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt 3900
aacatgtaat gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta 3960
tacatttaat acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgc 4020
gcggtgtcat ctatgttact agatcgatgt cgaatcgatc aacctgcatt aatgaatcgg 4080
ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 4140
ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 4200
acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 4260
aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 4320
tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 4380
aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 4440
gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc 4500
acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 4560
accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 4620
ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 4680
gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 4740
gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 4800
ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 4860
gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 4920
cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgacattaa cctataaaaa 4980
taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg 5040
acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca 5100
agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc 5160
atcagagcag attgtactga gagtgcacca tatggacata ttgtcgttag aacgcggcta 5220
caattaatac ataaccttat gtatcataca catacgattt aggtgacact atagaacggc 5280
gcgccaagct tggatctcct gcagcccggg ggatccgccc acgtacggta ccatctgcta 5340
atattttaaa tcacatgcaa gagaggaggc atggttccat tttctacctt cacattattt 5400
gagaaaaacg aacttgttct gtgttttatt tttgcccttc acattagtac aacgtggaag 5460
actcatggtt acacagaatc atacataagt acaatgcttg tccctaagaa aacaagcact 5520
cgttgtattg aacctttacg gctcatgcgg ccgcgaattc actagtgatt gaattcgcgg 5580
ccgcttagtc cgacttggcc ttggcggccg cggccgactc tttgagcgtg aagatctgcg 5640
ccgtctcggg cacagcgccg tagttgacaa agaggtgcgc ggtcttgaag aaggccgtga 5700
tgatgggctc gtcgttcctg cgcacgaggt gcgggtacgc ggccgcaaag tgcttggtgg 5760
cttcgttgag cttgtagtgc ggaatgatcg ggaacaagtg gtggacctgg tgcgtgccaa 5820
tgtggtggct caggttgtcc acgaacgcgc cgtacgagcg gtcgacgctc gagaggttgc 5880
ccttgacgta cgtccactcc gagtcgccgt accacggcgt cgcttcgtcg ttgtggtgca 5940
agaaggtcgt aatgacgagg aacgaagcaa agacaaagag cggcgcatag tagtagaggc 6000
ccatgacggc aaagccgagc gagtatgtga ggtacgcgta cgcggcgaag aaggcggccc 6060
agacgccgag cgacacgatg acggccgacg cgcggcgaag gaggagcggg tcccacgggt 6120
caaagtggct catcgtgcgc ggggcatacc cgaccttcaa gtagacaaac cacgcaccgc 6180
cgagcgtgta gacccattgg cgcacgtcct ggaggtcctt gaccgaccgg tgcgggtaaa 6240
agatctcgtc cttatcaatg ttgcccgtgt tcttgtggtg gtggcggtgc gtcacgcgcc 6300
agctctcgaa cggcgtcaaa atcgcagagt gcatgatgca gccgatgata aagttgacgc 6360
tgtggtagcg cgagaaggcc gagtggccgc agtcgtggcc gaccgtgaag aagccccaga 6420
agatgacgcc ctgcacgtag atgtaggtgg cgcaaacgag cgcgtggagc agaacgttat 6480
cggcaatgaa cggcgtcgag cgcgccgcgt agagcagcgc cgccgaggcc gacgcgttga 6540
agatcgcgcg ggccgtgtag tagagcgaga ggccgaggtt cgactcaaag cacgcgttcg 6600
ggatcgagtg cttgagctcc gtgagcgtcg ggaactcgac cttcgtctta tcctcagtca 6660
tgcggccgct gaagtattgc ttcttagtta acctttcctt tctctctcag ctatgtgaat 6720
tcattttgct ttcgtcacaa tttatatagt gaaattggat ctttggagtt aacgccttca 6780
caggattatc gtgttagaac aatgcttttt catgttctaa ttagtagtac attacaaatg 6840
tgcactctat tcaataagca tcttttggca cgttaataaa tcatgtgaaa aaaaaatact 6900
actatttcaa agaaagtgtt gtaaaaagaa acggaaagag agctggcttc agttgttgag 6960
acttgtttgc tagtaaaaat ggtgtgaaga gtgattcatg gtgaggtggt ttttcgtccc 7020
tttctgtttg catgaaaaac aaatggcaag agatgacgta ggattccttc ccttaacgat 7080
tatctgtttt taatttcaaa tatacatata ggaatttatg aattactaag gttgtaaaat 7140
atgctggtca tttatttatg gctaaaatat ttttttttct cgtaaatata aaaatattta 7200
aaatttattt ttatcatatt ttttatcctt ataaaattat gtgtacaacc tatataaaaa 7260
aatatcatat ttaatattga ttatatgttt aatcaatata aaaaatcatt atcatatatt 7320
tagatttatt cgaatataca tctaaacaaa aaataacata ttttaatttt atgaagaaaa 7380
aaaaatattt tatcctttat ttatttaaga ttaattaata gttatgtatt gtggaaagac 7440
ttttacacat gcaatagata tactgaatca attagatgcc aatgctgagt tggaaatcac 7500
ttgaggaggg gaggagactt gccaatgctt ttcagtttca tttaaatgat ttagtggagg 7560
agatagagta gtgataaagg catgccccaa ttttggagtg tatatatgag tggaaataag 7620
agagggatag agagaaaaaa taaagagagt aaaaataatt aatgtgaaat gatatgataa 7680
aaaaataaag aaagagataa agagaaaaat gaaatgagag atagatgaaa tagagagtag 7740
atacatgttt gtttaggttt tttttaggaa ataacacatt tttttctcat cacttattac 7800
tcactgtcaa tttcctctct ttcaatcata atgatatgat ttgtttaaca aaaatgtgaa 7860
aaaacatata aagtaaaata tttttataaa ttgataaata aaaatttaca aaatttattt 7920
cttattaaat tgaatagaaa atgaaagaaa agaaaagaaa aagtatatat aaaatgatat 7980
agctttaaaa agaataaatt tttcatatca gtcttttttt aataatttag aaatatttaa 8040
gtatatagca aaaatataat gtactttaca tatgcataaa taataatttg aaaatagaac 8100
taatagaata gagaaaaaag taatataata attaactata tgaaaattta gaagggacaa 8160
tatttttaat taagaatata aacaatattt cttttcatgt aatgagggac ggatgtacgg 8220
ggccagtgtt ggagtcaaag ccaaaatagt cacggggaaa ttaatgcact gcatgactat 8280
tcgaaaaaat tcactagcct tacttagatg ttagattaat agctaggggg tgcagataat 8340
tttgaaaggc atgaaaaaca ttaatttgta cattgcaagc ttttgatgac aagctttgca 8400
attgttcaca ctaccttatg ccatttataa atagagtgat tggcatatga aggaaatcat 8460
gagagtcgaa gcgaaaaaca aagcttgaga gtgtaggaaa aatacagttt ttttggtaaa 8520
aatacagtat ttgaatagga gcgaaaaata tcctttcaaa atgatccttt tctttttttt 8580
tttttttctt gttgttcttg gtcagttatt caaaggaaaa gggattgaaa taaaaacttg 8640
catgtgggat cgtacgtcga gtcgacctgc a 8671
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