Patent application title: DELTA-8 DESATURASE AND ITS USE IN MAKING POLYUNSATURATED FATTY ACIDS
Inventors:
Howard Glenn Damude (Hockessin, DE, US)
Quinn Qun Zhu (West Chester, PA, US)
Assignees:
E.I. DU PONT DE NEMOURS AND COMPANY
IPC8 Class: AC07F902FI
USPC Class:
554 84
Class name: Fatty compounds having an acid moiety which contains the carbonyl of a carboxylic acid, salt, ester, or amide group bonded directly to one end of an acyclic chain of at least seven (7) uninterrupted carbons, wherein any additional carbonyl in the acid moiety is (1) part of an aldehyde or ketone group, (2) bonded directly to a noncarbon atom which is between the additional carbonyl and the chain, or (3) attached indirectly to the chain via ionic bonding phosphorus containing nitrogen containing
Publication date: 2011-07-28
Patent application number: 20110184200
Abstract:
Isolated nucleic acid fragments and recombinant constructs comprising
such fragments encoding a delta-8 desaturase along with a method of
making long-chain polyunsaturated fatty acids (PUFAs) using this delta-8
desaturase in plants and oleaginous yeast are disclosed.Claims:
1. Oil or a by-product obtained from a seed comprising a recombinant
construct, wherein the recombinant construct comprises at least one
regulatory sequence operably linked to a polynucleotide selected from the
group consisting of: (a) a nucleotide sequence encoding a polypeptide
having delta-8 desaturase activity, wherein the polypeptide has at least
80% amino acid identity, based on the Clustal V method of alignment, when
compared to an amino acid sequence as set forth in SEQ ID NO:16; (b) a
nucleotide sequence encoding a polypeptide having delta-8 desaturase
activity, wherein the nucleotide sequence has at least 80% sequence
identity, based on the BLASTN method of alignment, when compared to a
nucleotide sequence as set forth in SEQ ID NO:15; and (c) a complement of
the nucleotide sequence of (a) or (b), wherein the complement and the
nucleotide sequence consist of the same number of nucleotides and are
100% complementary.
2. The oil or by-product of claim 1, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of: (i) the nucleotide sequence set forth in SEQ ID NO:15; (ii) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:16; and (iii) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that differs from the amino acid sequences in (ii) by at least one conservative amino acid substitution.
3. The oil or by-product of claim 1, wherein the seed is from an oilseed plant selected from the group consisting of soybean, Brassica species, sunflower, maize, cotton, flax and safflower.
4. The oil or by-product of claim 1, wherein the seed is from an oilseed plant selected from the group consisting of soybean, Brassica species, sunflower, maize, cotton, flax, and safflower and further wherein the seed produces a polyunsaturated fatty acid selected from the group consisting of arachidonic acid, eicosadienoic acid, eicosapentaenoic acid, eicosatetraenoic acid, eicosatrienoic acid, dihomo-gamma-linolenic acid, docosapentaenoic acid and docosahexaenoic acid.
5. The oil or by-product of claim 1, wherein the by-product is lecithin.
6. Oil or a by-product obtained from a seed comprising: (a) a first recombinant DNA construct comprising an isolated polynucleotide encoding at least one delta-8 desaturase polypeptide, operably linked to at least one regulatory sequence; and (b) at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-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.
7. The oil or by-product of claim 6, wherein the seed is from an oilseed plant selected from the group consisting of soybean, Brassica species, sunflower, maize, cotton, flax and safflower.
8. The oil or by-product of claim 6, wherein the seed is from an oilseed plant selected from the group consisting of soybean, Brassica species, sunflower, maize, cotton, flax, and safflower and further wherein the seed produces a polyunsaturated fatty acid selected from the group consisting of arachidonic acid, eicosadienoic acid, eicosapentaenoic acid, eicosatetraenoic acid, eicosatrienoic acid, dihomo-gamma-linolenic acid, docosapentaenoic acid and docosahexaenoic acid.
9. The oil or by-product of claim 6, wherein the by-product is lecithin.
10. Oil obtained by a method comprising: (a) transforming a cell with a recombinant construct, wherein the recombinant construct comprises at least one regulatory sequence operably linked to a polynucleotide selected from the group consisting of (i) a nucleotide sequence encoding a polypeptide having delta-8 desaturase activity, wherein the polypeptide has at least 80% amino acid identity, based on the Clustal V method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:16, (ii) a nucleotide sequence encoding a polypeptide having delta-8 desaturase activity, wherein the nucleotide sequence has at least 80% sequence identity, based on the BLASTN method of alignment, when compared to a nucleotide sequence as set forth in SEQ ID NO:15, and (iii) a complement of the nucleotide sequence of (a) or (b), wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary; and (b) selecting those transformed cells that make long-chain polyunsaturated fatty acids.
11. The oil of claim 10, wherein the cell is a yeast cell or a plant cell.
12. The oil of claim 11, wherein the yeast cell is an oleaginous yeast cell selected from the group consisting of Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces yeast cells.
13. The oil of claim 11, wherein the plant cell is an oilseed plant selected from the group consisting of soybean, Brassica species, sunflower, maize, cotton, flax and safflower plant cells.
14. Oil obtained by a method comprising: (a) transforming a soybean cell with a first recombinant DNA construct comprising an isolated polynucleotide encoding at least one delta-8 desaturase polypeptide, operably linked to at least one regulatory sequence and at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-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 a soybean 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 soybean plant.
15. Food or feed which incorporates the oil or by-product of claim 1.
16. Food or feed which incorporates the oil or by-product of claim 6.
17. Food or feed which incorporates the oil of claim 10.
18. Food or feed which incorporates the oil of claim 14.
19. Food or feed comprising an ingredient derived from the processing of a seed comprising a recombinant construct, wherein the recombinant construct comprises at least one regulatory sequence operably linked to a polynucleotide selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide having delta-8 desaturase activity, wherein the polypeptide has at least 80% amino acid identity, based on the Clustal V method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:16; (b) a nucleotide sequence encoding a polypeptide having delta-8 desaturase activity, wherein the nucleotide sequence has at least 80% sequence identity, based on the BLASTN method of alignment, when compared to a nucleotide sequence as set forth in SEQ ID NO:15; and (c) a complement of the nucleotide sequence of (a) or (b), wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.
20. Food or feed comprising an ingredient derived from the processing of a seed comprising: (a) a first recombinant DNA construct comprising an isolated polynucleotide encoding at least one delta-8 desaturase polypeptide, operably linked to at least one regulatory sequence; and (b) at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-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.
Description:
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser. No. 11/737,772, filed Apr. 20, 2007, which claims benefit of U.S. Provisional Application No. 60/795,810, filed Apr. 28, 2006, and U.S. Provisional Application No. 60/837,789, filed Aug. 15, 2006; the entire contents of all of which are herein incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention pertains to a polynucleotide sequence encoding a delta-8 desaturase and the use of this desaturase in making long-chain polyunsaturated fatty acids (PUFAs).
BACKGROUND OF THE INVENTION
[0003] Fatty acids (lipids) are water-insoluble organic biomolecules that can be extracted from cells and tissues by nonpolar solvents such as chloroform, ether or benzene. Lipids have several important biological functions, serving as (1) structural components of membranes; (2) storage and transport forms of metabolic
[0004] fuels; (3) a protective coating on the surface of many organisms; and, (4) cell-surface components concerned in cell recognition, species specificity and tissue immunity. More specifically, polyunsaturated fatty acids (PUFAs) are important components of the plasma membrane of the cell, where they may be found in such forms as phospholipids and also can be found in triglycerides. PUFAs also serve as precursors to other molecules of importance in human beings and animals, including the prostacyclins, leukotrienes and prostaglandins. There are two main families of PUFAs (i.e., the omega-3 fatty acids and the omega-6 fatty acids).
[0005] The human body is capable of producing most of the PUFAs which it requires to function. However, eicosapentaenoic acid (EPA; 20:5, delta-5,8,11,14,17) and docosahexaenoic acid (DHA; 22:6, delta-4,7,10,13,16,19) cannot be synthesized efficiently by the human body and thus must be supplied through the diet. Since the human body cannot produce adequate quantities of these PUFAs, they are called essential fatty acids. Because of their important roles in human health and nutrition, EPA and DHA are the subject of much interest as discussed herein.
[0006] DHA is a fatty acid of the omega-3 series according to the location of the last double bond in the methyl end. It is synthesized via alternating steps of desaturation and elongation (see FIG. 12). Production of DHA is important because of its beneficial effect on human health. For example, increased intake of DHA has been shown to be beneficial or have a positive effect in inflammatory disorders (e.g., rheumatoid arthritis), Type II diabetes, hypertension, atherosclerosis, depression, myocardial infarction, thrombosis, some cancers and for prevention of the onset of degenerative disorders such as Alzheimer's disease. Currently the major sources of DHA are oils from fish and algae.
[0007] EPA and arachidonic acid (AA or ARA; 20:4, delta-5,8,11,14) are both delta-5 essential fatty acids. EPA belongs to the omega-3 series with five double bonds in the acyl chain, is found in marine food, and is abundant in oily fish from the North Atlantic. Beneficial or positive effects of increased intake of EPA have been shown in patients with coronary heart disease, high blood pressure, inflammatory disorders, lung and kidney diseases, Type II diabetes, obesity, ulcerative colitis, Crohn's disease, anorexia nervosa, burns, osteoarthritis, osteoporosis, attention deficit/hyperactivity disorder and early stages of colorectal cancer (see, for example, the review of McColl, J., NutraCos. 2(4):35-40 (2003)).
[0008] AA belongs to the omega-6 series with four double bonds. The lack of a double bond in the omega-3 position confers on AA different properties than those found in EPA. The eicosanoids produced from AA have strong inflammatory and platelet aggregating properties, whereas those derived from EPA have anti-inflammatory and anti-platelet aggregating properties. AA is recognized as the principal omega-6 fatty acid found in the human brain and an important component of breast milk and many infant formulas, based on its role in early neurological and visual development. AA can be obtained from some foods (such as meat, fish, and eggs), but the concentration is low.
[0009] Gamma-linolenic acid (GLA; 18:3, delta-6,9,12) is another essential fatty acid found in mammals. GLA is the metabolic intermediate for very long-chain omega-6 fatty acids and for various active molecules. In mammals, formation of long-chain PUFAs is rate-limited by delta-6 desaturation. Many physiological and pathological conditions such as aging, stress, diabetes, eczema, and some infections have been shown to depress the delta-6 desaturation step. In addition, GLA is readily catabolized from the oxidation and rapid cell division associated with certain disorders (e.g., cancer or inflammation). As described above, research has shown that various omega fatty acids reduce the risk of heart disease, have a positive effect on children's development and on certain mental illnesses, autoimmune diseases and joint complaints. However, although there are many health benefits associated with a diet supplemented with these fatty acids, it is recognized that different PUFAs exert different physiological effects in the body (e.g., most notably, the opposing physiological effects of GLA and AA). Thus, production of oils using recombinant means is expected to have several advantages over production from natural sources. For example, recombinant organisms having preferred characteristics for oil production can be used, since the naturally occurring fatty acid profile of the host can be altered by the introduction of new biosynthetic pathways in the host and/or by the suppression of undesired pathways, thereby resulting in increased levels of production of desired PUFAs (or conjugated forms thereof) and decreased production of undesired PUFAs. Optionally, recombinant organisms can provide PUFAs in particular forms which may have specific uses; or, oil production can be manipulated such that the ratio of omega-3 to omega-6 fatty acids so produced is modified and/or a specific PUFA is produced without significant accumulation of other PUFA downstream or upstream products (e.g., production of oils comprising AA and lacking GLA).
[0010] The mechanism of PUFA synthesis frequently occurs via the delta-6 desaturation pathway. For example, long-chain PUFA synthesis in mammals proceeds predominantly by a delta-6 desaturation pathway, in which the first step is the delta-6 desaturation of linoleic acid (LA; 18:2, delta-9,12) and alpha-linolenic acid (ALA; 18:3, delta-9,12,15) to yield gamma-linolenic acid (GLA; 18:3, delta-6,9,12)) and stearidonic acid (STA; 18:4, delta-6,9,12,15), respectively. Further fatty acid elongation and desaturation steps give rise to arachidonic acid (AA or ARA) and eicosapentaenoic acid (EPA). Accordingly, genes encoding delta-6 desaturases, delta-6 elongase components (also identified as C18/20 elongases) and delta-5 desaturases have been cloned from a variety of organisms including higher plants, algae, mosses, fungi, nematodes and humans. Humans can synthesize long-chain PUFAs from the essential fatty acids, LA and ALA; however biosynthesis of long-chain PUFAs is somewhat limited (they are regulated by dietary and hormonal changes), and LA and ALA must be obtained from the diet.
[0011] PCT Publication No. WO 02/26946 (published Apr. 4, 2002) describes isolated nucleic acid molecules encoding FAD4, FAD5, FAD5-2 and FAD6 fatty acid desaturase family members which are expressed in long-chain PUFA-producing organisms, e.g., Thraustochytrium, Pythium irregulare, Schizichytrium and Crypthecodinium. It is indicated that constructs containing the desaturase genes can be used in any expression system including plants, animals, and microorganisms for the production of cells capable of producing long-chain PUFAs.
[0012] PCT Publication No. WO 98/55625 (published Dec. 19, 1998) describes the production of PUFAs by expression of polyketide-like synthesis genes in plants.
[0013] PCT Publication No. WO 98/46764 (published Oct. 22, 1998) describes compositions and methods for preparing long-chain fatty acids in plants, plant parts and plant cells which utilize nucleic acid sequences and constructs encoding fatty acid desaturases, including delta-5 desaturases, delta-6 desaturases and delta-12 desaturases.
[0014] U.S. Pat. No. 6,075,183 (issued to Knutzon et al. on Jun. 13, 2000) describes methods and compositions for synthesis of long-chain PUFAs in plants.
[0015] U.S. Pat. No. 6,459,018 (issued to Knutzon et al. on Oct. 1, 2002) describes a method for producing STA in plant seed utilizing a construct comprising a DNA sequence encoding a delta-6 desaturase.
[0016] Spychalla et al. (Proc. Natl. Acad. Sci. USA, 94:1142-1147 (1997)) describes the isolation and characterization of a cDNA from Caenorhabditis elegans that, when expressed in Arabidopsis, encodes a fatty acid desaturase which can catalyze the introduction of an omega-3 double bond into a range of 18- and 20-carbon fatty acids.
[0017] An alternate pathway for the biosynthesis of AA and EPA operates in some organisms (i.e., the delta-9 elongase/delta-8 desaturase pathway). Here LA and ALA are first elongated to eicosadienoic acid (EDA; 20:2, delta-11,14) and eicosatrienoic acid (EtrA; 20:3, delta-11,14,17), respectively, by a delta-9 elongase. Subsequent delta-8 and delta-5 desaturation of these products yields AA and EPA. The delta-8 pathway is present inter alia, in euglenoid species where it is the dominant pathway for formation of 20-carbon PUFAs.
[0018] PCT Publication No. WO 2000/34439 (published Jun. 15, 2000) discloses amino acid and nucleic acid sequences for delta-5 and delta-8 desaturase enzymes. Based on the information presented in Applicants' Assignee's co-pending application having Provisional Application No. 60/583,041 filed Jun. 25, 2004 (U.S. application Ser. No. 11/166,003 filed Jun. 24, 2005 (PCT Publication No. WO 2006/012325 and WO 2006/012326; published Feb. 2, 2006)), it is apparent that the delta-8 desaturase nucleotide and amino acid sequences of PCT Publication No. WO 2000/34439 are not correct. However, the correct sequence is set forth in corresponding U.S. Pat. No. 6,825,017 (issued to Browse et al. on Nov. 30, 2004) that describes desaturases, in particular, delta-5 and delta-8 desaturases and their use in synthesizing PUFAs. Browse discloses the same delta-8 desaturase in U.S. Publication No. 2006090221 (published on Apr. 27, 2006).
[0019] Applicants' Assignee's co-pending application having U.S. application Ser. No. 11/166,003 filed Jun. 24, 2005 (PCT Publication No. WO 2006/012325 and WO 2006/012326; published Feb. 2, 2006) concerns a Eulgena gracilis delta-8 desaturase.
[0020] Wallis et al. (Arch. Biochem. and Biophys. 365(2):307-316 (May 1999)) describes the cloning of a gene that appears to encode a delta-8 desaturase in Euglena gracilis. This sequence appears to be the same sequence disclosed in PCT Publication No. WO 2000/34439.
[0021] Qi et al. (Nat. Biotech. 22(6):739-45 (2004)) describes the production of long-chain PUFAs using, among other things, a delta-8 desaturase from Euglena gracilis; however, the complete sequence of the delta-8 desaturase is not provided.
[0022] PCT Publication No. WO 2004/057001 (published Jul. 8, 2004) discloses amino acid and nucleic acid sequences for a delta-8 desaturase enzyme from Euglena gracilis.
[0023] PCT Publication No. WO 2005/103253 (published Apr. 22, 2005) discloses amino acid and nucleic acid sequences for a delta-8 desaturase enzyme from Pavlova salina (see also U.S. Publication No. 2005/0273885).
[0024] Sayanova et al. (FEBS Lett. 580:1946-1952 (2006)) describes the isolation of and characterization of a cDNA from the free living soil amoeba Acanthamoeba castellanii that, when expressed in Arabidopsis, encodes a C20 delta-8 desaturase.
[0025] An expansive study of PUFAs from natural sources and from chemical synthesis are not sufficient for commercial needs. Therefore, it is of interest to find alternative means to allow production of commercial quantities of PUFAs. Biotechnology offers an attractive route for producing long-chain PUFAs in a safe, cost efficient manner in microorganisms and plants.
[0026] With respect to microorganisms, many algae, bacteria, molds and yeast can synthesize oils in the ordinary course of cellular metabolism. Thus, oil production involves cultivating the microorganism in a suitable culture medium to allow for oil synthesis, followed by separation of the microorganism from the fermentation medium and treatment for recovery of the intracellular oil. Attempts have been made to optimize production of fatty acids by fermentive means involving varying such parameters as microorganisms used, media and conditions that permit oil production. However, these efforts have proved largely unsuccessful in improving yield of oil or the ability to control the characteristics of the oil composition produced. One class of microorganisms that has not been previously examined as a production platform for PUFAs (prior to work by the Applicants' Assignee), however, are the oleaginous yeasts. These organisms can accumulate oil up to 80% of their dry cell weight. The technology for growing oleaginous yeast with high oil content is well developed (for example, see EP 0 005 277 B1; Ratledge, C., Prog. Ind. Microbiol. 16:119-206 (1982)), and may offer a cost advantage compared to commercial micro-algae fermentation for production of omega-3 or omega-6 PUFAs. Whole yeast cells may also represent a convenient way of encapsulating omega-3 or omega-6 PUFA-enriched oils for use in functional foods and animal feed supplements.
[0027] Applicants' Assignee's co-pending application having U.S. Provisional Application No. 60/739,989 filed Nov. 23, 2005 (Attorney Docket No. BB-1562), discloses a delta-9 elongase from Eulgena gracilis.
[0028] WO 02/077213 (published Oct. 3, 2002) describes isolated nucleic acid molecules encoding a fatty acid elongase with specificity for linoleic acid or alpha-linolenic acid from Isochrysis galbana (i.e., delta-9 elongase).
[0029] U.S. Pat. No. 6,403,349 (issued Jun. 11, 2002) concerns the identification of nucleotide and amino acid sequences of an elongase gene derived from Mortierella alpina.
[0030] PCT Publication No. WO 2004/101757 and PCT Publication No. WO 2004/101753 (published Nov. 25, 2004) concern the production of PUFAs in oleaginous yeasts and are Applicants' Assignee's copending applications.
[0031] PCT Publication No. WO 2004/071467 (published Aug. 26, 2004) concerns the production of PUFAs in plants, while PCT Publication No. WO 2004/071178 (published Aug. 26, 2004) concerns annexin promoters and their use in expression of transgenes in plants; both are Applicants' Assignee's copending applications.
[0032] Applicants' Assignee's copending applications having U.S. application Ser. No. 10/985,109 and U.S. application Ser. No. 10/985,254 filed Nov. 10, 2004 (PCT Publication No. 2005/047479 and PCT Publication No. 2005/047480; published May 26, 2005) concerns delta-15 desaturase genes suitable for increasing levels of omega-3 fatty acids.
[0033] Applicants' Assignee's copending applications also include U.S. application Ser. No. 11/265,761 filed Nov. 2, 2005, U.S. application Ser. No. 11/264,784 filed Nov. 1, 2005, and U.S. application Ser. No. 11/264,737 filed Nov. 1, 2005 (methods of making EPA, ARA and DHA, respectively, in Yarrowia lipolytica), each claiming benefit of an earlier provisional filing date of Nov. 4, 2004.
SUMMARY OF THE INVENTION
[0034] The invention concerns an isolated polynucleotide comprising: [0035] (a) a nucleotide sequence encoding a polypeptide having delta-8 desaturase activity, wherein the polypeptide has at least 80% amino acid identity, based on the Clustal V method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:16; [0036] (b) a nucleotide sequence encoding a polypeptide having delta-8 desaturase activity, wherein the nucleotide sequence has at least 80% sequence identity, based on the BLASTN method of alignment, when compared to a nucleotide sequence as set forth in SEQ ID NO:15; or [0037] (c) a complement of the nucleotide sequence of (a) or (b), wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.
[0038] In a second embodiment, the invention concerns codon optimization, specifically, an isolated nucleic acid molecule which encodes a delta-8 desaturase enzyme as set forth in SEQ ID NO:57 wherein at least 162 codons are codon-optimized for expression in Yarrowia sp.
[0039] In a third embodiment, the invention concerns a recombinant DNA construct comprising any of the polynucleotides of the invention operably linked to at least one regulatory sequence.
[0040] In a fourth embodiment, the invention concerns a cell comprising the recombinant DNA construct of the invention. Of interest are cells selected from the group consisting of plants and yeast.
[0041] In a fifth embodiment, the invention concerns a transformed Yarrowia sp. comprising the recombinant construct of the invention.
[0042] In a sixth embodiment, the invention 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.
[0043] In a seventh embodiment, the invention concerns a method for producing a transformed plant comprising transforming a plant cell with a polynucleotide of the invention and regenerating a plant from the transformed plant cell. A preferred plant is soybean.
[0044] In an eighth embodiment, the invention concerns a method for producing yeast comprising transforming a yeast cell with a polynucleotide of the invention and growing yeast from the transformed yeast cell and, in particular, oleaginous yeast.
[0045] In a ninth embodiment, the invention concerns a seed comprising the recombinant construct of the invention.
[0046] In a tenth embodiment, the invention concerns method for making long-chain polyunsaturated fatty acids in a cell comprising: [0047] (a) transforming a cell with the recombinant construct of the invention; [0048] (b) selecting those transformed cells that make long-chain polyunsaturated fatty acids.
[0049] In an eleventh embodiment, the invention concerns oil obtained from seed comprising the recombinant construct of the invention or yeast comprising the recombinant construct of the invention.
[0050] In a twelfth embodiment, the invention concerns a method for making long-chain polyunsaturated fatty acids in a plant cell comprising: [0051] (a) transforming a cell with the recombinant construct of the invention; and [0052] (b) selecting those transformed cells that make long-chain polyunsaturated fatty acids.
[0053] In a thirteenth embodiment, the invention concerns a method for producing at least one polyunsaturated fatty acid in a soybean cell comprising: [0054] (a) transforming a soybean cell with a first recombinant DNA construct comprising an isolated polynucleotide encoding at least one delta-8 desaturase polypeptide, operably linked to at least one regulatory sequence and at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-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; [0055] (b) regenerating a soybean plant from the transformed cell of step (a); and [0056] (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 soybean plant.
[0057] In a fourteenth embodiment, the invention concerns an oilseed plant comprising: [0058] (a) a first recombinant DNA construct comprising an isolated polynucleotide encoding at least one delta-8 desaturase polypeptide, operably linked to at least one regulatory sequence; and [0059] (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-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.
[0060] Also of interest are seeds obtained from such oilseed plants and oil obtained from these seeds.
[0061] In a fifteenth embodiment, the invention concerns food or feed which incorporates oil of the invention.
[0062] In a sixteenth embodiment, the invention concerns food or feed comprising an ingredient derived from the processing of the seeds of the invention.
[0063] In an seventeenth embodiment, the invention concerns an isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having delta-8 desaturase activity, wherein the nucleotide sequence has at least 90% sequence identity, based on the BLASTN method of alignment, when compared to a nucleotide sequence as set forth in SEQ ID NO:15.
[0064] In an eighteenth embodiment, the invention concerns method for making long-chain polyunsaturated fatty acids in a cell having a reduced level of by-product fatty acids, said method comprising: [0065] (a) transforming a host cell with at least one recombinant DNA construct comprising an isolated polynucleotide encoding at least two delta-8 desaturases operably linked to at least one regulatory sequence; and [0066] (b) selecting those transformed host cells obtained having a reduced level of by-product fatty acids, when compared to the level of such by-product fatty acids in a transformed host cell having at least one recombinant DNA construct comprising an isolated polynucleotide encoding one delta-8 desaturase operably linked to a regulatory sequence
Biological Deposits
[0067] The following plasmids have been deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, and bear the following designations, Accession Numbers and dates of deposit (Table 1).
TABLE-US-00001 TABLE 1 ATCC Deposits Plasmid Accession Number Date of Deposit pKR72 PTA-6019 May 28, 2004 pKR578 PTA-6280 Nov. 4, 2004 pKR903 PTA-7494 Apr. 12, 2006
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS
[0068] The invention can be more fully understood from the following detailed description and the accompanying drawings and Sequence Listing, which form a part of this application.
[0069] The sequence descriptions summarize the Sequences Listing attached hereto. The Sequence Listing contains one letter codes for nucleotide sequence characters and the single and three letter codes for amino acids as defined in the IUPAC-IUB standards described in Nucleic Acids Research 13:3021-3030 (1985) and in the Biochemical Journal 219(2):345-373 (1984).
[0070] FIG. 1 is a map of plasmid pY121 (yeast expression vector).
[0071] FIG. 2 is a map of plasmid pKR902 (soybean expression vector).
[0072] FIG. 3 is a map of plasmid pKR903 (soybean expression vector).
[0073] FIG. 4 is a map of plasmid pY118 (Yarrowia expression vector)
[0074] FIG. 5 is a map of plasmid pKR973 (soybean expression vector).
[0075] FIG. 6 is a map of plasmid pKR912 (soybean expression vector).
[0076] FIG. 7 is a map of plasmid pKR983 (soybean expression vector).
[0077] FIGS. 8A and 8B show a table of the fatty acid profiles from somatic soybean embryos expressing the Pavlova lutheri delta-8 desaturase and Isochrysis galbana delta-9 elongase (see Example 12).
[0078] FIGS. 9-A, 9-B, 9-C and 9-D are maps of plasmids pEgD9ES, pDMW263, pZUF17 and pZUFmEgD9ES.
[0079] FIG. 10 shows a chromatogram of the lipid profile of an Euglena gracilis cell extract as described in the Examples.
[0080] FIGS. 11-A, 11-B, 11-C and 11-D is a map of plasmids pPiD8S, pEXPGUS1-C and pZGD5T-CP and pZUFmE9SP8S.
[0081] FIG. 12 is a representative omega-3 and omega-6 fatty acid pathway providing for the conversion of myristic acid through various intermediates to docosahexaenoic acid (DHA).
[0082] FIGS. 13A and 13B show a Clustal V alignment (with default parameters) of SEQ ID NO:16 (the amino acid sequence of the delta-8 desaturase of the instant invention), SEQ ID NO:76 (the amino acid sequence of Pavlova salina delta-8 desaturase sequence disclosed as SEQ ID NO:1 in PCT Publication No. WO 2005/103253; published Apr. 22, 2005), SEQ ID NO:77 (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:17 (the amino acid sequence for the Rhizopus stolonifer delta-6 fatty acid desaturase (NCBI Accession No. ABB96724 (GI 83027409), locus ABB96724, CDS DQ291156; Zhang et al., unpublished)) and SEQ ID NO:2 (the amino acid sequence for the Rhizopus stolonifer delta-6 fatty acid desaturase (NCBI Accession No. AAX22052 (GI 60499699), locus AAX22052, CDS AY795076; Lu et al., unpublished)).
[0083] FIG. 14 shows the average fatty acid profile for the ten best EPA events of soybean embryogenic suspension culture (cv. Jack) transformed with the Ascl fragments of pKR973 (SEQ ID NO:45, FIG. 5) and pKR983 (SEQ ID NO:56; FIG. 7) (see Example 22).
[0084] SEQ ID NO:1 is the sequence of the T7 primer.
[0085] SEQ ID NO:2 is the amino acid sequence for the Rhizopus stolonifer delta-6 fatty acid desaturase (NCBI Accession No. AAX22052 (GI 60499699), locus AAX22052, CDS AY795076; Lu et al., unpublished).
[0086] SEQ ID NO:3 is the sequence of a portion of the cDNA insert from clone eps1c.pk002.f22 (5' end of cDNA insert).
[0087] SEQ ID NO:4 is nucleotide sequence of the fully sequenced EST eps1c.pk002.f22:fis (full insert sequence--FIS).
[0088] SEQ ID NO:5 is the deduced amino acid sequence of SEQ ID NO:4 (clone eps1c.pk002.f22:fis).
[0089] SEQ ID NO:6 is the sequence of the SeqE primer.
[0090] SEQ ID NO:7 is the sequence of the SeqW primer.
[0091] SEQ ID NO:8 is the amino acid sequence of the Mortierella alpina delta-6 desaturase (NCBI Accession No. BAC82361 (GI 34221934), locus BAC82361, CDS AB070557; Sakuradani and Shimizu, Biosci. Biotechnol. Biochem. 67:704-711 (2003)).
[0092] SEQ ID NO:9 is the sequence of the AP1 universal primer.
[0093] SEQ ID NO:10 is the sequence of the GSP PvDES primer.
[0094] SEQ ID NO:11 is the sequence of the M13-28Rev primer.
[0095] SEQ ID NO:12 is the sequence of the PvDES seq primer.
[0096] SEQ ID NO:13 is the full 5' end sequence from genome walk of Pavlova lutheri delta-8 desaturase.
[0097] SEQ ID NO:14 is the nucleotide sequence of the Pavlova lutheri delta-8 desaturase of the instant invention.
[0098] SEQ ID NO:15 is the nucleotide sequence of the CDS of SEQ ID NO:14 (Pavlova lutheri delta-8 desaturase of the instant invention).
[0099] SEQ ID NO:16 is the deduced amino acid sequence of SEQ ID NO:15 (delta-8 desaturase of the instant invention).
[0100] SEQ ID NO:17 is the amino acid sequence for the Rhizopus stolonifer delta-6 fatty acid desaturase (NCBI Accession No. ABB96724 (GI 83027409), locus ABB96724, CDS DQ291156; Zhang et al., unpublished).
[0101] SEQ ID NO:18 is the sequence of the PvDES5'Not-1 primer. SEQ ID NO:19 is the sequence of the PvDES3'Not-1 primer.
[0102] SEQ ID NO:20 is the sequence of the GSP PvDES-2 primer.
[0103] SEQ ID NO:21 is the sequence of pY121. SEQ ID NO:22 is the sequence of pKR123r. SEQ ID NO:23 is the sequence of pKR900. SEQ ID NO:24 is the sequence of pKR925. SEQ ID NO:25 is the sequence of pKR902. SEQ ID NO:26 is the sequence of pKR607.
[0104] SEQ ID NO:27 is the sequence of pKR903. SEQ ID NO:28 is the sequence of the GPDsense primer.
[0105] SEQ ID NO:29 is the sequence of the GPDantisense primer.
[0106] SEQ ID NO:30 is the sequence of pY5-22GPD.
[0107] SEQ ID NO:31 is the sequence of pY118.
[0108] SEQ ID NO:32 is the nucleotide sequence of the CDS of Euglena gracilis delta-9 elongase.
[0109] SEQ ID NO:33 is the sequence of oEugEL1-1 primer.
[0110] SEQ ID NO:34 is the sequence of oEugEL1-2 primer.
[0111] SEQ ID NO:35 is the sequence of pKR906.
[0112] SEQ ID NO:36 is the sequence of pKR132.
[0113] SEQ ID NO:37 is the sequence of pKR953.
[0114] SEQ ID NO:38 is the sequence of pKR287.
[0115] SEQ ID NO:39 is the nucleotide sequence of the CDS of Mortierella alpina delta-5 desaturase, which is described in U.S. Pat. No. 6,075,183 and PCT Publication Nos. WO 2004/071467 and WO 2005/0479479.
[0116] SEQ ID NO:40 is the sequence of pKR277.
[0117] SEQ ID NO:41 is the sequence of pKR952.
[0118] SEQ ID NO:42 is the sequence of pKR457.
[0119] SEQ ID NO:43 is the sequence of the modified Kti/NotI/Kti3'Salb3' cassette.
[0120] SEQ ID NO:44 is the sequence of pKR970.
[0121] SEQ ID NO:45 is the sequence of pKR973.
[0122] SEQ ID NO:46 is the sequence of pKR72.
[0123] SEQ ID NO:47 is the sequence of pKR912.
[0124] SEQ ID NO:48 is the sequence of pKR886r.
[0125] SEQ ID NO:49 is the sequence of pKR271.
[0126] SEQ ID NO:50 is the sequence of pKR226.
[0127] SEQ ID NO:51 is the sequence of the oCon-1 primer.
[0128] SEQ ID NO:52 is the sequence of the oCon-2 primer.
[0129] SEQ ID NO:53 is the sequence of pKR179.
[0130] SEQ ID NO:54 is the sequence of pKR226.
[0131] SEQ ID NO:55 is the sequence of pKR582.
[0132] SEQ ID NO:56 is the sequence of pKR983.
[0133] SEQ ID NO:57 is the nucleotide sequence for the synthetic (codon-optimized) delta-8 desaturase derived from Pavlova lutheri codon-optimized for expression in Yarrowia lipolytica.
[0134] SEQ ID NO:58 is the sequence of pPiD8S.
[0135] SEQ ID NO:59 is the nucleotide sequence of the long-chain PUFA elongation enzyme from Isochrysis galbana (NCBI Accession No. AAL37626 (GI 17226123), locus AAL37626, CDS AF390174).
[0136] SEQ ID NO:60 is the 5' sequence of the cDNA insert from clone eeg1c.pk001.n5.f, while SEQ ID NO:61 is the 3' sequence of the cDNA insert from clone eeg1c.pk001.n5.f.
[0137] SEQ ID NO:61 is the 3' sequence of the cDNA insert from clone eeg1c.pk001.n5.f.
[0138] SEQ ID NO:62 is the sequence aligned from SEQ ID NO:60 and SEQ ID NO:61 (full cDNA sequence excluding polyA tail).
[0139] SEQ ID NO:63 is nucleotide sequence of the M13F universal primer.
[0140] SEQ ID NO:64 is the deduced amino acid sequence of SEQ ID NO:63 (delta-9 elongase--clone eeg1c.pk001.n5.f).
[0141] SEQ ID NO:65 amino acid sequence of the long-chain PUFA elongation enzyme from Isochrysis galbana (NCBI Accession No. AAL37626 (GI 17226123), locus AAL37626, CDS AF390174).
[0142] SEQ ID NO:66 is the nucleotide sequence of the synthetic (codon-optimized) delta-9 elongase derived from Isochrysis galbana codon-optimized for expression in Yarrowia lipolytica.
[0143] SEQ ID NO:67 is the sequence of pEgD9ES.
[0144] SEQ ID NO:68 is the sequence of pDMW263.
[0145] SEQ ID NO:69 is the sequence of pZUF17.
[0146] SEQ ID NO:70 is the sequence of pZUFmEgD9ES.
[0147] SEQ ID NO:71 is the sequence of pZUFmE9SP8S.
[0148] SEQ ID NO:72 is the sequence of pEXPGUS1-C.
[0149] SEQ ID NO:73 is the sequence of pZGD5T-CP.
[0150] SEQ ID NO:74 is the sequence of pYZDE2-S.
[0151] SEQ ID NO:75 is the sequence of pY5-22.
[0152] SEQ ID NO:76 is the amino acid sequence of Pavlova salina delta-8 desaturase sequence disclosed as SEQ ID NO:1 in PCT Publication No. WO 2005/103253 (published Apr. 22, 2005).
[0153] SEQ ID NO:77 is the amino acid sequence of Eulgena gracilis delta-8 desaturase sequence disclosed as SEQ ID NO:2 in Applicants' Assignee's co-pending application having application Ser. No. 11/166,003 filed Jun. 24, 2005 (PCT Publication No. WO 2006/012325; published Feb. 2, 2006).
[0154] SEQ ID NO:78 is the sequence of pY5-30.
DETAILED DESCRIPTION OF THE INVENTION
[0155] All patents, patent applications, and publications cited herein are incorporated by reference in their entirety.
[0156] In the context of this disclosure, a number of terms shall be utilized.
[0157] 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 .sub.c22. 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" (-6 or n-6) versus "omega-3 fatty acids" (-3 or n-3) are provided in PCT Publication No. WO 2004/101757.
[0158] Fatty acids are described herein by a simple notation system of "X:Y", wherein the number before the colon indicates the number of carbon atoms in the fatty acid and the number after the colon is the number of double bonds that are present. 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 linolenic fatty acids. 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.
[0159] A representative pathway is illustrated in FIG. 12, 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.
[0160] 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 metabolic pathway (delta-15 desaturase, delta-9 elongase, delta-8 desaturase, delta-5 desaturase and delta-17 desaturase), EDA, ERA, 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.
[0161] 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 metabolic pathway (delta-15 desaturase, delta-9 elongase, delta-8 desaturase, delta-5 desaturase and delta-17 desaturase), sciadonic acid (SCI) and juniperonic acid (JUP) also can be produced by the action of a delta-5 desaturase on either EDA or ERA, 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.
[0162] A metabolic pathway, in a biochemical sense, can be regarded as a series of chemical reactions occurring within a cell, catalyzed by enzymes, to achieve either the formation of a metabolic product to be used or stored by the cell, or the initiation of another metabolic pathway (then called a flux generating step). Many of these pathways are elaborate, and involve a step by step modification of the initial substance to shape it into a product having the exact chemical structure desired.
[0163] 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, the abbreviations that will be used throughout the remainder of the specification, and each compounds' chemical name.
TABLE-US-00002 TABLE 2 Nomenclature of Polyunsaturated Fatty Acids Common Shorthand Name Abbreviation Chemical Name Notation oleic -- cis-9-octadecenoic 18:1 linoleic LA cis-9,12-octadecadienoic 18:2 {dot over (u)}-6 gamma- GLA cis-6,9,12- 18:3 {dot over (u)}-6 linolenic octadecatrienoic eicosadienoic EDA cis-11,14-eicosadienoic 20:2 {dot over (u)}-6 dihomo- DGLA or cis-8,11,14-eicosatrienoic 20:3 w-6 gamma- HGLA linolenic sciadonic SCI cis-5,11,14-eicosatrienoic 20:3b w-6 arachidonic AA or ARA cis-5,8,11,14- 20:4 w-6 eicosatetraenoic alpha-linolenic ALA cis-9,12,15- 18:3 w-3 octadecatrienoic stearidonic STA cis-6,9,12,15- 18:4 w-3 octadecatetraenoic eicosatrienoic ETrA or ERA cis-11,14,17- 20:3 w-3 eicosatrienoic eicosa- ETA cis-8,11,14,17- 20:4 w-3 tetraenoic eicosatetraenoic juniperonic JUP cis-5,11,14,17- 20:4b w-3 eicosatrienoic eicosa- EPA cis-5,8,11,14,17- 20:5 w-3 pentaenoic eicosapentaenoic docosa- DPA cis-7,10,13,16,19- 22:5 w-3 pentaenoic docosapentaenoic docosa- DHA cis-4,7,10,13,6,19- 22:6 w-3 hexaenoic docosahexaenoic
[0164] The term "essential fatty acid" refers to a particular PUFA that an organism must ingest in order to survive, being unable to synthesize the particular essential fatty acid de novo. For example, mammals can not synthesize the essential fatty acid LA. Other essential fatty acids include, but are not limited to, GLA, DGLA, AA, EPA and DHA.
[0165] The term "fat" refers to a lipid substance that is solid at 25° C. and usually saturated.
[0166] The term "oil" refers to a lipid substance that is liquid at 25° C. and usually polyunsaturated. PUFAs are found in the oils of some algae, oleaginous yeasts and filamentous fungi. "Microbial oils" or "single cell oils" are those oils naturally produced by microorganisms during their lifespan. Such oils can contain long-chain PUFAs.
[0167] The term "PUFA biosynthetic pathway" refers to a metabolic process that converts oleic acid to LA, EDA, GLA, DGLA, AA, ALA, STA, ETrA, ETA, EPA, DPA and DHA. This process is well described in the literature (e.g., see PCT Publication No. WO 2005/003322). Simplistically, this process involves elongation of the carbon chain through the addition of carbon atoms and desaturation of the molecule through the addition of double bonds, via a series of special desaturation and elongation enzymes (i.e., "PUFA biosynthetic pathway enzymes") present in the endoplasmic reticulim membrane. More specifically, "PUFA biosynthetic pathway 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.
[0168] "Desaturase" is a polypeptide which can desaturate one or more fatty acids to produce a mono- or poly-unsaturated fatty acid or precursor which is of interest. Of particular interest herein are delta-8 desaturases that will desaturate a fatty acid between the eighth and ninth carbon atom numbered from the carboxyl-terminal end of the molecule and that can, for example, catalyze the conversion of EDA to DGLA and/or ETrA to ETA. Other useful fatty acid desaturases include, for example, (1) delta-5 desaturases that catalyze the conversion of DGLA to AA and/or ETA to EPA; (2) delta-6 desaturases that catalyze the conversion of LA to GLA and/or ALA to STA; (3) delta-4 desaturases that catalyze the conversion of DPA to DHA; (4) delta-12 desaturases that catalyze the conversion of oleic acid to LA; (5) delta-15 desaturases that catalyze the conversion of LA to ALA and/or GLA to STA; (6) delta-17 desaturases that catalyze the conversion of AA to EPA and/or DGLA to ETA; and (7) delta-9 desaturases that catalyze the conversion of palmitate to palmitoleic acid (16:1) and/or stearate to oleic acid (18:1).
[0169] The term "elongase system" refers to a suite of four enzymes that are responsible for elongation of a fatty acid carbon chain to produce a fatty acid that is two carbons longer than the fatty acid substrate that the elongase system acts upon. More specifically, the process of elongation occurs in association with fatty acid synthase, whereby CoA is the acyl carrier (Lassner et al., Plant Cell 8:281-292 (1996)). In the first step, which has been found to be both substrate-specific and also rate-limiting, malonyl-CoA is condensed with a long-chain acyl-CoA to yield carbon dioxide (CO2) and a a-ketoacyl-CoA (where the acyl moiety has been elongated by two carbon atoms). Subsequent reactions include reduction to a-hydroxyacyl-CoA, dehydration to an enoyl-CoA and a second reduction to yield the elongated acyl-CoA. Examples of reactions catalyzed by elongase systems are the conversion of GLA to DGLA, STA to ETA, LA to EDA, ALA to ERA and EPA to DPA.
[0170] For the purposes herein, an enzyme catalyzing the first condensation reaction (i.e., conversion of malonyl-CoA to a-ketoacyl-CoA) will be referred to generically as an "elongase". In general, the substrate selectivity of elongases is somewhat broad but segregated by both chain length and the degree of unsaturation. Accordingly, elongases can have different specificities. For example, a C14/16 elongase will utilize a C14 substrate (e.g., myristic), a C16/18 elongase will utilize a C16 substrate (e.g., palmitate), a .sub.c18/20 elongase will utilize a C18 substrate (e.g., GLA, STA) and a C20/22 elongase will utilize a C20 substrate (e.g., EPA). In like manner, and of particular interest herein, a "delta-9 elongase" is able to catalyze the conversion of LA and ALA to EDA and ETrA. It is important to note that some elongases have broad specificity and thus a single enzyme may be capable of catalyzing several elongase reactions (e.g., thereby acting as both a C16/18 elongase and a .sub.Cl8/20 elongase).
[0171] In preferred embodiments, it is desirable to empirically determine the specificity of a fatty acid elongase by transforming a suitable host with the gene for the fatty acid elongase and determining its effect on the fatty acid profile of the host. Fatty acid elongases from different species can display great variability in substrate specificity. For example, Mortierella alpina delta-6 elongase acts as a C18/20 elongase (elongation of GLA to DGLA) in yeast, but can additionally act as a C20/22 elongation of LA or ALA to EDA or ETrA, respectively, in soybean
[0172] The term "delta-9 elongase/delta-8 desaturase pathway" refers to a elongase for the elongation of EPA to DPA or as a delta-9 elongase for the biosynthetic pathway for production of long-chain PUFAs. This pathway, at a minimum, comprises a delta-9 elongase and a delta-8 desaturase, thereby enabling biosynthesis of DGLA and/or ETA from LA and ALA, respectively.
[0173] The term "delta-9 elongase" refers to an enzyme that is capable of catalyzing at least one elongase reaction such as the elongation of linoleic (LA) or alpha-linolenic acid (ALA) to EDA or ETrA, respectively. It may act as a C16/18 elongase, C18/20 elongase and C20/22 elongase and may have alternate, but not preferred, specificities for delta-5 and delta-6 fatty acids such as EPA and/or GLA, respectively.
[0174] The term "delta-8 desaturase" refers to an enzyme that is capable of catalyzing at least one desaturation reaction such as the desaturation of eicosadienoic acid (EDA) or eicosatrienoic acid (ETrA) to DGLA or ETA, respectively. It acts as a C20 desaturase.
[0175] The terms "polynucleotide", "polynucleotide sequence", "nucleic acid sequence", "nucleic acid fragment" and "isolated nucleic acid fragment" are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. Nucleotides (usually found in their 5'-monophosphate form) are referred to by a single letter designation as follows: "A" for adenylate or deoxyadenylate (for RNA or DNA, respectively), "C" for cytidylate or deoxycytidylate, "G" for guanylate or deoxyguanylate, "U" for uridylate, "T" for deoxythymidylate, "R" for purines (A or G), "Y" for pyrimidines (C or T), "K" for G or T, "H" for A or C or T, "I" for inosine, and "N" for any nucleotide.
[0176] 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.
[0177] 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.
[0178] 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.
[0179] 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. One set of preferred conditions involves a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set of stringent conditions involves the use of higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions involves the use of two final washes in 0.1×SSC, 0.1% SDS at 65° C.
[0180] "Sequence identity" or "identity" in the context of nucleic acid or polypeptide sequences refers to the nucleic acid bases or amino acid residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
[0181] Thus, "percentage of sequence identity" refers to the valued determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the results by 100 to yield the percentage of sequence identity. Useful examples of percent sequence identities include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 50% to 100%. These identities can be determined using any of the programs described herein.
[0182] Sequence alignments and percent identity or similarity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the MegAlign® program of the LASARGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences are performed using the Clustal V method of alignment (Higgins, D. G. and Sharp, P. M. (1989) Comput. App!. Biosci. 5:151-153; Higgins, D. G. et al. (1992) Comput. App!. Biosci. 8:189-191) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4.
[0183] It is well understood by one skilled in the art that many levels of sequence identity are useful in identifying polypeptides, from other species, wherein such polypeptides have the same or similar function or activity. Useful examples of percent identities include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 55% to 100%. Indeed, any integer amino acid identity from 50% to 100% may be useful in describing the present invention. Also, of interest is any full-length or partial complement of this isolated nucleotide fragment.
[0184] "Gene" refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. "Native gene" refers to a gene as found in nature with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. A "foreign" gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A "transgene" is a gene that has been introduced into the genome by a transformation procedure.
[0185] 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.
[0186] An "allele" is one of several alternative forms of a gene occupying a given locus on a chromosome. When all the alleles present at a given locus on a chromosome are the same that plant is homozygous at that locus. If the alleles present at a given locus on a chromosome differ that plant is heterozygous at that locus.
[0187] "Coding sequence" refers to a DNA sequence that codes for a specific amino acid sequence. "Regulatory sequences" refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include, but are not limited to: promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures.
[0188] "Promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancer" is a DNA sequence that can stimulate promoter activity, and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of some variation may have identical promoter activity. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro, J. K., and Goldberg, R. B. Biochemistry of Plants 15:1-82 (1989).
[0189] "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)).
[0190] "3' non-coding sequences", "transcription terminator" or "termination sequences" refer to DNA sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor. The use of different 3' non-coding sequences is exemplified by Ingelbrecht, I. L., et al. Plant Cell 1:671-680 (1989).
[0191] "RNA transcript" refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript. A RNA transcript is referred to as the mature RNA when it is a RNA sequence derived from post-transcriptional processing of the primary transcript. "Messenger RNA" or "mRNA" refers to the RNA that is without introns and that can be translated into protein by the cell. "cDNA" refers to a DNA that is complementary to, and synthesized from, a mRNA template using the enzyme reverse transcriptase. The cDNA can be single-stranded or converted into double-stranded form using the Klenow fragment of DNA polymerase I. "Sense" RNA refers to RNA transcript that includes the mRNA and can be translated into protein within a cell or in vitro. "Antisense RNA" refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA, and that blocks the expression of a target gene (U.S. Pat. No. 5,107,065). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence. "Functional RNA" refers to antisense RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect on cellular processes. The terms "complement" and "reverse complement" are used interchangeably herein with respect to mRNA transcripts, and are meant to define the antisense RNA of the message.
[0192] The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is regulated by the other. For example, a promoter is operably linked with a coding sequence when it is capable of regulating the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation. In another example, the complementary RNA regions of the invention can be operably linked, either directly or indirectly, 5' to the target mRNA, or 3' to the target mRNA, or within the target mRNA, or a first complementary region is 5' and its complement is 3' to the target mRNA.
[0193] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989). Transformation methods are well known to those skilled in the art and are described below.
[0194] "PCR" or "polymerase chain reaction" is a technique for the synthesis of large quantities of specific DNA segments and consists of a series of repetitive cycles (Perkin Elmer Cetus Instruments, Norwalk, Conn.). Typically, the double-stranded DNA is heat denatured, the two primers complementary to the 3' boundaries of the target segment are annealed at low temperature and then extended at an intermediate temperature. One set of these three consecutive steps is referred to as a "cycle".
[0195] The term "recombinant" refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.
[0196] The terms "plasmid", "vector" and "cassette" refer to an extra chromosomal element often carrying genes that are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA fragments. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell. "Transformation cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitates transformation of a particular host cell. "Expression cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.
[0197] 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.
[0198] 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]).
[0199] The term "expression cassette" as used herein, refers to a discrete nucleic acid fragment into which a nucleic acid sequence or fragment can be moved.
[0200] "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.
[0201] "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.
[0202] "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. Recent work has described the use of "hairpin" structures that incorporate all, or part, of an mRNA encoding sequence in a complementary orientation that results in a potential "stem-loop" structure for the expressed RNA (PCT Publication No. WO 99/53050, published Oct. 21, 1999; PCT Publication No. WO 02/00904, published Jan. 3, 2002). This increases the frequency of co-suppression in the recovered transgenic plants. Another variation describes the use of plant viral sequences to direct the suppression, or "silencing", of proximal mRNA encoding sequences (PCT Publication No. WO 98/36083, published Aug. 20, 1998). Both of these co-suppressing phenomena have not been elucidated mechanistically, although genetic evidence has begun to unravel this complex situation (Elmayan et al., Plant Cell 10:1747-1757 (1998)).
[0203] 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). Generally, the cellular oil content of these 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. Microbiol. 57:419-25 (1991)).
[0204] The term "oleaginous yeast" refers to those microorganisms classified as yeasts that make oil. It is not uncommon for oleaginous microorganisms to accumulate in excess of about 25% of their dry cell weight as oil. Examples of oleaginous yeast include, but are no means limited to, the following genera: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.
[0205] The "Clustal V method of alignment" corresponds to the alignment method labeled Clustal V (described by Higgins and Sharp, CABIOS. 5:151-153 (1989)) and found in the MegAlign® program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). The "default parameters" are the parameters preset by the manufacturer of the program. For multiple alignments, they correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10; and, for pairwise alignments, they are KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. 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.
[0206] "BLASTN method of alignment" is an algorithm provided by the National Center for Biotechnology Information (NCBI) to compare nucleotide sequences using default parameters.
[0207] "Progeny" comprises any subsequent generation of a plant.
[0208] The present invention concerns an isolated polynucleotide comprising: [0209] (a) a nucleotide sequence encoding a polypeptide having delta-8 desaturase activity, wherein the polypeptide has at least 80% amino acid identity, based on the Clustal V method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:16; [0210] (b) a nucleotide sequence encoding a polypeptide having delta-8 desaturase activity, wherein the nucleotide sequence has at least 90% sequence identity, based on the BLASTN method of alignment, when compared to a nucleotide sequence as set forth in SEQ ID NO:15; or [0211] (c) a complement of the nucleotide sequence of (a) or (b), wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.
[0212] In still another aspect, this invention concerns an isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having delta-8 desaturase activity, wherein the nucleotide sequence has at least 80% sequence identity, based on the BLASTN method of alignment, when compared to a nucleotide sequence as set forth in SEQ ID NO:15.
[0213] It was found that a comparison of SEQ ID NO:15 and SEQ ID NO:57 using the BLASTN method of alignment with default parameters showed that these sequences had at least 86% sequence identity.
[0214] This delta-8 desaturase may be used alone or in combination with other desaturase and elongase components to produce various omega-6 and omega-3 PUFAs, including e.g., DGLA, ETA, AA, EPA, DPA and/or DHA (FIG. 12). One skilled in the art will recognize the appropriate combinations of the delta-8 desaturase of the invention herein in conjunction with 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, based on the particular host cell (and its native PUFA profile and/or desaturase and/or elongase profile), the availability of substrate, and the desired end product(s).
[0215] At times, it may be desirable to minimize by-product fatty acids. The relative abundance of by-product fatty acids could be decreased by increasing total delta-8 desaturase activity. One approach to minimize by-product fatty acids would be to express more than one delta-8 desaturase (i.e., the same or different delta-8 desaturase). For instance, the presence of sciadonic acid (SCI) and/or juniperonic acid (JUP), commonly found in the seed lipids of gymnosperms (Wolff et al., Lipids 35(1):1-22 (2000)), such as those in the Pinaceae family (pine), might be considered by-product fatty acids of a delta-6 desaturase or delta-9-elongase pathway. Although these fatty acids are considered to have various health-enhancing properties themselves (Nakane et al., Biol. Pharm. Bull. 23: 758-761 (2000)), their presence as by-product fatty acids in an engineered PUFA pathway, such as in an oilseed crop, may not be desirable depending on the application.
[0216] In another embodiment, this invention concerns a recombinant construct comprising the polynucleotide of the invention operably linked to at least one regulatory sequence.
[0217] As was noted above, a promoter is a DNA sequence that directs cellular machinery of a plant to produce RNA from the contiguous coding sequence downstream (3') of the promoter. The promoter region influences the rate, developmental stage, and cell type in which the RNA transcript of the gene is made. The RNA transcript is processed to produce mRNA which serves as a template for translation of the RNA sequence into the amino acid sequence of the encoded polypeptide. The 5' non-translated leader sequence is a region of the mRNA upstream of the protein coding region that may play a role in initiation and translation of the mRNA. The 3' transcription termination/polyadenylation signal is a non-translated region downstream of the protein coding region that functions in the plant cell to cause termination of the RNA transcript and the addition of polyadenylate nucleotides to the 3' end of the RNA.
[0218] The origin of the promoter chosen to drive expression of the 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, Kunitz trypsin inhibitor 3 promoter, annexin promoter, Glyl promoter, beta subunit of beta conglycinin promoter, P34/Gly Bd m 30K promoter, albumin promoter, Leg A1 promoter and Leg A2 promoter.
[0219] The annexin, or P34, promoter is described in PCT Publication No. WO 2004/071178 (published Aug. 26, 2004). The level of activity of the annexin promoter is comparable to that of many known strong promoters, such as: (1) the CaMV 35S promoter (Atanassova et al., Plant Mol. Biol. 37:275-285 (1998); Battraw and Hall, Plant Mol. Biol. 15:527-538 (1990); Holtorf et al., Plant Mol. Biol. 29:637-646 (1995); Jefferson et al., EMBO J. 6:3901-3907 (1987); Wilmink et al., Plant Mol. Biol. 28:949-955 (1995)); (2) the Arabidopsis oleosin promoters (Plant et al., Plant Mol. Biol. 25:193-205 (1994); Li, Texas A&M University Ph.D. dissertation, pp. 107-128 (1997)); (3) the Arabidopsis ubiquitin extension protein promoters (Callis et al., J. Biol. Chem. 265(21):12486-93 (1990)); (4) a tomato ubiquitin gene promoter (Rollfinke et al., Gene. 211(2):267-76 (1998)); (5) a soybean heat shock protein promoter (Schoffl et al., Mol Gen Genet. 217(2-3):246-53 (1989)); and, (6) a maize H3 histone gene promoter (Atanassova et al., Plant Mol. Biol. 37(2):275-85 (1989)).
[0220] Another useful feature of the annexin promoter is its expression profile in developing seeds. The annexin promoter is most active in developing seeds at early stages (before 10 days after pollination) and is largely quiescent in later stages. The expression profile of the annexin promoter is different from that of many seed-specific promoters, e.g., seed storage protein promoters, which often provide highest activity in later stages of development (Chen et al., Dev. Genet. 10:112-122 (1989); Ellerstrom et al., Plant Mol. Biol. 32:1019-1027 (1996); Keddie et al., Plant Mol. Biol. 24:327-340 (1994); Plant et al., (supra); Li, (supra)). The annexin promoter has a more conventional expression profile but remains distinct from other known seed specific promoters. Thus, the annexin promoter will be a very attractive candidate when overexpression, or suppression, of a gene in embryos is desired at an early developing stage. For example, it may be desirable to overexpress a gene regulating early embryo development or a gene involved in the metabolism prior to seed maturation.
[0221] Following identification of an appropriate promoter suitable for expression of a specific coding sequence, the promoter is then operably linked in a sense orientation using conventional means well known to those skilled in the art.
[0222] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J. et al., In Molecular Cloning: A Laboratory Manual; 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").
[0223] 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.
[0224] The recombinant constructs of the invention may be introduced into one plant cell; or, alternatively, each construct may be introduced into separate plant cells.
[0225] Expression in a plant cell may be accomplished in a transient or stable fashion as is described above.
[0226] 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.
[0227] Plant parts include differentiated and undifferentiated tissues including, but not limited to the following: roots, stems, shoots, leaves, pollen, seeds, tumor tissue and various forms of cells and culture (e.g., single cells, protoplasts, embryos and callus tissue). The plant tissue may be in plant or in a plant organ, tissue or cell culture.
[0228] 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) is 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.
[0229] Thus, this invention also concerns a method for transforming a cell, comprising transforming a cell with the recombinant construct of the invention and selecting those cells transformed with the recombinant construct of Claim 5.
[0230] Also of interest is a method for producing a transformed plant comprising transforming a plant cell with the polynucleotide of the instant invention and regenerating a plant from the transformed plant cell.
[0231] Methods for transforming dicots (primarily by use of Agrobacterium tumefaciens) and obtaining transgenic plants have been published, among others, for: cotton (U.S. Pat. No. 5,004,863; U.S. Pat. No. 5,159,135); soybean (U.S. Pat. No. 5,569,834; U.S. Pat. No. 5,416,011); Brassica (U.S. Pat. No. 5,463,174); peanut (Cheng et al. Plant Cell Rep. 15:653-657 (1996); McKently et al. Plant Cell Rep. 14:699-703 (1995)); papaya (Ling, K. et al. Bio/technology 9:752-758 (1991)); and pea (Grant et al. Plant Cell Rep. 15:254-258 (1995)). For a review of other commonly used methods of plant transformation see Newell, C. A. (Mol. Biotechnol. 16:53-65 (2000)). One of these methods of transformation uses Agrobacterium rhizogenes (Tepfler, M. and Casse-Delbart, F. Microbiol. Sci. 4:24-28 (1987)). Transformation of soybeans using direct delivery of DNA has been published using PEG fusion (PCT Publication No. WO 92/17598), electroporation (Chowrira, G. M. et al., Mol. Biotechnol. 3:17-23 (1995); Christou, P. et al., Proc. Natl. Acad. Sci. U.S.A. 84:3962-3966 (1987)), microinjection, or particle bombardment (McCabe, D. E. et. al., Bio/Technology 6:923 (1988); Christou et al., Plant Physiol. 87:671-674 (1988)).
[0232] 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.
[0233] 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.), 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).
[0234] Examples of oilseed plants include, but are not limited to, soybean, Brassica species, sunflower, maize, cotton, flax and safflower.
[0235] Examples of polyunsaturated fatty acids having at least twenty carbon atoms and five or more carbon-carbon double bonds include, but are not limited to, omega-3 fatty acids such as EPA, DPA and DHA. Seeds obtained from such plants are also within the scope of this invention as well as oil obtained from such seeds.
[0236] In one embodiment this invention concerns an oilseed plant comprising: a) a first recombinant DNA construct comprising an isolated polynucleotide encoding a delta-8 desaturase polypeptide, operably linked to at least one regulatory sequence; and 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-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] Such 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.
[0238] 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.
[0239] In another aspect, this invention concerns a method for making long-chain polyunsaturated fatty acids in a plant cell comprising: [0240] (a) transforming a cell with the recombinant construct of the invention; and [0241] (b) selecting those transformed cells that make long-chain polyunsaturated fatty acids.
[0242] In still another aspect, this invention concerns a method for producing at least one polyunsaturated fatty acid in a soybean cell comprising: [0243] (a) transforming a soybean cell with a first recombinant DNA construct comprising an isolated polynucleotide encoding a delta-8 desaturase polypeptide, operably linked to at least one regulatory sequence and at least one additional recombinant DNA construct comprising an isolated polynucleotide, operably linked to at least one regulatory sequence, encoding a polypeptide selected from the group consisting of a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8 desaturase, a delta-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; [0244] (b) regenerating a soybean plant from the transformed cell of step (a); and [0245] (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 soybean plant.
[0246] 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
[0247] 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).
[0248] 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. 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 from soybean oil through alteration of its physical properties. This is most commonly achieved through catalytic hydrogenation.
[0249] 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. 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.
[0250] 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).
[0251] Hydrogenated oils have also become 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.
[0252] 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. Partially hydrogenated oils, such as soybean oil, are widely used as ingredients for soft spreads, margarine and shortenings for baking and frying.
[0253] 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. For example, palmitate is converted to its unsaturated derivative (palmitoleic acid (16:1)) by the action of a delta-9 desaturase. Similarly, palmitate is elongated by a C16/18 fatty acid elongase to form stearic acid (18:0), which can be converted to its unsaturated derivative by a delta-9 desaturase to thereby yield oleic acid (18:1).
[0254] Triacylglycerols (the primary storage unit for fatty acids) are formed by the esterification of two molecules of acyl-CoA to glycerol-3-phosphate to yield 1,2-diacylglycerol phosphate (commonly identified as phosphatidic acid). The phosphate is then removed, by phosphatidic acid phosphatase, to yield 1,2-diacylglycerol. Triacylglycerol is formed upon the addition of a third fatty acid by the action of a diacylglycerol-acyl transferase.
[0255] Many microorganisms, including algae, bacteria, molds and yeasts, can synthesize PUFAs and omega fatty acids in the ordinary course of cellular metabolism. Particularly well-studied are fungi including Schizochytrium aggregatm, species of the genus Thraustochytrium and Morteriella alpina. Additionally, many dinoflagellates (Dinophyceaae) naturally produce high concentrations of PUFAs. As such, a variety of genes involved in oil production have been identified through genetic means and the DNA sequences of some of these genes are publicly available. See, for example, AY131238, Y055118, AY055117, AF296076, AF007561, L11421, NM 031344, AF465283, AF465281, AF110510, AF465282, AF419296, AB052086, AJ250735, AF126799, AF126798 (delta-6 desaturases); AF199596, AF226273, AF320509, AB072976, AF489588, AJ510244, AF419297, AF07879, AF067654, AB022097 (delta-5 desaturases); AAG36933, AF110509, AB020033, AAL13300, AF417244, AF161219, AY332747, AAG36933, AF110509, X86736, AF240777, AB007640, AB075526, AP002063 (delta-12 desaturases); NP 441622, BAA18302, BAA02924, AAL36934 (delta-15 desaturases); AF338466, AF438199, E11368, E11367, D83185, U90417, AF085500, AY504633, NM 069854, AF230693 (delta-9 desaturases); AF390174 (delta-9 elongase); AF139720 and CQ831420 (delta-8 desaturase); and AX464731, NM--119617, NM--134255, NM--134383, NM--134382, NM--068396, NM--068392, NM--070713, NM--068746, NM--064685 (elongases).
[0256] Additionally, the patent literature provides many additional DNA sequences of genes (and/or details concerning several of the genes above and their methods of isolation) involved in PUFA production (e.g., PCT Publication No. WO 02/077213 (delta-9 elongases); PCT Publication No. WO 00/34439, WO 04/057001 and U.S. Pat. No. 6,825,017 (delta-8 desaturases); U.S. Pat. No. 5,968,809 (delta-6 desaturases); U.S. Pat. No. 5,972,664 and U.S. Pat. No. 6,075,183 (delta-5 desaturases); PCT Publication No. WO 94/11516, U.S. Pat. No. 5,443,974, PCT Publication No. WO 03/099216 and PCT Publication No. WO 05/047485 (delta-12 desaturases); PCT Publication No. WO 93/11245 (delta-15 desaturases); PCT Publication No. WO 91/13972 and U.S. Pat. No. 5,057,419 (delta-9 desaturases); U.S. Publication No. 2003/0196217 A1 (delta-17 desaturase); and PCT Publication No. WO 00/12720 and PCT Publication No. WO 2002/077213, U.S. Pat. No. 6,403,349, U.S. Pat. No. 6,677,145, and U.S. Publication No. 2004/0111763 (C14/16, C16/18 and C18/20 elongases)). Each of these patents and applications are herein incorporated by reference in their entirety.
[0257] As will be obvious to one skilled in the art, the particular functionalities required to be introduced into a microbial host organism for production of a particular PUFA final product 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). LA, GLA, EDA, DGLA, AA, ALA, STA, ETrA, ETA, EPA, DPA and DHA may all be produced in oleaginous yeasts, by introducing various combinations of the following PUFA enzyme functionalities: 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/or a 020/22 elongase. One skilled in the art will be able to identify various candidate genes encoding each of the above enzymes, according to publicly available literature (e.g., GenBank), the patent literature, and experimental analysis of microorganisms having the ability to produce PUFAs. The 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. In some embodiments, manipulation of genes endogenous to the host is preferred; for other purposes, it is necessary to introduce heterologous genes.
[0258] Although the particular source of the desaturase and elongase genes introduced into the host is not critical to the invention, 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. For example, the polypeptide may have to compete for substrate with other enzymes in the host cell. Analyses of the KM and specific activity of the polypeptide are therefore considered in determining the suitability of a given polypeptide for modifying PUFA production in a given host cell. The polypeptide used in a particular host cell is one that can function under the biochemical conditions present in the intended host cell but otherwise can be any polypeptide having desaturase or elongase activity capable of modifying the desired PUFA.
[0259] In some cases, the host organism in which it is desirable to produce PUFAs will possess endogenous genes encoding some PUFA biosynthetic pathway enzymes. For example, oleaginous yeast can typically produce 18:2 fatty acids (and some have the additional capability of synthesizing 18:3 fatty acids); thus, oleaginous yeast typically possess native delta-12 desaturase activity and may also have delta-15 desaturases. In some embodiments, therefore, expression of the native desaturase enzyme is preferred over a heterologous (or "foreign") enzyme since (1) the native enzyme is optimized for interaction with other enzymes and proteins within the cell, and (2) heterologous genes are unlikely to share the same codon preference in the host organism. Additionally, advantages are incurred when the sequence of the native gene is known, as it permits facile disruption of the endogenous gene by targeted disruption.
[0260] In many instances, however, the appropriate desaturases and elongases are not present in the host organism of choice to enable production of the desired PUFA products. Thus, it is necessary to introduce heterologous genes. In one embodiment of the present invention, work was conducted toward the goal of the development of an oleaginous yeast that accumulates oils enriched in long-chain omega-3 and/or omega-6 fatty acids via expression of a delta-9 elongase/delta-8 desaturase pathway, to enable production of EDA, DGLA, ARA, ALA, ETrA, ETA, EPA, DPA and/or DHA.
[0261] In order to express genes encoding the delta-9 elongase/delta-8 desaturase pathway for the biosynthesis of long-chain PUFAs (e.g., AA and EPA) in these organisms, it was therefore necessary to (1) identify a suitable delta-9 elongase and delta-8 desaturase that functioned relatively efficiently in oleaginous yeast based on substrate-feeding trials, and, (2) subject the delta-9 elongase and delta-8 desaturase gene to codon-optimization techniques (infra) to further enhance the expression of the heterologous enzymes in the alternate oleaginous yeast host, to thereby enable maximal production of omega-3 and/or omega-6 fatty acids.
[0262] It will be obvious to one of skill in the art that heterologous genes will be expressed with variable efficiencies in an alternate host. Thus, omega-3 and/or omega-6 PUFA production may be optimized by selection of a particular desaturase or elongase whose level of expression in a heterologous host is preferred relative to the expression of an alternate desaturase or elongase in the host organism of interest. Furthermore, it may be desirable to modify the expression of particular PUFA biosynthetic pathway enzymes to achieve optimal conversion efficiency of each, according to the specific PUFA product composition of interest. A variety of genetic engineering techniques are available to optimize expression of a particular enzyme. Two such techniques include codon optimization and gene mutation, as described below. Genes produced by, for example, either of these two methods, having desaturase and/or elongase activity(s) would be useful in the invention herein for synthesis of omega-3 and/or omega-6 PUFAs.
[0263] As will be appreciated by one skilled in the art, it is frequently useful to modify a portion of the codons encoding a particular polypeptide that is to be expressed in a foreign host, such that the modified polypeptide uses codons that are preferred by the alternate host. Use of host-preferred codons can substantially enhance the expression of the foreign gene encoding the polypeptide.
[0264] 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 desaturase or elongase activity can be synthesized in whole or in part using the codons preferred in the host species. All (or portions) of the DNA also can be synthesized to remove any destabilizing sequences or regions of secondary structure that would be present in the transcribed mRNA. All (or portions) of the DNA also can be synthesized to alter the base composition to one more preferable in the desired host cell.
[0265] In the present invention, it is desirable to modify a portion of the codons encoding the polypeptide having delta-8 desaturase activity, to enhance the expression of the gene in a host organism including, but not limited to, a plant, plant parts and/or oleaginous yeast Yarrowia lipolytica. The nucleic acid sequence of the native gene (i.e., the Pavlova lutheri delta-8 desaturase defined herein as SEQ ID NOs:14, 15 and 16) is modified to employ host-preferred codons. This wildtype desaturase has 423 amino acids (SEQ ID NO:16); in the codon-optimized gene (SEQ ID NO:57), 166 bp of the 1272 bp coding region (13.1%) and 161 codons are codon-optimized (38.1%) and the translation initiation site is modified.
[0266] The skilled artisan will appreciate that modulation of the Pavlova lutheri delta-8 desaturase as well as numerous other heterologous delta-8 desaturases from variable sources can be codon-optimized to improve their expression in an oleaginous yeast host (e.g, see Example 18 herein, wherein a synthetic codon-optimized delta-8 desaturase derived from Pavlova lutheri was created for expression in Yarrowia lipolytica). The present invention comprises the complete sequence of the synthetic codon-optimized gene as reported in the accompanying Sequence Listing (SEQ ID NO:57), the complement of those complete sequences, and substantial portions of those sequences. Furthermore, the codon-optimization method described in PCT Publication No. WO 2004/101753 and described herein for optimization of the Pavlova lutheri delta-8 desaturase is equally applicable to other genes in the omega-3/omega-6 fatty acid biosynthetic pathway.
[0267] Methods for synthesizing sequences and bringing sequences together are well established in the literature. For example, in vitro mutagenesis and selection, site-directed mutagenesis, error prone PCR (Melnikov et al., Nucleic Acids Research, 27(4):1056-1062 (February 1999)), "gene shuffling" or other means can be employed to obtain mutations of naturally occurring desaturase or elongase genes (wherein such mutations may include deletions, insertions and point mutations, or combinations thereof). This would permit production of a polypeptide having desaturase or elongase activity, respectively, in vivo with more desirable physical and kinetic parameters for function in the host cell such as a longer half-life or a higher rate of production of a desired PUFA. Or, if desired, the regions of a polypeptide of interest (i.e., a desaturase or an elongase) important for enzymatic activity can be determined through routine mutagenesis, expression of the resulting mutant polypeptides and determination of their activities. An overview of these techniques are described in PCT Publication No. WO 2004/101757. All such mutant proteins and nucleotide sequences encoding them that are derived from the codon-optimized gene described herein are within the scope of the present invention.
[0268] Microbial production of omega-3 and/or omega-6 fatty acids has several advantages. For example, (1) many microbes are known with greatly simplified oil compositions compared with those of higher organisms, making purification of desired components easier, (2) microbial production is not subject to fluctuations caused by external variables, such as weather and food supply, (3) microbially produced oil is substantially free of contamination by environmental pollutants, (4) microbes can provide PUFAs in particular forms which may have specific uses, and (5) microbial oil production can be manipulated by controlling culture conditions, notably by providing particular substrates for microbially expressed enzymes, or by addition of compounds/genetic engineering to suppress undesired biochemical pathways.
[0269] In addition to these advantages, production of omega-3 and/or omega-6 fatty acids from recombinant microbes provides the ability to alter the naturally occurring microbial fatty acid profile by providing new biosynthetic pathways in the host or by suppressing undesired pathways, thereby increasing levels of desired PUFAs, or conjugated forms thereof, and decreasing levels of undesired PUFAs. For example, it is possible to modify the ratio of omega-3 to omega-6 fatty acids so produced, produce either omega-3 or omega-6 fatty acids exclusively while eliminating production of the alternate omega fatty acid, or engineer production of a specific PUFA without significant accumulation of other PUFA downstream or upstream products (e.g., enable biosynthesis of AA, EPA and/or DHA via the delta-9 elongase/delta-8 desaturase pathway, thereby avoiding synthesis of GLA and/or STA).
[0270] The genes and gene products described herein may be produced in heterologous microbial host cells, particularly in the cells of oleaginous yeasts (e.g., Yarrowia lipolytica). Expression in recombinant microbial hosts may be useful for the production of various PUFA pathway intermediates, or for the modulation of PUFA pathways already existing in the host for the synthesis of new products heretofore not possible using the host.
[0271] 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 preferred desaturase and/or elongase sequences. These chimeric genes could then be introduced into appropriate microorganisms via transformation to provide high-level expression of the encoded enzymes.
[0272] Accordingly, it is expected that introduction of chimeric genes encoding a PUFA biosynthetic pathway, under the control of the appropriate promoters will result in increased production of omega-3 and/or omega-6 fatty acids. It is contemplated that it will be useful to express various combinations of these PUFA desaturase and elongase genes together in a host microorganism. It will be obvious to one skilled in the art that the particular genes included within a particular expression cassette(s) will depend on the host cell, its ability to synthesize PUFAs using native desaturases and elongases, the availability of substrate and the desired end product(s). For example, it may be desirable for an expression cassette to be constructed comprising genes encoding one or more of the following enzymatic activities: 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/or a C20/22 elongase. As such, the present invention encompasses a method of producing PUFAs comprising exposing a fatty acid substrate to the PUFA enzyme(s) described herein, such that the substrate is converted to the desired fatty acid product. Thus, each PUFA gene and corresponding enzyme product described herein (e.g., a wildtype, codon-optimized, synthetic and/or mutant enzyme having appropriate desaturase or elongase activity) can be used directly or indirectly for the production of PUFAs. Direct production of PUFAs occurs wherein the fatty acid substrate is converted directly into the desired fatty acid product without any intermediate steps or pathway intermediates. For example, production of AA would occur in a host cell which produces or which is provided DGLA, by adding or introducing into said cell an expression cassette that provides delta-5 desaturase activity. Similarly, expression of the delta-8 desaturase of the invention permits the direct synthesis of EDA and ETrA (when provided LA and ALA, respectively, as substrate). Thus, for example, the present invention may encompass a method of producing either EDA or ETrA, respectively, comprising: [0273] a) providing a host organism including, but not limited to, an oleaginous yeast comprising: (i) a gene encoding a delta-8 desaturase polypeptide as set forth in SEQ ID NO:16 or SEQ ID NO:57; and (ii) a source of desaturase substrate consisting of either EDA or ETrA, respectively; and, [0274] b) growing the yeast of step (a) in the presence of a suitable fermentable carbon source wherein the gene encoding a delta-8 desaturase polypeptide is expressed and EDA is converted to DGLA or ETrA is converted to ETA, respectively; and, [0275] c) optionally recovering the DGLA or ETA, respectively, of step (b).
[0276] In some preferred embodiments, the nucleotide sequence of a gene encoding a delta-8 desaturase polypeptide is set forth in SEQ ID NO:57 wherein at least 162 codons have been optimized for expression in Yarrowia.
[0277] In contrast, multiple genes encoding the PUFA biosynthetic pathway may be used in combination, such that a series of reactions occur to produce a desired PUFA. For example, expression cassette(s) encoding delta-9 elongase, delta-8 desaturase, delta-5 desaturase and delta-17 desaturase activity would enable a host cell that naturally produces LA, to instead produce ARA (such that LA is converted to EDA by delta-9 elongase; EDA may then be converted to DGLA by a delta-8 desaturase; DGLA is then converted to ARA by a delta-5 desaturase). In a related manner, expression of the delta-8 desaturase of the invention enables the direct/indirect production of ETA, EPA, DPA and/or DHA as down-stream PUFAs, if subsequent desaturase and elongation reactions are catalyzed. In a preferred embodiment, wherein the host cell is an oleaginous yeast, expression cassettes encoding each of the enzymes necessary for PUFA biosynthesis will need to be introduced into the organism, since naturally produced PUFAs in these organisms are limited to 18:2 fatty acids (i.e., LA), and less commonly, 18:3 fatty acids (i.e., ALA). Alternatively, substrate feeding may be required.
[0278] 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 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.
[0279] Initiation control regions or promoters which are useful to drive expression of desaturase and/or elongase 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, alternatively, 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. The transcriptional initiation regulatory regions can be obtained, for example, from (1) genes in the glycolytic pathway, such as alcohol dehydrogenase, glyceraldehyde-3-phosphate-dehydrogenase (PCT Publication No. WO 2005/003310), phosphoglycerate mutase (PCT Publication No. WO 2005/003310), fructose-bisphosphate aldolase (PCT Publication No. WO 2005/049805), phosphoglucose-isomerase, phosphoglycerate kinase, glycerol-3-phosphate O-acyltransferase (PCT Publication No. WO 2006/031937), etc.; or (2) regulatable genes such as acid phosphatase, lactase, metallothionein, glucoamylase, the translation elongation factor EF1-α (TEF) protein (U.S. Pat. No. 6,265,185), ribosomal protein S7 (U.S. Pat. No. 6,265,185), ammonium transporter proteins (U.S. application Ser. No. 11/185,301), export proteins, etc. 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.
[0280] 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, as demonstrated in the invention herein in Yarrowia lipolytica, 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.
[0281] 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, the termination region is derived from a yeast gene, particularly Saccharomyces, Schizosaccharomyces, Candida, Yarrowia or Kluyveromyces. The 3'-regions of mammalian genes encoding a-interferon and a-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.
[0282] 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 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 in the host organism 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 PUFA biosynthetic pathway enzymes.
[0283] Once the DNA encoding a desaturase or elongase polypeptide suitable for expression in an oleaginous yeast has been obtained, 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.
[0284] A 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. Toward this end, it is desirable to identify a sequence within the genome that is present in multiple copies.
[0285] Schmid-Berger et al. (J. Bact. 176(9):2477-2482 (1994)) discovered the first retrotransposon-like element Ylt1 in Yarrowia lipolytica. This retrotransposon is characterized by the presence of long terminal repeats (LTRs; each approximately 700 bp in length) called zeta regions. Ylt1 and solo zeta elements were present in a dispersed manner within the genome in at least 35 copies/genome and 50-60 copies/genome, respectively; both elements were determined to function as sites of homologous recombination. Further, work by Juretzek et al. (Yeast 18:97-113 (2001)) demonstrated that gene expression could be dramatically increased by targeting plasmids into the repetitive regions of the yeast genome (using linear DNA with LTR zeta regions at both ends), as compared to the expression obtained using low-copy plasmid transformants. Thus, zeta-directed integration can be ideal as a means to ensure multiple integration of plasmid DNA into Yarrowia lipolytica, thereby permitting high-level gene expression. Unfortunately, however, not all strains of Yarrowia lipolytica possess zeta regions (e.g., the strain identified as ATCC Accession No. 20362). When the strain lacks such regions, it is also possible to integrate plasmid DNA comprising expression cassettes into alternate loci to reach the desired copy number for the expression cassette. For example, preferred alternate loci include: 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, the Lip1 gene locus (GenBank Accession No. Z50020) and/or the Lip2 gene locus (GenBank Accession No. AJ012632).
[0286] 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.
[0287] 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 constructs 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 can be experimentally determined so that all introduced genes are expressed at the necessary levels to provide for synthesis of the desired products.
[0288] Constructs comprising the gene of interest may be introduced into a 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)).
[0289] For convenience, a host cell that has been manipulated by any method to take up a DNA sequence (e.g., an expression cassette) will be referred to as "transformed" or "recombinant" herein. The transformed host will have at least one copy of the expression construct and may have two or more, depending upon whether the gene is integrated into the genome, amplified or is present on an extrachromosomal element having multiple copy numbers.
[0290] The transformed host cell can be identified by various selection techniques, as described in PCT Publication No. WO 04/101757. 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 could be integrated into a different locus of the Yarrowia genome to thereby produce a new strain having a Ura+phenotype. Subsequent integration would produce 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.
[0291] Following transformation, substrates suitable for the recombinantly expressed desaturases and/or elongases (and optionally other PUFA enzymes that are expressed within the host cell) may be produced by the host either naturally or transgenically, or they may be provided exogenously.
[0292] 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 may require metabolic engineering directly within the PUFA biosynthetic pathway or additional manipulation of pathways that contribute carbon to the PUFA biosynthetic pathway.
[0293] 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.
[0294] 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. The endogenous desaturase activity can be reduced or eliminated by, for example, (1) providing a cassette for transcription of antisense sequences to the delta-15 desaturase transcription product, (2) disrupting the delta-15 desaturase gene through insertion, substitution and/or deletion of all or part of the target gene; or (3) using a host cell which naturally has [or has been mutated to have] low or no delta-15 desaturase activity. Inhibition of undesired desaturase pathways can also be accomplished through the use of specific desaturase inhibitors such as those described in U.S. Pat. No. 4,778,630.
[0295] Alternatively, it may be desirable to maximize production of omega-3 fatty acids (and minimize synthesis of omega-6 fatty acids). Thus, one could utilize a host microorganism wherein the delta-12 desaturase activity that permits conversion of oleic acid to LA is removed or inhibited, using any of the means described above (see also e.g., PCT Publication No. WO 2004/104167, herein incorporated entirely by reference). 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).
[0296] Beyond the immediate PUFA biosynthetic pathway, it is expected that manipulation of several other enzymatic pathways leading to the biosynthesis of precursor fatty acids may contribute to the overall net biosynthesis of specific PUFAs. Identification and manipulation of these related pathways will be useful in the future.
[0297] Additional copies of desaturase and elongase genes may be introduced into the host to increase the output of omega-3 and/or omega-6 fatty acid biosynthetic pathways. Expression of the desaturase or elongase genes also can be increased at the transcriptional level through the use of a stronger promoter (either regulated or constitutive) to cause increased expression, by removing/deleting destabilizing sequences from either the mRNA or the encoded protein, or by adding stabilizing sequences to the mRNA (U.S. Pat. No. 4,910,141). Yet another approach to increase expression of the desaturase or elongase genes, as demonstrated in the instant invention, is to increase the translational efficiency of the encoded mRNAs by replacement of codons in the native gene with those for optimal gene expression in the selected host microorganism.
[0298] Conversely, 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). For gene disruption, a foreign DNA fragment (typically a selectable marker gene) is inserted into the structural gene to be disrupted in order to interrupt its coding sequence and thereby functionally inactivate the gene. Transformation of the disruption cassette into the host cell results in replacement of the functional native gene by homologous recombination with the non-functional disrupted gene (see, for example: Hamilton et al. J. Bacteriol. 171:4617-4622 (1989); Balbas et al. Gene 136:211-213 (1993); Gueldener et al. Nucleic Acids Res. 24:2519-2524 (1996); and Smith et al. Methods Mol. Cell. Biol. 5:270-277 (1996)).
[0299] Antisense technology is another method of down-regulating genes when the sequence of the target gene is known. To accomplish this, a nucleic acid segment from the desired gene is cloned and operably linked to a promoter such that the anti-sense strand of RNA will be transcribed. This construct is then introduced into the host cell and the antisense strand of RNA is produced. Antisense RNA inhibits gene expression by preventing the accumulation of mRNA that encodes the protein of interest. The person skilled in the art will know that special considerations are associated with the use of antisense technologies in order to reduce expression of particular genes. For example, the proper level of expression of antisense genes may require the use of different chimeric genes utilizing different regulatory elements known to the skilled artisan.
[0300] Although targeted gene disruption and antisense technology offer effective means of down-regulating genes where the sequence is known, other less specific methodologies have been developed that are not sequence-based (e.g., mutagenesis via UV radiation/chemical agents or use of transposable elements/transposons; see PCT Publication No. WO 2004/101757).
[0301] 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 methods described above. For example, the present invention provides methods whereby genes encoding key enzymes in the biosynthetic pathways are introduced into oleaginous yeasts for the production of omega-3 and/or omega-6 fatty acids. It will be particularly useful to express these 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.
[0302] Microbial host cells for production of omega fatty acids may include microbial 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.
[0303] 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).
[0304] Most preferred is the oleaginous yeast Yarrowia lipolytica; and, in a further embodiment, most preferred are the Yarrowia lipolytica strains designated as ATCC Accession Nos. 20362, 8862, 18944, 76982 and/or LGAM S(7)1 (Papanikolaou, S., and Aggelis, G., Bioresour. Technol. 82(1):43-9 (2002)).
[0305] The transformed microbial host cell is grown under conditions that optimize desaturase and elongase activities and produce the greatest and the most economical yield of the preferred 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. Microorganisms of interest, such as oleaginous yeast, are 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.)).
[0306] Fermentation media in the present invention must contain a suitable carbon source. Suitable carbon sources are taught in 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.
[0307] 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 microorganism and promotion of the enzymatic pathways necessary for PUFA production. Particular attention is given to several metal ions (e.g., Fe+2, Cu+2, 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)).
[0308] 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 particular microorganism 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.
[0309] 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. 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.
[0310] The PUFAs may be found in the host microorganism as free fatty acids or in esterified forms such as acylglycerols, phospholipids, sulfolipids or glycolipids, and may be extracted from the host cell 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)).
[0311] 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.
[0312] 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 oils of the invention and the yeast oils of the invention comprising long-chain PUFAs will function in food and feed products to impart the health benefits of current formulations. More specifically, oils of the invention containing omega-3 and/or omega-6 fatty acids 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.
[0313] Additionally the present 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 present 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.
[0314] A "food analog" is a food-like product manufactured to resemble its food counterpart, whether meat, cheese, milk or the like, and is intended to have the appearance, taste, and texture of its counterpart. Thus, the term "food" as used herein also encompasses food analogs. Food analogs can be made use processes well known to those skilled in the art. U.S. Pat. Nos. 6,355,296 B1 and 6,187,367 B1 describe emulsified meat analogs and emulsified meat extenders. U.S. Pat. No. 5,206,050 B1 describes soy protein curd useful for cooked food analogs (also can be used as a process to form a curd useful to make food analogs). U.S. Pat. No. 4,284,656 to Hwa describes a soy protein curd useful for food analogs. U.S. Pat. No. 3,988,485 to Hibbert et al. describes a meat-like protein food formed from spun vegetable protein fibers. U.S. Pat. No. 3,950,564 to Puski et al. describes a process of making a soy based meat substitute and U.S. Pat. No. 3,925,566 to Reinhart et al. describes a simulated meat product. For example, soy protein that has been processed to impart a structure, chunk or fiber for use as a food ingredient is called "textured soy protein" (TSP). TSPS are frequently made to resemble meat, seafood, or poultry in structure and appearance when hydrated.
[0315] 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.
[0316] 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).
[0317] 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.
[0318] 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.
[0319] 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.
[0320] A snack food product comprises any of the above or below described food products.
[0321] A fried food product comprises any of the above or below described food products that has been fried.
[0322] The beverage can be in a liquid or in a dry powdered form.
[0323] For example, there can be mentioned non-carbonated drinks such as fruit juices, fresh, frozen, canned or concentrate; flavored or plain milk drinks, etc. Adult and infant nutritional formulas are well known in the art and commercially available (e.g., Similac®, Ensure®, Jevity®, and Alimentum® from Ross Products Division, Abbott Laboratories).
[0324] Infant formulas are liquids or reconstituted powders fed to infants and young children. "Infant formula" is defined herein as an enteral nutritional product which can be substituted for human breast milk in feeding infants and typically is composed of a desired percentage of fat mixed with desired percentages of carbohydrates and proteins in an aquous solution (e.g., see U.S. Pat. No. 4,670,285). Based on the worldwide composition studies, as well as levels specified by expert groups, average human breast milk typically contains about 0.20% to 0.40% of total fatty acids (assuming about 50% of calories from fat); and, generally the ratio of DHA to ARA would range from about 1:1 to 1:2 (see, e.g., formulations of Enfamil LIPIL® (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.
[0325] 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.
[0326] Additional food products into which the long-chain 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.
[0327] 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 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). For example, more concentrated formulations comprising ARA, EPA or DHA 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.
[0328] Animal feeds are generically defined herein as products intended for use as feed or for mixing in feed for animals other than humans. The long-chain PUFA containing oils of the invention can be used as an ingredient in various animal feeds.
[0329] 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 which concerns the propagation, cultivation or farming of aquatic organisms and/or animals in fresh or marine waters.
EXAMPLES
[0330] The present invention is further defined in the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.
[0331] 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:
[0332] Yarrowia lipolytica strains with ATCC Accession Nos. 20362, 76982 and 90812 were purchased from the American Type Culture Collection (Rockville, Md.). Yarrowia lipolytica strains were typically grown at 28° C. on YPD agar (1% yeast extract, 2% bactopeptone, 2% glucose, 2% agar).
[0333] Transformation of Yarrowia lipolytica was performed according to the method of Chen, D. C. et al. (Appl. Microbiol. Biotechnol. 48(2):232-235 (1997)), unless otherwise noted. Briefly, Yarrowia was streaked onto a YPD plate and grown at 30° C. for approximately 18 h. Several large loopfuls of cells were scraped from the plate and resuspended in 1 mL of transformation buffer containing: 2.25 mL of 50% PEG, average MW 3350; 0.125 mL of 2 M Li acetate, pH 6.0; 0.125 mL of 2 M DTT; and 50 g sheared salmon sperm DNA. Then, approximately 500 ng of linearized plasmid DNA was incubated in 100 L of resuspended cells, and maintained at 39° C. for 1 h 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.
[0334] For selection of transformants, minimal medium ("MM") was generally used; the composition of MM is as follows: 0.17% yeast nitrogen base (Difco Laboratories, Detroit, Mich.) without ammonium sulfate or amino acids, 2% glucose, 0.1% proline, pH 6.1). Supplements of uracil were added as appropriate to a final concentration of 0.01% (thereby producing "MMU" selection media, prepared with 20 g/L agar).
[0335] Alternatively, transformants were selected on 5-fluoroorotic acid ("FOA"; also 5-fluorouracil-6-carboxylic acid monohydrate) selection media, comprising: 0.17% yeast nitrogen base (Difco Laboratories, Detroit, Mich.) without ammonium sulfate or amino acids, 2% glucose, 0.1% proline, 75 mg/L uracil, 75 mg/L uridine, 900 mg/L FOA (Zymo Research Corp., Orange, Calif.) and 20 g/L agar.
Fatty Acid Analysis of Yarrowia lipolytica:
[0336] For fatty acid analysis, cells were collected by centrifugation and lipids were extracted as described in Bligh, E. G. & Dyer, W. J. (Can. J. Biochem. Physiol. 37:911-917 (1959)). Fatty acid methyl esters were prepared by transesterification of the lipid extract with sodium methoxide (Roughan, G. and Nishida, I., Arch Biochem Biophys. 276(1):38-46 (1990)) and subsequently analyzed with a Hewlett-Packard 6890 GC fitted with a 30 m×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.
[0337] 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 L of 1%) was added to the sample, and then the sample was vortexed and rocked for 20 min. After adding 3 drops of 1 M NaCl and 400 L hexane, the sample was vortexed and spun. The upper layer was removed and analyzed by GC as described above.
Example 1
Pavlova Lutheri (CCMP459) cDNA Synthesis, Library Construction and Sequencing
[0338] A cDNA library of Pavlova lutheri (CCMP459) was synthesized as described in PCT Publication No. WO 2004/071467 ((published Aug. 26, 2004). Briefly, frozen pellets of Pav459 were obtained from Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP, 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 manufacturers 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' SalI/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.
[0339] 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 hours 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.
[0340] Plasmids were end-sequenced in 384-well plates, using vector-primed T7 primer (SEQ ID NO:1) and the ABI Big Dye 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 ABI 3700 automated sequencers.
Example 2
Identification of Delta-8 Desaturase Enzyme Homologs from Pavlova Lutheri cDNA Library Eps1c
[0341] cDNA clones encoding Pavlova lutheri delta-8 desaturase homologs (hereby called delta-8 desaturases) were identified by conducting BLAST (Basic Local Alignment Search Tool; Altschul et al., J. Mol. Biol. 215:403-410 (1993)) searches for similarity to sequences contained in the BLAST "nr" database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the last major release of the SWISS-PROT protein sequence database, EMBL, and DDBJ databases). The cDNA sequences obtained in Example 1 were analyzed for similarity to all publicly available DNA sequences contained in the "nr" database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). The DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the "nr" database using the BLASTX algorithm (Gish and States, Nat. Genet. 3:266-272 (1993)) provided by the NCBI. For convenience, the P-value (probability) of observing a match of a cDNA sequence to a sequence contained in the searched databases merely by chance as calculated by BLAST are reported herein as "pLog" values, 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.
[0342] The BLASTX search using the nucleotide sequence from clone eps1c.pk002.f22 revealed similarity of the protein encoded by the cDNA to the delta-6 desaturase from Rhizopus stolonifer (SEQ ID NO:2) (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:3 (5' end of cDNA insert). Subsequently, the full insert sequence (eps1c.pk002.f22:fis) was obtained and is shown in SEQ ID NO:4. Sequence for the deduced amino acid sequence (from nucleotide 1 of SEQ ID NO:4 to the first stop codon at nucleotide 864 of SEQ ID NO:4) is shown in SEQ ID NO:5. Full insert sequencing was carried out using a modified transposition protocol. Clones identified for FIS 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:6) and SeqW (SEQ ID NO:7).
[0343] Sequence data was collected (ABI Prism Collections software) and assembled using the Phrap sequence assembly program (P. Green, University of Washington, Seattle). Assemblies are viewed by the Consed sequence editor (D. Gordon, University of Washington, Seattle) for final editing.
[0344] The amino acid sequence set forth in SEQ ID NO:5 was evaluated by BLASTP, yielding a pLog value of 19.52 (E value of 3e-20) versus the delta-6 desaturase from Mortierella alpina (SEQ ID NO:8) (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 alpina and other fatty acid desaturases, the Pavlova lutheri delta-8 desaturase was not full length and was lacking sequence at the 5' end.
Example 3
Cloning a Full-length Delta-8 Desaturase from Pavlova Lutheri Genomic DNA
[0345] 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 μg 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.
[0346] For the primary PCR, the Advantage®-GC Genomic 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 Genomic 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 AP1 (10 μM, SEQ ID NO:9) and 1 μL of GSP PvDES (10 μM, SEQ ID NO:10). 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.
[0347] 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 Zymoclean® 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:1) and M13-28Rev (SEQ ID NO:11) primers as described above. Additional sequence was then obtained using a gene-specific sequencing primer PvDES seq (SEQ ID NO:12) that was derived from the newly acquired sequence data. The full 5' end sequence obtained by genome walking is shown in SEQ ID NO:13. The sequence of the overlapping regions of the genomic sequence (SEQ ID NO:13) and the fully sequenced EST eps1c.pk002.f22:fis (SEQ ID NO:4) 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:4) was lacking 459 bp when compared to the genomic sequence (SEQ ID NO:13). 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:13) was combined with the 3' end of the EST sequence (SEQ ID NO:4) to give SEQ ID NO:14. Using EditSeq® 6.1 sequence analysis software (DNASTAR Inc., Madison, Wis.), an ORF was identified (SEQ ID NO:15). The amino acid sequence coded for by SEQ ID NO:15 is shown in SEQ ID NO:16.
[0348] The amino acid sequence set forth in SEQ ID NO:16 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:17) (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:76) 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 LASARGENE 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 LASARGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.) with the default parameters for pairwise alignment (KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5 and GAP LENGTH PENALTY=10). BLAST scores and probabilities indicate that the instant nucleic acid fragment (SEQ ID NO:15) encodes an entire Pavlova lutheri delta-8 desaturase.
[0349] FIGS. 13A and 13B show a Clustal V alignment (with default parameters) of SEQ ID NO:16 (the amino acid sequence of the delta-8 desaturase of the instant invention), SEQ ID NO:76 (the amino acid sequence of Pavlova salina delta-8 desaturase sequence disclosed as SEQ ID NO:1 in PCT Publication No. WO 2005/103253; published Apr. 22, 2005), SEQ ID NO:77 (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:17 (the amino acid sequence for the Rhizopus stolonifer delta-6 fatty acid desaturase (NCBI Accession No. ABB96724 (GI 83027409), locus ABB96724, CDS DQ291156; Zhang et al., unpublished)) and SEQ ID NO:2 (the amino acid sequence for the Rhizopus stolonifer delta-6 fatty acid desaturase (NCBI Accession No. AAX22052 (GI 60499699), locus AAX22052, CDS AY795076; Lu et al., unpublished)). The results of the Clustal V alignment show that SEQ ID NO:16 is 76.4%, 22.6%, 22.2%, and 22.2% identical to SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:17 and SEQ ID NO:2, respectively.
Example 4
Cloning the Pavlova Lutheri Delta-8 Desaturase from Pavlova Lutheri cDNA
[0350] Pavlova lutheri (CCMP459) was obtained from COMP and grown in 250 mL flasks containing 50 mL of F/2-Si medium (made using F/2 Family Medium Kit-KIT20F2 and Filtered Seqwater-SEA2 from CCMP) at 26° C. with shaking at 150 rpm. Cultures were transferred to new medium on a weekly basis using 1:4 (old culture:new medium) dilution.
[0351] Cultures from 28 flasks (1400 mL) were combined, cells were pelleted by centrifugation at 1,800×g for 10 min, washed once with water and re-centrifuged. Total RNA was extracted from the resulting pellet using the RNA STAT-60® reagent (TEL-TEST, Inc., Friendswood, Tex.) and following the manufacturer's protocol provided. In this way, 2.6 mg of total RNA (2.6 mg/mL) was obtained from the pellet. The mRNA was isolated from 1.25 mg of total RNA using the mRNA Purification Kit (Amersham Biosciences, Piscataway, N.J.) following the manufacturer's protocol provided. In this way, 112 μg of mRNA was obtained.
[0352] cDNA was synthesized from 224 ng of mRNA using the SuperScript® First-Strand Synthesis System for RT-PCR Kit (Invitrogen® Life Technologies, Carlsbad, Calif.) with the provided oligo(dT) primer according to the manufacturer's protocol. After RNase H treatment as per the protocol, the Pavlova lutheri delta-8 desaturase was amplified from the resulting cDNA with oligonucleotide primers PvDES5'Not-1 (SEQ ID NO:18) and PvDES3'Not-1 (SEQ ID NO:19) using the conditions described below.
[0353] cDNA (2 μL) from the reaction described above was combined with 50 μmol of PvDES5'Not-1 (SEQ ID NO:18), 50 pmol of PvDES3'Not-1 (SEQ ID NO:19), 1 μL of PCR nucleotide mix (10 mM, Promega, Madison, Wis.), 5 μL of 10×PCR buffer (Invitrogen Corporation), 1.5 μL of MgCl2 (50 mM, Invitrogen Corporation), 0.5 μL of Taq polymerase (Invitrogen Corporation) and water to 50 μL. The reaction conditions were 94° C. for 3 min followed by 35 cycles of 94° C. for 45 sec, 55° C. for 45 sec and 72° C. for 1 min. The PCR was finished at 72° C. for 7 min and then held at 4° C. The PCR reaction was analyzed by agarose gel electrophoresis on 5 μL and a DNA band with molecular weight around 1.3 kb was observed. The remaining 45 μL of product was separated by agarose gel electrophoresis and the DNA purified using the Zymoclean® 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. Multiple clones were sequenced using the T7 (SEQ ID NO:1), M13-28Rev (SEQ ID NO:11)
and PvDes-2 (SEQ ID NO:20) oligonucleotides. The sequence of the clones tested were identical to that of SEQ ID NO:15 and one of the correct clones (pLF113) was chosen for further expression studies.
Example 5
Cloning the Pavlova Lutheri Delta-8 Desaturase into a Yeast Expression Vector
[0354] The yeast episomal plasmid (YEp)-type vector pRS425 (Christianson et al., Gene 110:119-122 (1992)) contains sequences from the Saccharomyces cerevisiae 2 endogenous plasmid, a LEU2 selectable marker and sequences based on the backbone of a multifunctional phagemid, pBluescript II SK(+). The Saccharomyces cerevisiae strong, constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter was cloned between the SaclI and SpeI sites of pRS425 in the same way as described by Jia et al. (Physiol. Genom. 3:83-92 (2000)) to produce pGPD-425. A NotI site was introduced into the BamHI site of pGPD-425, thus giving a NotI site flanked by BamHI sites, and this plasmid was called pY-75. The Pavlova lutheri delta-8 desaturase was released from pLF113 (from Example 4) by digestion with NotI and cloned into the NotI site of pY75 to produce pY121 (SEQ ID NO:21; FIG. 1).
Example 6
Cloning the Pavlova Lutheri Delta-8 Desaturase into a Yarrowia Expression Vector
[0355] The Yarrowia GPD promoter was amplified from plasmid pYZDE2-S (SEQ ID NO:74) using oligonucleotides GPDsense (SEQ ID NO:28) and GPDantisense (SEQ ID NO:29). The "Yarrowia GPD" promoter within this chimeric gene 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 (WO 2005/003310). The resulting DNA fragment was digested with SalI/NotI and cloned into the SalI/NotI fragment of pY5-22 (SEQ ID NO:75) thus replacing the TEF promoter and giving pY5-22GPD (SEQ ID NO: 30).
[0356] The Pavlova lutheri delta-8 desaturase was released from pLF113 (from Example 4) by digestion with NotI and cloned into the NotI site of pY5-22GPD to produce pY118 (SEQ ID NO:31; FIG. 4).
Example 7
Cloning the Pavlova Lutheri Delta-8 Desaturase into a Soybean Expression Vector
[0357] Vector pKR123r (SEQ ID NO:22), which was previously described in PCT Publication No. WO 2004/071467 (published Aug. 26, 2004; the contents of which are hereby incorporated by reference), contains a NotI site flanked by the Kunitz soybean Trypsin Inhibitor (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 (KTi/NotI/KTi3' cassette). The Pavlova lutheri delta-8 desaturase (SEQ ID NO:15) was released from pLF113 (from Example 4) by digestion with NotI and cloned into the NotI site of pKR123r to produce pKR900 (SEQ ID NO:23).
[0358] Plasmid pKR72 (ATCC Accession No. PTA-6019; SEQ ID NO:46, 7085 bp sequence), a derivative of pKS123 which was previously described in PCT Publication No. WO 2002/008269 (the contents of which are hereby incorporated by reference), contains the hygromycin B phosphotransferase gene (HPT) (Gritz, L. and Davies, J., Gene 25:179-188 (1983)), flanked by the T7 promoter and transcription terminator (T7prom/hpt/T7term cassette), and a bacterial origin of replication (ori) for selection and replication in bacteria (e.g., E. coli). In addition, pKR72 also contains the hygromycin B phosphotransferase gene, flanked by the 35S promoter (Odell et al., Nature 313:810-812 (1985)) and NOS 3' transcription terminator (Depicker et al., J. Mol. Appl. Genet. 1:561-570 (1982)) (35S/hpt/NOS3' cassette) for selection in plants such as soybean. pKR72 also contains a NotI restriction site, flanked by the promoter for the 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. The βcon/NotI/Phas3' cassette in plasmid pKR72 was removed by digestion with HindIII and the fragment containing the HPT gene was re-ligated to give pKR325 (SEQ ID NO:24), previously described in PCT Publication No. WO 2006/012325 (the contents of which are hereby incorporated by reference).
[0359] Plasmid pKR900 (SEQ ID NO:23) was then digested with SbfI and the fragment containing the Pavlova lutheri delta-8 desaturase was cloned into the SbfI site of pKR325 to produce pKR902 (SEQ ID NO:25). A schematic depiction of pK902 is shown in FIG. 2.
Example 8
Cloning the Pavlova Lutheri Delta-8 Desaturase into a Soybean Expression Vector and Co-Expression with the Isochrysis Galbana Delta-9 Elongase
[0360] Plasmid pKR900 (from Example 7; SEQ ID NO:23) was digested with SbfI and the fragment containing the Pavlova lutheri delta-8 desaturase was cloned into the SbfI site of pKR607 (SEQ ID NO:26), previously described in PCT Publication No. WO 2006/012325 (the contents of which are hereby incorporated by reference) to give pKR903 (SEQ ID NO:27). In this way, the Pavlova lutheri delta-8 desaturase is co-expressed with the Isochrysis galbana delta-9 elongase behind strong, seed-specific promoters. A schematic depiction of pK903 (ATCC Accession No. PTA-7494) is shown in FIG. 3.
Example 9
Functional Analysis of the Pavlova Lutheri Delta-8 Desaturase in Saccharomyces Cerevisiae
[0361] Saccharomyces cerevisiae expression plasmids pY121 and pY75 (from Example 5) were transformed into Saccharomyces cerevisiae INVSC1 (Invitrogen Corporation) using standard lithium acetate transformation procedures. Transformants were selected on DOBA media supplemented with CSM-leu (Qbiogene, Carlsbad, Calif.). Transformants were evaluated for delta-6, delta-8 and delta-5 desaturase activities in the following way. Transformants from each plate were inoculated into 2 mL of DOB medium supplemented with CSM-leu (Qbiogene) and 0.2% tergitol. Cells were grown for 1 day at 30° C. after which, 0.1 mL was transferred to 3 mL of the same medium supplemented with either linoleic acid [LA-18:2(9,12)], a-linolenic acid [ALA-18:3(9,12,15)], dihomo-gamma-linolenic acid [DGLA-20:3(8,11,14)], eicosadienoic acid [EDA-20:2(11,14)] or eicosatrienoic acid [ERA-20:3(11,14,17)] to 0.175 mM. 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 30 min. at 50° C. after which 500 μL of 1M sodium chloride and 100 μL of heptane were added. After thorough mixing and centrifugation, fatty acid methyl esters (FAMEs) were analyzed by GC as described supra. In so doing, no desaturation activity for any of the substrates tested could be detected.
Example 10
Functional Analysis of the Pavlova Lutheri Delta-8 Desaturase in Yarrowia Lipolytica
[0362] A uracil ura3 auxotrophic strain of Yarrowia lipolytica (strain Y2224) was used for functional assays. To produce Y2224, Yarrowia lipolytica (ATCC Accession No. 20362) cells from a YPD plate were streaked onto a minimal medium 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 minimal medium plates containing 200 mg/mL 5-FOA and minimal medium plates lacking uracil and uridine to confirm uracil ura3 auxotrophy. One confirmed auxotroph was designated Y2224.
[0363] Yarrowia lipolytica strain Y2224 was grown at 28° C. on YPD agar (1% yeast extract, 2% bactopeptone, 2% glucose, 2% agar). For selection of transformants, minimal medium (0.17% yeast nitrogen base (DIFCO Laboratories, Detroit, Mich.) without ammonium sulfate or amino acids, 2% glucose, 0.1% proline, pH 6.1) was used. Supplements of adenine, leucine, lysine and/or uracil are added to a final concentration of 0.01%.
Transformation of Yarrowia Lipolytica
[0364] Plasmid pY118, containing the Pavlova lutheri delta-8 desaturase, or pY5-22GPD, the vector control, were transformed into Yarrowia lipolytica strain Y2224 as described in the General Methods.
[0365] Briefly, Yarrowia lipolytica Strain #2224 was streaked onto a YPD plate and grown at 30° C. for approximately 18 h. Several large loopfuls of cells were scraped from the plate and resuspended in 1 mL of transformation buffer containing: 0.25 mL of 50% PEG, average MW 3350; 0.125 mL of 2 M Li acetate, pH 6.0; 0.125 mL of 2M DTT; and 50 μg sheared salmon sperm DNA. About 500 ng of pY118 or pY5-22GPD plasmid DNA were incubated in 100 μL of resuspended cells, and maintained at 39° C. for 1 h with vortex mixing at 15 min intervals. The cells were plated onto minimal media plates lacking uracil and maintained at 30° C. for 2 to 3 days
[0366] Single colonies of transformant Yarrowia lipolytica containing pY118 or pY5-22GPD were grown in 3 mL minimal media lacking uracil supplemented with 0.2% tergitol at 30° C. for 1 day. After this, 0.1 mL was transferred to 3 mL of the same medium supplemented with either no fatty acid a-linolenic acid [ALA-18:3(9,12,15)], dihomo-gamma-linolenic acid [DGLA-20:3(8,11,14)], eicosadienoic acid [EDA-20:2(11,14)] or eicosatrienoic acid [ERA-20:3(11,14,17)] to 0.175 mM. 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 as described supra. FAMEs from cells containing pY118 were analyzed by GC as for cells containing pY121 in Example 9. In so doing, no desaturation activity for any of the substrates tested could be detected.
Example 11
Transformation of Somatic Soybean Embryo Cultures
Culture Conditions:
[0367] Soybean embryogenic suspension cultures (cv. Jack) were 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 were subcultured every 7 days to two weeks by inoculating approximately 35 mg of tissue into 35 mL of fresh liquid SB196 (the preferred subculture interval is every 7 days).
[0368] Soybean embryogenic suspension cultures were transformed with the soybean expression plasmids by the method of particle gun bombardment (Klein et al., Nature, 327:70 (1987)) using a DuPont Biolistic PDS1000/HE instrument (helium retrofit) for all transformations. Soybean Embryogenic Suspension Culture Initiation:
[0369] Soybean cultures were initiated twice each month with 5-7 days between each initiation. Pods with immature seeds from available soybean plants 45-55 days after planting were picked, removed from their shells and placed into a sterilized magenta box. The soybean seeds were sterilized by shaking them for 15 min in a 5% Clorox solution with 1 drop of ivory soap (i.e., 95 mL of autoclaved distilled water plus 5 mL Clorox and 1 drop of soap, mixed well). Seeds were rinsed using 2 1-liter bottles of sterile distilled water and those less than 4 mm were placed on individual microscope slides. The small end of the seed was cut and the cotyledons pressed out of the seed coat. Cotyledons were transferred to plates containing SB1 medium (25-30 cotyledons per plate). Plates were wrapped with fiber tape and stored for 8 weeks. After this time secondary embryos were cut and placed into SB196 liquid media for 7 days.
Preparation of DNA for Bombardment:
[0370] Either an intact plasmid or a DNA plasmid fragment containing the genes of interest and the selectable marker gene were used for bombardment. Fragments from soybean expression plasmids pKR902 and pKR903 were obtained by gel isolation of digested plasmids. In each case, 100 g of plasmid DNA was used in 0.5 mL of the specific enzyme mix described below. Plasmids were digested with AscI (100 units) in NEBuffer 4 (20 mM Tris-acetate, 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM dithiothreitol, pH 7.9), 100 g/mL BSA, and 5 mM beta-mercaptoethanol at 37° C. for 1.5 hr. The resulting DNA fragments were separated by gel electrophoresis on 1% SeaPlaque GTG agarose (BioWhitaker Molecular Applications) and the DNA fragments containing gene cassettes were cut from the agarose gel. DNA was purified from the agarose using the GELase digesting enzyme following the manufacturer's protocol.
[0371] A 50 pL aliquot of sterile distilled water containing 3 mg of gold particles was added to 5 pL of a 1 pg/pL DNA solution (either intact plasmid or DNA fragment prepared as described above), 50 pL 2.5M CaCl2 and 20 pL of 0.1 M spermidine. The mixture was shaken 3 min on level 3 of a vortex shaker and spun for 10 sec in a bench microfuge. After a wash with 400 pL of 100% ethanol, the pellet was suspended by sonication in 40 pL of 100% ethanol. Five pL of DNA suspension was dispensed to each flying disk of the Biolistic PDS1000/HE instrument disk. Each 5 pL aliquot contained approximately 0.375 mg gold particles per bombardment (i.e., per disk).
Tissue Preparation and Bombardment with DNA:
[0372] Approximately 150-200 mg of 7 day old embryonic suspension cultures were placed in an empty, sterile 60×15 mm petri dish and the dish was covered with plastic mesh. Tissue was bombarded 1 or 2 shots per plate with membrane rupture pressure set at 1100 PSI and the chamber was evacuated to a vacuum of 27-28 inches of mercury. Tissue was placed approximately 3.5 inches from the retaining/stopping screen.
Selection of Transformed Embryos:
[0373] Transformed embryos were selected using hygromycin as the selectable marker. Specifically, following bombardment, the tissue was placed into fresh SB196 media and cultured as described above. Six days post-bombardment, the SB196 is exchanged with fresh SB196 containing 30 mg/L hygromycin. The selection media was refreshed weekly. Four to six weeks post-selection, green, transformed tissue may was observed growing from untransformed, necrotic embryogenic clusters. Isolated, green tissue was removed and inoculated into multiwell plates to generate new, clonally propagated, transformed embryogenic suspension cultures.
Embryo Maturation:
[0374] Embryos were cultured for four-six 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 E/m2s.
[0375] After this time embryo clusters were removed to a solid agar media, SB166, for 1-2 weeks. Clusters were then subcultured to medium SB103 for 3 weeks. During this period, individual embryos were removed from the clusters and screened for alterations in their fatty acid compositions as described supra.
Media Recipes:
TABLE-US-00004 [0376] 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 Stock Number 1000 mL 500 mL 1 MS Fe EDTA 100x Stock Na2EDTA* 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)
[0377] 1 package MS salts (Gibco/BRL--Cat. No. 11117-066)
[0378] 1 mL B5 vitamins 1000× stock
[0379] 31.5 g sucrose
[0380] 2 mL 2,4-D (20 mg/L final concentration)
[0381] pH 5.7
[0382] 8 g TC agar
SB 166 Solid Medium (Per Liter)
[0383] 1 package MS salts (Gibco/BRL--Cat. No. 11117-066)
[0384] 1 mL B5 vitamins 1000× stock
[0385] 60 g maltose
[0386] 750 mg MgCl2 hexahydrate
[0387] 5 g activated charcoal
[0388] pH 5.7
[0389] 2 g gelrite
SB 103 Solid Medium (Per Liter)
[0390] 1 package MS salts (Gibco/BRL--Cat. No. 11117-066)
[0391] 1 mL B5 vitamins 1000× stock
[0392] 60 g maltose
[0393] 750 mg MgCl2 hexahydrate
[0394] pH 5.7
[0395] 2 g gelrite
SB 71-4 Solid Medium (Per Liter)
[0396] 1 bottle Gamborg's B5 salts w/sucrose (Gibco/BRL--Cat. No. 21153-036)
[0397] pH 5.7
[0398] 5 g TC agar
2,4-D Stock
[0399] Obtain premade from Phytotech Cat. No. D 295--concentration 1 mg/mL
B5 Vitamins Stock (Per 100 mL)
[0400] Store aliquots at -20° C.
[0401] 10 g myo-inositol
[0402] 100 mg nicotinic acid
[0403] 100 mg pyridoxine HCL
[0404] 1 g thiamine
If the solution does not dissolve quickly enough, apply a low level of heat via the hot stir plate.
Example 12
Functional Analysis of the Pavlova Lutheri Delta-8 Desaturase in Somatic Soybean Embryos
[0405] Mature somatic soybean embryos are a good model for zygotic embryos. While in the globular embryo state in liquid culture, somatic soybean embryos contain very low amounts of triacylglycerol or storage proteins typical of maturing, zygotic soybean embryos. At this developmental stage, the ratio of total triacylglyceride to total polar lipid (phospholipids and glycolipid) is about 1:4, as is typical of zygotic soybean embryos at the developmental stage from which the somatic embryo culture was initiated. At the globular stage as well, the mRNAs for the prominent seed proteins, a'-subunit of 3-conglycinin, kunitz trypsin inhibitor 3, and seed lectin are essentially absent. Upon transfer to hormone-free media to allow differentiation to the maturing somatic embryo state, triacylglycerol becomes the most abundant lipid class. As well, mRNAs for a'-subunit of 3-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.
[0406] Transgenic somatic soybean embryos containing pKR902 (Example 7) or pKR903 (Example 8) were analyzed in the following way. Fatty acid methyl esters were prepared from single, matured, somatic soy embryos by transesterification. Individual embryos were placed in a vial containing 50 pL 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 pL injected from hexane layer) were separated and quantified using a Hewlett-Packard 6890 Gas Chromatograph fitted with an Omegawax 320 fused silica capillary column (Catalog #24152, Supelco Inc.). The oven temperature was programmed to hold at 220° 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.). Routinely, 5-10 embryos per event were analyzed by GC, using the methodology described above.
[0407] Embryo fatty acid profiles for 20 events (5 embryos each) containing pKR902 (Example 7--Pavlova lutheri delta-8 desaturase only) were obtained. No delta-6 desaturase activity (i.e., conversion of LA to GLA or ALA to STA) could be detected in any of the events analyzed.
[0408] Embryo fatty acid profiles for 6 lines containing pKR903 (Example 8--Pavlova lutheri delta-8 desaturase and Isochrysis galbana delta-9 elongase) are shown in FIGS. 8A and 8B. Calculated overall % desaturation, % desaturation for n3 and n-6 substrates and desaturation ratios are also shown in FIGS. 8A and 8B.
[0409] In summary of FIGS. 8A and 8B, the Pavlova lutheri delta-8 desaturase works well in soybean to convert both EDA and ERA to DGLA and ETA, respectively. The line with the highest average DGLA content (1890-3-5) had embryos with an average DGLA content of 20.7% and an average ETA content of 3.9%. The highest DGLA and ETA content for an individual embryo from this line was 26.3% and 5.4%, respectively. The highest average overall % desaturation (calculation described below) was 72.7% with the highest overall % desaturation for an individual embryo being 83.1%. When broken down into % desaturation for the n-6 and n-3 substrates, the highest average % desaturation was 80.5% and 47.9% for EDA and ERA, respectively. The highest % desaturation for an individual embryo was 89.9% and 55.9% for EDA and ERA, respectively. The Pavlova lutheri delta-8 desaturase has a preference for EDA over ERA with the average desaturation ratio ranging from 1.7 to 3.3. Interestingly, some GLA accumulates in embryos were the delta-8 desaturase is expressed well.
[0410] Furthermore, in summary of FIGS. 8A and 8B, the overall % desaturation (C20% delta-8 desaturation) was calculated by dividing the sum of the wt. % for DGLA and ETA by the sum of the wt. % for EDA, DGLA, ERA and ETA and multiplying by 100 to express as a %. The individual n-6 delta-8 desaturation (EDA % delta-8 desaturation) was calculated by dividing the sum of the wt. % for DGLA by the sum of the wt. % for EDA and DGLA and multiplying by 100 to express as a %. Similarly, the individual n-3 delta-8 desaturation (ERA % delta-8 desaturation) shown was calculated by dividing the sum of the wt. % for ETA by the sum of the wt. % for ERA and ETA and multiplying by 100 to express as a %. The ratio of delta-8 desaturation for n-6 versus n-3 substrates (ratio EDA/ERA % desaturation) was obtained by dividing the EDA % delta-8 desaturation by the ERA % delta-8 desaturation.
Example 13
Cloning the Pavlova lutheri Delta-8 Desaturase into a Soybean Expression Vector Containing the Euglena Gracilis Delta-9 Elongase and Mortierella Alpina Delta-5 Desaturase
[0411] The Euglena gracilis delta-9 elongase (SEQ ID NO:32) was amplified with oligonucleotide primers oEugEL1-1 (SEQ ID NO:33) and oEugEL1-2 (SEQ ID NO:34) using the VentR®DNA Polymerase (Cat. 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:35).
[0412] Plasmid pKR906 was digested with NotI and the fragment containing the Euglena gracilis delta-9 elongase was cloned into plasmid pKR132 (SEQ ID NO:36, which is described in PCT Publication No. WO 2004/071467) to give pKR953 (SEQ ID NO:37).
[0413] Vector pKR287 (SEQ ID NO:38; which is described in PCT Publication No. WO 2004/071467, published Aug. 26, 2004; the contents of which are hereby incorporated by reference), contains the Mortierella alpina delta-5 desaturase (SEQ ID NO:39), which is described in U.S. Pat. No. 6,075,183 and PCT Publication Nos. WO 2004/071467 and WO 2005/047479 (the contents of which are hereby incorporated by reference), flanked by the soybean glycinin Gy1 promoter and the pea leguminA2 3' termination region (Gy1/MaD5/legA2 cassette). Vector pKR287 was digested with SbfI/BsiWI and the fragment containing the Gy1/MaD5/legA2 cassette was cloned into the SbfI/BsiWI fragment of pKR277 (SEQ ID NO:40; which is described in PCT Publication No. WO 2004/071467, the contents of which are hereby incorporated by reference) to produce pK952 (SEQ ID NO:41).
[0414] Vector pKR457 (SEQ ID NO:42), which was previously described in PCT Publication No. WO 2005/047479 (the contents of which are hereby incorporated by reference), contains a NotI site flanked by the Kunitz soybean Trypsin Inhibitor (KTi) promoter (Jofuku et al., Plant Cell 1:1079-1093 (1989)) and the KTi 3' termination region, the isolation of which is described in U.S. Pat. No. 6,372,965, followed by the soy albumin transcription terminator, which was previously described in PCT Publication No. WO 2004/071467 (Kti/NotI/Kti3'Salb3' cassette). Through a number of sub-cloning steps, sequences containing Asp718 restriction sites were added to the 5' and 3' ends of the Kti/NotI/Kti3'Salb3' cassette to give SEQ ID NO:43. After cloning the NotI fragment of pLF113 (Example 4), containing the Pavlova lutheri delta-8 desaturase, into the modified Kti/NotI/Kti3'Salb3' cassette (SEQ ID NO:43), the DNA fragment was digested with Asp718 and cloned into the SbfI site of pKR952 (SEQ ID NO:41) to give pKR970 (SEQ ID NO:44).
[0415] Plasmid pKR953 (SEQ ID NO:37) was digested with PstI and the fragment containing the Euglena gracilis delta-9 elongase was cloned into the SbfI site of pKR970 (SEQ ID NO:44) to give pKR973 (SEQ ID NO:45, FIG. 5).
[0416] In this way, the Pavlova lutheri delta-8 desaturase could be co-expressed with the Mortierella alpina delta-5 desaturase and the Euglena gracilis delta-9 elongase behind strong, seed-specific promoters.
Example 14
[0417] Cloning the Euglena Gracilis Delta-9 Elongase into a Soybean Expression Vector
[0418] The gene for the Euglena gracilis delta-9 elongase (SEQ ID NO:32) is released from pKR906 (SEQ ID NO:35) by digestion with NotI and cloned into the NotI site of pKR72 (SEQ ID NO:46 and has ATCC Accession No. PTA-6019) to produce pKR912 (SEQ ID NO:47). A schematic depiction of pKR912 is shown in FIG. 6.
Example 15
Construction of a Vector Containing the Saprolegnia Diclina Delta-17 Desaturase and Fusarium Moniliforme Delta-15 Desaturase
[0419] Vector pKR886r (SEQ ID NO:48) was made by cloning the PstI fragment, containing the Ann/Sdd17/BD30 cassette from pKR271 (SEQ ID NO:49, which is described in PCT Publication No. WO 2004/071467) into the SbfI site of pKR226 (SEQ ID NO:50, which is described in PCT Publication No. WO 2004/071467).
[0420] The βcon/NotI/Phas3' cassette in plasmid pKR72 (SEQ ID NO:46 and has ATCC Accession No. PTA-6019) was amplified using oligonucleotide primers oCon1 (SEQ ID NO:51) and oCon-2 (SEQ ID NO:52) using the VentR® DNA Polymerase (Cat. No. MO254S, New England Biolabs Inc., Beverly, Mass.) following the manufacturer's protocol. The resulting DNA fragment was digested with XbaI and cloned into the XbaI site of pUC19, to produce pKR179 (SEQ ID NO:53).
[0421] The Fusarium monoliforme delta-15 desaturase was released from plasmid pKR578 (SEQ ID NO:54, which is described in PCT Publication No. WO 2005/047479 and has ATCC Accession No. PTA-6280) by digestion with NotI and was cloned into the NotI site of plasmid pKR179 to give pKR582 (SEQ ID NO:55).
[0422] Vector pKR582 was digested with PstI and the fragment containing the Fusarium monoliforme delta-15 desaturase was cloned into the SbfI site of pKR886r (SEQ ID NO:48) to give pKR983 (SEQ ID NO:56). A schematic depiction of pKR983 is shown in FIG. 7.
Example 16
Co-Expressinq Other Promoter/Gene/Terminator Cassette Combinations
[0423] 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 Publication Nos. WO 2004/071467 and WO 2004/071178 describe the isolation of a number of promoter and transcription terminator sequences for use in embryo-specific expression in soybean. Furthermore, PCT Publication Nos. WO 2004/071467, WO 2005/047479 and WO 2006/012325 describe the synthesis of multiple promoter/gene/terminator cassette combinations by ligating individual promoters, genes and transcription terminators together in unique combinations. Generally, a NotI site flanked by the suitable promoter (such as those listed in, but not limited to, Table 4) and a transcription terminator (such as those listed in, but not limited to, Table 5) 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 6 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.
[0424] In addition, PCT Publication Nos. WO 2004/071467, WO 2005/047479 and WO 2006/012325 describe the further linking together of individual gene cassettes in unique combinations, along with suitable selectable marker cassettes, in order to obtain the desired phenotypic expression. Although this is done mainly using different restriction enzymes sites, one skilled in the art can appreciate that a number of techniques can be utilized to achieve the desired promoter/gene/transcription terminator combination. In so doing, any combination of embryo-specific promoter/gene/transcription terminator cassettes can be achieved. One skilled in the art can also appreciate that these cassettes can be located on individual DNA fragments or on multiple fragments where co-expression of genes is the outcome of co-transformation of multiple DNA fragments.
TABLE-US-00005 TABLE 4 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 WO 2004/071467 glycinin Gy1 soybean WO 2004/071467 albumin 2S soybean U.S. Pat. No. 6,177,613 legumin A1 pea Rerie et al., Mol. Gen. Genet. 225: 148-157 (1991) β-conglycinin β-subunit soybean WO 2004/071467 BD30 (also called P34) soybean WO 2004/071467 legumin A2 pea Rerie et al., Mol. Gen. Genet. 225: 148-157 (1991)
TABLE-US-00006 TABLE 5 Transcription Terminators Transcription Terminator Organism Reference phaseolin 3' bean WO 2004/071467 kunitz trypsin inhibitor 3' soybean WO 2004/071467 BD30 (also called P34) 3' soybean WO 2004/071467 legumin A2 3' pea WO 2004/071467 albumin 2S 3' soybean WO 2004/071467
TABLE-US-00007 TABLE 6 EPA Biosynthetic Pathway Genes Gene Organism Reference delta-6 desaturase Saprolegnia diclina WO 2002/081668 delta-6 desaturase Mortierella alpina U.S. Pat. No. 5,968,809 elongase Mortierella alpina WO 2000/12720 U.S. Pat. No. 6,403,349 delta-5 desaturase Mortierella alpina U.S. Pat. No. 6,075,183 delta-5 desaturase Saprolegnia diclina WO 2002/081668 delta-15 desaturase Fusarium moniliforme WO 2005/047479 delta-17 desaturase Saprolegnia diclina WO 2002/081668 elongase Thraustochytrium WO 2002/08401 aureum U.S. Pat. No. 6,677,145 elongase Pavlova sp. Pereira et al., Biochem. J. 384: 357-366 (2004) delta-4 desaturase Schizochytrium WO 2002/090493 aggregatum delta-9 elongase Isochrysis galbana WO 2002/077213 delta-9 elongase Euglena gracilis U.S. Provisional Application No. 60/739,989 delta-8 desaturase Euglena gracilis WO 2000/34439 U.S. Pat. No. 6,825,017 WO 2004/057001 WO 2006/012325 delta-8 desaturase Acanthamoeba Sayanova et al., FEBS Lett. castellanii 580: 1946-1952 (2006) delta-8 desaturase Pavlova salina WO 2005/103253 delta-8 desaturase Pavlova lutheri instant application
Example 17
Synthesis of a Codon-Optimized Delta-8 Desaturase Gene Derived from Pavlova Lutheri in Yarrowia lipolytica
[0425] The codon usage of the delta-8 desaturase gene of Pavlova lutheri (SEQ ID NO:14; Example 4, supra) was optimized for expression in Yarrowia lipolytica, in a manner similar to that described in PCT Publication No. WO 2004/101753. Specifically, a codon-optimized delta-8 desaturase gene (designated "PiD8S"; SEQ ID NO:57) was designed based on the coding sequence of the delta-8 desaturase gene of the instant invention (SEQ ID NO:14), 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 Brewer, J., Gene 265(1-2):11-23 (2001)). In addition to modification of the translation initiation site, 161 bp of the 1272 bp coding region were modified (13.1%) and 161 codons were optimized (38.1%). None of the modifications in the codon-optimized gene changed the amino acid sequence of the encoded protein (SEQ ID NO:16). The designed PiD8S gene was synthesized by GenScript Corporation (Piscataway, N.J.) and cloned into pUC57 (GenBank Accession No. Y14837) to generate pPiD8S (SEQ ID NO:58; FIG. 11-A).
Example 18
Construction of Plasmid pZUFmEgD9ES, Comprising a Codon-Optimized Delta-9 Elongase Gene Derived from Euglena Gracilis
[0426] The present Example describes the construction of plasmid pZUFmEgD9ES (SEQ ID NO:70), comprising a synthetic delta-9 elongase gene (derived from Euglena gracilis) that was codon-optimized for Yarrowia lipolytica (designated herein as "EgD9S" or "EgD9ES"). Plasmid pZUFmEgD9ES (SEQ ID NO: 70; FIG. 9-D) was constructed by three-way ligation using fragments from plasmids pEgD9ES, pDMW263 and pZUF17 (SEQ ID NO:67, SEQ ID NO:68 and SEQ ID NO:69, respectively; FIGS. 9-A, 9-B and 9-C, respectively). This plasmid was utilized to construct plasmid pZUFmE9SP8S (SEQ ID NO:71) comprising the synthetic codon-optimized PiD8S from Example 17 and EgD9S as described herein in Example 19, infra.
Euglena Gracilis Growth Conditions, Lipid Profile and mRNA Isolation:
[0427] Euglena gracilis was obtained from Dr. Richard Triemer's lab at Michigan State University (East Lansing, Mich.). From 10 mL of actively growing culture, a 1 mL aliquot was transferred into 250 mL of Euglena gracilis (Eg) Medium in a 500 mL glass bottle. Eg medium was made by combining 1 g of sodium acetate, 1 g of beef extract (U126-01, Difco Laboratories, Detroit, Mich.), 2 g of Bacto® tryptone (0123-17-3, Difco Laboratories) and 2 g of Bacto® yeast extract (0127-17-9, Difco Laboratories) in 970 mL of water. After filter sterilizing, 30 mL of soil-water supernatant (15-3790, Carolina Biological Supply Company, Burlington, N.C.) was aseptically added to give the final Eg medium. Euglena gracilis cultures were grown at 23° C. with a 16 h light, 8 h dark cycle for 2 weeks with no agitation.
[0428] After 2 weeks, 10 mL of culture was removed for lipid analysis and centrifuged at 1,800×g for 5 min. The pellet was washed once with water and re-centrifuged. The resulting pellet was dried for 5 min under vacuum, resuspended in 100 L of trimethylsulfonium hydroxide (TMSH) and incubated at room temperature for 15 min with shaking. After this, 0.5 mL of hexane was added and the vials were incubated for 15 min at room temperature with shaking. Fatty acid methyl esters (5 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 (Supelco Inc., Cat. No. 24152). 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 (Nu-Chek Prep, Inc. Cat. No. U-99-A) and the resulting chromatogram is shown in FIG. 10.
[0429] The remaining 2 week culture (240 mL) was pelleted by centrifugation at 1,800×g for 10 min, washed once with water and re-centrifuged. Total RNA was extracted from the resulting pellet using the RNA STAT-60® reagent (TEL-TEST, Inc., Friendswood, Tex.) and following the manufacturer's protocol provided (use 5 mL of reagent, dissolved RNA in 0.5 mL of water). In this way, 1 mg of total RNA (2 mg/mL) was obtained from the pellet. The mRNA was isolated from 1 mg of total RNA using the mRNA Purification Kit (Amersham Biosciences, Piscataway, N.J.) following the manufacturer's protocol provided. In this way, 85 g of mRNA was obtained.
Euglena Gracilis cDNA Synthesis, Library Construction and Sequencing:
[0430] A cDNA library was generated using the Cloneminer® cDNA Library Construction Kit (Cat. No. 18249-029, Invitrogen Corporation, Carlsbad, Calif.) and following the manufacturer's protocol provided (Version B, 25-0608). Using the non-radiolabeling method, cDNA was synthesized from 3.2 g of mRNA (described above) using the Biotin-attB2-Oligo(dT) primer. After synthesis of the first and second strand, the attB1 adapter was added, ligated and the cDNA was size fractionated using column chromatography. DNA from fractions 7 and 8 (size ranging from ˜800-1500 bp) were concentrated, recombined into pDONR® 222 and transformed into E. coli ElectroMAX® DH10B® T1 Phage-Resistant cells (Invitrogen Corporation). The Euglena gracilis library was named eeg1c.
[0431] For sequencing, clones first were recovered from archived glycerol cultures grown/frozen in 384-well freezing media plates, and replicated with a sterile 384 pin replicator (Genetix, Boston, Mass.) in 384-well microtiter plates containing LB+75 g/mL Kanamycin (replicated plates). Plasmids then were isolated, using the Templiphi DNA sequencing template amplification kit method (Amersham Biosciences) following the manufacturer's protocol. Briefly, the Templiphi method uses bacteriophage φ29 DNA polymerase to amplify circular single-stranded or double-stranded DNA by isothermal rolling circle amplification (Dean et al., Genome Res. 11:1095-1099 (2001); Nelson et al., Biotechniques 32:S44-S47 (2002)). After growing 20 h at 37° C., cells from the replicated plate were added to 5 L of dilution buffer and denatured at 95° C. for 3 min to partially lyse cells and release the denatured template. 5 L of Templiphi premix then were added to each sample and the resulting reaction mixture was incubated at 30° C. for 16 h, then at 65° C. for 10 min to inactivate the φ29 DNA polymerase activity. DNA quantification with the PicoGreen® dsDNA Quantitation Reagent (Molecular Probes) was performed after diluting the amplified samples 1:3 in distilled water.
[0432] The amplified products then were denatured at 95° C. for 10 min and end-sequenced in 384-well plates, using the M13F universal primer (SEQ ID NO:63), and the ABI Big Dye version 3.1 Prism Sequencing Kit. For the sequencing reaction, 100-200 ng of templates and 6.4 pmol of primers were used, and the following reaction conditions were repeated 25 times: 96° 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 ABI 3730xl automated sequencers.
Identification of Long-Chain Polyunsaturated Fatty Acid Elongation Enzyme Homologs from Euglena Gracilis cDNA Library eeg1c:
[0433] cDNA clones encoding long-chain polyunsaturated fatty acid elongation enzyme homologs (i.e., LC-PUFA ELO homologs or delta-9 elongases) were identified and analyzed by conducting BLAST searches for similarity to sequences contained in the BLAST "nr" database, as described supra (see Example 2).
[0434] The BLASTX search using the nucleotide sequences from clone eeg1c.pk001.n5.f revealed similarity of the protein encoded by the cDNA to the long-chain PUFA elongation enzyme from Isochrysis galbana (SEQ ID NO:59) (NCBI Accession No. AAL37626 (GI 17226123), locus AAL37626, CDS AF390174; Qi et al., FEBS Lett. 510(3):159-165 (2002)). The sequence of a portion of the cDNA insert from clone eeg1c.pk001.n5.f is shown in SEQ ID NO:60 (5' end of cDNA insert). Additional sequence was obtained from the 3' end of the cDNA insert of eeg1c.pk001.n5.1 as described above, but using the poly(A) tail-primed WobbleT oligonucleotides. Briefly, the WobbleT primer is an equimolar mix of 21 mer poly(T)A, poly(T)C and poly(T)G, used to sequence the 3' end of cDNA clones.
[0435] The 3' end sequence is shown in SEQ ID NO:61. Both the 5' and 3' sequences were aligned using Sequencher® (Version 4.2, Gene Codes Corporation, Ann Arbor, Mich.) and the resulting sequence for the cDNA is shown in SEQ ID NO:62. Sequence for the coding sequence from the cDNA in eeg1c.pk001.n5.f and the corresponding deduced amino acid sequence is shown in SEQ ID NO:32 and SEQ ID NO:64, respectively.
[0436] The amino acid sequence set forth in SEQ ID NO:64 was evaluated by BLASTP, yielding a pLog value of 38.70 (E value of 2e-39) versus the Isochrysis galbana sequence (SEQ ID NO:65). The Euglena gracilis delta-9 elongase is 39.4% identical to the Isochrysis galbana delta-9 elongase sequence 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 LASARGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.) with the default parameters for pairwise alignment (KTUPLE=2). The Euglena gracilis delta-9 elongase is 31.8% identical to the Isochrysis galbana delta-9 elongase 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 LASARGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.) with the default parameters for pairwise alignment (KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5 and GAP LENGTH PENALTY=10). BLAST scores and probabilities indicate that the instant nucleic acid fragment (SEQ ID NO:32) encode an entire Euglena gracilis delta-9 elongase.
Synthesis of the Codon-Optimized Delta-9 Elongase Gene:
[0437] The codon usage of the delta-9 elongase gene of Euglena gracilis (SEQ ID NOs:32 and 64) was optimized for expression in Yarrowia lipolytica, in a manner similar to that described supra (see Example 17) and WO 2004/101753. Specifically, a codon-optimized delta-9 elongase gene (designated "EgD9S"), SEQ ID NO:66) was designed, based on the coding sequence of the delta-9 elongase (clone eeg1c.pk001.n5.f), according to the Yarrowia codon usage pattern, the consensus sequence around the `ATG` translation initiation codon, and the general rules of RNA stability (Guhaniyogi, G. and Brewer, J., supra)). In addition to the modification of the translation initiation site, 117 bp of the 777 bp coding region were modified (15.1%) and 106 codons were optimized (40.9%). None of the modifications in the codon-optimized gene changed the amino acid sequence of the encoded protein (SEQ ID NO:64). The designed EgD9S (also "EgD9ES") gene was synthesized by GenScript Corporation (Piscataway, N.J.) and was cloned into pUC57 (GenBank Accession No. Y14837) to generate pEgD9ES (SEQ ID NO:67; FIG. 9-A)
Construction of Plasmid pDMW263:
[0438] Plasmid pY5-30 (SEQ ID NO:78) (previously described in PCT Publication No. WO 2005/003310 (the contents of which are hereby incorporated by reference) is a shuttle plasmid that can replicate both in E. coli and Yarrowia lipolytica. Plasmid pY5-30 contains the following: a Yarrowia autonomous replication sequence (ARS18); a ColE1 plasmid origin of replication; an ampicillin-resistance gene (AmpR) for selection in E. coli; a Yarrowia LEU2 gene for selection in Yarrowia; and a chimeric TEF::GUS::XPR gene. Plasmid pDMW263 (SEQ ID NO:68; FIG. 9-B) was created from pY5-30, by replacing the TEF promoter with the Yarrowia lipolytica FBAINm promoter (PCT Publication No. WO 2005/049805) using techniques well known to one skilled in the art. Briefly, this promoter refers to a modified promoter which is located in the 5' upstream untranslated region in front of the `ATG` translation initiation codon of the fructose-bisphosphate aldolase enzyme (E.C. 4.1.2.13) encoded by the fba1 gene and that is necessary for expression, plus a portion of 5' coding region that has an intron, wherein FBAINm has a 52 bp deletion between the ATG translation initiation codon and the intron of the FBAIN promoter (thereby including only 22 amino acids of the N-terminus) and a new translation consensus motif after the intron. Table 7 summarizes the components of pDMW263.
TABLE-US-00008 TABLE 7 Components of Plasmid pDMW263 RE Sites and Nucleotides Within Description of Fragment and SEQ ID NO: 68 Chimeric Gene Components 4992-4296 ARS18 sequence (GenBank Accession No. A17608) SalI/SaclI FBAINm::GUS::XPR, comprising: (8505-2014) FBAINm: Yarrowia lipolytica FBAINm promoter (WO 2005/049805) GUS: E. coli gene encoding-glucuronidase (Jefferson, R. A., Nature. 14: 342: 837-838 (1989)) XPR: ~100 bp of the 3' region of the Yarrowia Xpr gene (GenBank Accession No. M17741) 6303-8505 Yarrowia Leu2 gene (GenBank Accession No. AF260230)
Construction of Plasmid pZUF17:
[0439] Plasmid pZUF17 (SEQ ID NO:69; FIG. 9-C) possesses a similar backbone to that of pDMW236. However, the plasmid comprises a Yarrowia Ura3 gene for selection in Yarrowia and a chimeric FBAIN::D17S::Pex20 gene, instead of the LEU2 gene and chimeric FBAINm::GUS::XPR gene of pDMW263. Table 8 summarizes the components of pZUF17.
TABLE-US-00009 TABLE 8 Components of Plasmid pZUF17 RE Sites and Nucleotides Within Description of Fragment and SEQ ID NO: 69 Chimeric Gene Components 2866-4170 ARS18 sequence (GenBank Accession No. A17608) CalI/PacI FBAIN::D17S::Pex20, comprising: (5750-8165) FBAIN: Yarrowia lipolytica FBAIN promoter (WO 2005/049805) .sub.Δ17S: synthetic {umlaut over (A)}17 desaturase gene derived from Saprolegnia diclina (US 2003/0196217 A1), codon- optimized for Yarrowia lipolytica (WO 2004/101757) Pex20: Pex20 terminator sequence of Yarrowia Pex20 gene (GenBank Accession No. AF054613) 5703-4216 Yarrowia Ura3 gene (GenBank Accession No. AJ306421)
Final Construction of Plasmid pZUFmEgD9ES:
[0440] The NcoI/NotI fragment from plasmid pEgD9ES (SEQ ID NO:67; FIG. 9-A; comprising EgD9ES) and the SalI/NcoI fragment from pDMW263 (SEQ ID NO:68; FIG. 9-B; comprising the Yarrowia lipolytica FBAINm promoter) were used directionally to replace the SaII/NotI fragment of pZUF17 (SEQ ID NO:69; FIG. 9-C). This resulted in generation of pZUFmEgD9ES (SEQ ID NO:70; FIG. 9-D), comprising a chimeric FBAINm::EgD9ES::Pex20 gene.
Example 19
Construction of Plasmid pZUFmE9SP8S, Comprising the Codon-Optimized Delta-8 Desaturase Gene Derived from Pavlova Lutheri and the Codon-Optimized Delta-9 Elongase Gene Derived From Euglena Gracilis
[0441] The present Example describes the construction of plasmid pZUFmE9SP8S (SEQ ID NO:71), comprising the synthetic codon-optimized PiD8S from Example 17 and the synthetic codon-optimized EgD9ES from Example 18. Plasmid pZUFmE9SP8S (SEQ ID NO:71; FIG. 11-D) was constructed by four-way ligation using fragments from plasmids pPiD8S, pZUFmEgD9ES, pEXPGUS1-C and pZGD5T-CP (SEQ ID NO:58, SEQ ID NO:70, SEQ ID NO:72 and SEQ ID NO:73, respectively; FIGS. 11-A, 9-D, 11-B and 11-C, respectively). This plasmid was utilized to test functional co-expression of PiD8S and EgD9ES, as described in Example 20, infra.
Plasmid pEXPGUS1-C:
[0442] Plasmid pEXPGUS1-C (SEQ ID NO:72; FIG. 11-B) comprises a chimeric EXP1::GUS::XPR gene (nucleotides 953-3963 of SEQ ID NO:72). The "EXP1" promoter within this chimeric gene refers to the 5' upstream untranslated -1000 to -1 by region in front of the `ATG` translation initiation codon of a protein encoded by the Yarrowia lipolytica "YALI0C12034g" gene (GenBank Accession No. XM--501745) and that is necessary for expression. Based on significant homology of "YALI0C12034g" to the sp|Q12207 Saccharomyces cerevisiae non-classical export protein 2 (whose function is involved in a novel pathway of export of proteins that lack a cleavable signal sequence), this gene was designated as the exp1 gene, encoding a protein designated as EXP1 (U.S. application Ser. No. 11/265,761). "GUS" and "XPR" are defined as described above in Table 7.
Plasmid pZGD5T-CP:
[0443] Plasmid pZGD5T-CP (SEQ ID NO:73; FIG. 11-C) comprises a chimeric GPD::MAD5::Pex16 gene (nucleotides 3200-346 of SEQ ID NO:73). The "GPD" promoter within this chimeric gene 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). The "MAD5" coding region of the chimeric gene corresponds to the Mortierella alpina.sub.Δ5 desaturase gene (GenBank Accession No. AF067654), while "Pex16" refers to the Pex16 terminator of the Yarrowia Pex16 gene (GenBank Accession No. U75433).
Final Construction of Plasmid pZUFmE9SP8S:
[0444] The NcoI/NotI fragment of plasmid pPiD8S (SEQ ID NO:58; FIG. 11-A; comprising the synthetic delta-8 desaturase gene of the present invention (i.e., PiD8S), the ClaI/NcoI fragment from pEXPGUS1-C (SEQ ID NO:72; FIG. 11-B; comprising the EXP1 promoter), and the NotI/PacI fragment from plasmid pZGD5T-CP (SEQ ID NO:73; FIG. 11-C; comprising the Pex16 terminator) were used directionally to replace the ClaI/PacI fragment of pZUFmEgD9ES (SEQ ID NO:70; FIG. 9-D) to generate pZUFmE9SP8S (SEQ ID NO:71; FIG. 11-D).
Example 20
Functional Expression of Plasmid pZUFmE9SP8S in Yarrowia Lipolytica
[0445] The present Example describes expression of plasmid pZUFmE9SP8S, comprising the chimeric FBAINm::EgD9ES::Pex20 gene and the chimeric EXP::PiD8S::Pex16 gene. Expression of pZUFmE9SP8S in Yarrowia lipolytica led to the production of up to 2.8% EDA and 0.5% of DGLA.
[0446] Specifically, pZUFmE9SP8S (SEQ ID NO:71; FIG. 11-D) was transformed into Yarrowia Lipolytica Y20362U (an autonomous Ura-mutant of ATCC Accession No. 20362, that was generated by selecting for FOA resistance) as described supra. The transformant cells were plated onto MM selection media plates and maintained at 30° C. for 2 to 3 days. Fifteen (15) 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.
[0447] GC analyses showed that there were about 2.8% EDA (C20:2) and 0.3% of DGLA (C20:3) of total lipids produced in 13 of these 15 transformants, wherein the conversion efficiency of EDA to DGLA in these 13 strains was at an average rate of about 9.7%. Strain #7 produced 2.8% EDA and 0.5% of DGLA, with a conversion efficiency of about 15%. The term "conversion efficiency" refers to the efficiency by which a particular enzyme (e.g., the codon-optimized delta-8 desaturase identified herein as PiD8S) can convert substrate (i.e., EDA) to product (i.e., DGLA). 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.
Example 21
Chlorsulfuron Selection (ALS) and Plant Regeneration Chlorsulfuron (ALS) Selection
[0448] Following bombardment, the tissue is divided between 2 flasks with fresh SB196 media and cultured as described in Example 11. 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 may be observed growing from untransformed, necrotic embryogenic clusters. Isolated, green tissue is removed and inoculated into multiwell plates containing SB196 to generate new, clonally propagated, transformed embryogenic suspension cultures.
Regeneration of Soybean Somatic Embryos into Plants:
[0449] In order to obtain whole plants from embryogenic suspension cultures, the tissue must be regenerated. Embyros are matured as described in Example 11. After subculturing on medium SB103 for 3 weeks, individual embryos can be removed from the clusters and screened for alterations in their fatty acid compositions as described in Example 12. It should be noted that any detectable phenotype, resulting from the expression of the genes of interest, could be screened at this stage. This would include, but not be limited to, alterations in fatty acid profile, protein profile and content, carbohydrate content, growth rate, viability, or the ability to develop normally into a soybean plant.
[0450] 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 24-cell pack tray, covered with clear plastic dome. After 2 weeks the dome is removed and plants hardened off for a further week. If plantlets looked hardy they are transplanted to 10'' pot of Redi-Earth with up to 3 plantlets per pot. After 10 to 16 weeks, mature seeds are harvested, chipped and analyzed for fatty acids as described in Example 12.
[0451] Media recipes can be found in Example 11 and chlorsulfuron stock is 1 mg/mL in 0.01 N ammonium hydroxide.
Example 22
Co-Expression of the Euglena Gracilis Delta-9 Elongase with the Pavlova Lutheri (CCMP459) Delta-8 Desaturase, the Mortierella Alpina Delta-5 Desaturase, the Saprolegnia Diclina Delta-17 Desaturase and the Fusarium Moniliforme Delta-15 Desaturase in Soybean Embryos Transformed with Soybean Expression Vectors pKR973 and pKR983
[0452] Soybean embryogenic suspension culture (cv. Jack) was transformed with the AscI fragments of pKR973 (SEQ ID NO:45, FIG. 5) and pKR983 (SEQ ID NO:56; FIG. 7) (fragments containing the expression cassettes), as described for production in Example 11. Transformants were selected using chlorsulfuron as described in Example 21 and embryos were matured as described in Example 11. A subset of soybean embryos generated from each event (ten embryos per event) were harvested and analyzed for fatty acid composition as described in Example 12. Fatty acids were identified by comparison of retention times to those for authentic standards.
[0453] In this way, 243 events transformed with pKR973 and pKR983 were analyzed. From the 243 events analyzed, 117 were identified that produced EPA in at least one embryo out of ten analyzed at a relative abundance greater than 1.0% of the total fatty acids. Of these, 15 were identified that produced EPA in at least one embryo out of ten analyzed at a relative abundance greater than 10.0% of the total fatty acids. The average fatty acid profile for the ten best EPA events (average of seven to ten individual embryos) is shown in FIG. 14. Fatty acids are identified as 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic acid), LA, GLA, ALA, EDA, DGLA, ARA, ERA, JUN, ETA, EPA and DPA; and, fatty acid compositions listed in FIG. 14 are expressed as a weight percent (wt. %) of total fatty acids. For FIG. 14, fatty acids listed as "others" include: 18:2 (5,9), STA, 20:0, 20:1(11), 20:2 (7,11) or 20:2 (8,11) and 20:3 (5,11,14). Each of these fatty acids is present at a relative abundance of less than 1% of the total fatty acids. The activity of the Pavlova lutheri (CCMP459) delta-8 desaturase is expressed as percent delta-8 desaturation (% Desat), calculated according to the following formula: ([product]/[substrate+product])*100.
[0454] More specifically, the combined percent delta-8 desaturation for EDA and ERA is shown as "Total delta-8% Desat", determined as: ([DGLA+ARA+ERA+ETA+EPA+DPA]/[EDA+DGLA+ARA+ERA+JUN+ETA+EPA+DPA])*100.
[0455] In summary of FIG. 14, the Pavlova lutheri (CCMP459) delta-8 desaturase functioned in soybean to convert both EDA and ERA to DGLA and ETA, respectively, and these were further converted to other LC-PUFAs. Line AFS 4802-3-14, the high EPA line with the highest average overall % delta-8 desaturation, had overall % delta-8 desaturation of 82.5%.
Sequence CWU
1
78119DNAArtificial SequenceT7 primer 1ggaaacagct atgaccatg
192459PRTRhizopus stolonifer 2Met Ser
Thr Leu Asp Arg Gln Ser Ile Phe Thr Ile Lys Glu Leu Glu1 5
10 15Ser Ile Ser Gln Arg Ile His Asp
Gly Asp Glu Glu Ala Met Lys Phe 20 25
30Ile Ile Ile Asp Lys Lys Val Tyr Asp Val Thr Glu Phe Ile Glu
Asp 35 40 45His Pro Gly Gly Ala
Gln Val Leu Leu Thr His Val Gly Lys Asp Ala 50 55
60Ser Asp Val Phe His Ala Met His Pro Glu Ser Ala Tyr Glu
Val Leu65 70 75 80Asn
Asn Tyr Phe Val Gly Asp Val Gln Glu Thr Val Val Thr Glu Lys
85 90 95Ser Ser Ser Ala Gln Phe Ala
Val Glu Met Arg Gln Leu Arg Asp Gln 100 105
110Leu Lys Lys Glu Gly Tyr Phe His Ser Ser Lys Leu Phe Tyr
Ala Tyr 115 120 125Lys Val Leu Ser
Thr Leu Ala Ile Cys Ile Ala Gly Leu Ser Leu Leu 130
135 140Tyr Ala Tyr Gly Arg Thr Ser Thr Leu Ala Val Val
Ala Ser Ala Ile145 150 155
160Thr Val Gly Ile Phe Trp Gln Gln Cys Gly Trp Leu Ala His Asp Phe
165 170 175Gly His His Gln Cys
Phe Glu Asp Arg Thr Trp Asn Asp Val Leu Val 180
185 190Val Phe Leu Gly Asn Phe Cys Gln Gly Phe Ser Leu
Ser Trp Trp Lys 195 200 205Asn Lys
His Asn Thr His His Ala Ser Thr Asn Val His Gly Gln Asp 210
215 220Pro Asp Ile Asp Thr Ala Pro Val Leu Leu Trp
Asp Glu Tyr Ala Ser225 230 235
240Ala Ala Tyr Tyr Ala Ser Leu Asp Gln Glu Pro Thr Met Val Ser Arg
245 250 255Phe Leu Ala Glu
Gln Val Leu Pro His Gln Thr Arg Tyr Phe Phe Phe 260
265 270Ile Leu Ala Phe Ala Arg Leu Ser Trp Ala Leu
Gln Ser Leu Ser Tyr 275 280 285Ser
Phe Lys Lys Glu Ser Ile Asn Lys Ser Arg Gln Leu Asn Leu Phe 290
295 300Glu Arg Val Cys Ile Val Gly His Trp Ala
Leu Phe Ala Phe Cys Ile305 310 315
320Tyr Ser Trp Cys Ser Asn Val Tyr His Met Val Leu Phe Phe Leu
Val 325 330 335Ser Gln Ala
Thr Thr Gly Tyr Thr Leu Ala Leu Val Phe Ala Leu Asn 340
345 350His Asn Gly Met Pro Val Ile Thr Glu Glu
Lys Ala Glu Ser Met Glu 355 360
365Phe Phe Glu Ile Gln Val Ile Thr Gly Arg Asp Val Thr Leu Ser Pro 370
375 380Leu Gly Asp Trp Phe Met Gly Gly
Leu Asn Tyr Gln Ile Glu His His385 390
395 400Val Phe Pro Asn Met Pro Arg His Asn Leu Pro Thr
Val Lys Pro Met 405 410
415Val Lys Ser Leu Cys Gln Lys Tyr Asp Ile Asn Tyr His Asp Thr Gly
420 425 430Phe Leu Lys Gly Thr Leu
Glu Val Leu Gln Thr Leu Asp Ile Thr Ser 435 440
445Lys Leu Ser Leu Gln Leu Ser Lys Lys Ser Phe 450
4553695DNAPavlova lutheri 3agggccaagg gtgccaacca ccttccacgt
gagactacac accgtaggcc gatgggcaag 60ggtggagacg gcggcgcgca ggcggtgagc
gggaccgacg cgtctctcgc tgaggtgagc 120tccgtcgata gcaagagcgt gcacgtcgtg
ctctacggca agcgcgtgga tgtcacaaag 180ttccagggct acgacgtggc ctggtggcgc
gcgcgccata acacgcacca cgtgtgcacc 240aacgaggatg gttcggaccc ggacatcaag
acggcgcccc tgctcatcta cgtgcgagag 300aacccgtcca ttgccaagcg gctcaacttc
ttccagcgct ggcagcagta ctactatgtg 360ccgaccatgg ccatcctcga cctctactgg
cgcctggagt ccatcgcgta cgtggctgtg 420cgcctgccta agatgtggat gcaggccgcc
gctcttgccg ctcactacgc gctcctgtgc 480tgggtcttcg cagcgcatct caacctcatc
cctctcatga tggttgcacg cggcttcgcg 540acgggcatcg ttgtctttgc aacccactat
ggtgaggaca tcctcgaccg cgagcacgtc 600gagggcatga cgctcgtcga gcagaccgcc
aagacctccc gtaacatcac gggcggctgg 660ctagtgaacg tgctcacggg cttcatctcc
ctgca 69541106DNAPavlova lutheri
4agggccaagg gtgccaacca ccttccacgt gagactacac accgtaggcc gatgggcaag
60ggtggagacg gcggcgcgca ggcggtgagc gggaccgacg cgtctctcgc tgaggtgagc
120tccgtcgata gcaagagcgt gcacgtcgtg ctctacggca agcgcgtgga tgtcacaaag
180ttccagggct acgacgtggc ctggtggcgc gcgcgccata acacgcacca cgtgtgcacc
240aacgaggatg gttcggaccc ggacatcaag acggcgcccc tgctcatcta cgtgcgagag
300aacccgtcca ttgccaagcg gctcaacttc ttccagcgct ggcagcagta ctactatgtg
360ccgaccatgg ccatcctcga cctctactgg cgcctggagt ccatcgcgta cgtggctgtg
420cgcctgccta agatgtggat gcaggccgcc gctcttgccg ctcactacgc gctcctgtgc
480tgggtcttcg cagcgcatct caacctcatc cctctcatga tggttgcacg cggcttcgcg
540acgggcatcg ttgtctttgc aacccactat ggtgaggaca tcctcgaccg cgagcacgtc
600gagggcatga cgctcgtcga gcagaccgcc aagacctccc gtaacatcac gggcggctgg
660ctagtgaacg tgctcacggg cttcatctcc ctgcagaccg agcatcacct cttccccatg
720atgcccaccg gcaacctaat gactatccag cccgaggtac gcgacttctt caagaagcat
780ggcctcgagt accgcgaggg caacctcttc cagtgcgtgc accagaacat caaggctctc
840gccttcgagc acctcctcca ctgagcgtca ccactcaagc gtcctaagtg cacaggtact
900gtcttctgac cgatggccgc gcggctccct cggctggcag tggggccaac gagtggcctc
960gcgggatcgg gcacgatcgg gcctccatga aacttcagtg ttcagagaca agccgacaac
1020ctccgcatcg tgagaaatct tttaaagcag tatgttccat cacgccgctt ttgcagtcaa
1080taacattacc caaaaaaaaa aaaaaa
11065287PRTPavlova lutheri 5Arg Ala Lys Gly Ala Asn His Leu Pro Arg Glu
Thr Thr His Arg Arg1 5 10
15Pro Met Gly Lys Gly Gly Asp Gly Gly Ala Gln Ala Val Ser Gly Thr
20 25 30Asp Ala Ser Leu Ala Glu Val
Ser Ser Val Asp Ser Lys Ser Val His 35 40
45Val Val Leu Tyr Gly Lys Arg Val Asp Val Thr Lys Phe Gln Gly
Tyr 50 55 60Asp Val Ala Trp Trp Arg
Ala Arg His Asn Thr His His Val Cys Thr65 70
75 80Asn Glu Asp Gly Ser Asp Pro Asp Ile Lys Thr
Ala Pro Leu Leu Ile 85 90
95Tyr Val Arg Glu Asn Pro Ser Ile Ala Lys Arg Leu Asn Phe Phe Gln
100 105 110Arg Trp Gln Gln Tyr Tyr
Tyr Val Pro Thr Met Ala Ile Leu Asp Leu 115 120
125Tyr Trp Arg Leu Glu Ser Ile Ala Tyr Val Ala Val Arg Leu
Pro Lys 130 135 140Met Trp Met Gln Ala
Ala Ala Leu Ala Ala His Tyr Ala Leu Leu Cys145 150
155 160Trp Val Phe Ala Ala His Leu Asn Leu Ile
Pro Leu Met Met Val Ala 165 170
175Arg Gly Phe Ala Thr Gly Ile Val Val Phe Ala Thr His Tyr Gly Glu
180 185 190Asp Ile Leu Asp Arg
Glu His Val Glu Gly Met Thr Leu Val Glu Gln 195
200 205Thr Ala Lys Thr Ser Arg Asn Ile Thr Gly Gly Trp
Leu Val Asn Val 210 215 220Leu Thr Gly
Phe Ile Ser Leu Gln Thr Glu His His Leu Phe Pro Met225
230 235 240Met Pro Thr Gly Asn Leu Met
Thr Ile Gln Pro Glu Val Arg Asp Phe 245
250 255Phe Lys Lys His Gly Leu Glu Tyr Arg Glu Gly Asn
Leu Phe Gln Cys 260 265 270Val
His Gln Asn Ile Lys Ala Leu Ala Phe Glu His Leu Leu His 275
280 285620DNAArtificial Sequenceprimer SeqE
6cgacacactc caatctttcc
20720DNAArtificial Sequenceprimer SeqW 7ggtggctgga gttagacatc
208457PRTMortierella alpina 8Met Ala
Ala Ala Pro Ser Val Arg Thr Phe Thr Arg Ala Glu Ile Leu1 5
10 15Asn Ala Glu Ala Leu Asn Glu Gly
Lys Lys Asp Ala Glu Ala Pro Phe 20 25
30Leu Met Ile Ile Asp Asn Lys Val Tyr Asp Val Arg Glu Phe Val
Pro 35 40 45Asp His Pro Gly Gly
Ser Val Ile Leu Thr His Val Gly Lys Asp Gly 50 55
60Thr Asp Val Phe Asp Thr Phe His Pro Glu Ala Ala Trp Glu
Thr Leu65 70 75 80Ala
Asn Phe Tyr Val Gly Asp Ile Asp Glu Ser Asp Arg Ala Ile Lys
85 90 95Asn Asp Asp Phe Ala Ala Glu
Val Arg Lys Leu Arg Thr Leu Phe Gln 100 105
110Ser Leu Gly Tyr Tyr Asp Ser Ser Lys Ala Tyr Tyr Ala Phe
Lys Val 115 120 125Ser Phe Asn Leu
Cys Ile Trp Gly Leu Ser Thr Phe Ile Val Ala Lys 130
135 140Trp Gly Gln Thr Ser Thr Leu Ala Asn Val Leu Ser
Ala Ala Leu Leu145 150 155
160Gly Leu Phe Trp Gln Gln Cys Gly Trp Leu Ala His Asp Phe Leu His
165 170 175His Gln Val Phe Gln
Asp Arg Phe Trp Gly Asp Leu Phe Gly Ala Phe 180
185 190Leu Gly Gly Val Cys Gln Gly Phe Ser Ser Ser Trp
Trp Lys Asp Lys 195 200 205His Asn
Thr His His Ala Ala Pro Asn Val His Gly Glu Asp Pro Asp 210
215 220Ile Asp Thr His Pro Leu Leu Thr Trp Ser Glu
His Ala Leu Glu Met225 230 235
240Phe Ser Asp Val Pro Asp Glu Glu Leu Thr Arg Met Trp Ser Arg Phe
245 250 255Met Val Leu Asn
Gln Thr Trp Phe Tyr Phe Pro Ile Leu Ser Phe Ala 260
265 270Arg Leu Ser Trp Cys Leu Gln Ser Ile Met Phe
Val Leu Pro Asn Gly 275 280 285Gln
Ala His Lys Pro Ser Gly Ala Arg Val Pro Ile Ser Leu Val Glu 290
295 300Gln Leu Ser Leu Ala Met His Trp Thr Trp
Tyr Leu Ala Thr Met Phe305 310 315
320Leu Phe Ile Lys Asp Pro Val Asn Met Ile Val Tyr Phe Leu Val
Ser 325 330 335Gln Ala Val
Cys Gly Asn Leu Leu Ala Ile Val Phe Ser Leu Asn His 340
345 350Asn Gly Met Pro Val Ile Ser Lys Glu Glu
Ala Val Asp Met Asp Phe 355 360
365Phe Thr Lys Gln Ile Ile Thr Gly Arg Asp Val His Pro Gly Leu Phe 370
375 380Ala Asn Trp Phe Thr Gly Gly Leu
Asn Tyr Gln Ile Glu His His Leu385 390
395 400Phe Pro Ser Met Pro Arg His Asn Phe Ser Lys Ile
Gln Pro Ala Val 405 410
415Glu Thr Leu Cys Lys Lys Tyr Gly Val Arg Tyr His Thr Thr Gly Met
420 425 430Ile Glu Gly Thr Ala Glu
Val Phe Ser Arg Leu Asn Glu Val Ser Lys 435 440
445Ala Ala Ser Lys Met Gly Lys Ala Gln 450
455922DNAArtificial Sequenceprimer AP1 9gtaatacgac tcactatagg gc
221020DNAArtificial Sequenceprimer
GSP PvDES 10ctgcgaagac ccagcacagg
201122DNAArtificial Sequenceprimer M13-28Rev 11gtaatacgac
tcactatagg gc
221221DNAArtificial Sequenceprimer PavDes seq 12ttgtggcgct caatcatctc c
21131294DNAPavlova lutheri
13ctttgcgagc gcggcgcaga cgattgcggc ccgtagtgat cgcggtgcgc attgctgtgt
60ttctagtttt gctgacgccc ggcccgataa tgacaccttc tcccgtttga aatactaata
120agtaactata ttataatatt caaaggtggc gactatggat ctccttttct aaagttcagc
180ggaattggga atcggagaaa tttcgagata tgtcataatc acgtgctcta tctcgaatga
240accgcggccg gtgagcgatt actcgggaag ccaattccta ttaacgagtc aggggggatc
300tttgaggtga gtcggccacg cagagagagc aaggaatcat cctcatccgc cgttctcgag
360aaagagccaa gggtgccaac caccttccac gtgagactac acaccgtagg ccgatgggca
420agggtggaga cggcggcgcg caggcggcga gcgggaccga cgcatctctc gctgaggtga
480gctccgtcga tagcaagagc gtgcgcgtcg tgctctacgg caagcgcgtg gatgtcacaa
540agttccagag ggcacacccg ggcgggagca aggtgttccg catcttccag gagcgcgacg
600cgacggagca gttcgagtct taccactcgc ccaaggccat caagatgatg gagggcatgc
660tcaagaagtc ggaggatgcg cccgcttccg tgcccctgcc ctcgcggtcc accatgggca
720cggagttcaa ggagatgatt gagcgccaca agagggctgg tctctacgac ccttgcccgt
780tggacgagct gttcaagctc accatcgtcc ttgcgcccat cttcgtgggc gcctatctcg
840tgcggagcgg cgtctcgccc ctcgcgggcg cgctctccat gggctttggc ttctacctcg
900acggctggct tgctcacrac tacctgcatc acgcagtctt caagggctcg gtcaacacgc
960tcgtcaaggc gaacaacgcc atgggatacg ccctcggctt cctccagggc tacgacgtgg
1020cctggtggcg cgcgcgccat aacacgcacc acgtgtgcac caacgaggat ggttcggacc
1080cggacatcaa gacggcgccc ctgctcatct acgtgcgaga gaacccgtcc attgccaagc
1140ggctcaactt cttccagcgc tggcagcagt actactatgt gccgaccatg gccatcctcg
1200acctctactg gcgcctggag tccatcgcgt acgtggctgt gcgcctgcct aagatgtgga
1260tgcaggccgc cgctcttgcc gctcactacg cgct
1294141927DNAPavlova lutheri 14ctttgcgagc gcggcgcaga cgattgcggc
ccgtagtgat cgcggtgcgc attgctgtgt 60ttctagtttt gctgacgccc ggcccgataa
tgacaccttc tcccgtttga aatactaata 120agtaactata ttataatatt caaaggtggc
gactatggat ctccttttct aaagttcagc 180ggaattggga atcggagaaa tttcgagata
tgtcataatc acgtgctcta tctcgaatga 240accgcggccg gtgagcgatt actcgggaag
ccaattccta ttaacgagtc aggggggatc 300tttgaggtga gtcggccacg cagagagagc
aaggaatcat cctcatccgc cgttctcgag 360aaagagccaa gggtgccaac caccttccac
gtgagactac acaccgtagg ccgatgggca 420agggtggaga cggcggcgcg caggcggtga
gcgggaccga cgcgtctctc gctgaggtga 480gctccgtcga tagcaagagc gtgcacgtcg
tgctctacgg caagcgcgtg gatgtcacaa 540agttccagaa ggcacacccg ggcgggagca
aggtgttccg catcttccag gagcgcgacg 600cgacggagca gttcgagtct taccactcgc
ccaaggccat caagatgatg gagggcatgc 660tcaagaagtc ggaggatgcg cccgcttccg
tgcccctgcc ctcgcggtcc accatgggca 720cggagttcaa ggagatgatt gagcgccaca
agagggctgg tctctacgac ccttgcccgt 780tggacgagct gttcaagctc accatcgtcc
ttgcgcccat cttcgtgggc gcctatctcg 840tgcggagcgg cgtctcgccc ctcgcgggcg
cgctctccat gggctttggc ttctacctcg 900acggctggct tgctcacgac tacctgcatc
acgcagtctt caagggctcg gtcaacacgc 960tcgtcaaggc gaacaacgcc atgggatacg
ccctcggctt cctccagggc tacgacgtgg 1020cctggtggcg cgcgcgccat aacacgcacc
acgtgtgcac caacgaggat ggttcggacc 1080cggacatcaa gacggcgccc ctgctcatct
acgtgcgaga gaacccgtcc attgccaagc 1140ggctcaactt cttccagcgc tggcagcagt
actactatgt gccgaccatg gccatcctcg 1200acctctactg gcgcctggag tccatcgcgt
acgtggctgt gcgcctgcct aagatgtgga 1260tgcaggccgc cgctcttgcc gctcactacg
cgctcctgtg ctgggtcttc gcagcgcatc 1320tcaacctcat ccctctcatg atggttgcac
gcggcttcgc gacgggcatc gttgtctttg 1380caacccacta tggtgaggac atcctcgacc
gcgagcacgt cgagggcatg acgctcgtcg 1440agcagaccgc caagacctcc cgtaacatca
cgggcggctg gctagtgaac gtgctcacgg 1500gcttcatctc cctgcagacc gagcatcacc
tcttccccat gatgcccacc ggcaacctaa 1560tgactatcca gcccgaggta cgcgacttct
tcaagaagca tggcctcgag taccgcgagg 1620gcaacctctt ccagtgcgtg caccagaaca
tcaaggctct cgccttcgag cacctcctcc 1680actgagcgtc accactcaag cgtcctaagt
gcacaggtac tgtcttctga ccgatggccg 1740cgcggctccc tcggctggca gtggggccaa
cgagtggcct cgcgggatcg ggcacgatcg 1800ggcctccatg aaacttcagt gttcagagac
aagccgacaa cctccgcatc gtgagaaatc 1860ttttaaagca gtatgttcca tcacgccgct
tttgcagtca ataacattac ccaaaaaaaa 1920aaaaaaa
1927151269DNAPavlova lutheri
15atgggcaagg gtggagacgg cggcgcgcag gcggtgagcg ggaccgacgc gtctctcgct
60gaggtgagct ccgtcgatag caagagcgtg cacgtcgtgc tctacggcaa gcgcgtggat
120gtcacaaagt tccagaaggc acacccgggc gggagcaagg tgttccgcat cttccaggag
180cgcgacgcga cggagcagtt cgagtcttac cactcgccca aggccatcaa gatgatggag
240ggcatgctca agaagtcgga ggatgcgccc gcttccgtgc ccctgccctc gcggtccacc
300atgggcacgg agttcaagga gatgattgag cgccacaaga gggctggtct ctacgaccct
360tgcccgttgg acgagctgtt caagctcacc atcgtccttg cgcccatctt cgtgggcgcc
420tatctcgtgc ggagcggcgt ctcgcccctc gcgggcgcgc tctccatggg ctttggcttc
480tacctcgacg gctggcttgc tcacgactac ctgcatcacg cagtcttcaa gggctcggtc
540aacacgctcg tcaaggcgaa caacgccatg ggatacgccc tcggcttcct ccagggctac
600gacgtggcct ggtggcgcgc gcgccataac acgcaccacg tgtgcaccaa cgaggatggt
660tcggacccgg acatcaagac ggcgcccctg ctcatctacg tgcgagagaa cccgtccatt
720gccaagcggc tcaacttctt ccagcgctgg cagcagtact actatgtgcc gaccatggcc
780atcctcgacc tctactggcg cctggagtcc atcgcgtacg tggctgtgcg cctgcctaag
840atgtggatgc aggccgccgc tcttgccgct cactacgcgc tcctgtgctg ggtcttcgca
900gcgcatctca acctcatccc tctcatgatg gttgcacgcg gcttcgcgac gggcatcgtt
960gtctttgcaa cccactatgg tgaggacatc ctcgaccgcg agcacgtcga gggcatgacg
1020ctcgtcgagc agaccgccaa gacctcccgt aacatcacgg gcggctggct agtgaacgtg
1080ctcacgggct tcatctccct gcagaccgag catcacctct tccccatgat gcccaccggc
1140aacctaatga ctatccagcc cgaggtacgc gacttcttca agaagcatgg cctcgagtac
1200cgcgagggca acctcttcca gtgcgtgcac cagaacatca aggctctcgc cttcgagcac
1260ctcctccac
126916423PRTPavlova lutheri 16Met Gly Lys Gly Gly Asp Gly Gly Ala Gln Ala
Val Ser Gly Thr Asp1 5 10
15Ala Ser Leu Ala Glu Val Ser Ser Val Asp Ser Lys Ser Val His Val
20 25 30Val Leu Tyr Gly Lys Arg Val
Asp Val Thr Lys Phe Gln Lys Ala His 35 40
45Pro Gly Gly Ser Lys Val Phe Arg Ile Phe Gln Glu Arg Asp Ala
Thr 50 55 60Glu Gln Phe Glu Ser Tyr
His Ser Pro Lys Ala Ile Lys Met Met Glu65 70
75 80Gly Met Leu Lys Lys Ser Glu Asp Ala Pro Ala
Ser Val Pro Leu Pro 85 90
95Ser Arg Ser Thr Met Gly Thr Glu Phe Lys Glu Met Ile Glu Arg His
100 105 110Lys Arg Ala Gly Leu Tyr
Asp Pro Cys Pro Leu Asp Glu Leu Phe Lys 115 120
125Leu Thr Ile Val Leu Ala Pro Ile Phe Val Gly Ala Tyr Leu
Val Arg 130 135 140Ser Gly Val Ser Pro
Leu Ala Gly Ala Leu Ser Met Gly Phe Gly Phe145 150
155 160Tyr Leu Asp Gly Trp Leu Ala His Asp Tyr
Leu His His Ala Val Phe 165 170
175Lys Gly Ser Val Asn Thr Leu Val Lys Ala Asn Asn Ala Met Gly Tyr
180 185 190Ala Leu Gly Phe Leu
Gln Gly Tyr Asp Val Ala Trp Trp Arg Ala Arg 195
200 205His Asn Thr His His Val Cys Thr Asn Glu Asp Gly
Ser Asp Pro Asp 210 215 220Ile Lys Thr
Ala Pro Leu Leu Ile Tyr Val Arg Glu Asn Pro Ser Ile225
230 235 240Ala Lys Arg Leu Asn Phe Phe
Gln Arg Trp Gln Gln Tyr Tyr Tyr Val 245
250 255Pro Thr Met Ala Ile Leu Asp Leu Tyr Trp Arg Leu
Glu Ser Ile Ala 260 265 270Tyr
Val Ala Val Arg Leu Pro Lys Met Trp Met Gln Ala Ala Ala Leu 275
280 285Ala Ala His Tyr Ala Leu Leu Cys Trp
Val Phe Ala Ala His Leu Asn 290 295
300Leu Ile Pro Leu Met Met Val Ala Arg Gly Phe Ala Thr Gly Ile Val305
310 315 320Val Phe Ala Thr
His Tyr Gly Glu Asp Ile Leu Asp Arg Glu His Val 325
330 335Glu Gly Met Thr Leu Val Glu Gln Thr Ala
Lys Thr Ser Arg Asn Ile 340 345
350Thr Gly Gly Trp Leu Val Asn Val Leu Thr Gly Phe Ile Ser Leu Gln
355 360 365Thr Glu His His Leu Phe Pro
Met Met Pro Thr Gly Asn Leu Met Thr 370 375
380Ile Gln Pro Glu Val Arg Asp Phe Phe Lys Lys His Gly Leu Glu
Tyr385 390 395 400Arg Glu
Gly Asn Leu Phe Gln Cys Val His Gln Asn Ile Lys Ala Leu
405 410 415Ala Phe Glu His Leu Leu His
42017459PRTRhizopus stolonifer 17Met Ser Thr Leu Asp Arg Gln Ser
Ile Phe Thr Ile Lys Glu Leu Glu1 5 10
15Ser Ile Ser Gln Arg Ile His Asp Gly Asp Glu Glu Ala Met
Lys Phe 20 25 30Ile Ile Ile
Asp Lys Lys Val Tyr Asp Val Thr Glu Phe Ile Glu Asp 35
40 45His Pro Gly Gly Ala Gln Val Leu Leu Thr His
Val Gly Lys Asp Ala 50 55 60Ser Asp
Val Phe His Ala Met His Pro Glu Ser Ala Tyr Glu Val Leu65
70 75 80Asn Asn Tyr Phe Val Gly Asp
Val Gln Glu Thr Val Val Thr Glu Lys 85 90
95Ser Ser Ser Ala Gln Phe Ala Val Glu Met Arg Gln Leu
Arg Asp Gln 100 105 110Leu Lys
Lys Glu Gly Tyr Phe His Ser Ser Lys Leu Phe Tyr Ala Tyr 115
120 125Lys Val Leu Ser Thr Leu Ala Ile Cys Ile
Ala Gly Leu Ser Pro Leu 130 135 140Tyr
Ala Tyr Gly Arg Thr Ser Thr Leu Ala Val Val Ala Ser Ala Ile145
150 155 160Thr Val Gly Ile Phe Trp
Gln Gln Cys Gly Trp Leu Ala His Asp Phe 165
170 175Gly His His Gln Cys Phe Glu Asp Arg Thr Trp Asn
Asp Val Leu Val 180 185 190Val
Phe Leu Gly Asn Phe Cys Gln Gly Phe Ser Leu Ser Trp Trp Lys 195
200 205Asn Lys His Asn Thr His His Ala Ser
Thr Asn Val His Gly Gln Asp 210 215
220Pro Asp Ile Asp Thr Ala Pro Val Leu Leu Trp Asp Glu Tyr Ala Ser225
230 235 240Ala Ala Tyr Tyr
Ala Ser Leu Asp Gln Glu Pro Thr Met Val Ser Arg 245
250 255Phe Leu Ala Glu Gln Val Leu Pro His Gln
Thr Arg Tyr Phe Phe Phe 260 265
270Ile Leu Ala Phe Ala Arg Leu Ser Trp Ala Leu Gln Ser Leu Ser Tyr
275 280 285Ser Phe Lys Lys Glu Ser Ile
Asn Lys Ser Arg Gln Leu Asn Leu Phe 290 295
300Glu Arg Val Cys Ile Val Gly His Trp Ala Leu Ser Ala Phe Cys
Ile305 310 315 320Tyr Ser
Trp Cys Ser Asn Val Tyr His Met Val Leu Phe Phe Leu Val
325 330 335Ser Gln Ala Thr Thr Gly Tyr
Thr Leu Ala Leu Val Phe Ala Leu Asn 340 345
350His Asn Gly Met Pro Val Ile Thr Glu Glu Lys Ala Glu Ser
Met Glu 355 360 365Phe Phe Glu Ile
Gln Val Ile Thr Gly Arg Asp Val Thr Leu Ser Pro 370
375 380Leu Gly Asp Trp Phe Met Gly Gly Leu Asn Tyr Gln
Ile Glu His His385 390 395
400Val Phe Pro Asn Met Pro Arg His Asn Leu Pro Thr Val Lys Pro Met
405 410 415Val Lys Ser Leu Cys
Gln Lys Tyr Asp Ile Asn Tyr His Asp Thr Gly 420
425 430Phe Leu Lys Gly Thr Leu Glu Val Leu Gln Thr Leu
Asp Ile Thr Ser 435 440 445Lys Leu
Ser Leu Gln Leu Ser Lys Lys Ser Phe 450
4551830DNAArtificial Sequenceprimer PvDES5'Not-1 18gcggccgcac catgggcaag
ggtggagacg 301927DNAArtificial
Sequenceprimer PvDES3'Not-1 19gcggccgctc agtggaggag gtgctcg
272020DNAArtificial Sequenceprimer GSP PvDES-2
20gcatccacat cttaggcagg
20218801DNAArtificial Sequenceplasmid pY121 21ggccgcaggg cggatccccc
gggctgcagg aattcgatat caagcttatc gataccgtcg 60acctcgaggg ggggcccggt
acccaattcg ccctatagtg agtcgtatta cgcgcgctca 120ctggccgtcg ttttacaacg
tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc 180cttgcagcac atcccccttt
cgccagctgg cgtaatagcg aagaggcccg caccgatcgc 240ccttcccaac agttgcgcag
cctgaatggc gaatggcgcg acgcgccctg tagcggcgca 300ttaagcgcgg cgggtgtggt
ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta 360gcgcccgctc ctttcgcttt
cttcccttcc tttctcgcca cgttcgccgg ctttccccgt 420caagctctaa atcgggggct
ccctttaggg ttccgattta gtgctttacg gcacctcgac 480cccaaaaaac ttgattaggg
tgatggttca cgtagtgggc catcgccctg atagacggtt 540tttcgccctt tgacgttgga
gtccacgttc tttaatagtg gactcttgtt ccaaactgga 600acaacactca accctatctc
ggtctattct tttgatttat aagggatttt gccgatttcg 660gcctattggt taaaaaatga
gctgatttaa caaaaattta acgcgaattt taacaaaata 720ttaacgttta caatttcctg
atgcggtatt ttctccttac gcatctgtgc ggtatttcac 780accgcatatc gacggtcgag
gagaacttct agtatatcca catacctaat attattgcct 840tattaaaaat ggaatcccaa
caattacatc aaaatccaca ttctcttcaa aatcaattgt 900cctgtacttc cttgttcatg
tgtgttcaaa aacgttatat ttataggata attatactct 960atttctcaac aagtaattgg
ttgtttggcc gagcggtcta aggcgcctga ttcaagaaat 1020atcttgaccg cagttaactg
tgggaatact caggtatcgt aagatgcaag agttcgaatc 1080tcttagcaac cattattttt
ttcctcaaca taacgagaac acacaggggc gctatcgcac 1140agaatcaaat tcgatgactg
gaaatttttt gttaatttca gaggtcgcct gacgcatata 1200cctttttcaa ctgaaaaatt
gggagaaaaa ggaaaggtga gaggccggaa ccggcttttc 1260atatagaata gagaagcgtt
catgactaaa tgcttgcatc acaatacttg aagttgacaa 1320tattatttaa ggacctattg
ttttttccaa taggtggtta gcaatcgtct tactttctaa 1380cttttcttac cttttacatt
tcagcaatat atatatatat ttcaaggata taccattcta 1440atgtctgccc ctatgtctgc
ccctaagaag atcgtcgttt tgccaggtga ccacgttggt 1500caagaaatca cagccgaagc
cattaaggtt cttaaagcta tttctgatgt tcgttccaat 1560gtcaagttcg atttcgaaaa
tcatttaatt ggtggtgctg ctatcgatgc tacaggtgtc 1620ccacttccag atgaggcgct
ggaagcctcc aagaaggttg atgccgtttt gttaggtgct 1680gtggctggtc ctaaatgggg
taccggtagt gttagacctg aacaaggttt actaaaaatc 1740cgtaaagaac ttcaattgta
cgccaactta agaccatgta actttgcatc cgactctctt 1800ttagacttat ctccaatcaa
gccacaattt gctaaaggta ctgacttcgt tgttgtcaga 1860gaattagtgg gaggtattta
ctttggtaag agaaaggaag acgatggtga tggtgtcgct 1920tgggatagtg aacaatacac
cgttccagaa gtgcaaagaa tcacaagaat ggccgctttc 1980atggccctac aacatgagcc
accattgcct atttggtcct tggataaagc taatcttttg 2040gcctcttcaa gattatggag
aaaaactgtg gaggaaacca tcaagaacga attccctaca 2100ttgaaggttc aacatcaatt
gattgattct gccgccatga tcctagttaa gaacccaacc 2160cacctaaatg gtattataat
caccagcaac atgtttggtg atatcatctc cgatgaagcc 2220tccgttatcc caggttcctt
gggtttgttg ccatctgcgt ccttggcctc tttgccagac 2280aagaacaccg catttggttt
gtacgaacca tgccacggtt ctgctccaga tttgccaaag 2340aataaggttg accctatcgc
cactatcttg tctgctgcaa tgatgttgaa attgtcattg 2400aacttgcctg aagaaggtaa
ggccattgaa gatgcagtta aaaaggtttt ggatgcaggt 2460atcagaactg gtgatttagg
tggttccaac agtaccaccg aagtcggtga tgctgtcgcc 2520gaagaagtta agaaaatcct
tgcttaaaaa gattctcttt ttttatgata tttgtacata 2580aactttataa atgaaattca
taatagaaac gacacgaaat tacaaaatgg aatatgttca 2640tagggtagac gaaactatat
acgcaatcta catacattta tcaagaagga gaaaaaggag 2700gatagtaaag gaatacaggt
aagcaaattg atactaatgg ctcaacgtga taaggaaaaa 2760gaattgcact ttaacattaa
tattgacaag gaggagggca ccacacaaaa agttaggtgt 2820aacagaaaat catgaaacta
cgattcctaa tttgatattg gaggattttc tctaaaaaaa 2880aaaaaataca acaaataaaa
aacactcaat gacctgacca tttgatggag tttaagtcaa 2940taccttcttg aagcatttcc
cataatggtg aaagttccct caagaatttt actctgtcag 3000aaacggcctt acgacgtagt
cgatatggtg cactctcagt acaatctgct ctgatgccgc 3060atagttaagc cagccccgac
acccgccaac acccgctgac gcgccctgac gggcttgtct 3120gctcccggca tccgcttaca
gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 3180gttttcaccg tcatcaccga
aacgcgcgag acgaaagggc ctcgtgatac gcctattttt 3240ataggttaat gtcatgataa
taatggtttc ttagtatgat ccaatatcaa aggaaatgat 3300agcattgaag gatgagacta
atccaattga ggagtggcag catatagaac agctaaaggg 3360tagtgctgaa ggaagcatac
gataccccgc atggaatggg ataatatcac aggaggtact 3420agactacctt tcatcctaca
taaatagacg catataagta cgcatttaag cataaacacg 3480cactatgccg ttcttctcat
gtatatatat atacaggcaa cacgcagata taggtgcgac 3540gtgaacagtg agctgtatgt
gcgcagctcg cgttgcattt tcggaagcgc tcgttttcgg 3600aaacgctttg aagttcctat
tccgaagttc ctattctcta gaaagtatag gaacttcaga 3660gcgcttttga aaaccaaaag
cgctctgaag acgcactttc aaaaaaccaa aaacgcaccg 3720gactgtaacg agctactaaa
atattgcgaa taccgcttcc acaaacattg ctcaaaagta 3780tctctttgct atatatctct
gtgctatatc cctatataac ctacccatcc acctttcgct 3840ccttgaactt gcatctaaac
tcgacctcta cattttttat gtttatctct agtattactc 3900tttagacaaa aaaattgtag
taagaactat tcatagagtg aatcgaaaac aatacgaaaa 3960tgtaaacatt tcctatacgt
agtatataga gacaaaatag aagaaaccgt tcataatttt 4020ctgaccaatg aagaatcatc
aacgctatca ctttctgttc acaaagtatg cgcaatccac 4080atcggtatag aatataatcg
gggatgcctt tatcttgaaa aaatgcaccc gcagcttcgc 4140tagtaatcag taaacgcggg
aagtggagtc aggctttttt tatggaagag aaaatagaca 4200ccaaagtagc cttcttctaa
ccttaacgga cctacagtgc aaaaagttat caagagactg 4260cattatagag cgcacaaagg
agaaaaaaag taatctaaga tgctttgtta gaaaaatagc 4320gctctcggga tgcatttttg
tagaacaaaa aagaagtata gattctttgt tggtaaaata 4380gcgctctcgc gttgcatttc
tgttctgtaa aaatgcagct cagattcttt gtttgaaaaa 4440ttagcgctct cgcgttgcat
ttttgtttta caaaaatgaa gcacagattc ttcgttggta 4500aaatagcgct ttcgcgttgc
atttctgttc tgtaaaaatg cagctcagat tctttgtttg 4560aaaaattagc gctctcgcgt
tgcatttttg ttctacaaaa tgaagcacag atgcttcgtt 4620caggtggcac ttttcgggga
aatgtgcgcg gaacccctat ttgtttattt ttctaaatac 4680attcaaatat gtatccgctc
atgagacaat aaccctgata aatgcttcaa taatattgaa 4740aaaggaagag tatgagtatt
caacatttcc gtgtcgccct tattcccttt tttgcggcat 4800tttgccttcc tgtttttgct
cacccagaaa cgctggtgaa agtaaaagat gctgaagatc 4860agttgggtgc acgagtgggt
tacatcgaac tggatctcaa cagcggtaag atccttgaga 4920gttttcgccc cgaagaacgt
tttccaatga tgagcacttt taaagttctg ctatgtggcg 4980cggtattatc ccgtattgac
gccgggcaag agcaactcgg tcgccgcata cactattctc 5040agaatgactt ggttgagtac
tcaccagtca cagaaaagca tcttacggat ggcatgacag 5100taagagaatt atgcagtgct
gccataacca tgagtgataa cactgcggcc aacttacttc 5160tgacaacgat cggaggaccg
aaggagctaa ccgctttttt gcacaacatg ggggatcatg 5220taactcgcct tgatcgttgg
gaaccggagc tgaatgaagc cataccaaac gacgagcgtg 5280acaccacgat gcctgtagca
atggcaacaa cgttgcgcaa actattaact ggcgaactac 5340ttactctagc ttcccggcaa
caattaatag actggatgga ggcggataaa gttgcaggac 5400cacttctgcg ctcggccctt
ccggctggct ggtttattgc tgataaatct ggagccggtg 5460agcgtgggtc tcgcggtatc
attgcagcac tggggccaga tggtaagccc tcccgtatcg 5520tagttatcta cacgacgggg
agtcaggcaa ctatggatga acgaaataga cagatcgctg 5580agataggtgc ctcactgatt
aagcattggt aactgtcaga ccaagtttac tcatatatac 5640tttagattga tttaaaactt
catttttaat ttaaaaggat ctaggtgaag atcctttttg 5700ataatctcat gaccaaaatc
ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 5760tagaaaagat caaaggatct
tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 5820aaacaaaaaa accaccgcta
ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 5880tttttccgaa ggtaactggc
ttcagcagag cgcagatacc aaatactgtc cttctagtgt 5940agccgtagtt aggccaccac
ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 6000taatcctgtt accagtggct
gctgccagtg gcgataagtc gtgtcttacc gggttggact 6060caagacgata gttaccggat
aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 6120agcccagctt ggagcgaacg
acctacaccg aactgagata cctacagcgt gagctatgag 6180aaagcgccac gcttcccgaa
gggagaaagg cggacaggta tccggtaagc ggcagggtcg 6240gaacaggaga gcgcacgagg
gagcttccag ggggaaacgc ctggtatctt tatagtcctg 6300tcgggtttcg ccacctctga
cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 6360gcctatggaa aaacgccagc
aacgcggcct ttttacggtt cctggccttt tgctggcctt 6420ttgctcacat gttctttcct
gcgttatccc ctgattctgt ggataaccgt attaccgcct 6480ttgagtgagc tgataccgct
cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg 6540aggaagcgga agagcgccca
atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt 6600aatgcagctg gcacgacagg
tttcccgact ggaaagcggg cagtgagcgc aacgcaatta 6660atgtgagtta cctcactcat
taggcacccc aggctttaca ctttatgctt ccggctccta 6720tgttgtgtgg aattgtgagc
ggataacaat ttcacacagg aaacagctat gaccatgatt 6780acgccaagcg cgcaattaac
cctcactaaa gggaacaaaa gctggagctc caccgcggga 6840tttcgaaact aagttcttgg
tgttttaaaa ctaaaaaaaa gactaactat aaaagtagaa 6900tttaagaagt ttaagaaata
gatttacaga attacaatca atacctaccg tctttatata 6960cttattagtc aagtagggga
ataatttcag ggaactggtt tcaacctttt ttttcagctt 7020tttccaaatc agagagagca
gaaggtaata gaaggtgtaa gaaaatgaga tagatacatg 7080cgtgggtcaa ttgccttgtg
tcatcattta ctccaggcag gttgcatcac tccattgagg 7140ttgtgcccgt tttttgcctg
tttgtgcccc tgttctctgt agttgcgcta agagaatgga 7200cctatgaact gatggttggt
gaagaaaaca atattttggt gctgggattc tttttttttc 7260tggatgccag cttaaaaagc
gggctccatt atatttagtg gatgccagga ataaactgtt 7320cacccagaca cctacgatgt
tatatattct gtgtaacccg ccccctattt tgggcatgta 7380cgggttacag cagaattaaa
aggctaattt tttgactaaa taaagttagg aaaatcacta 7440ctattaatta tttacgtatt
ctttgaaatg gcagtattga taatgataaa ctcgaaatca 7500ctagtggatc cgcccagcgg
ccgcaccatg ggcaagggtg gagacggcgg cgcgcaggcg 7560gtgagcggga ccgacgcgtc
tctcgctgag gtgagctccg tcgatagcaa gagcgtgcac 7620gtcgtgctct acggcaagcg
cgtggatgtc acaaagttcc agaaggcaca cccgggcggg 7680agcaaggtgt tccgcatctt
ccaggagcgc gacgcgacgg agcagttcga gtcttaccac 7740tcgcccaagg ccatcaagat
gatggagggc atgctcaaga agtcggagga tgcgcccgct 7800tccgtgcccc tgccctcgcg
gtccaccatg ggcacggagt tcaaggagat gattgagcgc 7860cacaagaggg ctggtctcta
cgacccttgc ccgttggacg agctgttcaa gctcaccatc 7920gtccttgcgc ccatcttcgt
gggcgcctat ctcgtgcgga gcggcgtctc gcccctcgcg 7980ggcgcgctct ccatgggctt
tggcttctac ctcgacggct ggcttgctca cgactacctg 8040catcacgcag tcttcaaggg
ctcggtcaac acgctcgtca aggcgaacaa cgccatggga 8100tacgccctcg gcttcctcca
gggctacgac gtggcctggt ggcgcgcgcg ccataacacg 8160caccacgtgt gcaccaacga
ggatggttcg gacccggaca tcaagacggc gcccctgctc 8220atctacgtgc gagagaaccc
gtccattgcc aagcggctca acttcttcca gcgctggcag 8280cagtactact atgtgccgac
catggccatc ctcgacctct actggcgcct ggagtccatc 8340gcgtacgtgg ctgtgcgcct
gcctaagatg tggatgcagg ccgccgctct tgccgctcac 8400tacgcgctcc tgtgctgggt
cttcgcagcg catctcaacc tcatccctct catgatggtt 8460gcacgcggct tcgcgacggg
catcgttgtc tttgcaaccc actatggtga ggacatcctc 8520gaccgcgagc acgtcgaggg
catgacgctc gtcgagcaga ccgccaagac ctcccgtaac 8580atcacgggcg gctggctagt
gaacgtgctc acgggcttca tctccctgca gaccgagcat 8640cacctcttcc ccatgatgcc
caccggcaac ctaatgacta tccagcccga ggtacgcgac 8700ttcttcaaga agcatggcct
cgagtaccgc gagggcaacc tcttccagtg cgtgcaccag 8760aacatcaagg ctctcgcctt
cgagcacctc ctccactgag c 8801224993DNAArtificial
Sequenceplasmid pKR123r 22ctagacctgc aggatataat gagccgtaaa caaagatgat
taagtagtaa ttaatacgta 60ctagtaaaag tggcaaaaga taacgagaaa gaaccaattt
ctttgcattc ggccttagcg 120gaaggcatat ataagctttg attattttat ttagtgtaat
gatttcgtac aaccaaagca 180tttatttagt actctcacac ttgtgtcgcg gccgcttggg
gggctatgga agactttctt 240agttagttgt gtgaataagc aatgttggga gaatcgggac
tacttatagg ataggaataa 300aacagaaaag tattaagtgc taatgaaata tttagactga
taattaaaat cttcacgtat 360gtccacttga tataaaaacg tcaggaataa aggaagtaca
gtagaattta aaggtactct 420ttttatatat acccgtgttc tctttttggc tagctagttg
cataaaaaat aatctatatt 480tttatcatta ttttaaatat cttatgagat ggtaaatatt
tatcataatt ttttttacta 540ttatttatta tttgtgtgtg taatacatat agaagttaat
tacaaatttt atttactttt 600tcattatttt gatatgattc accattaatt tagtgttatt
atttataata gttcatttta 660atctttttgt atatattatg cgtgcagtac ttttttccta
catataacta ctattacatt 720ttatttatat aatattttta ttaatgaatt ttcgtgataa
tatgtaatat tgttcattat 780tatttcagat tttttaaaaa tatttgtgtt attatttatg
aaatatgtaa tttttttagt 840atttgatttt atgatgataa agtgttctaa attcaaaaga
agggggaaag cgtaaacatt 900aaaaaacgtc atcaaacaaa aacaaaatct tgttaataaa
gataaaactg tttgttttga 960tcactgttat ttcgtaatat aaaaacatta tttatattta
tattgttgac aaccaaattt 1020gcctatcaaa tctaaccaat ataatgcatg cgtggcaggt
aatgtactac catgaactta 1080agtcatgaca taataaaccg tgaatctgac caatgcatgt
acctanctaa attgtatttg 1140tgacacgaag caaatgattc aattcacaat ggagatggga
aacaaataat gaagaaccca 1200gaactaagaa agcttttctg aaaaataaaa taaaggcaat
gtcaaaagta tactgcatca 1260tcagtccaga aagcacatga tattttttta tcagtatcaa
tgcagctagt tttattttac 1320aatatcgata tagctagttt aaatatattg cagctagatt
tataaatatt tgtgttatta 1380tttatcattt gtgtaatcct gtttttagta ttttagttta
tatatgatga taatgtattc 1440caaatttaaa agaagggaaa taaatttaaa caagaaaaaa
agtcatcaaa caaaaaacaa 1500atgaaagggt ggaaagatgt taccatgtaa tgtgaatgtt
acagtatttc ttttattata 1560gagttaacaa attaactaat atgattttgt taataatgat
aaaatatttt ttttattatt 1620atttcataat ataaaaatag tttacttaat ataaaaaaaa
ttctatcgtt cacaacaaag 1680ttggccacct aatttaacca tgcatgtacc catggaccat
attaggtaac catcaaacct 1740gatgaagaga taaagagatg aagacttaag tcataacaca
aaaccataaa aaacaaaaat 1800acaatcaacc gtcaatctga ccaatgcatg aaaaagctgc
aatagtgagt ggcgacacaa 1860agcacatgat tttcttacaa cggagataaa accaaaaaaa
tatttcatga acaacctaga 1920acaaataaag cttttatata ataaatatat aaataaataa
aggctatgga ataatatact 1980tcaatatatt tggattaaat aaattgttgg cggggttgat
atatttatac acacctaaag 2040tcacttcaat ctcattttca cttaactttt attttttttt
tctttttatt tatcataaag 2100agaatattga taatatactt tttaacatat ttttatgaca
ttttttattg gtgaaaactt 2160attaaaaatc ataaattttg taagttagat ttatttaaag
agttcctctt cttattttaa 2220attttttaat aaatttttaa ataactaaaa tttgtgttaa
aaatgttaaa aaatgtgtta 2280ttaacccttc tcttcgagga cgtacgtcta gagtcgacct
gcaggcatgc aagcttggcg 2340taatcatggt catagctgtt tcctgtgtga aattgttatc
cgctcacaat tccacacaac 2400atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
aatgagtgag ctaactcaca 2460ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
acctgtcgtg ccagctgcat 2520taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
ttgggcgctc ttccgcttcc 2580tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
gagcggtatc agctcactca 2640aaggcggtaa tacggttatc cacagaatca ggggataacg
caggaaagaa catgtgagca 2700aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
tgctggcgtt tttccatagg 2760ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
gtcagaggtg gcgaaacccg 2820acaggactat aaagatacca ggcgtttccc cctggaagct
ccctcgtgcg ctctcctgtt 2880ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
cttcgggaag cgtggcgctt 2940tctcatagct cacgctgtag gtatctcagt tcggtgtagg
tcgttcgctc caagctgggc 3000tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
tatccggtaa ctatcgtctt 3060gagtccaacc cggtaagaca cgacttatcg ccactggcag
cagccactgg taacaggatt 3120agcagagcga ggtatgtagg cggtgctaca gagttcttga
agtggtggcc taactacggc 3180tacactagaa ggacagtatt tggtatctgc gctctgctga
agccagttac cttcggaaaa 3240agagttggta gctcttgatc cggcaaacaa accaccgctg
gtagcggtgg tttttttgtt 3300tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
aagatccttt gatcttttct 3360acggggtctg acgctcagtg gaacgaaaac tcacgttaag
ggattttggt catgagatta 3420tcaaaaagga tcttcaccta gatcctttta aattaaaaat
gaagttttaa atcaatctaa 3480agtatatatg agtaaacttg gtctgacagt taccaatgct
taatcagtga ggcacctatc 3540tcagcgatct gtctatttcg ttcatccata gttgcctgac
tccccgtcgt gtagataact 3600acgatacggg agggcttacc atctggcccc agtgctgcaa
tgataccgcg agacccacgc 3660tcaccggctc cagatttatc agcaataaac cagccagccg
gaagggccga gcgcagaagt 3720ggtcctgcaa ctttatccgc ctccatccag tctattaatt
gttgccggga agctagagta 3780agtagttcgc cagttaatag tttgcgcaac gttgttgcca
ttgctacagg catcgtggtg 3840tcacgctcgt cgtttggtat ggcttcattc agctccggtt
cccaacgatc aaggcgagtt 3900acatgatccc ccatgttgtg caaaaaagcg gttagctcct
tcggtcctcc gatcgttgtc 3960agaagtaagt tggccgcagt gttatcactc atggttatgg
cagcactgca taattctctt 4020actgtcatgc catccgtaag atgcttttct gtgactggtg
agtactcaac caagtcattc 4080tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
cgtcaatacg ggataatacc 4140gcgccacata gcagaacttt aaaagtgctc atcattggaa
aacgttcttc ggggcgaaaa 4200ctctcaagga tcttaccgct gttgagatcc agttcgatgt
aacccactcg tgcacccaac 4260tgatcttcag catcttttac tttcaccagc gtttctgggt
gagcaaaaac aggaaggcaa 4320aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
gaatactcat actcttcctt 4380tttcaatatt attgaagcat ttatcagggt tattgtctca
tgagcggata catatttgaa 4440tgtatttaga aaaataaaca aataggggtt ccgcgcacat
ttccccgaaa agtgccacct 4500gacgtctaag aaaccattat tatcatgaca ttaacctata
aaaataggcg tatcacgagg 4560ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc
tctgacacat gcagctcccg 4620gagacggtca cagcttgtct gtaagcggat gccgggagca
gacaagcccg tcagggcgcg 4680tcagcgggtg ttggcgggtg tcggggctgg cttaactatg
cggcatcaga gcagattgta 4740ctgagagtgc accatatgcg gtgtgaaata ccgcacagat
gcgtaaggag aaaataccgc 4800atcaggcgcc attcgccatt caggctgcgc aactgttggg
aagggcgatc ggtgcgggcc 4860tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg
caaggcgatt aagttgggta 4920acgccagggt tttcccagtc acgacgttgt aaaacgacgg
ccagtgaatt cgagctcggt 4980acccggggat cct
4993236276DNAArtificial Sequenceplasmid pKR900
23ggccgcgaca caagtgtgag agtactaaat aaatgctttg gttgtacgaa atcattacac
60taaataaaat aatcaaagct tatatatgcc ttccgctaag gccgaatgca aagaaattgg
120ttctttctcg ttatcttttg ccacttttac tagtacgtat taattactac ttaatcatct
180ttgtttacgg ctcattatat cctgcaggtc tagaggatcc ccgggtaccg agctcgaatt
240cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
300gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
360gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg tattttctcc
420ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg
480atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
540cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
600gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
660tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
720ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
780cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
840gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
900ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
960tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
1020aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
1080ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
1140agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
1200gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
1260gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
1320gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
1380tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
1440ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
1500cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
1560gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
1620cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
1680tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
1740aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
1800aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
1860gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
1920cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
1980ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
2040accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
2100tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
2160cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
2220gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
2280ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
2340cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
2400tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
2460ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
2520ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
2580ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
2640gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
2700acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
2760ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
2820tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagcttgcat
2880gcctgcaggt cgactctaga cgtacgtcct cgaagagaag ggttaataac acatttttta
2940acatttttaa cacaaatttt agttatttaa aaatttatta aaaaatttaa aataagaaga
3000ggaactcttt aaataaatct aacttacaaa atttatgatt tttaataagt tttcaccaat
3060aaaaaatgtc ataaaaatat gttaaaaagt atattatcaa tattctcttt atgataaata
3120aaaagaaaaa aaaaataaaa gttaagtgaa aatgagattg aagtgacttt aggtgtgtat
3180aaatatatca accccgccaa caatttattt aatccaaata tattgaagta tattattcca
3240tagcctttat ttatttatat atttattata taaaagcttt atttgttcta ggttgttcat
3300gaaatatttt tttggtttta tctccgttgt aagaaaatca tgtgctttgt gtcgccactc
3360actattgcag ctttttcatg cattggtcag attgacggtt gattgtattt ttgtttttta
3420tggttttgtg ttatgactta agtcttcatc tctttatctc ttcatcaggt ttgatggtta
3480cctaatatgg tccatgggta catgcatggt taaattaggt ggccaacttt gttgtgaacg
3540atagaatttt ttttatatta agtaaactat ttttatatta tgaaataata ataaaaaaaa
3600tattttatca ttattaacaa aatcatatta gttaatttgt taactctata ataaaagaaa
3660tactgtaaca ttcacattac atggtaacat ctttccaccc tttcatttgt tttttgtttg
3720atgacttttt ttcttgttta aatttatttc ccttctttta aatttggaat acattatcat
3780catatataaa ctaaaatact aaaaacagga ttacacaaat gataaataat aacacaaata
3840tttataaatc tagctgcaat atatttaaac tagctatatc gatattgtaa aataaaacta
3900gctgcattga tactgataaa aaaatatcat gtgctttctg gactgatgat gcagtatact
3960tttgacattg cctttatttt atttttcaga aaagctttct tagttctggg ttcttcatta
4020tttgtttccc atctccattg tgaattgaat catttgcttc gtgtcacaaa tacaatttag
4080ntaggtacat gcattggtca gattcacggt ttattatgtc atgacttaag ttcatggtag
4140tacattacct gccacgcatg cattatattg gttagatttg ataggcaaat ttggttgtca
4200acaatataaa tataaataat gtttttatat tacgaaataa cagtgatcaa aacaaacagt
4260tttatcttta ttaacaagat tttgtttttg tttgatgacg ttttttaatg tttacgcttt
4320cccccttctt ttgaatttag aacactttat catcataaaa tcaaatacta aaaaaattac
4380atatttcata aataataaca caaatatttt taaaaaatct gaaataataa tgaacaatat
4440tacatattat cacgaaaatt cattaataaa aatattatat aaataaaatg taatagtagt
4500tatatgtagg aaaaaagtac tgcacgcata atatatacaa aaagattaaa atgaactatt
4560ataaataata acactaaatt aatggtgaat catatcaaaa taatgaaaaa gtaaataaaa
4620tttgtaatta acttctatat gtattacaca cacaaataat aaataatagt aaaaaaaatt
4680atgataaata tttaccatct cataagatat ttaaaataat gataaaaata tagattattt
4740tttatgcaac tagctagcca aaaagagaac acgggtatat ataaaaagag tacctttaaa
4800ttctactgta cttcctttat tcctgacgtt tttatatcaa gtggacatac gtgaagattt
4860taattatcag tctaaatatt tcattagcac ttaatacttt tctgttttat tcctatccta
4920taagtagtcc cgattctccc aacattgctt attcacacaa ctaactaaga aagtcttcca
4980tagcccccca agcggccgca ccatgggcaa gggtggagac ggcggcgcgc aggcggtgag
5040cgggaccgac gcgtctctcg ctgaggtgag ctccgtcgat agcaagagcg tgcacgtcgt
5100gctctacggc aagcgcgtgg atgtcacaaa gttccagaag gcacacccgg gcgggagcaa
5160ggtgttccgc atcttccagg agcgcgacgc gacggagcag ttcgagtctt accactcgcc
5220caaggccatc aagatgatgg agggcatgct caagaagtcg gaggatgcgc ccgcttccgt
5280gcccctgccc tcgcggtcca ccatgggcac ggagttcaag gagatgattg agcgccacaa
5340gagggctggt ctctacgacc cttgcccgtt ggacgagctg ttcaagctca ccatcgtcct
5400tgcgcccatc ttcgtgggcg cctatctcgt gcggagcggc gtctcgcccc tcgcgggcgc
5460gctctccatg ggctttggct tctacctcga cggctggctt gctcacgact acctgcatca
5520cgcagtcttc aagggctcgg tcaacacgct cgtcaaggcg aacaacgcca tgggatacgc
5580cctcggcttc ctccagggct acgacgtggc ctggtggcgc gcgcgccata acacgcacca
5640cgtgtgcacc aacgaggatg gttcggaccc ggacatcaag acggcgcccc tgctcatcta
5700cgtgcgagag aacccgtcca ttgccaagcg gctcaacttc ttccagcgct ggcagcagta
5760ctactatgtg ccgaccatgg ccatcctcga cctctactgg cgcctggagt ccatcgcgta
5820cgtggctgtg cgcctgccta agatgtggat gcaggccgcc gctcttgccg ctcactacgc
5880gctcctgtgc tgggtcttcg cagcgcatct caacctcatc cctctcatga tggttgcacg
5940cggcttcgcg acgggcatcg ttgtctttgc aacccactat ggtgaggaca tcctcgaccg
6000cgagcacgtc gagggcatga cgctcgtcga gcagaccgcc aagacctccc gtaacatcac
6060gggcggctgg ctagtgaacg tgctcacggg cttcatctcc ctgcagaccg agcatcacct
6120cttccccatg atgcccaccg gcaacctaat gactatccag cccgaggtac gcgacttctt
6180caagaagcat ggcctcgagt accgcgaggg caacctcttc cagtgcgtgc accagaacat
6240caaggctctc gccttcgagc acctcctcca ctgagc
6276245303DNAArtificial Sequenceprimer pKR325 24agcttggatc tcctgcagga
tctggccggc cggatctcgt acggatccgt cgacggcgcg 60cccgatcatc cggatatagt
tcctcctttc agcaaaaaac ccctcaagac ccgtttagag 120gccccaaggg gttatgctag
ttattgctca gcggtggcag cagccaactc agcttccttt 180cgggctttgt tagcagccgg
atcgatccaa gctgtacctc actattcctt tgccctcgga 240cgagtgctgg ggcgtcggtt
tccactatcg gcgagtactt ctacacagcc atcggtccag 300acggccgcgc ttctgcgggc
gatttgtgta cgcccgacag tcccggctcc ggatcggacg 360attgcgtcgc atcgaccctg
cgcccaagct gcatcatcga aattgccgtc aaccaagctc 420tgatagagtt ggtcaagacc
aatgcggagc atatacgccc ggagccgcgg cgatcctgca 480agctccggat gcctccgctc
gaagtagcgc gtctgctgct ccatacaagc caaccacggc 540ctccagaaga agatgttggc
gacctcgtat tgggaatccc cgaacatcgc ctcgctccag 600tcaatgaccg ctgttatgcg
gccattgtcc gtcaggacat tgttggagcc gaaatccgcg 660tgcacgaggt gccggacttc
ggggcagtcc tcggcccaaa gcatcagctc atcgagagcc 720tgcgcgacgg acgcactgac
ggtgtcgtcc atcacagttt gccagtgata cacatgggga 780tcagcaatcg cgcatatgaa
atcacgccat gtagtgtatt gaccgattcc ttgcggtccg 840aatgggccga acccgctcgt
ctggctaaga tcggccgcag cgatcgcatc catagcctcc 900gcgaccggct gcagaacagc
gggcagttcg gtttcaggca ggtcttgcaa cgtgacaccc 960tgtgcacggc gggagatgca
ataggtcagg ctctcgctga attccccaat gtcaagcact 1020tccggaatcg ggagcgcggc
cgatgcaaag tgccgataaa cataacgatc tttgtagaaa 1080ccatcggcgc agctatttac
ccgcaggaca tatccacgcc ctcctacatc gaagctgaaa 1140gcacgagatt cttcgccctc
cgagagctgc atcaggtcgg agacgctgtc gaacttttcg 1200atcagaaact tctcgacaga
cgtcgcggtg agttcaggct tttccatggg tatatctcct 1260tcttaaagtt aaacaaaatt
atttctagag ggaaaccgtt gtggtctccc tatagtgagt 1320cgtattaatt tcgcgggatc
gagatcgatc caattccaat cccacaaaaa tctgagctta 1380acagcacagt tgctcctctc
agagcagaat cgggtattca acaccctcat atcaactact 1440acgttgtgta taacggtcca
catgccggta tatacgatga ctggggttgt acaaaggcgg 1500caacaaacgg cgttcccgga
gttgcacaca agaaatttgc cactattaca gaggcaagag 1560cagcagctga cgcgtacaca
acaagtcagc aaacagacag gttgaacttc atccccaaag 1620gagaagctca actcaagccc
aagagctttg ctaaggccct aacaagccca ccaaagcaaa 1680aagcccactg gctcacgcta
ggaaccaaaa ggcccagcag tgatccagcc ccaaaagaga 1740tctcctttgc cccggagatt
acaatggacg atttcctcta tctttacgat ctaggaagga 1800agttcgaagg tgaaggtgac
gacactatgt tcaccactga taatgagaag gttagcctct 1860tcaatttcag aaagaatgct
gacccacaga tggttagaga ggcctacgca gcaggtctca 1920tcaagacgat ctacccgagt
aacaatctcc aggagatcaa ataccttccc aagaaggtta 1980aagatgcagt caaaagattc
aggactaatt gcatcaagaa cacagagaaa gacatatttc 2040tcaagatcag aagtactatt
ccagtatgga cgattcaagg cttgcttcat aaaccaaggc 2100aagtaataga gattggagtc
tctaaaaagg tagttcctac tgaatctaag gccatgcatg 2160gagtctaaga ttcaaatcga
ggatctaaca gaactcgccg tgaagactgg cgaacagttc 2220atacagagtc ttttacgact
caatgacaag aagaaaatct tcgtcaacat ggtggagcac 2280gacactctgg tctactccaa
aaatgtcaaa gatacagtct cagaagacca aagggctatt 2340gagacttttc aacaaaggat
aatttcggga aacctcctcg gattccattg cccagctatc 2400tgtcacttca tcgaaaggac
agtagaaaag gaaggtggct cctacaaatg ccatcattgc 2460gataaaggaa aggctatcat
tcaagatgcc tctgccgaca gtggtcccaa agatggaccc 2520ccacccacga ggagcatcgt
ggaaaaagaa gacgttccaa ccacgtcttc aaagcaagtg 2580gattgatgtg acatctccac
tgacgtaagg gatgacgcac aatcccacta tccttcgcaa 2640gacccttcct ctatataagg
aagttcattt catttggaga ggacacgctc gagctcattt 2700ctctattact tcagccataa
caaaagaact cttttctctt cttattaaac catgaaaaag 2760cctgaactca ccgcgacgtc
tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc 2820gacctgatgc agctctcgga
gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg 2880cgtggatatg tcctgcgggt
aaatagctgc gccgatggtt tctacaaaga tcgttatgtt 2940tatcggcact ttgcatcggc
cgcgctcccg attccggaag tgcttgacat tggggaattc 3000agcgagagcc tgacctattg
catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg 3060cctgaaaccg aactgcccgc
tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct 3120gcggccgatc ttagccagac
gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa 3180tacactacat ggcgtgattt
catatgcgcg attgctgatc cccatgtgta tcactggcaa 3240actgtgatgg acgacaccgt
cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt 3300tgggccgagg actgccccga
agtccggcac ctcgtgcacg cggatttcgg ctccaacaat 3360gtcctgacgg acaatggccg
cataacagcg gtcattgact ggagcgaggc gatgttcggg 3420gattcccaat acgaggtcgc
caacatcttc ttctggaggc cgtggttggc ttgtatggag 3480cagcagacgc gctacttcga
gcggaggcat ccggagcttg caggatcgcc gcggctccgg 3540gcgtatatgc tccgcattgg
tcttgaccaa ctctatcaga gcttggttga cggcaatttc 3600gatgatgcag cttgggcgca
gggtcgatgc gacgcaatcg tccgatccgg agccgggact 3660gtcgggcgta cacaaatcgc
ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa 3720gtactcgccg atagtggaaa
ccgacgcccc agcactcgtc cgagggcaaa ggaatagtga 3780ggtacctaaa gaaggagtgc
gtcgaagcag atcgttcaaa catttggcaa taaagtttct 3840taagattgaa tcctgttgcc
ggtcttgcga tgattatcat ataatttctg ttgaattacg 3900ttaagcatgt aataattaac
atgtaatgca tgacgttatt tatgagatgg gtttttatga 3960ttagagtccc gcaattatac
atttaatacg cgatagaaaa caaaatatag cgcgcaaact 4020aggataaatt atcgcgcgcg
gtgtcatcta tgttactaga tcgatgtcga atcgatcaac 4080ctgcattaat gaatcggcca
acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 4140gcttcctcgc tcactgactc
gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 4200cactcaaagg cggtaatacg
gttatccaca gaatcagggg ataacgcagg aaagaacatg 4260tgagcaaaag gccagcaaaa
ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 4320cataggctcc gcccccctga
cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 4380aacccgacag gactataaag
ataccaggcg tttccccctg gaagctccct cgtgcgctct 4440cctgttccga ccctgccgct
taccggatac ctgtccgcct ttctcccttc gggaagcgtg 4500gcgctttctc aatgctcacg
ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 4560ctgggctgtg tgcacgaacc
ccccgttcag cccgaccgct gcgccttatc cggtaactat 4620cgtcttgagt ccaacccggt
aagacacgac ttatcgccac tggcagcagc cactggtaac 4680aggattagca gagcgaggta
tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 4740tacggctaca ctagaaggac
agtatttggt atctgcgctc tgctgaagcc agttaccttc 4800ggaaaaagag ttggtagctc
ttgatccggc aaacaaacca ccgctggtag cggtggtttt 4860tttgtttgca agcagcagat
tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 4920ttttctacgg ggtctgacgc
tcagtggaac gaaaactcac gttaagggat tttggtcatg 4980acattaacct ataaaaatag
gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat 5040gacggtgaaa acctctgaca
catgcagctc ccggagacgg tcacagcttg tctgtaagcg 5100gatgccggga gcagacaagc
ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc 5160tggcttaact atgcggcatc
agagcagatt gtactgagag tgcaccatat ggacatattg 5220tcgttagaac gcggctacaa
ttaatacata accttatgta tcatacacat acgatttagg 5280tgacactata gaacggcgcg
cca 5303258898DNAArtificial
Sequenceplasmid pKR902 25ggatctggcc ggccggatct cgtacggatc cgtcgacggc
gcgcccgatc atccggatat 60agttcctcct ttcagcaaaa aacccctcaa gacccgttta
gaggccccaa ggggttatgc 120tagttattgc tcagcggtgg cagcagccaa ctcagcttcc
tttcgggctt tgttagcagc 180cggatcgatc caagctgtac ctcactattc ctttgccctc
ggacgagtgc tggggcgtcg 240gtttccacta tcggcgagta cttctacaca gccatcggtc
cagacggccg cgcttctgcg 300ggcgatttgt gtacgcccga cagtcccggc tccggatcgg
acgattgcgt cgcatcgacc 360ctgcgcccaa gctgcatcat cgaaattgcc gtcaaccaag
ctctgataga gttggtcaag 420accaatgcgg agcatatacg cccggagccg cggcgatcct
gcaagctccg gatgcctccg 480ctcgaagtag cgcgtctgct gctccataca agccaaccac
ggcctccaga agaagatgtt 540ggcgacctcg tattgggaat ccccgaacat cgcctcgctc
cagtcaatga ccgctgttat 600gcggccattg tccgtcagga cattgttgga gccgaaatcc
gcgtgcacga ggtgccggac 660ttcggggcag tcctcggccc aaagcatcag ctcatcgaga
gcctgcgcga cggacgcact 720gacggtgtcg tccatcacag tttgccagtg atacacatgg
ggatcagcaa tcgcgcatat 780gaaatcacgc catgtagtgt attgaccgat tccttgcggt
ccgaatgggc cgaacccgct 840cgtctggcta agatcggccg cagcgatcgc atccatagcc
tccgcgaccg gctgcagaac 900agcgggcagt tcggtttcag gcaggtcttg caacgtgaca
ccctgtgcac ggcgggagat 960gcaataggtc aggctctcgc tgaattcccc aatgtcaagc
acttccggaa tcgggagcgc 1020ggccgatgca aagtgccgat aaacataacg atctttgtag
aaaccatcgg cgcagctatt 1080tacccgcagg acatatccac gccctcctac atcgaagctg
aaagcacgag attcttcgcc 1140ctccgagagc tgcatcaggt cggagacgct gtcgaacttt
tcgatcagaa acttctcgac 1200agacgtcgcg gtgagttcag gcttttccat gggtatatct
ccttcttaaa gttaaacaaa 1260attatttcta gagggaaacc gttgtggtct ccctatagtg
agtcgtatta atttcgcggg 1320atcgagatcg atccaattcc aatcccacaa aaatctgagc
ttaacagcac agttgctcct 1380ctcagagcag aatcgggtat tcaacaccct catatcaact
actacgttgt gtataacggt 1440ccacatgccg gtatatacga tgactggggt tgtacaaagg
cggcaacaaa cggcgttccc 1500ggagttgcac acaagaaatt tgccactatt acagaggcaa
gagcagcagc tgacgcgtac 1560acaacaagtc agcaaacaga caggttgaac ttcatcccca
aaggagaagc tcaactcaag 1620cccaagagct ttgctaaggc cctaacaagc ccaccaaagc
aaaaagccca ctggctcacg 1680ctaggaacca aaaggcccag cagtgatcca gccccaaaag
agatctcctt tgccccggag 1740attacaatgg acgatttcct ctatctttac gatctaggaa
ggaagttcga aggtgaaggt 1800gacgacacta tgttcaccac tgataatgag aaggttagcc
tcttcaattt cagaaagaat 1860gctgacccac agatggttag agaggcctac gcagcaggtc
tcatcaagac gatctacccg 1920agtaacaatc tccaggagat caaatacctt cccaagaagg
ttaaagatgc agtcaaaaga 1980ttcaggacta attgcatcaa gaacacagag aaagacatat
ttctcaagat cagaagtact 2040attccagtat ggacgattca aggcttgctt cataaaccaa
ggcaagtaat agagattgga 2100gtctctaaaa aggtagttcc tactgaatct aaggccatgc
atggagtcta agattcaaat 2160cgaggatcta acagaactcg ccgtgaagac tggcgaacag
ttcatacaga gtcttttacg 2220actcaatgac aagaagaaaa tcttcgtcaa catggtggag
cacgacactc tggtctactc 2280caaaaatgtc aaagatacag tctcagaaga ccaaagggct
attgagactt ttcaacaaag 2340gataatttcg ggaaacctcc tcggattcca ttgcccagct
atctgtcact tcatcgaaag 2400gacagtagaa aaggaaggtg gctcctacaa atgccatcat
tgcgataaag gaaaggctat 2460cattcaagat gcctctgccg acagtggtcc caaagatgga
cccccaccca cgaggagcat 2520cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa
gtggattgat gtgacatctc 2580cactgacgta agggatgacg cacaatccca ctatccttcg
caagaccctt cctctatata 2640aggaagttca tttcatttgg agaggacacg ctcgagctca
tttctctatt acttcagcca 2700taacaaaaga actcttttct cttcttatta aaccatgaaa
aagcctgaac tcaccgcgac 2760gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc
tccgacctga tgcagctctc 2820ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga
gggcgtggat atgtcctgcg 2880ggtaaatagc tgcgccgatg gtttctacaa agatcgttat
gtttatcggc actttgcatc 2940ggccgcgctc ccgattccgg aagtgcttga cattggggaa
ttcagcgaga gcctgaccta 3000ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac
ctgcctgaaa ccgaactgcc 3060cgctgttctg cagccggtcg cggaggccat ggatgcgatc
gctgcggccg atcttagcca 3120gacgagcggg ttcggcccat tcggaccgca aggaatcggt
caatacacta catggcgtga 3180tttcatatgc gcgattgctg atccccatgt gtatcactgg
caaactgtga tggacgacac 3240cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg
ctttgggccg aggactgccc 3300cgaagtccgg cacctcgtgc acgcggattt cggctccaac
aatgtcctga cggacaatgg 3360ccgcataaca gcggtcattg actggagcga ggcgatgttc
ggggattccc aatacgaggt 3420cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg
gagcagcaga cgcgctactt 3480cgagcggagg catccggagc ttgcaggatc gccgcggctc
cgggcgtata tgctccgcat 3540tggtcttgac caactctatc agagcttggt tgacggcaat
ttcgatgatg cagcttgggc 3600gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg
actgtcgggc gtacacaaat 3660cgcccgcaga agcgcggccg tctggaccga tggctgtgta
gaagtactcg ccgatagtgg 3720aaaccgacgc cccagcactc gtccgagggc aaaggaatag
tgaggtacct aaagaaggag 3780tgcgtcgaag cagatcgttc aaacatttgg caataaagtt
tcttaagatt gaatcctgtt 3840gccggtcttg cgatgattat catataattt ctgttgaatt
acgttaagca tgtaataatt 3900aacatgtaat gcatgacgtt atttatgaga tgggttttta
tgattagagt cccgcaatta 3960tacatttaat acgcgataga aaacaaaata tagcgcgcaa
actaggataa attatcgcgc 4020gcggtgtcat ctatgttact agatcgatgt cgaatcgatc
aacctgcatt aatgaatcgg 4080ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
tccgcttcct cgctcactga 4140ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
gctcactcaa aggcggtaat 4200acggttatcc acagaatcag gggataacgc aggaaagaac
atgtgagcaa aaggccagca 4260aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
ttccataggc tccgcccccc 4320tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
cgaaacccga caggactata 4380aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
tctcctgttc cgaccctgcc 4440gcttaccgga tacctgtccg cctttctccc ttcgggaagc
gtggcgcttt ctcaatgctc 4500acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
aagctgggct gtgtgcacga 4560accccccgtt cagcccgacc gctgcgcctt atccggtaac
tatcgtcttg agtccaaccc 4620ggtaagacac gacttatcgc cactggcagc agccactggt
aacaggatta gcagagcgag 4680gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
aactacggct acactagaag 4740gacagtattt ggtatctgcg ctctgctgaa gccagttacc
ttcggaaaaa gagttggtag 4800ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
ttttttgttt gcaagcagca 4860gattacgcgc agaaaaaaag gatctcaaga agatcctttg
atcttttcta cggggtctga 4920cgctcagtgg aacgaaaact cacgttaagg gattttggtc
atgacattaa cctataaaaa 4980taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt
gatgacggtg aaaacctctg 5040acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
gcggatgccg ggagcagaca 5100agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
ggctggctta actatgcggc 5160atcagagcag attgtactga gagtgcacca tatggacata
ttgtcgttag aacgcggcta 5220caattaatac ataaccttat gtatcataca catacgattt
aggtgacact atagaacggc 5280gcgccaagct tggatctcct gcaggatata atgagccgta
aacaaagatg attaagtagt 5340aattaatacg tactagtaaa agtggcaaaa gataacgaga
aagaaccaat ttctttgcat 5400tcggccttag cggaaggcat atataagctt tgattatttt
atttagtgta atgatttcgt 5460acaaccaaag catttattta gtactctcac acttgtgtcg
cggccgctca gtggaggagg 5520tgctcgaagg cgagagcctt gatgttctgg tgcacgcact
ggaagaggtt gccctcgcgg 5580tactcgaggc catgcttctt gaagaagtcg cgtacctcgg
gctggatagt cattaggttg 5640ccggtgggca tcatggggaa gaggtgatgc tcggtctgca
gggagatgaa gcccgtgagc 5700acgttcacta gccagccgcc cgtgatgtta cgggaggtct
tggcggtctg ctcgacgagc 5760gtcatgccct cgacgtgctc gcggtcgagg atgtcctcac
catagtgggt tgcaaagaca 5820acgatgcccg tcgcgaagcc gcgtgcaacc atcatgagag
ggatgaggtt gagatgcgct 5880gcgaagaccc agcacaggag cgcgtagtga gcggcaagag
cggcggcctg catccacatc 5940ttaggcaggc gcacagccac gtacgcgatg gactccaggc
gccagtagag gtcgaggatg 6000gccatggtcg gcacatagta gtactgctgc cagcgctgga
agaagttgag ccgcttggca 6060atggacgggt tctctcgcac gtagatgagc aggggcgccg
tcttgatgtc cgggtccgaa 6120ccatcctcgt tggtgcacac gtggtgcgtg ttatggcgcg
cgcgccacca ggccacgtcg 6180tagccctgga ggaagccgag ggcgtatccc atggcgttgt
tcgccttgac gagcgtgttg 6240accgagccct tgaagactgc gtgatgcagg tagtcgtgag
caagccagcc gtcgaggtag 6300aagccaaagc ccatggagag cgcgcccgcg aggggcgaga
cgccgctccg cacgagatag 6360gcgcccacga agatgggcgc aaggacgatg gtgagcttga
acagctcgtc caacgggcaa 6420gggtcgtaga gaccagccct cttgtggcgc tcaatcatct
ccttgaactc cgtgcccatg 6480gtggaccgcg agggcagggg cacggaagcg ggcgcatcct
ccgacttctt gagcatgccc 6540tccatcatct tgatggcctt gggcgagtgg taagactcga
actgctccgt cgcgtcgcgc 6600tcctggaaga tgcggaacac cttgctcccg cccgggtgtg
ccttctggaa ctttgtgaca 6660tccacgcgct tgccgtagag cacgacgtgc acgctcttgc
tatcgacgga gctcacctca 6720gcgagagacg cgtcggtccc gctcaccgcc tgcgcgccgc
cgtctccacc cttgcccatg 6780gtgcggccgc ttggggggct atggaagact ttcttagtta
gttgtgtgaa taagcaatgt 6840tgggagaatc gggactactt ataggatagg aataaaacag
aaaagtatta agtgctaatg 6900aaatatttag actgataatt aaaatcttca cgtatgtcca
cttgatataa aaacgtcagg 6960aataaaggaa gtacagtaga atttaaaggt actcttttta
tatatacccg tgttctcttt 7020ttggctagct agttgcataa aaaataatct atatttttat
cattatttta aatatcttat 7080gagatggtaa atatttatca taattttttt tactattatt
tattatttgt gtgtgtaata 7140catatagaag ttaattacaa attttattta ctttttcatt
attttgatat gattcaccat 7200taatttagtg ttattattta taatagttca ttttaatctt
tttgtatata ttatgcgtgc 7260agtacttttt tcctacatat aactactatt acattttatt
tatataatat ttttattaat 7320gaattttcgt gataatatgt aatattgttc attattattt
cagatttttt aaaaatattt 7380gtgttattat ttatgaaata tgtaattttt ttagtatttg
attttatgat gataaagtgt 7440tctaaattca aaagaagggg gaaagcgtaa acattaaaaa
acgtcatcaa acaaaaacaa 7500aatcttgtta ataaagataa aactgtttgt tttgatcact
gttatttcgt aatataaaaa 7560cattatttat atttatattg ttgacaacca aatttgccta
tcaaatctaa ccaatataat 7620gcatgcgtgg caggtaatgt actaccatga acttaagtca
tgacataata aaccgtgaat 7680ctgaccaatg catgtaccta nctaaattgt atttgtgaca
cgaagcaaat gattcaattc 7740acaatggaga tgggaaacaa ataatgaaga acccagaact
aagaaagctt ttctgaaaaa 7800taaaataaag gcaatgtcaa aagtatactg catcatcagt
ccagaaagca catgatattt 7860ttttatcagt atcaatgcag ctagttttat tttacaatat
cgatatagct agtttaaata 7920tattgcagct agatttataa atatttgtgt tattatttat
catttgtgta atcctgtttt 7980tagtatttta gtttatatat gatgataatg tattccaaat
ttaaaagaag ggaaataaat 8040ttaaacaaga aaaaaagtca tcaaacaaaa aacaaatgaa
agggtggaaa gatgttacca 8100tgtaatgtga atgttacagt atttctttta ttatagagtt
aacaaattaa ctaatatgat 8160tttgttaata atgataaaat atttttttta ttattatttc
ataatataaa aatagtttac 8220ttaatataaa aaaaattcta tcgttcacaa caaagttggc
cacctaattt aaccatgcat 8280gtacccatgg accatattag gtaaccatca aacctgatga
agagataaag agatgaagac 8340ttaagtcata acacaaaacc ataaaaaaca aaaatacaat
caaccgtcaa tctgaccaat 8400gcatgaaaaa gctgcaatag tgagtggcga cacaaagcac
atgattttct tacaacggag 8460ataaaaccaa aaaaatattt catgaacaac ctagaacaaa
taaagctttt atataataaa 8520tatataaata aataaaggct atggaataat atacttcaat
atatttggat taaataaatt 8580gttggcgggg ttgatatatt tatacacacc taaagtcact
tcaatctcat tttcacttaa 8640cttttatttt ttttttcttt ttatttatca taaagagaat
attgataata tactttttaa 8700catattttta tgacattttt tattggtgaa aacttattaa
aaatcataaa ttttgtaagt 8760tagatttatt taaagagttc ctcttcttat tttaaatttt
ttaataaatt tttaaataac 8820taaaatttgt gttaaaaatg ttaaaaaatg tgttattaac
ccttctcttc gaggacgtac 8880gtctagagtc gacctgca
8898267887DNAArtificial Sequenceplasmid pKR607
26ggccgcagta tatcttaaat tctttaatac ggtgtactag gatattgaac tggttcttga
60tgatgaaaac ctgggccgag attgcagcta tttatagtca taggtcttgt taacatgcat
120ggacatttgg ccacggggtg gcatgcagtt tgacgggtgt tgaaataaac aaaaatgagg
180tggcggaaga gaatacgagt ttgaggttgg gttagaaaca acaaatgtga gggctcatga
240tgggttgagt tggtgaatgt tttgggctgc tcgattgaca cctttgtgag tacgtgttgt
300tgtgcatggc ttttggggtc cagttttttt ttcttgacgc ggcgatcctg atcagctagt
360ggataagtga tgtccactgt gtgtgattgc gtttttgttt gaattttatg aacttagaca
420ttgctatgca aaggatactc tcattgtgtt ttgtcttctt ttgttccttg gctttttctt
480atgatccaag agactagtca gtgttgtggc attcgagact accaagatta attatgatgg
540gggaaggata agtaactgat tagtacggac tgttaccaaa ttaattaata agcggcaaat
600gaagggcatg gatcaaaagc ttggatctcc tgcaggatct ggccggccgg atctcgtacg
660gatccgtcga cggcgcgccc gatcatccgg atatagttcc tcctttcagc aaaaaacccc
720tcaagacccg tttagaggcc ccaaggggtt atgctagtta ttgctcagcg gtggcagcag
780ccaactcagc ttcctttcgg gctttgttag cagccggatc gatccaagct gtacctcact
840attcctttgc cctcggacga gtgctggggc gtcggtttcc actatcggcg agtacttcta
900cacagccatc ggtccagacg gccgcgcttc tgcgggcgat ttgtgtacgc ccgacagtcc
960cggctccgga tcggacgatt gcgtcgcatc gaccctgcgc ccaagctgca tcatcgaaat
1020tgccgtcaac caagctctga tagagttggt caagaccaat gcggagcata tacgcccgga
1080gccgcggcga tcctgcaagc tccggatgcc tccgctcgaa gtagcgcgtc tgctgctcca
1140tacaagccaa ccacggcctc cagaagaaga tgttggcgac ctcgtattgg gaatccccga
1200acatcgcctc gctccagtca atgaccgctg ttatgcggcc attgtccgtc aggacattgt
1260tggagccgaa atccgcgtgc acgaggtgcc ggacttcggg gcagtcctcg gcccaaagca
1320tcagctcatc gagagcctgc gcgacggacg cactgacggt gtcgtccatc acagtttgcc
1380agtgatacac atggggatca gcaatcgcgc atatgaaatc acgccatgta gtgtattgac
1440cgattccttg cggtccgaat gggccgaacc cgctcgtctg gctaagatcg gccgcagcga
1500tcgcatccat agcctccgcg accggctgca gaacagcggg cagttcggtt tcaggcaggt
1560cttgcaacgt gacaccctgt gcacggcggg agatgcaata ggtcaggctc tcgctgaatt
1620ccccaatgtc aagcacttcc ggaatcggga gcgcggccga tgcaaagtgc cgataaacat
1680aacgatcttt gtagaaacca tcggcgcagc tatttacccg caggacatat ccacgccctc
1740ctacatcgaa gctgaaagca cgagattctt cgccctccga gagctgcatc aggtcggaga
1800cgctgtcgaa cttttcgatc agaaacttct cgacagacgt cgcggtgagt tcaggctttt
1860ccatgggtat atctccttct taaagttaaa caaaattatt tctagaggga aaccgttgtg
1920gtctccctat agtgagtcgt attaatttcg cgggatcgag atcgatccaa ttccaatccc
1980acaaaaatct gagcttaaca gcacagttgc tcctctcaga gcagaatcgg gtattcaaca
2040ccctcatatc aactactacg ttgtgtataa cggtccacat gccggtatat acgatgactg
2100gggttgtaca aaggcggcaa caaacggcgt tcccggagtt gcacacaaga aatttgccac
2160tattacagag gcaagagcag cagctgacgc gtacacaaca agtcagcaaa cagacaggtt
2220gaacttcatc cccaaaggag aagctcaact caagcccaag agctttgcta aggccctaac
2280aagcccacca aagcaaaaag cccactggct cacgctagga accaaaaggc ccagcagtga
2340tccagcccca aaagagatct cctttgcccc ggagattaca atggacgatt tcctctatct
2400ttacgatcta ggaaggaagt tcgaaggtga aggtgacgac actatgttca ccactgataa
2460tgagaaggtt agcctcttca atttcagaaa gaatgctgac ccacagatgg ttagagaggc
2520ctacgcagca ggtctcatca agacgatcta cccgagtaac aatctccagg agatcaaata
2580ccttcccaag aaggttaaag atgcagtcaa aagattcagg actaattgca tcaagaacac
2640agagaaagac atatttctca agatcagaag tactattcca gtatggacga ttcaaggctt
2700gcttcataaa ccaaggcaag taatagagat tggagtctct aaaaaggtag ttcctactga
2760atctaaggcc atgcatggag tctaagattc aaatcgagga tctaacagaa ctcgccgtga
2820agactggcga acagttcata cagagtcttt tacgactcaa tgacaagaag aaaatcttcg
2880tcaacatggt ggagcacgac actctggtct actccaaaaa tgtcaaagat acagtctcag
2940aagaccaaag ggctattgag acttttcaac aaaggataat ttcgggaaac ctcctcggat
3000tccattgccc agctatctgt cacttcatcg aaaggacagt agaaaaggaa ggtggctcct
3060acaaatgcca tcattgcgat aaaggaaagg ctatcattca agatgcctct gccgacagtg
3120gtcccaaaga tggaccccca cccacgagga gcatcgtgga aaaagaagac gttccaacca
3180cgtcttcaaa gcaagtggat tgatgtgaca tctccactga cgtaagggat gacgcacaat
3240cccactatcc ttcgcaagac ccttcctcta tataaggaag ttcatttcat ttggagagga
3300cacgctcgag ctcatttctc tattacttca gccataacaa aagaactctt ttctcttctt
3360attaaaccat gaaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa
3420agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca
3480gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct
3540acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc
3600ttgacattgg ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg
3660tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg
3720ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac
3780cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc
3840atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc
3900tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg
3960atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga
4020gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt
4080ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag
4140gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct
4200tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc
4260gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga
4320ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga
4380gggcaaagga atagtgaggt acctaaagaa ggagtgcgtc gaagcagatc gttcaaacat
4440ttggcaataa agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata
4500atttctgttg aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat
4560gagatgggtt tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa
4620aatatagcgc gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatcg
4680atgtcgaatc gatcaacctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
4740gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
4800ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
4860acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
4920cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
4980caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
5040gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
5100tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc agttcggtgt
5160aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
5220ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
5280cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
5340tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
5400tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
5460ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
5520aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
5580aagggatttt ggtcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
5640tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
5700cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
5760ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
5820accatatgga catattgtcg ttagaacgcg gctacaatta atacataacc ttatgtatca
5880tacacatacg atttaggtga cactatagaa cggcgcgcca agcttgttga aacatccctg
5940aagtgtctca ttttatttta tttattcttt gctgataaaa aaataaaata aaagaagcta
6000agcacacggt caaccattgc tctactgcta aaagggttat gtgtagtgtt ttactgcata
6060aattatgcag caaacaagac aactcaaatt aaaaaatttc ctttgcttgt ttttttgttg
6120tctctgactt gactttcttg tggaagttgg ttgtataagg attgggacac cattgtcctt
6180cttaatttaa ttttattctt tgctgataaa aaaaaaaatt tcatatagtg ttaaataata
6240atttgttaaa taaccaaaaa gtcaaatatg tttactctcg tttaaataat tgagattcgt
6300ccagcaaggc taaacgattg tatagattta tgacaatatt tactttttta tagataaatg
6360ttatattata ataaatttat atacatatat tatatgttat ttattattat tttaaatcct
6420tcaatatttt atcaaaccaa ctcataattt tttttttatc tgtaagaagc aataaaatta
6480aatagaccca ctttaaggat gatccaacct ttatacagag taagagagtt caaatagtac
6540cctttcatat acatatcaac taaaatatta gaaatatcat ggatcaaacc ttataaagac
6600attaaataag tggataagta taatatataa atgggtagta tataatatat aaatggatac
6660aaacttctct ctttataatt gttatgtctc cttaacatcc taatataata cataagtggg
6720taatatataa tatataaatg gagacaaact tcttccatta taattgttat gtcttcttaa
6780cacttatgtc tcgttcacaa tgctaaggtt agaattgttt agaaagtctt atagtacaca
6840tttgtttttg tactatttga agcattccat aagccgtcac gattcagatg atttataata
6900ataagaggaa atttatcata gaacaataag gtgcatagat agagtgttaa tatatcataa
6960catcctttgt ttattcatag aagaagtgag atggagctca gttattatac tgttacatgg
7020tcggatacaa tattccatgc tctccatgag ctcttacacc tacatgcatt ttagttcata
7080cttgcggccg ctaaagctgc ttaccagcct tagcggattt cttggtggcc aggttgtcct
7140ggtaaaagaa gtgacagaac aggagaaaga cagatccgac gtaggcgtag ttgaaagccc
7200aggagaacag cttgcccttg tcagagttga agcagggaac gttgatgtag tcccagacca
7260ggagaaagcc accgacgaac tggcaaatct gcatggcagt gatcagaggc ttggccttga
7320acttgtagcc agcggcagtc agtccatagt aggtgtacat gatggtgtga atgaacgagt
7380taaagaacat gaagatccac acaccctcgt tgtgcagtcg aatgccgagg tagacgtccc
7440agggagctcc aaagtgatgg aaggcctgca gaaaggacac tcgcttgccc ttgaggacca
7500gccaagcggt gtcgaggtac tccacgtact tagaatagta gaaggccttg gcagtccagg
7560tgaacagctt ggagtcccag acaggagagg gacactgaaa gagaggctgg ggagtatcac
7620cggtctgtct tcgcagccag gctccagtac cgtagtccca gccgagagcg gtggcagtca
7680cgtagaagga cagggcagag aagagagcca ggaggacgtt gtaccagatc atggaggttc
7740ggtaggctcc tttcttctcg tccacgagac cagagtttcg caggagaggc ttcaggagca
7800ggtaggagaa ggtgccaatg aggatttcgg gatcggtgac ggcagcccag attcgctcgc
7860cagcgtcgtt ggccagagcc atggtgc
78872711482DNAArtificial Sequenceplasmid pKR903 27ggatctggcc ggccggatct
cgtacggatc cgtcgacggc gcgcccgatc atccggatat 60agttcctcct ttcagcaaaa
aacccctcaa gacccgttta gaggccccaa ggggttatgc 120tagttattgc tcagcggtgg
cagcagccaa ctcagcttcc tttcgggctt tgttagcagc 180cggatcgatc caagctgtac
ctcactattc ctttgccctc ggacgagtgc tggggcgtcg 240gtttccacta tcggcgagta
cttctacaca gccatcggtc cagacggccg cgcttctgcg 300ggcgatttgt gtacgcccga
cagtcccggc tccggatcgg acgattgcgt cgcatcgacc 360ctgcgcccaa gctgcatcat
cgaaattgcc gtcaaccaag ctctgataga gttggtcaag 420accaatgcgg agcatatacg
cccggagccg cggcgatcct gcaagctccg gatgcctccg 480ctcgaagtag cgcgtctgct
gctccataca agccaaccac ggcctccaga agaagatgtt 540ggcgacctcg tattgggaat
ccccgaacat cgcctcgctc cagtcaatga ccgctgttat 600gcggccattg tccgtcagga
cattgttgga gccgaaatcc gcgtgcacga ggtgccggac 660ttcggggcag tcctcggccc
aaagcatcag ctcatcgaga gcctgcgcga cggacgcact 720gacggtgtcg tccatcacag
tttgccagtg atacacatgg ggatcagcaa tcgcgcatat 780gaaatcacgc catgtagtgt
attgaccgat tccttgcggt ccgaatgggc cgaacccgct 840cgtctggcta agatcggccg
cagcgatcgc atccatagcc tccgcgaccg gctgcagaac 900agcgggcagt tcggtttcag
gcaggtcttg caacgtgaca ccctgtgcac ggcgggagat 960gcaataggtc aggctctcgc
tgaattcccc aatgtcaagc acttccggaa tcgggagcgc 1020ggccgatgca aagtgccgat
aaacataacg atctttgtag aaaccatcgg cgcagctatt 1080tacccgcagg acatatccac
gccctcctac atcgaagctg aaagcacgag attcttcgcc 1140ctccgagagc tgcatcaggt
cggagacgct gtcgaacttt tcgatcagaa acttctcgac 1200agacgtcgcg gtgagttcag
gcttttccat gggtatatct ccttcttaaa gttaaacaaa 1260attatttcta gagggaaacc
gttgtggtct ccctatagtg agtcgtatta atttcgcggg 1320atcgagatcg atccaattcc
aatcccacaa aaatctgagc ttaacagcac agttgctcct 1380ctcagagcag aatcgggtat
tcaacaccct catatcaact actacgttgt gtataacggt 1440ccacatgccg gtatatacga
tgactggggt tgtacaaagg cggcaacaaa cggcgttccc 1500ggagttgcac acaagaaatt
tgccactatt acagaggcaa gagcagcagc tgacgcgtac 1560acaacaagtc agcaaacaga
caggttgaac ttcatcccca aaggagaagc tcaactcaag 1620cccaagagct ttgctaaggc
cctaacaagc ccaccaaagc aaaaagccca ctggctcacg 1680ctaggaacca aaaggcccag
cagtgatcca gccccaaaag agatctcctt tgccccggag 1740attacaatgg acgatttcct
ctatctttac gatctaggaa ggaagttcga aggtgaaggt 1800gacgacacta tgttcaccac
tgataatgag aaggttagcc tcttcaattt cagaaagaat 1860gctgacccac agatggttag
agaggcctac gcagcaggtc tcatcaagac gatctacccg 1920agtaacaatc tccaggagat
caaatacctt cccaagaagg ttaaagatgc agtcaaaaga 1980ttcaggacta attgcatcaa
gaacacagag aaagacatat ttctcaagat cagaagtact 2040attccagtat ggacgattca
aggcttgctt cataaaccaa ggcaagtaat agagattgga 2100gtctctaaaa aggtagttcc
tactgaatct aaggccatgc atggagtcta agattcaaat 2160cgaggatcta acagaactcg
ccgtgaagac tggcgaacag ttcatacaga gtcttttacg 2220actcaatgac aagaagaaaa
tcttcgtcaa catggtggag cacgacactc tggtctactc 2280caaaaatgtc aaagatacag
tctcagaaga ccaaagggct attgagactt ttcaacaaag 2340gataatttcg ggaaacctcc
tcggattcca ttgcccagct atctgtcact tcatcgaaag 2400gacagtagaa aaggaaggtg
gctcctacaa atgccatcat tgcgataaag gaaaggctat 2460cattcaagat gcctctgccg
acagtggtcc caaagatgga cccccaccca cgaggagcat 2520cgtggaaaaa gaagacgttc
caaccacgtc ttcaaagcaa gtggattgat gtgacatctc 2580cactgacgta agggatgacg
cacaatccca ctatccttcg caagaccctt cctctatata 2640aggaagttca tttcatttgg
agaggacacg ctcgagctca tttctctatt acttcagcca 2700taacaaaaga actcttttct
cttcttatta aaccatgaaa aagcctgaac tcaccgcgac 2760gtctgtcgag aagtttctga
tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc 2820ggagggcgaa gaatctcgtg
ctttcagctt cgatgtagga gggcgtggat atgtcctgcg 2880ggtaaatagc tgcgccgatg
gtttctacaa agatcgttat gtttatcggc actttgcatc 2940ggccgcgctc ccgattccgg
aagtgcttga cattggggaa ttcagcgaga gcctgaccta 3000ttgcatctcc cgccgtgcac
agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc 3060cgctgttctg cagccggtcg
cggaggccat ggatgcgatc gctgcggccg atcttagcca 3120gacgagcggg ttcggcccat
tcggaccgca aggaatcggt caatacacta catggcgtga 3180tttcatatgc gcgattgctg
atccccatgt gtatcactgg caaactgtga tggacgacac 3240cgtcagtgcg tccgtcgcgc
aggctctcga tgagctgatg ctttgggccg aggactgccc 3300cgaagtccgg cacctcgtgc
acgcggattt cggctccaac aatgtcctga cggacaatgg 3360ccgcataaca gcggtcattg
actggagcga ggcgatgttc ggggattccc aatacgaggt 3420cgccaacatc ttcttctgga
ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt 3480cgagcggagg catccggagc
ttgcaggatc gccgcggctc cgggcgtata tgctccgcat 3540tggtcttgac caactctatc
agagcttggt tgacggcaat ttcgatgatg cagcttgggc 3600gcagggtcga tgcgacgcaa
tcgtccgatc cggagccggg actgtcgggc gtacacaaat 3660cgcccgcaga agcgcggccg
tctggaccga tggctgtgta gaagtactcg ccgatagtgg 3720aaaccgacgc cccagcactc
gtccgagggc aaaggaatag tgaggtacct aaagaaggag 3780tgcgtcgaag cagatcgttc
aaacatttgg caataaagtt tcttaagatt gaatcctgtt 3840gccggtcttg cgatgattat
catataattt ctgttgaatt acgttaagca tgtaataatt 3900aacatgtaat gcatgacgtt
atttatgaga tgggttttta tgattagagt cccgcaatta 3960tacatttaat acgcgataga
aaacaaaata tagcgcgcaa actaggataa attatcgcgc 4020gcggtgtcat ctatgttact
agatcgatgt cgaatcgatc aacctgcatt aatgaatcgg 4080ccaacgcgcg gggagaggcg
gtttgcgtat tgggcgctct tccgcttcct cgctcactga 4140ctcgctgcgc tcggtcgttc
ggctgcggcg agcggtatca gctcactcaa aggcggtaat 4200acggttatcc acagaatcag
gggataacgc aggaaagaac atgtgagcaa aaggccagca 4260aaaggccagg aaccgtaaaa
aggccgcgtt gctggcgttt ttccataggc tccgcccccc 4320tgacgagcat cacaaaaatc
gacgctcaag tcagaggtgg cgaaacccga caggactata 4380aagataccag gcgtttcccc
ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 4440gcttaccgga tacctgtccg
cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc 4500acgctgtagg tatctcagtt
cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 4560accccccgtt cagcccgacc
gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 4620ggtaagacac gacttatcgc
cactggcagc agccactggt aacaggatta gcagagcgag 4680gtatgtaggc ggtgctacag
agttcttgaa gtggtggcct aactacggct acactagaag 4740gacagtattt ggtatctgcg
ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 4800ctcttgatcc ggcaaacaaa
ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 4860gattacgcgc agaaaaaaag
gatctcaaga agatcctttg atcttttcta cggggtctga 4920cgctcagtgg aacgaaaact
cacgttaagg gattttggtc atgacattaa cctataaaaa 4980taggcgtatc acgaggccct
ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg 5040acacatgcag ctcccggaga
cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca 5100agcccgtcag ggcgcgtcag
cgggtgttgg cgggtgtcgg ggctggctta actatgcggc 5160atcagagcag attgtactga
gagtgcacca tatggacata ttgtcgttag aacgcggcta 5220caattaatac ataaccttat
gtatcataca catacgattt aggtgacact atagaacggc 5280gcgccaagct tgttgaaaca
tccctgaagt gtctcatttt attttattta ttctttgctg 5340ataaaaaaat aaaataaaag
aagctaagca cacggtcaac cattgctcta ctgctaaaag 5400ggttatgtgt agtgttttac
tgcataaatt atgcagcaaa caagacaact caaattaaaa 5460aatttccttt gcttgttttt
ttgttgtctc tgacttgact ttcttgtgga agttggttgt 5520ataaggattg ggacaccatt
gtccttctta atttaatttt attctttgct gataaaaaaa 5580aaaatttcat atagtgttaa
ataataattt gttaaataac caaaaagtca aatatgttta 5640ctctcgttta aataattgag
attcgtccag caaggctaaa cgattgtata gatttatgac 5700aatatttact tttttataga
taaatgttat attataataa atttatatac atatattata 5760tgttatttat tattatttta
aatccttcaa tattttatca aaccaactca taattttttt 5820tttatctgta agaagcaata
aaattaaata gacccacttt aaggatgatc caacctttat 5880acagagtaag agagttcaaa
tagtaccctt tcatatacat atcaactaaa atattagaaa 5940tatcatggat caaaccttat
aaagacatta aataagtgga taagtataat atataaatgg 6000gtagtatata atatataaat
ggatacaaac ttctctcttt ataattgtta tgtctcctta 6060acatcctaat ataatacata
agtgggtaat atataatata taaatggaga caaacttctt 6120ccattataat tgttatgtct
tcttaacact tatgtctcgt tcacaatgct aaggttagaa 6180ttgtttagaa agtcttatag
tacacatttg tttttgtact atttgaagca ttccataagc 6240cgtcacgatt cagatgattt
ataataataa gaggaaattt atcatagaac aataaggtgc 6300atagatagag tgttaatata
tcataacatc ctttgtttat tcatagaaga agtgagatgg 6360agctcagtta ttatactgtt
acatggtcgg atacaatatt ccatgctctc catgagctct 6420tacacctaca tgcattttag
ttcatacttg cggccgctaa agctgcttac cagccttagc 6480ggatttcttg gtggccaggt
tgtcctggta aaagaagtga cagaacagga gaaagacaga 6540tccgacgtag gcgtagttga
aagcccagga gaacagcttg cccttgtcag agttgaagca 6600gggaacgttg atgtagtccc
agaccaggag aaagccaccg acgaactggc aaatctgcat 6660ggcagtgatc agaggcttgg
ccttgaactt gtagccagcg gcagtcagtc catagtaggt 6720gtacatgatg gtgtgaatga
acgagttaaa gaacatgaag atccacacac cctcgttgtg 6780cagtcgaatg ccgaggtaga
cgtcccaggg agctccaaag tgatggaagg cctgcagaaa 6840ggacactcgc ttgcccttga
ggaccagcca agcggtgtcg aggtactcca cgtacttaga 6900atagtagaag gccttggcag
tccaggtgaa cagcttggag tcccagacag gagagggaca 6960ctgaaagaga ggctggggag
tatcaccggt ctgtcttcgc agccaggctc cagtaccgta 7020gtcccagccg agagcggtgg
cagtcacgta gaaggacagg gcagagaaga gagccaggag 7080gacgttgtac cagatcatgg
aggttcggta ggctcctttc ttctcgtcca cgagaccaga 7140gtttcgcagg agaggcttca
ggagcaggta ggagaaggtg ccaatgagga tttcgggatc 7200ggtgacggca gcccagattc
gctcgccagc gtcgttggcc agagccatgg tgcggccgca 7260gtatatctta aattctttaa
tacggtgtac taggatattg aactggttct tgatgatgaa 7320aacctgggcc gagattgcag
ctatttatag tcataggtct tgttaacatg catggacatt 7380tggccacggg gtggcatgca
gtttgacggg tgttgaaata aacaaaaatg aggtggcgga 7440agagaatacg agtttgaggt
tgggttagaa acaacaaatg tgagggctca tgatgggttg 7500agttggtgaa tgttttgggc
tgctcgattg acacctttgt gagtacgtgt tgttgtgcat 7560ggcttttggg gtccagtttt
tttttcttga cgcggcgatc ctgatcagct agtggataag 7620tgatgtccac tgtgtgtgat
tgcgtttttg tttgaatttt atgaacttag acattgctat 7680gcaaaggata ctctcattgt
gttttgtctt cttttgttcc ttggcttttt cttatgatcc 7740aagagactag tcagtgttgt
ggcattcgag actaccaaga ttaattatga tgggggaagg 7800ataagtaact gattagtacg
gactgttacc aaattaatta ataagcggca aatgaagggc 7860atggatcaaa agcttggatc
tcctgcaggt cgactctaga cgtacgtcct cgaagagaag 7920ggttaataac acatttttta
acatttttaa cacaaatttt agttatttaa aaatttatta 7980aaaaatttaa aataagaaga
ggaactcttt aaataaatct aacttacaaa atttatgatt 8040tttaataagt tttcaccaat
aaaaaatgtc ataaaaatat gttaaaaagt atattatcaa 8100tattctcttt atgataaata
aaaagaaaaa aaaaataaaa gttaagtgaa aatgagattg 8160aagtgacttt aggtgtgtat
aaatatatca accccgccaa caatttattt aatccaaata 8220tattgaagta tattattcca
tagcctttat ttatttatat atttattata taaaagcttt 8280atttgttcta ggttgttcat
gaaatatttt tttggtttta tctccgttgt aagaaaatca 8340tgtgctttgt gtcgccactc
actattgcag ctttttcatg cattggtcag attgacggtt 8400gattgtattt ttgtttttta
tggttttgtg ttatgactta agtcttcatc tctttatctc 8460ttcatcaggt ttgatggtta
cctaatatgg tccatgggta catgcatggt taaattaggt 8520ggccaacttt gttgtgaacg
atagaatttt ttttatatta agtaaactat ttttatatta 8580tgaaataata ataaaaaaaa
tattttatca ttattaacaa aatcatatta gttaatttgt 8640taactctata ataaaagaaa
tactgtaaca ttcacattac atggtaacat ctttccaccc 8700tttcatttgt tttttgtttg
atgacttttt ttcttgttta aatttatttc ccttctttta 8760aatttggaat acattatcat
catatataaa ctaaaatact aaaaacagga ttacacaaat 8820gataaataat aacacaaata
tttataaatc tagctgcaat atatttaaac tagctatatc 8880gatattgtaa aataaaacta
gctgcattga tactgataaa aaaatatcat gtgctttctg 8940gactgatgat gcagtatact
tttgacattg cctttatttt atttttcaga aaagctttct 9000tagttctggg ttcttcatta
tttgtttccc atctccattg tgaattgaat catttgcttc 9060gtgtcacaaa tacaatttag
ntaggtacat gcattggtca gattcacggt ttattatgtc 9120atgacttaag ttcatggtag
tacattacct gccacgcatg cattatattg gttagatttg 9180ataggcaaat ttggttgtca
acaatataaa tataaataat gtttttatat tacgaaataa 9240cagtgatcaa aacaaacagt
tttatcttta ttaacaagat tttgtttttg tttgatgacg 9300ttttttaatg tttacgcttt
cccccttctt ttgaatttag aacactttat catcataaaa 9360tcaaatacta aaaaaattac
atatttcata aataataaca caaatatttt taaaaaatct 9420gaaataataa tgaacaatat
tacatattat cacgaaaatt cattaataaa aatattatat 9480aaataaaatg taatagtagt
tatatgtagg aaaaaagtac tgcacgcata atatatacaa 9540aaagattaaa atgaactatt
ataaataata acactaaatt aatggtgaat catatcaaaa 9600taatgaaaaa gtaaataaaa
tttgtaatta acttctatat gtattacaca cacaaataat 9660aaataatagt aaaaaaaatt
atgataaata tttaccatct cataagatat ttaaaataat 9720gataaaaata tagattattt
tttatgcaac tagctagcca aaaagagaac acgggtatat 9780ataaaaagag tacctttaaa
ttctactgta cttcctttat tcctgacgtt tttatatcaa 9840gtggacatac gtgaagattt
taattatcag tctaaatatt tcattagcac ttaatacttt 9900tctgttttat tcctatccta
taagtagtcc cgattctccc aacattgctt attcacacaa 9960ctaactaaga aagtcttcca
tagcccccca agcggccgca ccatgggcaa gggtggagac 10020ggcggcgcgc aggcggtgag
cgggaccgac gcgtctctcg ctgaggtgag ctccgtcgat 10080agcaagagcg tgcacgtcgt
gctctacggc aagcgcgtgg atgtcacaaa gttccagaag 10140gcacacccgg gcgggagcaa
ggtgttccgc atcttccagg agcgcgacgc gacggagcag 10200ttcgagtctt accactcgcc
caaggccatc aagatgatgg agggcatgct caagaagtcg 10260gaggatgcgc ccgcttccgt
gcccctgccc tcgcggtcca ccatgggcac ggagttcaag 10320gagatgattg agcgccacaa
gagggctggt ctctacgacc cttgcccgtt ggacgagctg 10380ttcaagctca ccatcgtcct
tgcgcccatc ttcgtgggcg cctatctcgt gcggagcggc 10440gtctcgcccc tcgcgggcgc
gctctccatg ggctttggct tctacctcga cggctggctt 10500gctcacgact acctgcatca
cgcagtcttc aagggctcgg tcaacacgct cgtcaaggcg 10560aacaacgcca tgggatacgc
cctcggcttc ctccagggct acgacgtggc ctggtggcgc 10620gcgcgccata acacgcacca
cgtgtgcacc aacgaggatg gttcggaccc ggacatcaag 10680acggcgcccc tgctcatcta
cgtgcgagag aacccgtcca ttgccaagcg gctcaacttc 10740ttccagcgct ggcagcagta
ctactatgtg ccgaccatgg ccatcctcga cctctactgg 10800cgcctggagt ccatcgcgta
cgtggctgtg cgcctgccta agatgtggat gcaggccgcc 10860gctcttgccg ctcactacgc
gctcctgtgc tgggtcttcg cagcgcatct caacctcatc 10920cctctcatga tggttgcacg
cggcttcgcg acgggcatcg ttgtctttgc aacccactat 10980ggtgaggaca tcctcgaccg
cgagcacgtc gagggcatga cgctcgtcga gcagaccgcc 11040aagacctccc gtaacatcac
gggcggctgg ctagtgaacg tgctcacggg cttcatctcc 11100ctgcagaccg agcatcacct
cttccccatg atgcccaccg gcaacctaat gactatccag 11160cccgaggtac gcgacttctt
caagaagcat ggcctcgagt accgcgaggg caacctcttc 11220cagtgcgtgc accagaacat
caaggctctc gccttcgagc acctcctcca ctgagcggcc 11280gcgacacaag tgtgagagta
ctaaataaat gctttggttg tacgaaatca ttacactaaa 11340taaaataatc aaagcttata
tatgccttcc gctaaggccg aatgcaaaga aattggttct 11400ttctcgttat cttttgccac
ttttactagt acgtattaat tactacttaa tcatctttgt 11460ttacggctca ttatatcctg
ca 114822818DNAArtificial
SequenceGPDsense primer 28atacgagatc gtcaaggg
182926DNAArtificial SequenceGPDantisense primer
29gcggccgcgg attgatgtgt gtttaa
26306970DNAArtificial Sequenceplasmid pY5-22GPD 30tcgacgcagt aggatgtcct
gcacgggtct ttttgtgggg tgtggagaaa ggggtgcttg 60gagatggaag ccggtagaac
cgggctgctt gtgcttggag atggaagccg gtagaaccgg 120gctgcttggg gggatttggg
gccgctgggc tccaaagagg ggtaggcatt tcgttggggt 180tacgtaattg cggcatttgg
gtcctgcgcg catgtcccat tggtcagaat tagtccggat 240aggagactta tcagccaatc
acagcgccgg atccacctgt aggttgggtt gggtgggagc 300acccctccac agagtagagt
caaacagcag cagcaacatg atagttgggg gtgtgcgtgt 360taaaggaaaa aaaagaagct
tgggttatat tcccgctcta tttagaggtt gcgggataga 420cgccgacgga gggcaatggc
gccatggaac cttgcggata tcgatacgcc gcggcggact 480gcgtccgaac cagctccagc
agcgtttttt ccgggccatt gagccgactg cgaccccgcc 540aacgtgtctt ggcccacgca
ctcatgtcat gttggtgttg ggaggccact ttttaagtag 600cacaaggcac ctagctcgca
gcaaggtgtc cgaaccaaag aagcggctgc agtggtgcaa 660acggggcgga aacggcggga
aaaagccacg ggggcacgaa ttgaggcacg ccctcgaatt 720tgagacgagt cacggcccca
ttcgcccgcg caatggctcg ccaacgcccg gtcttttgca 780ccacatcagg ttaccccaag
ccaaaccttt gtgttaaaaa gcttaacata ttataccgaa 840cgtaggtttg ggcgggcttg
ctccgtctgt ccaaggcaac atttatataa gggtctgcat 900cgccggctca attgaatctt
ttttcttctt ctcttctcta tattcattct tgaattaaac 960acacatcaat ccgcggccgc
caccgcggcc cgagattccg gcctcttcgg ccgccaagcg 1020acccgggtgg acgtctagag
gtacctagca attaacagat agtttgccgg tgataattct 1080cttaacctcc cacactcctt
tgacataacg atttatgtaa cgaaactgaa atttgaccag 1140atattgtgtc cgcggtggag
ctccagcttt tgttcccttt agtgagggtt aatttcgagc 1200ttggcgtaat catggtcata
gctgtttcct gtgtgaaatt gttatccgct cacaattcca 1260cacaacgtac gagccggaag
cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 1320ctcacattaa ttgcgttgcg
ctcactgccc gctttccagt cgggaaacct gtcgtgccag 1380ctgcattaat gaatcggcca
acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 1440gcttcctcgc tcactgactc
gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 1500cactcaaagg cggtaatacg
gttatccaca gaatcagggg ataacgcagg aaagaacatg 1560tgagcaaaag gccagcaaaa
ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 1620cataggctcc gcccccctga
cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 1680aacccgacag gactataaag
ataccaggcg tttccccctg gaagctccct cgtgcgctct 1740cctgttccga ccctgccgct
taccggatac ctgtccgcct ttctcccttc gggaagcgtg 1800gcgctttctc atagctcacg
ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 1860ctgggctgtg tgcacgaacc
ccccgttcag cccgaccgct gcgccttatc cggtaactat 1920cgtcttgagt ccaacccggt
aagacacgac ttatcgccac tggcagcagc cactggtaac 1980aggattagca gagcgaggta
tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 2040tacggctaca ctagaaggac
agtatttggt atctgcgctc tgctgaagcc agttaccttc 2100ggaaaaagag ttggtagctc
ttgatccggc aaacaaacca ccgctggtag cggtggtttt 2160tttgtttgca agcagcagat
tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 2220ttttctacgg ggtctgacgc
tcagtggaac gaaaactcac gttaagggat tttggtcatg 2280agattatcaa aaaggatctt
cacctagatc cttttaaatt aaaaatgaag ttttaaatca 2340atctaaagta tatatgagta
aacttggtct gacagttacc aatgcttaat cagtgaggca 2400cctatctcag cgatctgtct
atttcgttca tccatagttg cctgactccc cgtcgtgtag 2460ataactacga tacgggaggg
cttaccatct ggccccagtg ctgcaatgat accgcgagac 2520ccacgctcac cggctccaga
tttatcagca ataaaccagc cagccggaag ggccgagcgc 2580agaagtggtc ctgcaacttt
atccgcctcc atccagtcta ttaattgttg ccgggaagct 2640agagtaagta gttcgccagt
taatagtttg cgcaacgttg ttgccattgc tacaggcatc 2700gtggtgtcac gctcgtcgtt
tggtatggct tcattcagct ccggttccca acgatcaagg 2760cgagttacat gatcccccat
gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 2820gttgtcagaa gtaagttggc
cgcagtgtta tcactcatgg ttatggcagc actgcataat 2880tctcttactg tcatgccatc
cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 2940tcattctgag aatagtgtat
gcggcgaccg agttgctctt gcccggcgtc aatacgggat 3000aataccgcgc cacatagcag
aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 3060cgaaaactct caaggatctt
accgctgttg agatccagtt cgatgtaacc cactcgtgca 3120cccaactgat cttcagcatc
ttttactttc accagcgttt ctgggtgagc aaaaacagga 3180aggcaaaatg ccgcaaaaaa
gggaataagg gcgacacgga aatgttgaat actcatactc 3240ttcctttttc aatattattg
aagcatttat cagggttatt gtctcatgag cggatacata 3300tttgaatgta tttagaaaaa
taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 3360ccacctgacg cgccctgtag
cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc 3420gtgaccgcta cacttgccag
cgccctagcg cccgctcctt tcgctttctt cccttccttt 3480ctcgccacgt tcgccggctt
tccccgtcaa gctctaaatc gggggctccc tttagggttc 3540cgatttagtg ctttacggca
cctcgacccc aaaaaacttg attagggtga tggttcacgt 3600agtgggccat cgccctgata
gacggttttt cgccctttga cgttggagtc cacgttcttt 3660aatagtggac tcttgttcca
aactggaaca acactcaacc ctatctcggt ctattctttt 3720gatttataag ggattttgcc
gatttcggcc tattggttaa aaaatgagct gatttaacaa 3780aaatttaacg cgaattttaa
caaaatatta acgcttacaa tttccattcg ccattcaggc 3840tgcgcaactg ttgggaaggg
cgatcggtgc gggcctcttc gctattacgc cagctggcga 3900aagggggatg tgctgcaagg
cgattaagtt gggtaacgcc agggttttcc cagtcacgac 3960gttgtaaaac gacggccagt
gaattgtaat acgactcact atagggcgaa ttgggtaccg 4020ggccccccct cgaggtcgat
ggtgtcgata agcttgatat cgaattcatg tcacacaaac 4080cgatcttcgc ctcaaggaaa
cctaattcta catccgagag actgccgaga tccagtctac 4140actgattaat tttcgggcca
ataatttaaa aaaatcgtgt tatataatat tatatgtatt 4200atatatatac atcatgatga
tactgacagt catgtcccat tgctaaatag acagactcca 4260tctgccgcct ccaactgatg
ttctcaatat ttaaggggtc atctcgcatt gtttaataat 4320aaacagactc catctaccgc
ctccaaatga tgttctcaaa atatattgta tgaacttatt 4380tttattactt agtattatta
gacaacttac ttgctttatg aaaaacactt cctatttagg 4440aaacaattta taatggcagt
tcgttcattt aacaatttat gtagaataaa tgttataaat 4500gcgtatggga aatcttaaat
atggatagca taaatgatat ctgcattgcc taattcgaaa 4560tcaacagcaa cgaaaaaaat
cccttgtaca acataaatag tcatcgagaa atatcaacta 4620tcaaagaaca gctattcaca
cgttactatt gagattatta ttggacgaga atcacacact 4680caactgtctt tctctcttct
agaaatacag gtacaagtat gtactattct cattgttcat 4740acttctagtc atttcatccc
acatattcct tggatttctc tccaatgaat gacattctat 4800cttgcaaatt caacaattat
aataagatat accaaagtag cggtatagtg gcaatcaaaa 4860agcttctctg gtgtgcttct
cgtatttatt tttattctaa tgatccatta aaggtatata 4920tttatttctt gttatataat
ccttttgttt attacatggg ctggatacat aaaggtattt 4980tgatttaatt ttttgcttaa
attcaatccc ccctcgttca gtgtcaactg taatggtagg 5040aaattaccat acttttgaag
aagcaaaaaa aatgaaagaa aaaaaaaatc gtatttccag 5100gttagacgtt ccgcagaatc
tagaatgcgg tatgcggtac attgttcttc gaacgtaaaa 5160gttgcgctcc ctgagatatt
gtacattttt gcttttacaa gtacaagtac atcgtacaac 5220tatgtactac tgttgatgca
tccacaacag tttgttttgt ttttttttgt tttttttttt 5280tctaatgatt cattaccgct
atgtatacct acttgtactt gtagtaagcc gggttattgg 5340cgttcaatta atcatagact
tatgaatctg cacggtgtgc gctgcgagtt acttttagct 5400tatgcatgct acttgggtgt
aatattggga tctgttcgga aatcaacgga tgctcaaccg 5460atttcgacag taattaatta
agtcatacac aagtcagctt tcttcgagcc tcatataagt 5520ataagtagtt caacgtatta
gcactgtacc cagcatctcc gtatcgagaa acacaacaac 5580atgccccatt ggacagatca
tgcggataca caggttgtgc agtatcatac atactcgatc 5640agacaggtcg tctgaccatc
atacaagctg aacaagcgct ccatacttgc acgctctcta 5700tatacacagt taaattacat
atccatagtc taacctctaa cagttaatct tctggtaagc 5760ctcccagcca gccttctggt
atcgcttggc ctcctcaata ggatctcggt tctggccgta 5820cagacctcgg ccgacaatta
tgatatccgt tccggtagac atgacatcct caacagttcg 5880gtactgctgt ccgagagcgt
ctcccttgtc gtcaagaccc accccggggg tcagaataag 5940ccagtcctca gagtcgccct
taggtcggtt ctgggcaatg aagccaacca caaactcggg 6000gtcggatcgg gcaagctcaa
tggtctgctt ggagtactcg ccagtggcca gagagccctt 6060gcaagacagc tcggccagca
tgagcagacc tctggccagc ttctcgttgg gagaggggac 6120taggaactcc ttgtactggg
agttctcgta gtcagagacg tcctccttct tctgttcaga 6180gacagtttcc tcggcaccag
ctcgcaggcc agcaatgatt ccggttccgg gtacaccgtg 6240ggcgttggtg atatcggacc
actcggcgat tcggtgacac cggtactggt gcttgacagt 6300gttgccaata tctgcgaact
ttctgtcctc gaacaggaag aaaccgtgct taagagcaag 6360ttccttgagg gggagcacag
tgccggcgta ggtgaagtcg tcaatgatgt cgatatgggt 6420tttgatcatg cacacataag
gtccgacctt atcggcaagc tcaatgagct ccttggtggt 6480ggtaacatcc agagaagcac
acaggttggt tttcttggct gccacgagct tgagcactcg 6540agcggcaaag gcggacttgt
ggacgttagc tcgagcttcg taggagggca ttttggtggt 6600gaagaggaga ctgaaataaa
tttagtctgc agaacttttt atcggaacct tatctggggc 6660agtgaagtat atgttatggt
aatagttacg agttagttga acttatagat agactggact 6720atacggctat cggtccaaat
tagaaagaac gtcaatggct ctctgggcgt cgcctttgcc 6780gacaaaaatg tgatcatgat
gaaagccagc aatgacgttg cagctgatat tgttgtcggc 6840caaccgcgcc gaaaacgcag
ctgtcagacc cacagcctcc aacgaagaat gtatcgtcaa 6900agtgatccaa gcacactcat
agttggagtc gtactccaaa ggcggcaatg acgagtcaga 6960cagatactcg
6970318253DNAArtificial
Sequenceplasmid pY118 31ggccgccacc gcggcccgag attccggcct cttcggccgc
caagcgaccc gggtggacgt 60ctagaggtac ctagcaatta acagatagtt tgccggtgat
aattctctta acctcccaca 120ctcctttgac ataacgattt atgtaacgaa actgaaattt
gaccagatat tgtgtccgcg 180gtggagctcc agcttttgtt ccctttagtg agggttaatt
tcgagcttgg cgtaatcatg 240gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
attccacaca acgtacgagc 300cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
agctaactca cattaattgc 360gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
tgccagctgc attaatgaat 420cggccaacgc gcggggagag gcggtttgcg tattgggcgc
tcttccgctt cctcgctcac 480tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
tcagctcact caaaggcggt 540aatacggtta tccacagaat caggggataa cgcaggaaag
aacatgtgag caaaaggcca 600gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
tttttccata ggctccgccc 660ccctgacgag catcacaaaa atcgacgctc aagtcagagg
tggcgaaacc cgacaggact 720ataaagatac caggcgtttc cccctggaag ctccctcgtg
cgctctcctg ttccgaccct 780gccgcttacc ggatacctgt ccgcctttct cccttcggga
agcgtggcgc tttctcatag 840ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
tccaagctgg gctgtgtgca 900cgaacccccc gttcagcccg accgctgcgc cttatccggt
aactatcgtc ttgagtccaa 960cccggtaaga cacgacttat cgccactggc agcagccact
ggtaacagga ttagcagagc 1020gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
cctaactacg gctacactag 1080aaggacagta tttggtatct gcgctctgct gaagccagtt
accttcggaa aaagagttgg 1140tagctcttga tccggcaaac aaaccaccgc tggtagcggt
ggtttttttg tttgcaagca 1200gcagattacg cgcagaaaaa aaggatctca agaagatcct
ttgatctttt ctacggggtc 1260tgacgctcag tggaacgaaa actcacgtta agggattttg
gtcatgagat tatcaaaaag 1320gatcttcacc tagatccttt taaattaaaa atgaagtttt
aaatcaatct aaagtatata 1380tgagtaaact tggtctgaca gttaccaatg cttaatcagt
gaggcaccta tctcagcgat 1440ctgtctattt cgttcatcca tagttgcctg actccccgtc
gtgtagataa ctacgatacg 1500ggagggctta ccatctggcc ccagtgctgc aatgataccg
cgagacccac gctcaccggc 1560tccagattta tcagcaataa accagccagc cggaagggcc
gagcgcagaa gtggtcctgc 1620aactttatcc gcctccatcc agtctattaa ttgttgccgg
gaagctagag taagtagttc 1680gccagttaat agtttgcgca acgttgttgc cattgctaca
ggcatcgtgg tgtcacgctc 1740gtcgtttggt atggcttcat tcagctccgg ttcccaacga
tcaaggcgag ttacatgatc 1800ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
ccgatcgttg tcagaagtaa 1860gttggccgca gtgttatcac tcatggttat ggcagcactg
cataattctc ttactgtcat 1920gccatccgta agatgctttt ctgtgactgg tgagtactca
accaagtcat tctgagaata 1980gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
cgggataata ccgcgccaca 2040tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
tcggggcgaa aactctcaag 2100gatcttaccg ctgttgagat ccagttcgat gtaacccact
cgtgcaccca actgatcttc 2160agcatctttt actttcacca gcgtttctgg gtgagcaaaa
acaggaaggc aaaatgccgc 2220aaaaaaggga ataagggcga cacggaaatg ttgaatactc
atactcttcc tttttcaata 2280ttattgaagc atttatcagg gttattgtct catgagcgga
tacatatttg aatgtattta 2340gaaaaataaa caaatagggg ttccgcgcac atttccccga
aaagtgccac ctgacgcgcc 2400ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
cgcagcgtga ccgctacact 2460tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
tcctttctcg ccacgttcgc 2520cggctttccc cgtcaagctc taaatcgggg gctcccttta
gggttccgat ttagtgcttt 2580acggcacctc gaccccaaaa aacttgatta gggtgatggt
tcacgtagtg ggccatcgcc 2640ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
ttctttaata gtggactctt 2700gttccaaact ggaacaacac tcaaccctat ctcggtctat
tcttttgatt tataagggat 2760tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
taacaaaaat ttaacgcgaa 2820ttttaacaaa atattaacgc ttacaatttc cattcgccat
tcaggctgcg caactgttgg 2880gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
tggcgaaagg gggatgtgct 2940gcaaggcgat taagttgggt aacgccaggg ttttcccagt
cacgacgttg taaaacgacg 3000gccagtgaat tgtaatacga ctcactatag ggcgaattgg
gtaccgggcc ccccctcgag 3060gtcgatggtg tcgataagct tgatatcgaa ttcatgtcac
acaaaccgat cttcgcctca 3120aggaaaccta attctacatc cgagagactg ccgagatcca
gtctacactg attaattttc 3180gggccaataa tttaaaaaaa tcgtgttata taatattata
tgtattatat atatacatca 3240tgatgatact gacagtcatg tcccattgct aaatagacag
actccatctg ccgcctccaa 3300ctgatgttct caatatttaa ggggtcatct cgcattgttt
aataataaac agactccatc 3360taccgcctcc aaatgatgtt ctcaaaatat attgtatgaa
cttattttta ttacttagta 3420ttattagaca acttacttgc tttatgaaaa acacttccta
tttaggaaac aatttataat 3480ggcagttcgt tcatttaaca atttatgtag aataaatgtt
ataaatgcgt atgggaaatc 3540ttaaatatgg atagcataaa tgatatctgc attgcctaat
tcgaaatcaa cagcaacgaa 3600aaaaatccct tgtacaacat aaatagtcat cgagaaatat
caactatcaa agaacagcta 3660ttcacacgtt actattgaga ttattattgg acgagaatca
cacactcaac tgtctttctc 3720tcttctagaa atacaggtac aagtatgtac tattctcatt
gttcatactt ctagtcattt 3780catcccacat attccttgga tttctctcca atgaatgaca
ttctatcttg caaattcaac 3840aattataata agatatacca aagtagcggt atagtggcaa
tcaaaaagct tctctggtgt 3900gcttctcgta tttattttta ttctaatgat ccattaaagg
tatatattta tttcttgtta 3960tataatcctt ttgtttatta catgggctgg atacataaag
gtattttgat ttaatttttt 4020gcttaaattc aatcccccct cgttcagtgt caactgtaat
ggtaggaaat taccatactt 4080ttgaagaagc aaaaaaaatg aaagaaaaaa aaaatcgtat
ttccaggtta gacgttccgc 4140agaatctaga atgcggtatg cggtacattg ttcttcgaac
gtaaaagttg cgctccctga 4200gatattgtac atttttgctt ttacaagtac aagtacatcg
tacaactatg tactactgtt 4260gatgcatcca caacagtttg ttttgttttt ttttgttttt
tttttttcta atgattcatt 4320accgctatgt atacctactt gtacttgtag taagccgggt
tattggcgtt caattaatca 4380tagacttatg aatctgcacg gtgtgcgctg cgagttactt
ttagcttatg catgctactt 4440gggtgtaata ttgggatctg ttcggaaatc aacggatgct
caaccgattt cgacagtaat 4500taattaagtc atacacaagt cagctttctt cgagcctcat
ataagtataa gtagttcaac 4560gtattagcac tgtacccagc atctccgtat cgagaaacac
aacaacatgc cccattggac 4620agatcatgcg gatacacagg ttgtgcagta tcatacatac
tcgatcagac aggtcgtctg 4680accatcatac aagctgaaca agcgctccat acttgcacgc
tctctatata cacagttaaa 4740ttacatatcc atagtctaac ctctaacagt taatcttctg
gtaagcctcc cagccagcct 4800tctggtatcg cttggcctcc tcaataggat ctcggttctg
gccgtacaga cctcggccga 4860caattatgat atccgttccg gtagacatga catcctcaac
agttcggtac tgctgtccga 4920gagcgtctcc cttgtcgtca agacccaccc cgggggtcag
aataagccag tcctcagagt 4980cgcccttagg tcggttctgg gcaatgaagc caaccacaaa
ctcggggtcg gatcgggcaa 5040gctcaatggt ctgcttggag tactcgccag tggccagaga
gcccttgcaa gacagctcgg 5100ccagcatgag cagacctctg gccagcttct cgttgggaga
ggggactagg aactccttgt 5160actgggagtt ctcgtagtca gagacgtcct ccttcttctg
ttcagagaca gtttcctcgg 5220caccagctcg caggccagca atgattccgg ttccgggtac
accgtgggcg ttggtgatat 5280cggaccactc ggcgattcgg tgacaccggt actggtgctt
gacagtgttg ccaatatctg 5340cgaactttct gtcctcgaac aggaagaaac cgtgcttaag
agcaagttcc ttgaggggga 5400gcacagtgcc ggcgtaggtg aagtcgtcaa tgatgtcgat
atgggttttg atcatgcaca 5460cataaggtcc gaccttatcg gcaagctcaa tgagctcctt
ggtggtggta acatccagag 5520aagcacacag gttggttttc ttggctgcca cgagcttgag
cactcgagcg gcaaaggcgg 5580acttgtggac gttagctcga gcttcgtagg agggcatttt
ggtggtgaag aggagactga 5640aataaattta gtctgcagaa ctttttatcg gaaccttatc
tggggcagtg aagtatatgt 5700tatggtaata gttacgagtt agttgaactt atagatagac
tggactatac ggctatcggt 5760ccaaattaga aagaacgtca atggctctct gggcgtcgcc
tttgccgaca aaaatgtgat 5820catgatgaaa gccagcaatg acgttgcagc tgatattgtt
gtcggccaac cgcgccgaaa 5880acgcagctgt cagacccaca gcctccaacg aagaatgtat
cgtcaaagtg atccaagcac 5940actcatagtt ggagtcgtac tccaaaggcg gcaatgacga
gtcagacaga tactcgtcga 6000cgcagtagga tgtcctgcac gggtcttttt gtggggtgtg
gagaaagggg tgcttggaga 6060tggaagccgg tagaaccggg ctgcttgtgc ttggagatgg
aagccggtag aaccgggctg 6120cttgggggga tttggggccg ctgggctcca aagaggggta
ggcatttcgt tggggttacg 6180taattgcggc atttgggtcc tgcgcgcatg tcccattggt
cagaattagt ccggatagga 6240gacttatcag ccaatcacag cgccggatcc acctgtaggt
tgggttgggt gggagcaccc 6300ctccacagag tagagtcaaa cagcagcagc aacatgatag
ttgggggtgt gcgtgttaaa 6360ggaaaaaaaa gaagcttggg ttatattccc gctctattta
gaggttgcgg gatagacgcc 6420gacggagggc aatggcgcca tggaaccttg cggatatcga
tacgccgcgg cggactgcgt 6480ccgaaccagc tccagcagcg ttttttccgg gccattgagc
cgactgcgac cccgccaacg 6540tgtcttggcc cacgcactca tgtcatgttg gtgttgggag
gccacttttt aagtagcaca 6600aggcacctag ctcgcagcaa ggtgtccgaa ccaaagaagc
ggctgcagtg gtgcaaacgg 6660ggcggaaacg gcgggaaaaa gccacggggg cacgaattga
ggcacgccct cgaatttgag 6720acgagtcacg gccccattcg cccgcgcaat ggctcgccaa
cgcccggtct tttgcaccac 6780atcaggttac cccaagccaa acctttgtgt taaaaagctt
aacatattat accgaacgta 6840ggtttgggcg ggcttgctcc gtctgtccaa ggcaacattt
atataagggt ctgcatcgcc 6900ggctcaattg aatctttttt cttcttctct tctctatatt
cattcttgaa ttaaacacac 6960atcaatccgc ggccgcacca tgggcaaggg tggagacggc
ggcgcgcagg cggtgagcgg 7020gaccgacgcg tctctcgctg aggtgagctc cgtcgatagc
aagagcgtgc acgtcgtgct 7080ctacggcaag cgcgtggatg tcacaaagtt ccagaaggca
cacccgggcg ggagcaaggt 7140gttccgcatc ttccaggagc gcgacgcgac ggagcagttc
gagtcttacc actcgcccaa 7200ggccatcaag atgatggagg gcatgctcaa gaagtcggag
gatgcgcccg cttccgtgcc 7260cctgccctcg cggtccacca tgggcacgga gttcaaggag
atgattgagc gccacaagag 7320ggctggtctc tacgaccctt gcccgttgga cgagctgttc
aagctcacca tcgtccttgc 7380gcccatcttc gtgggcgcct atctcgtgcg gagcggcgtc
tcgcccctcg cgggcgcgct 7440ctccatgggc tttggcttct acctcgacgg ctggcttgct
cacgactacc tgcatcacgc 7500agtcttcaag ggctcggtca acacgctcgt caaggcgaac
aacgccatgg gatacgccct 7560cggcttcctc cagggctacg acgtggcctg gtggcgcgcg
cgccataaca cgcaccacgt 7620gtgcaccaac gaggatggtt cggacccgga catcaagacg
gcgcccctgc tcatctacgt 7680gcgagagaac ccgtccattg ccaagcggct caacttcttc
cagcgctggc agcagtacta 7740ctatgtgccg accatggcca tcctcgacct ctactggcgc
ctggagtcca tcgcgtacgt 7800ggctgtgcgc ctgcctaaga tgtggatgca ggccgccgct
cttgccgctc actacgcgct 7860cctgtgctgg gtcttcgcag cgcatctcaa cctcatccct
ctcatgatgg ttgcacgcgg 7920cttcgcgacg ggcatcgttg tctttgcaac ccactatggt
gaggacatcc tcgaccgcga 7980gcacgtcgag ggcatgacgc tcgtcgagca gaccgccaag
acctcccgta acatcacggg 8040cggctggcta gtgaacgtgc tcacgggctt catctccctg
cagaccgagc atcacctctt 8100ccccatgatg cccaccggca acctaatgac tatccagccc
gaggtacgcg acttcttcaa 8160gaagcatggc ctcgagtacc gcgagggcaa cctcttccag
tgcgtgcacc agaacatcaa 8220ggctctcgcc ttcgagcacc tcctccactg agc
825332777DNAEuglena gracilis 32atggaggtgg
tgaatgaaat agtctcaatt gggcaggaag ttttacccaa agttgattat 60gcccaactct
ggagtgatgc cagtcactgt gaggtgcttt acttgtccat cgcatttgtc 120atcttgaagt
tcactcttgg cccccttggt ccaaaaggtc agtctcgtat gaagtttgtt 180ttcaccaatt
acaaccttct catgtccatt tattcgttgg gatcattcct ctcaatggca 240tatgccatgt
acaccatcgg tgttatgtct gacaactgcg agaaggcttt tgacaacaac 300gtcttcagga
tcaccacgca gttgttctat ttgagcaagt tcctggagta tattgactcc 360ttctatttgc
cactgatggg caagcctctg acctggttgc aattcttcca tcatttgggg 420gcaccgatgg
atatgtggct gttctataat taccgaaatg aagctgtttg gatttttgtg 480ctgttgaatg
gtttcatcca ctggatcatg tacggttatt attggaccag attgatcaag 540ctgaagttcc
ccatgccaaa atccctgatt acatcaatgc agatcattca attcaatgtt 600ggtttctaca
ttgtctggaa gtacaggaac attccctgtt atcgccaaga tgggatgagg 660atgtttggct
ggttcttcaa ttacttttat gttggcacag tcttgtgttt gttcttgaat 720ttctatgtgc
aaacgtatat cgtcaggaag cacaagggag ccaaaaagat tcagtga
7773330DNAArtificial Sequencesense oligonucleotide oEugEL1-1 primer
33agcggccgca ccatggaggt ggtgaatgaa
303430DNAArtificial Sequencesense oligonucleotide oEugEL1-2 primer
34tgcggccgct cactgaatct ttttggctcc
30354311DNAArtificial Sequenceplasmid pKR906 35agcggccgca ccatggaggt
ggtgaatgaa atagtctcaa ttgggcagga agttttaccc 60aaagttgatt atgcccaact
ctggagtgat gccagtcact gtgaggtgct ttacttgtcc 120atcgcatttg tcatcttgaa
gttcactctt ggcccccttg gtccaaaagg tcagtctcgt 180atgaagtttg ttttcaccaa
ttacaacctt ctcatgtcca tttattcgtt gggatcattc 240ctctcaatgg catatgccat
gtacaccatc ggtgttatgt ctgacaactg cgagaaggct 300tttgacaaca acgtcttcag
gatcaccacg cagttgttct atttgagcaa gttcctggag 360tatattgact ccttctattt
gccactgatg ggcaagcctc tgacctggtt gcaattcttc 420catcatttgg gggcaccgat
ggatatgtgg ctgttctata attaccgaaa tgaagctgtt 480tggatttttg tgctgttgaa
tggtttcatc cactggatca tgtacggtta ttattggacc 540agattgatca agctgaagtt
ccccatgcca aaatccctga ttacatcaat gcagatcatt 600caattcaatg ttggtttcta
cattgtctgg aagtacagga acattccctg ttatcgccaa 660gatgggatga ggatgtttgg
ctggttcttc aattactttt atgttggcac agtcttgtgt 720ttgttcttga atttctatgt
gcaaacgtat atcgtcagga agcacaaggg agccaaaaag 780attcagtgag cggccgcacc
tgaattccag cacactggcg gccgttacta gtggatccga 840gctcggtacc aagcttgatg
catagcttga gtattctaac gcgtcaccta aatagcttgg 900cgtaatcatg gtcatagctg
tttcctgtgt gaaattgtta tccgctcaca attccacaca 960acatacgagc cggaagcata
aagtgtaaag cctggggtgc ctaatgagtg agctaactca 1020cattaattgc gttgcgctca
ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 1080attaatgaat cggccaacgc
gcggggagag gcggtttgcg tattgggcgc tcttccgctt 1140cctcgctcac tgactcgctg
cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 1200caaaggcggt aatacggtta
tccacagaat caggggataa cgcaggaaag aacatgtgag 1260caaaaggcca gcaaaagccc
aggaaccgta aaaaggccgc gttgctggcg tttttccata 1320ggctccgccc ccctgacgag
catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 1380cgacaggact ataaagatac
caggcgtttc cccctggaag ctccctcgtg cgctctcctg 1440ttccgaccct gccgcttacc
ggatacctgt ccgcctttct cccttcggga agcgtggcgc 1500tttctcatag ctcacgctgt
aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 1560gctgtgtgca cgaacccccc
gttcagcccg accgctgcgc cttatccggt aactatcgtc 1620ttgagtccaa cccggtaaga
cacgacttat cgccactggc agcagccact ggtaacagga 1680ttagcagagc gaggtatgta
ggcggtgcta cagagttctt gaagtggtgg cctaactacg 1740gctacactag aaggacagta
tttggtatct gcgctctgct gaagccagtt accttcggaa 1800aaagagttgg tagctcttga
tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 1860tttgcaagca gcagattacg
cgcagaaaaa aaggatctca agaagatcct ttgatctttt 1920ctacggggtc tgacgctcag
tggaacgaaa actcacgtta agggattttg gtcatgagat 1980tatcaaaaag gatcttcacc
tagatccttt taaattaaaa atgaagtttt agcacgtgtc 2040agtcctgctc ctcggccacg
aagtgcacgc agttgccggc cgggtcgcgc agggcgaact 2100cccgccccca cggctgctcg
ccgatctcgg tcatggccgg cccggaggcg tcccggaagt 2160tcgtggacac gacctccgac
cactcggcgt acagctcgtc caggccgcgc acccacaccc 2220aggccagggt gttgtccggc
accacctggt cctggaccgc gctgatgaac agggtcacgt 2280cgtcccggac cacaccggcg
aagtcgtcct ccacgaagtc ccgggagaac ccgagccggt 2340cggtccagaa ctcgaccgct
ccggcgacgt cgcgcgcggt gagcaccgga acggcactgg 2400tcaacttggc catggtggcc
ctcctcacgt gctattattg aagcatttat cagggttatt 2460gtctcatgag cggatacata
tttgaatgta tttagaaaaa taaacaaata ggggttccgc 2520gcacatttcc ccgaaaagtg
ccacctgtat gcggtgtgaa ataccgcaca gatgcgtaag 2580gagaaaatac cgcatcagga
aattgtaagc gttaataatt cagaagaact cgtcaagaag 2640gcgatagaag gcgatgcgct
gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg 2700gtcagcccat tcgccgccaa
gctcttcagc aatatcacgg gtagccaacg ctatgtcctg 2760atagcggtcc gccacaccca
gccggccaca gtcgatgaat ccagaaaagc ggccattttc 2820caccatgata ttcggcaagc
aggcatcgcc atgggtcacg acgagatcct cgccgtcggg 2880catgctcgcc ttgagcctgg
cgaacagttc ggctggcgcg agcccctgat gctcttcgtc 2940cagatcatcc tgatcgacaa
gaccggcttc catccgagta cgtgctcgct cgatgcgatg 3000tttcgcttgg tggtcgaatg
ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc 3060atcagccatg atggatactt
tctcggcagg agcaaggtga gatgacagga gatcctgccc 3120cggcacttcg cccaatagca
gccagtccct tcccgcttca gtgacaacgt cgagcacagc 3180tgcgcaagga acgcccgtcg
tggccagcca cgatagccgc gctgcctcgt cttgcagttc 3240attcagggca ccggacaggt
cggtcttgac aaaaagaacc gggcgcccct gcgctgacag 3300ccggaacacg gcggcatcag
agcagccgat tgtctgttgt gcccagtcat agccgaatag 3360cctctccacc caagcggccg
gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa 3420cgatcctcat cctgtctctt
gatcagagct tgatcccctg cgccatcaga tccttggcgg 3480cgagaaagcc atccagttta
ctttgcaggg cttcccaacc ttaccagagg gcgccccagc 3540tggcaattcc ggttcgcttg
ctgtccataa aaccgcccag tctagctatc gccatgtaag 3600cccactgcaa gctacctgct
ttctctttgc gcttgcgttt tcccttgtcc agatagccca 3660gtagctgaca ttcatccggg
gtcagcaccg tttctgcgga ctggctttct acgtgaaaag 3720gatctaggtg aagatccttt
ttgataatct catgcctgac atttatattc cccagaacat 3780caggttaatg gcgtttttga
tgtcattttc gcggtggctg agatcagcca cttcttcccc 3840gataacggag accggcacac
tggccatatc ggtggtcatc atgcgccagc tttcatcccc 3900gatatgcacc accgggtaaa
gttcacggga gactttatct gacagcagac gtgcactggc 3960cagggggatc accatccgtc
gccccggcgt gtcaataata tcactctgta catccacaaa 4020cagacgataa cggctctctc
ttttataggt gtaaacctta aactgccgta cgtataggct 4080gcgcaactgt tgggaagggc
gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 4140agggggatgt gctgcaaggc
gattaagttg ggtaacgcca gggttttccc agtcacgacg 4200ttgtaaaacg acggccagtg
aattgtaata cgactcacta tagggcgaat tgggccctct 4260agatgcatgc tcgagcggcc
gccagtgtga tggatatctg cagaattcag g 4311363983DNAArtificial
Sequenceplasmid pKR132 36ctagagtcga cctgcaggca tgcaagcttg gcgtaatcat
ggtcatagct gtttcctgtg 60tgaaattgtt atccgctcac aattccacac aacatacgag
ccggaagcat aaagtgtaaa 120gcctggggtg cctaatgagt gagctaactc acattaattg
cgttgcgctc actgcccgct 180ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
tcggccaacg cgcggggaga 240ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
ctgactcgct gcgctcggtc 300gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
taatacggtt atccacagaa 360tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
agcaaaaggc caggaaccgt 420aaaaaggccg cgttgctggc gtttttccat aggctccgcc
cccctgacga gcatcacaaa 480aatcgacgct caagtcagag gtggcgaaac ccgacaggac
tataaagata ccaggcgttt 540ccccctggaa gctccctcgt gcgctctcct gttccgaccc
tgccgcttac cggatacctg 600tccgcctttc tcccttcggg aagcgtggcg ctttctcata
gctcacgctg taggtatctc 660agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
acgaaccccc cgttcagccc 720gaccgctgcg ccttatccgg taactatcgt cttgagtcca
acccggtaag acacgactta 780tcgccactgg cagcagccac tggtaacagg attagcagag
cgaggtatgt aggcggtgct 840acagagttct tgaagtggtg gcctaactac ggctacacta
gaaggacagt atttggtatc 900tgcgctctgc tgaagccagt taccttcgga aaaagagttg
gtagctcttg atccggcaaa 960caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
agcagattac gcgcagaaaa 1020aaaggatctc aagaagatcc tttgatcttt tctacggggt
ctgacgctca gtggaacgaa 1080aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
ggatcttcac ctagatcctt 1140ttaaattaaa aatgaagttt taaatcaatc taaagtatat
atgagtaaac ttggtctgac 1200agttaccaat gcttaatcag tgaggcacct atctcagcga
tctgtctatt tcgttcatcc 1260atagttgcct gactccccgt cgtgtagata actacgatac
gggagggctt accatctggc 1320cccagtgctg caatgatacc gcgagaccca cgctcaccgg
ctccagattt atcagcaata 1380aaccagccag ccggaagggc cgagcgcaga agtggtcctg
caactttatc cgcctccatc 1440cagtctatta attgttgccg ggaagctaga gtaagtagtt
cgccagttaa tagtttgcgc 1500aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
cgtcgtttgg tatggcttca 1560ttcagctccg gttcccaacg atcaaggcga gttacatgat
cccccatgtt gtgcaaaaaa 1620gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
agttggccgc agtgttatca 1680ctcatggtta tggcagcact gcataattct cttactgtca
tgccatccgt aagatgcttt 1740tctgtgactg gtgagtactc aaccaagtca ttctgagaat
agtgtatgcg gcgaccgagt 1800tgctcttgcc cggcgtcaat acgggataat accgcgccac
atagcagaac tttaaaagtg 1860ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
ggatcttacc gctgttgaga 1920tccagttcga tgtaacccac tcgtgcaccc aactgatctt
cagcatcttt tactttcacc 1980agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
caaaaaaggg aataagggcg 2040acacggaaat gttgaatact catactcttc ctttttcaat
attattgaag catttatcag 2100ggttattgtc tcatgagcgg atacatattt gaatgtattt
agaaaaataa acaaataggg 2160gttccgcgca catttccccg aaaagtgcca cctgacgtct
aagaaaccat tattatcatg 2220acattaacct ataaaaatag gcgtatcacg aggccctttc
gtctcgcgcg tttcggtgat 2280gacggtgaaa acctctgaca catgcagctc ccggagacgg
tcacagcttg tctgtaagcg 2340gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
gtgttggcgg gtgtcggggc 2400tggcttaact atgcggcatc agagcagatt gtactgagag
tgcaccatat gcggtgtgaa 2460ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc
gccattcgcc attcaggctg 2520cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
tattacgcca gctggcgaaa 2580gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
ggttttccca gtcacgacgt 2640tgtaaaacga cggccagtga attcgagctc ggtacccggg
gatcctctag acctgcaggc 2700caactgcgtt tggggctcca gattaaacga cgccgtttcg
ttcctttcgc ttcacggctt 2760aacgatgtcg tttctgtctg tgcccaaaaa ataaaggcat
ttgttatttg caccagatat 2820ttactaagtg caccctagtt tgacaagtag gcgataatta
caaatagatg cggtgcaaat 2880aataaatttt gaaggaaata attacaaaag aacagaactt
atatttactt tattttaaaa 2940aactaaaatg aaagaacaaa aaaagtaaaa aatacaaaaa
atgtgcttta accactttca 3000ttatttgtta cagaaagtat gattctactc aaattgatct
gttgtatctg gtgctgcctt 3060gtcacactgg cgatttcaat cccctaaaga tatggtgcaa
actgcgaagt gatcaatatc 3120tgctcggtta atttagatta attaataata ttcaacgtga
tgtaccaaaa aaagacaatt 3180ttttgctcca ttgacaaatt aaacctcatc aaggtaattt
ccaaacctat aagcaaaaaa 3240atttcacatt aattggcccg caatcctatt agtcttatta
tactagagta ggaaaaaaaa 3300caattacaca acttgtctta ttattctcta tgctaatgaa
tatttttccc ttttgttaga 3360aatcagtgtt tcctaattta ttgagtatta attccactca
ccgcatatat ttaccgttga 3420ataagaaaat tttacacata attcttttta agataaataa
tttttttata ctagatctta 3480tatgattacg tgaagccaag tgggttatac taatgatata
taatgtttga tagtaatcag 3540tttataaacc aaatgcatgg aaatgttacg tggaagcacg
taaattaaca agcattgaag 3600caaatgcagc caccgcacca aaaccacccc acttcacttc
cacgtaccat attccatgca 3660actacaacac cctaaaactt caataaatgc ccccaccttc
acttcacttc acccatcaat 3720agcaagcggc cgcgaagtta aaagcaatgt tgtcacttgt
cgtactaaca catgatgtga 3780tagtttatgc tagctagcta taacataagc tgtctctgag
tgtgttgtat attaataaag 3840atcatcactg gtgaatggtg atcgtgtacg taccctactt
agtaggcaat ggaagcactt 3900agagtgtgct ttgtgcatgg ccttgcctct gttttgagac
ttttgtaatg ttttcgagtt 3960taaatctttg cctttgcgta cgt
3983374771DNAArtificial Sequenceplasmid pKR953
37ggccgcgaag ttaaaagcaa tgttgtcact tgtcgtacta acacatgatg tgatagttta
60tgctagctag ctataacata agctgtctct gagtgtgttg tatattaata aagatcatca
120ctggtgaatg gtgatcgtgt acgtacccta cttagtaggc aatggaagca cttagagtgt
180gctttgtgca tggccttgcc tctgttttga gacttttgta atgttttcga gtttaaatct
240ttgcctttgc gtacgtctag agtcgacctg caggcatgca agcttggcgt aatcatggtc
300atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
360aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
420gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
480ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
540ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
600acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
660aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
720tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
780aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
840gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
900acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
960accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
1020ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
1080gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
1140gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
1200ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
1260gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
1320cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
1380cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
1440gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
1500tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
1560gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
1620agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
1680tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
1740agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
1800gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
1860catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
1920ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
1980atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
2040tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
2100cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
2160cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
2220atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
2280aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
2340ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
2400aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga
2460aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct
2520cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
2580agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
2640tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
2700ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgcca
2760ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
2820acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt
2880ttcccagtca cgacgttgta aaacgacggc cagtgaattc gagctcggta cccggggatc
2940ctctagacct gcaggccaac tgcgtttggg gctccagatt aaacgacgcc gtttcgttcc
3000tttcgcttca cggcttaacg atgtcgtttc tgtctgtgcc caaaaaataa aggcatttgt
3060tatttgcacc agatatttac taagtgcacc ctagtttgac aagtaggcga taattacaaa
3120tagatgcggt gcaaataata aattttgaag gaaataatta caaaagaaca gaacttatat
3180ttactttatt ttaaaaaact aaaatgaaag aacaaaaaaa gtaaaaaata caaaaaatgt
3240gctttaacca ctttcattat ttgttacaga aagtatgatt ctactcaaat tgatctgttg
3300tatctggtgc tgccttgtca cactggcgat ttcaatcccc taaagatatg gtgcaaactg
3360cgaagtgatc aatatctgct cggttaattt agattaatta ataatattca acgtgatgta
3420ccaaaaaaag acaatttttt gctccattga caaattaaac ctcatcaagg taatttccaa
3480acctataagc aaaaaaattt cacattaatt ggcccgcaat cctattagtc ttattatact
3540agagtaggaa aaaaaacaat tacacaactt gtcttattat tctctatgct aatgaatatt
3600tttccctttt gttagaaatc agtgtttcct aatttattga gtattaattc cactcaccgc
3660atatatttac cgttgaataa gaaaatttta cacataattc tttttaagat aaataatttt
3720tttatactag atcttatatg attacgtgaa gccaagtggg ttatactaat gatatataat
3780gtttgatagt aatcagttta taaaccaaat gcatggaaat gttacgtgga agcacgtaaa
3840ttaacaagca ttgaagcaaa tgcagccacc gcaccaaaac caccccactt cacttccacg
3900taccatattc catgcaacta caacacccta aaacttcaat aaatgccccc accttcactt
3960cacttcaccc atcaatagca agcggccgca ccatggaggt ggtgaatgaa atagtctcaa
4020ttgggcagga agttttaccc aaagttgatt atgcccaact ctggagtgat gccagtcact
4080gtgaggtgct ttacttgtcc atcgcatttg tcatcttgaa gttcactctt ggcccccttg
4140gtccaaaagg tcagtctcgt atgaagtttg ttttcaccaa ttacaacctt ctcatgtcca
4200tttattcgtt gggatcattc ctctcaatgg catatgccat gtacaccatc ggtgttatgt
4260ctgacaactg cgagaaggct tttgacaaca acgtcttcag gatcaccacg cagttgttct
4320atttgagcaa gttcctggag tatattgact ccttctattt gccactgatg ggcaagcctc
4380tgacctggtt gcaattcttc catcatttgg gggcaccgat ggatatgtgg ctgttctata
4440attaccgaaa tgaagctgtt tggatttttg tgctgttgaa tggtttcatc cactggatca
4500tgtacggtta ttattggacc agattgatca agctgaagtt ccccatgcca aaatccctga
4560ttacatcaat gcagatcatt caattcaatg ttggtttcta cattgtctgg aagtacagga
4620acattccctg ttatcgccaa gatgggatga ggatgtttgg ctggttcttc aattactttt
4680atgttggcac agtcttgtgt ttgttcttga atttctatgt gcaaacgtat atcgtcagga
4740agcacaaggg agccaaaaag attcagtgag c
4771385492DNAArtificial Sequenceplasmid pKR287 38ggccgcattt cgcaccaaat
caatgaaagt aataatgaaa agtctgaata agaatactta 60ggcttagatg cctttgttac
ttgtgtaaaa taacttgagt catgtacctt tggcggaaac 120agaataaata aaaggtgaaa
ttccaatgct ctatgtataa gttagtaata cttaatgtgt 180tctacggttg tttcaatatc
atcaaactct aattgaaact ttagaaccac aaatctcaat 240cttttcttaa tgaaatgaaa
aatcttaatt gtaccatgtt tatgttaaac accttacaat 300tggttggaga ggaggaccaa
ccgatgggac aacattggga gaaagagatt caatggagat 360ttggatagga gaacaacatt
ctttttcact tcaatacaag atgagtgcaa cactaaggat 420atgtatgaga ctttcagaag
ctacgacaac atagatgagt gaggtggtga ttcctagcaa 480gaaagacatt agaggaagcc
aaaatcgaac aaggaagaca tcaagggcaa gagacaggac 540catccatctc aggaaaagga
gctttgggat agtccgagaa gttgtacaag aaattttttg 600gagggtgagt gatgcattgc
tggtgacttt aactcaatca aaattgagaa agaaagaaaa 660gggagggggc tcacatgtga
atagaaggga aacgggagaa ttttacagtt ttgatctaat 720gggcatccca gctagtggta
acatattcac catgtttaac cttcacgtac gtctagagga 780tccccgggta ccgagctcga
attcactggc cgtcgtttta caacgtcgtg actgggaaaa 840ccctggcgtt acccaactta
atcgccttgc agcacatccc cctttcgcca gctggcgtaa 900tagcgaagag gcccgcaccg
atcgcccttc ccaacagttg cgcagcctga atggcgaatg 960gcgcctgatg cggtattttc
tccttacgca tctgtgcggt atttcacacc gcatatggtg 1020cactctcagt acaatctgct
ctgatgccgc atagttaagc cagccccgac acccgccaac 1080acccgctgac gcgccctgac
gggcttgtct gctcccggca tccgcttaca gacaagctgt 1140gaccgtctcc gggagctgca
tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag 1200acgaaagggc ctcgtgatac
gcctattttt ataggttaat gtcatgataa taatggtttc 1260ttagacgtca ggtggcactt
ttcggggaaa tgtgcgcgga acccctattt gtttattttt 1320ctaaatacat tcaaatatgt
atccgctcat gagacaataa ccctgataaa tgcttcaata 1380atattgaaaa aggaagagta
tgagtattca acatttccgt gtcgccctta ttcccttttt 1440tgcggcattt tgccttcctg
tttttgctca cccagaaacg ctggtgaaag taaaagatgc 1500tgaagatcag ttgggtgcac
gagtgggtta catcgaactg gatctcaaca gcggtaagat 1560ccttgagagt tttcgccccg
aagaacgttt tccaatgatg agcactttta aagttctgct 1620atgtggcgcg gtattatccc
gtattgacgc cgggcaagag caactcggtc gccgcataca 1680ctattctcag aatgacttgg
ttgagtactc accagtcaca gaaaagcatc ttacggatgg 1740catgacagta agagaattat
gcagtgctgc cataaccatg agtgataaca ctgcggccaa 1800cttacttctg acaacgatcg
gaggaccgaa ggagctaacc gcttttttgc acaacatggg 1860ggatcatgta actcgccttg
atcgttggga accggagctg aatgaagcca taccaaacga 1920cgagcgtgac accacgatgc
ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg 1980cgaactactt actctagctt
cccggcaaca attaatagac tggatggagg cggataaagt 2040tgcaggacca cttctgcgct
cggcccttcc ggctggctgg tttattgctg ataaatctgg 2100agccggtgag cgtgggtctc
gcggtatcat tgcagcactg gggccagatg gtaagccctc 2160ccgtatcgta gttatctaca
cgacggggag tcaggcaact atggatgaac gaaatagaca 2220gatcgctgag ataggtgcct
cactgattaa gcattggtaa ctgtcagacc aagtttactc 2280atatatactt tagattgatt
taaaacttca tttttaattt aaaaggatct aggtgaagat 2340cctttttgat aatctcatga
ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc 2400agaccccgta gaaaagatca
aaggatcttc ttgagatcct ttttttctgc gcgtaatctg 2460ctgcttgcaa acaaaaaaac
caccgctacc agcggtggtt tgtttgccgg atcaagagct 2520accaactctt tttccgaagg
taactggctt cagcagagcg cagataccaa atactgtcct 2580tctagtgtag ccgtagttag
gccaccactt caagaactct gtagcaccgc ctacatacct 2640cgctctgcta atcctgttac
cagtggctgc tgccagtggc gataagtcgt gtcttaccgg 2700gttggactca agacgatagt
taccggataa ggcgcagcgg tcgggctgaa cggggggttc 2760gtgcacacag cccagcttgg
agcgaacgac ctacaccgaa ctgagatacc tacagcgtga 2820gctatgagaa agcgccacgc
ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg 2880cagggtcgga acaggagagc
gcacgaggga gcttccaggg ggaaacgcct ggtatcttta 2940tagtcctgtc gggtttcgcc
acctctgact tgagcgtcga tttttgtgat gctcgtcagg 3000ggggcggagc ctatggaaaa
acgccagcaa cgcggccttt ttacggttcc tggccttttg 3060ctggcctttt gctcacatgt
tctttcctgc gttatcccct gattctgtgg ataaccgtat 3120taccgccttt gagtgagctg
ataccgctcg ccgcagccga acgaccgagc gcagcgagtc 3180agtgagcgag gaagcggaag
agcgcccaat acgcaaaccg cctctccccg cgcgttggcc 3240gattcattaa tgcagctggc
acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa 3300cgcaattaat gtgagttagc
tcactcatta ggcaccccag gctttacact ttatgcttcc 3360ggctcgtatg ttgtgtggaa
ttgtgagcgg ataacaattt cacacaggaa acagctatga 3420ccatgattac gccaagcttg
catgcctgca ggctagccta agtacgtact caaaatgcca 3480acaaataaaa aaaaagttgc
tttaataatg ccaaaacaaa ttaataaaac acttacaaca 3540ccggattttt tttaattaaa
atgtgccatt taggataaat agttaatatt tttaataatt 3600atttaaaaag ccgtatctac
taaaatgatt tttatttggt tgaaaatatt aatatgttta 3660aatcaacaca atctatcaaa
attaaactaa aaaaaaaata agtgtacgtg gttaacatta 3720gtacagtaat ataagaggaa
aatgagaaat taagaaattg aaagcgagtc taatttttaa 3780attatgaacc tgcatatata
aaaggaaaga aagaatccag gaagaaaaga aatgaaacca 3840tgcatggtcc cctcgtcatc
acgagtttct gccatttgca atagaaacac tgaaacacct 3900ttctctttgt cacttaattg
agatgccgaa gccacctcac accatgaact tcatgaggtg 3960tagcacccaa ggcttccata
gccatgcata ctgaagaatg tctcaagctc agcaccctac 4020ttctgtgacg tgtccctcat
tcaccttcct ctcttcccta taaataacca cgcctcaggt 4080tctccgcttc acaactcaaa
cattctctcc attggtcctt aaacactcat cagtcatcac 4140cgcggccgca tgggaacgga
ccaaggaaaa accttcacct gggaagagct ggcggcccat 4200aacaccaagg acgacctact
cttggccatc cgcggcaggg tgtacgatgt cacaaagttc 4260ttgagccgcc atcctggtgg
agtggacact ctcctgctcg gagctggccg agatgttact 4320ccggtctttg agatgtatca
cgcgtttggg gctgcagatg ccattatgaa gaagtactat 4380gtcggtacac tggtctcgaa
tgagctgccc atcttcccgg agccaacggt gttccacaaa 4440accatcaaga cgagagtcga
gggctacttt acggatcgga acattgatcc caagaataga 4500ccagagatct ggggacgata
cgctcttatc tttggatcct tgatcgcttc ctactacgcg 4560cagctctttg tgcctttcgt
tgtcgaacgc acatggcttc aggtggtgtt tgcaatcatc 4620atgggatttg cgtgcgcaca
agtcggactc aaccctcttc atgatgcgtc tcacttttca 4680gtgacccaca accccactgt
ctggaagatt ctgggagcca cgcacgactt tttcaacgga 4740gcatcgtacc tggtgtggat
gtaccaacat atgctcggcc atcaccccta caccaacatt 4800gctggagcag atcccgacgt
gtcgacgtct gagcccgatg ttcgtcgtat caagcccaac 4860caaaagtggt ttgtcaacca
catcaaccag cacatgtttg ttcctttcct gtacggactg 4920ctggcgttca aggtgcgcat
tcaggacatc aacattttgt actttgtcaa gaccaatgac 4980gctattcgtg tcaatcccat
ctcgacatgg cacactgtga tgttctgggg cggcaaggct 5040ttctttgtct ggtatcgcct
gattgttccc ctgcagtatc tgcccctggg caaggtgctg 5100ctcttgttca cggtcgcgga
catggtgtcg tcttactggc tggcgctgac cttccaggcg 5160aaccacgttg ttgaggaagt
tcagtggccg ttgcctgacg agaacgggat catccaaaag 5220gactgggcag ctatgcaggt
cgagactacg caggattacg cacacgattc gcacctctgg 5280accagcatca ctggcagctt
gaactaccag gctgtgcacc atctgttccc caacgtgtcg 5340cagcaccatt atcccgatat
tctggccatc atcaagaaca cctgcagcga gtacaaggtt 5400ccataccttg tcaaggatac
gttttggcaa gcatttgctt cacatttgga gcacttgcgt 5460gttcttggac tccgtcccaa
ggaagagtag gc 5492391338DNAMortierella
alpina 39atgggaacgg accaaggaaa aaccttcacc tgggaagagc tggcggccca
taacaccaag 60gacgacctac tcttggccat ccgcggcagg gtgtacgatg tcacaaagtt
cttgagccgc 120catcctggtg gagtggacac tctcctgctc ggagctggcc gagatgttac
tccggtcttt 180gagatgtatc acgcgtttgg ggctgcagat gccattatga agaagtacta
tgtcggtaca 240ctggtctcga atgagctgcc catcttcccg gagccaacgg tgttccacaa
aaccatcaag 300acgagagtcg agggctactt tacggatcgg aacattgatc ccaagaatag
accagagatc 360tggggacgat acgctcttat ctttggatcc ttgatcgctt cctactacgc
gcagctcttt 420gtgcctttcg ttgtcgaacg cacatggctt caggtggtgt ttgcaatcat
catgggattt 480gcgtgcgcac aagtcggact caaccctctt catgatgcgt ctcacttttc
agtgacccac 540aaccccactg tctggaagat tctgggagcc acgcacgact ttttcaacgg
agcatcgtac 600ctggtgtgga tgtaccaaca tatgctcggc catcacccct acaccaacat
tgctggagca 660gatcccgacg tgtcgacgtc tgagcccgat gttcgtcgta tcaagcccaa
ccaaaagtgg 720tttgtcaacc acatcaacca gcacatgttt gttcctttcc tgtacggact
gctggcgttc 780aaggtgcgca ttcaggacat caacattttg tactttgtca agaccaatga
cgctattcgt 840gtcaatccca tctcgacatg gcacactgtg atgttctggg gcggcaaggc
tttctttgtc 900tggtatcgcc tgattgttcc cctgcagtat ctgcccctgg gcaaggtgct
gctcttgttc 960acggtcgcgg acatggtgtc gtcttactgg ctggcgctga ccttccaggc
gaaccacgtt 1020gttgaggaag ttcagtggcc gttgcctgac gagaacggga tcatccaaaa
ggactgggca 1080gctatgcagg tcgagactac gcaggattac gcacacgatt cgcacctctg
gaccagcatc 1140actggcagct tgaactacca ggctgtgcac catctgttcc ccaacgtgtc
gcagcaccat 1200tatcccgata ttctggccat catcaagaac acctgcagcg agtacaaggt
tccatacctt 1260gtcaaggata cgttttggca agcatttgct tcacatttgg agcacttgcg
tgttcttgga 1320ctccgtccca aggaagag
1338402577DNAArtificial Sequenceplasmid pKR277 40agcttggatc
tcctgcagga tctggccggc cggatctcgt acggatccgt cgacggcgcg 60cccgatcatc
cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag 120gccccaaggg
gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt 180cgggctttgt
tagcagccgg atcgatccaa gctgtacctc actattcctt tgccctcgga 240cgagtgctgg
ggcgtcggtt tccactatcg gcgagtactt ctacacagcc atcggtccag 300acggccgcgc
ttctgcgggc gatttgtgta cgcccgacag tcccggctcc ggatcggacg 360attgcgtcgc
atcgaccctg cgcccaagct gcatcatcga aattgccgtc aaccaagctc 420tgatagagtt
ggtcaagacc aatgcggagc atatacgccc ggagccgcgg cgatcctgca 480agctccggat
gcctccgctc gaagtagcgc gtctgctgct ccatacaagc caaccacggc 540ctccagaaga
agatgttggc gacctcgtat tgggaatccc cgaacatcgc ctcgctccag 600tcaatgaccg
ctgttatgcg gccattgtcc gtcaggacat tgttggagcc gaaatccgcg 660tgcacgaggt
gccggacttc ggggcagtcc tcggcccaaa gcatcagctc atcgagagcc 720tgcgcgacgg
acgcactgac ggtgtcgtcc atcacagttt gccagtgata cacatgggga 780tcagcaatcg
cgcatatgaa atcacgccat gtagtgtatt gaccgattcc ttgcggtccg 840aatgggccga
acccgctcgt ctggctaaga tcggccgcag cgatcgcatc catagcctcc 900gcgaccggct
gcagaacagc gggcagttcg gtttcaggca ggtcttgcaa cgtgacaccc 960tgtgcacggc
gggagatgca ataggtcagg ctctcgctga attccccaat gtcaagcact 1020tccggaatcg
ggagcgcggc cgatgcaaag tgccgataaa cataacgatc tttgtagaaa 1080ccatcggcgc
agctatttac ccgcaggaca tatccacgcc ctcctacatc gaagctgaaa 1140gcacgagatt
cttcgccctc cgagagctgc atcaggtcgg agacgctgtc gaacttttcg 1200atcagaaact
tctcgacaga cgtcgcggtg agttcaggct tttccatggg tatatctcct 1260tcttaaagtt
aaacaaaatt atttctagag ggaaaccgtt gtggtctccc tatagtgagt 1320cgtattaatt
tcgcgggatc gagatctgat caacctgcat taatgaatcg gccaacgcgc 1380ggggagaggc
ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg 1440ctcggtcgtt
cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 1500cacagaatca
ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 1560gaaccgtaaa
aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 1620tcacaaaaat
cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 1680ggcgtttccc
cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 1740atacctgtcc
gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag 1800gtatctcagt
tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 1860tcagcccgac
cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 1920cgacttatcg
ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 1980cggtgctaca
gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt 2040tggtatctgc
gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 2100cggcaaacaa
accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 2160cagaaaaaaa
ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 2220gaacgaaaac
tcacgttaag ggattttggt catgacatta acctataaaa ataggcgtat 2280cacgaggccc
tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca 2340gctcccggag
acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca 2400gggcgcgtca
gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca 2460gattgtactg
agagtgcacc atatggacat attgtcgtta gaacgcggct acaattaata 2520cataacctta
tgtatcatac acatacgatt taggtgacac tatagaacgg cgcgcca
2577415364DNAArtificial Sequenceplasmid pKR952 41ggctagccta agtacgtact
caaaatgcca acaaataaaa aaaaagttgc tttaataatg 60ccaaaacaaa ttaataaaac
acttacaaca ccggattttt tttaattaaa atgtgccatt 120taggataaat agttaatatt
tttaataatt atttaaaaag ccgtatctac taaaatgatt 180tttatttggt tgaaaatatt
aatatgttta aatcaacaca atctatcaaa attaaactaa 240aaaaaaaata agtgtacgtg
gttaacatta gtacagtaat ataagaggaa aatgagaaat 300taagaaattg aaagcgagtc
taatttttaa attatgaacc tgcatatata aaaggaaaga 360aagaatccag gaagaaaaga
aatgaaacca tgcatggtcc cctcgtcatc acgagtttct 420gccatttgca atagaaacac
tgaaacacct ttctctttgt cacttaattg agatgccgaa 480gccacctcac accatgaact
tcatgaggtg tagcacccaa ggcttccata gccatgcata 540ctgaagaatg tctcaagctc
agcaccctac ttctgtgacg tgtccctcat tcaccttcct 600ctcttcccta taaataacca
cgcctcaggt tctccgcttc acaactcaaa cattctctcc 660attggtcctt aaacactcat
cagtcatcac cgcggccgca tgggaacgga ccaaggaaaa 720accttcacct gggaagagct
ggcggcccat aacaccaagg acgacctact cttggccatc 780cgcggcaggg tgtacgatgt
cacaaagttc ttgagccgcc atcctggtgg agtggacact 840ctcctgctcg gagctggccg
agatgttact ccggtctttg agatgtatca cgcgtttggg 900gctgcagatg ccattatgaa
gaagtactat gtcggtacac tggtctcgaa tgagctgccc 960atcttcccgg agccaacggt
gttccacaaa accatcaaga cgagagtcga gggctacttt 1020acggatcgga acattgatcc
caagaataga ccagagatct ggggacgata cgctcttatc 1080tttggatcct tgatcgcttc
ctactacgcg cagctctttg tgcctttcgt tgtcgaacgc 1140acatggcttc aggtggtgtt
tgcaatcatc atgggatttg cgtgcgcaca agtcggactc 1200aaccctcttc atgatgcgtc
tcacttttca gtgacccaca accccactgt ctggaagatt 1260ctgggagcca cgcacgactt
tttcaacgga gcatcgtacc tggtgtggat gtaccaacat 1320atgctcggcc atcaccccta
caccaacatt gctggagcag atcccgacgt gtcgacgtct 1380gagcccgatg ttcgtcgtat
caagcccaac caaaagtggt ttgtcaacca catcaaccag 1440cacatgtttg ttcctttcct
gtacggactg ctggcgttca aggtgcgcat tcaggacatc 1500aacattttgt actttgtcaa
gaccaatgac gctattcgtg tcaatcccat ctcgacatgg 1560cacactgtga tgttctgggg
cggcaaggct ttctttgtct ggtatcgcct gattgttccc 1620ctgcagtatc tgcccctggg
caaggtgctg ctcttgttca cggtcgcgga catggtgtcg 1680tcttactggc tggcgctgac
cttccaggcg aaccacgttg ttgaggaagt tcagtggccg 1740ttgcctgacg agaacgggat
catccaaaag gactgggcag ctatgcaggt cgagactacg 1800caggattacg cacacgattc
gcacctctgg accagcatca ctggcagctt gaactaccag 1860gctgtgcacc atctgttccc
caacgtgtcg cagcaccatt atcccgatat tctggccatc 1920atcaagaaca cctgcagcga
gtacaaggtt ccataccttg tcaaggatac gttttggcaa 1980gcatttgctt cacatttgga
gcacttgcgt gttcttggac tccgtcccaa ggaagagtag 2040gcggccgcat ttcgcaccaa
atcaatgaaa gtaataatga aaagtctgaa taagaatact 2100taggcttaga tgcctttgtt
acttgtgtaa aataacttga gtcatgtacc tttggcggaa 2160acagaataaa taaaaggtga
aattccaatg ctctatgtat aagttagtaa tacttaatgt 2220gttctacggt tgtttcaata
tcatcaaact ctaattgaaa ctttagaacc acaaatctca 2280atcttttctt aatgaaatga
aaaatcttaa ttgtaccatg tttatgttaa acaccttaca 2340attggttgga gaggaggacc
aaccgatggg acaacattgg gagaaagaga ttcaatggag 2400atttggatag gagaacaaca
ttctttttca cttcaataca agatgagtgc aacactaagg 2460atatgtatga gactttcaga
agctacgaca acatagatga gtgaggtggt gattcctagc 2520aagaaagaca ttagaggaag
ccaaaatcga acaaggaaga catcaagggc aagagacagg 2580accatccatc tcaggaaaag
gagctttggg atagtccgag aagttgtaca agaaattttt 2640tggagggtga gtgatgcatt
gctggtgact ttaactcaat caaaattgag aaagaaagaa 2700aagggagggg gctcacatgt
gaatagaagg gaaacgggag aattttacag ttttgatcta 2760atgggcatcc cagctagtgg
taacatattc accatgttta accttcacgt acggatccgt 2820cgacggcgcg cccgatcatc
cggatatagt tcctcctttc agcaaaaaac ccctcaagac 2880ccgtttagag gccccaaggg
gttatgctag ttattgctca gcggtggcag cagccaactc 2940agcttccttt cgggctttgt
tagcagccgg atcgatccaa gctgtacctc actattcctt 3000tgccctcgga cgagtgctgg
ggcgtcggtt tccactatcg gcgagtactt ctacacagcc 3060atcggtccag acggccgcgc
ttctgcgggc gatttgtgta cgcccgacag tcccggctcc 3120ggatcggacg attgcgtcgc
atcgaccctg cgcccaagct gcatcatcga aattgccgtc 3180aaccaagctc tgatagagtt
ggtcaagacc aatgcggagc atatacgccc ggagccgcgg 3240cgatcctgca agctccggat
gcctccgctc gaagtagcgc gtctgctgct ccatacaagc 3300caaccacggc ctccagaaga
agatgttggc gacctcgtat tgggaatccc cgaacatcgc 3360ctcgctccag tcaatgaccg
ctgttatgcg gccattgtcc gtcaggacat tgttggagcc 3420gaaatccgcg tgcacgaggt
gccggacttc ggggcagtcc tcggcccaaa gcatcagctc 3480atcgagagcc tgcgcgacgg
acgcactgac ggtgtcgtcc atcacagttt gccagtgata 3540cacatgggga tcagcaatcg
cgcatatgaa atcacgccat gtagtgtatt gaccgattcc 3600ttgcggtccg aatgggccga
acccgctcgt ctggctaaga tcggccgcag cgatcgcatc 3660catagcctcc gcgaccggct
gcagaacagc gggcagttcg gtttcaggca ggtcttgcaa 3720cgtgacaccc tgtgcacggc
gggagatgca ataggtcagg ctctcgctga attccccaat 3780gtcaagcact tccggaatcg
ggagcgcggc cgatgcaaag tgccgataaa cataacgatc 3840tttgtagaaa ccatcggcgc
agctatttac ccgcaggaca tatccacgcc ctcctacatc 3900gaagctgaaa gcacgagatt
cttcgccctc cgagagctgc atcaggtcgg agacgctgtc 3960gaacttttcg atcagaaact
tctcgacaga cgtcgcggtg agttcaggct tttccatggg 4020tatatctcct tcttaaagtt
aaacaaaatt atttctagag ggaaaccgtt gtggtctccc 4080tatagtgagt cgtattaatt
tcgcgggatc gagatctgat caacctgcat taatgaatcg 4140gccaacgcgc ggggagaggc
ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg 4200actcgctgcg ctcggtcgtt
cggctgcggc gagcggtatc agctcactca aaggcggtaa 4260tacggttatc cacagaatca
ggggataacg caggaaagaa catgtgagca aaaggccagc 4320aaaaggccag gaaccgtaaa
aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 4380ctgacgagca tcacaaaaat
cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 4440aaagatacca ggcgtttccc
cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 4500cgcttaccgg atacctgtcc
gcctttctcc cttcgggaag cgtggcgctt tctcaatgct 4560cacgctgtag gtatctcagt
tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 4620aaccccccgt tcagcccgac
cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 4680cggtaagaca cgacttatcg
ccactggcag cagccactgg taacaggatt agcagagcga 4740ggtatgtagg cggtgctaca
gagttcttga agtggtggcc taactacggc tacactagaa 4800ggacagtatt tggtatctgc
gctctgctga agccagttac cttcggaaaa agagttggta 4860gctcttgatc cggcaaacaa
accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 4920agattacgcg cagaaaaaaa
ggatctcaag aagatccttt gatcttttct acggggtctg 4980acgctcagtg gaacgaaaac
tcacgttaag ggattttggt catgacatta acctataaaa 5040ataggcgtat cacgaggccc
tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct 5100gacacatgca gctcccggag
acggtcacag cttgtctgta agcggatgcc gggagcagac 5160aagcccgtca gggcgcgtca
gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg 5220catcagagca gattgtactg
agagtgcacc atatggacat attgtcgtta gaacgcggct 5280acaattaata cataacctta
tgtatcatac acatacgatt taggtgacac tatagaacgg 5340cgcgccaagc ttggatctcc
tgca 5364425252DNAArtificial
Sequenceplasmid pKR457 42gtacgtgggc ggatcccccg ggctgcagga attcactggc
cgtcgtttta caacgtcgtg 60actgggaaaa ccctggcgtt acccaactta atcgccttgc
agcacatccc cctttcgcca 120gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
ccaacagttg cgcagcctga 180atggcgaatg gcgcctgatg cggtattttc tccttacgca
tctgtgcggt atttcacacc 240gcatatggtg cactctcagt acaatctgct ctgatgccgc
atagttaagc cagccccgac 300acccgccaac acccgctgac gcgccctgac gggcttgtct
gctcccggca tccgcttaca 360gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
gttttcaccg tcatcaccga 420aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
ataggttaat gtcatgataa 480taatggtttc ttagacgtca ggtggcactt ttcggggaaa
tgtgcgcgga acccctattt 540gtttattttt ctaaatacat tcaaatatgt atccgctcat
gagacaataa ccctgataaa 600tgcttcaata atattgaaaa aggaagagta tgagtattca
acatttccgt gtcgccctta 660ttcccttttt tgcggcattt tgccttcctg tttttgctca
cccagaaacg ctggtgaaag 720taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
catcgaactg gatctcaaca 780gcggtaagat ccttgagagt tttcgccccg aagaacgttt
tccaatgatg agcactttta 840aagttctgct atgtggcgcg gtattatccc gtattgacgc
cgggcaagag caactcggtc 900gccgcataca ctattctcag aatgacttgg ttgagtactc
accagtcaca gaaaagcatc 960ttacggatgg catgacagta agagaattat gcagtgctgc
cataaccatg agtgataaca 1020ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
ggagctaacc gcttttttgc 1080acaacatggg ggatcatgta actcgccttg atcgttggga
accggagctg aatgaagcca 1140taccaaacga cgagcgtgac accacgatgc ctgtagcaat
ggcaacaacg ttgcgcaaac 1200tattaactgg cgaactactt actctagctt cccggcaaca
attaatagac tggatggagg 1260cggataaagt tgcaggacca cttctgcgct cggcccttcc
ggctggctgg tttattgctg 1320ataaatctgg agccggtgag cgtgggtctc gcggtatcat
tgcagcactg gggccagatg 1380gtaagccctc ccgtatcgta gttatctaca cgacggggag
tcaggcaact atggatgaac 1440gaaatagaca gatcgctgag ataggtgcct cactgattaa
gcattggtaa ctgtcagacc 1500aagtttactc atatatactt tagattgatt taaaacttca
tttttaattt aaaaggatct 1560aggtgaagat cctttttgat aatctcatga ccaaaatccc
ttaacgtgag ttttcgttcc 1620actgagcgtc agaccccgta gaaaagatca aaggatcttc
ttgagatcct ttttttctgc 1680gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
agcggtggtt tgtttgccgg 1740atcaagagct accaactctt tttccgaagg taactggctt
cagcagagcg cagataccaa 1800atactgtcct tctagtgtag ccgtagttag gccaccactt
caagaactct gtagcaccgc 1860ctacatacct cgctctgcta atcctgttac cagtggctgc
tgccagtggc gataagtcgt 1920gtcttaccgg gttggactca agacgatagt taccggataa
ggcgcagcgg tcgggctgaa 1980cggggggttc gtgcacacag cccagcttgg agcgaacgac
ctacaccgaa ctgagatacc 2040tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
gagaaaggcg gacaggtatc 2100cggtaagcgg cagggtcgga acaggagagc gcacgaggga
gcttccaggg ggaaacgcct 2160ggtatcttta tagtcctgtc gggtttcgcc acctctgact
tgagcgtcga tttttgtgat 2220gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
cgcggccttt ttacggttcc 2280tggccttttg ctggcctttt gctcacatgt tctttcctgc
gttatcccct gattctgtgg 2340ataaccgtat taccgccttt gagtgagctg ataccgctcg
ccgcagccga acgaccgagc 2400gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
acgcaaaccg cctctccccg 2460cgcgttggcc gattcattaa tgcagctggc acgacaggtt
tcccgactgg aaagcgggca 2520gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
ggcaccccag gctttacact 2580ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
ataacaattt cacacaggaa 2640acagctatga ccatgattac gccaagcttg catgcctgca
ggtcgactcg acgtacgtcc 2700tcgaagagaa gggttaataa cacatttttt aacattttta
acacaaattt tagttattta 2760aaaatttatt aaaaaattta aaataagaag aggaactctt
taaataaatc taacttacaa 2820aatttatgat ttttaataag ttttcaccaa taaaaaatgt
cataaaaata tgttaaaaag 2880tatattatca atattctctt tatgataaat aaaaagaaaa
aaaaaataaa agttaagtga 2940aaatgagatt gaagtgactt taggtgtgta taaatatatc
aaccccgcca acaatttatt 3000taatccaaat atattgaagt atattattcc atagccttta
tttatttata tatttattat 3060ataaaagctt tatttgttct aggttgttca tgaaatattt
ttttggtttt atctccgttg 3120taagaaaatc atgtgctttg tgtcgccact cactattgca
gctttttcat gcattggtca 3180gattgacggt tgattgtatt tttgtttttt atggttttgt
gttatgactt aagtcttcat 3240ctctttatct cttcatcagg tttgatggtt acctaatatg
gtccatgggt acatgcatgg 3300ttaaattagg tggccaactt tgttgtgaac gatagaattt
tttttatatt aagtaaacta 3360tttttatatt atgaaataat aataaaaaaa atattttatc
attattaaca aaatcatatt 3420agttaatttg ttaactctat aataaaagaa atactgtaac
attcacatta catggtaaca 3480tctttccacc ctttcatttg ttttttgttt gatgactttt
tttcttgttt aaatttattt 3540cccttctttt aaatttggaa tacattatca tcatatataa
actaaaatac taaaaacagg 3600attacacaaa tgataaataa taacacaaat atttataaat
ctagctgcaa tatatttaaa 3660ctagctatat cgatattgta aaataaaact agctgcattg
atactgataa aaaaatatca 3720tgtgctttct ggactgatga tgcagtatac ttttgacatt
gcctttattt tatttttcag 3780aaaagctttc ttagttctgg gttcttcatt atttgtttcc
catctccatt gtgaattgaa 3840tcatttgctt cgtgtcacaa atacaattta gntaggtaca
tgcattggtc agattcacgg 3900tttattatgt catgacttaa gttcatggta gtacattacc
tgccacgcat gcattatatt 3960ggttagattt gataggcaaa tttggttgtc aacaatataa
atataaataa tgtttttata 4020ttacgaaata acagtgatca aaacaaacag ttttatcttt
attaacaaga ttttgttttt 4080gtttgatgac gttttttaat gtttacgctt tcccccttct
tttgaattta gaacacttta 4140tcatcataaa atcaaatact aaaaaaatta catatttcat
aaataataac acaaatattt 4200ttaaaaaatc tgaaataata atgaacaata ttacatatta
tcacgaaaat tcattaataa 4260aaatattata taaataaaat gtaatagtag ttatatgtag
gaaaaaagta ctgcacgcat 4320aatatataca aaaagattaa aatgaactat tataaataat
aacactaaat taatggtgaa 4380tcatatcaaa ataatgaaaa agtaaataaa atttgtaatt
aacttctata tgtattacac 4440acacaaataa taaataatag taaaaaaaat tatgataaat
atttaccatc tcataagata 4500tttaaaataa tgataaaaat atagattatt ttttatgcaa
ctagctagcc aaaaagagaa 4560cacgggtata tataaaaaga gtacctttaa attctactgt
acttccttta ttcctgacgt 4620ttttatatca agtggacata cgtgaagatt ttaattatca
gtctaaatat ttcattagca 4680cttaatactt ttctgtttta ttcctatcct ataagtagtc
ccgattctcc caacattgct 4740tattcacaca actaactaag aaagtcttcc atagcccccc
aagcggccgc gacacaagtg 4800tgagagtact aaataaatgc tttggttgta cgaaatcatt
acactaaata aaataatcaa 4860agcttatata tgccttccgc taaggccgaa tgcaaagaaa
ttggttcttt ctcgttatct 4920tttgccactt ttactagtac gtattaatta ctacttaatc
atctttgttt acggctcatt 4980atatccggtc tagaggatcc aaggccgcga agttaaaagc
aatgttgtca cttgtcgtac 5040taacacatga tgtgatagtt tatgctagct agctataaca
taagctgtct ctgagtgtgt 5100tgtatattaa taaagatcat cactggtgaa tggtgatcgt
gtacgtaccc tacttagtag 5160gcaatggaag cacttagagt gtgctttgtg catggccttg
cctctgtttt gagacttttg 5220taatgttttc gagtttaaat ctttgccttt gc
5252432635DNAArtificial Sequencemodified
Kti/NotI/Kti3'Salb3' cassette 43ggtaccgggc cccccctcga ggtcgcccgg
gggatccgcc ctaagcttcg tacgtcctcg 60aagagaaggg ttaataacac attttttaac
atttttaaca caaattttag ttatttaaaa 120atttattaaa aaatttaaaa taagaagagg
aactctttaa ataaatctaa cttacaaaat 180ttatgatttt taataagttt tcaccaataa
aaaatgtcat aaaaatatgt taaaaagtat 240attatcaata ttctctttat gataaataaa
aagaaaaaaa aaataaaagt taagtgaaaa 300tgagattgaa gtgactttag gtgtgtataa
atatatcaac cccgccaaca atttatttaa 360tccaaatata ttgaagtata ttattccata
gcctttattt atttatatat ttattatata 420aaagctttat ttgttctagg ttgttcatga
aatatttttt tggttttatc tccgttgtaa 480gaaaatcatg tgctttgtgt cgccactcac
tattgcagct ttttcatgca ttggtcagat 540tgacggttga ttgtattttt gttttttatg
gttttgtgtt atgacttaag tcttcatctc 600tttatctctt catcaggttt gatggttacc
taatatggtc catgggtaca tgcatggtta 660aattaggtgg ccaactttgt tgtgaacgat
agaatttttt ttatattaag taaactattt 720ttatattatg aaataataat aaaaaaaata
ttttatcatt attaacaaaa tcatattagt 780taatttgtta actctataat aaaagaaata
ctgtaacatt cacattacat ggtaacatct 840ttccaccctt tcatttgttt tttgtttgat
gacttttttt cttgtttaaa tttatttccc 900ttcttttaaa tttggaatac attatcatca
tatataaact aaaatactaa aaacaggatt 960acacaaatga taaataataa cacaaatatt
tataaatcta gctgcaatat atttaaacta 1020gctatatcga tattgtaaaa taaaactagc
tgcattgata ctgataaaaa aatatcatgt 1080gctttctgga ctgatgatgc agtatacttt
tgacattgcc tttattttat ttttcagaaa 1140agctttctta gttctgggtt cttcattatt
tgtttcccat ctccattgtg aattgaatca 1200tttgcttcgt gtcacaaata caatttagnt
aggtacatgc attggtcaga ttcacggttt 1260attatgtcat gacttaagtt catggtagta
cattacctgc cacgcatgca ttatattggt 1320tagatttgat aggcaaattt ggttgtcaac
aatataaata taaataatgt ttttatatta 1380cgaaataaca gtgatcaaaa caaacagttt
tatctttatt aacaagattt tgtttttgtt 1440tgatgacgtt ttttaatgtt tacgctttcc
cccttctttt gaatttagaa cactttatca 1500tcataaaatc aaatactaaa aaaattacat
atttcataaa taataacaca aatattttta 1560aaaaatctga aataataatg aacaatatta
catattatca cgaaaattca ttaataaaaa 1620tattatataa ataaaatgta atagtagtta
tatgtaggaa aaaagtactg cacgcataat 1680atatacaaaa agattaaaat gaactattat
aaataataac actaaattaa tggtgaatca 1740tatcaaaata atgaaaaagt aaataaaatt
tgtaattaac ttctatatgt attacacaca 1800caaataataa ataatagtaa aaaaaattat
gataaatatt taccatctca taagatattt 1860aaaataatga taaaaatata gattattttt
tatgcaacta gctagccaaa aagagaacac 1920gggtatatat aaaaagagta cctttaaatt
ctactgtact tcctttattc ctgacgtttt 1980tatatcaagt ggacatacgt gaagatttta
attatcagtc taaatatttc attagcactt 2040aatacttttc tgttttattc ctatcctata
agtagtcccg attctcccaa cattgcttat 2100tcacacaact aactaagaaa gtcttccata
gccccccaag cggccgcgac acaagtgtga 2160gagtactaaa taaatgcttt ggttgtacga
aatcattaca ctaaataaaa taatcaaagc 2220ttatatatgc cttccgctaa ggccgaatgc
aaagaaattg gttctttctc gttatctttt 2280gccactttta ctagtacgta ttaattacta
cttaatcatc tttgtttacg gctcattata 2340tccggtctag aggatccaag gccgcgaagt
taaaagcaat gttgtcactt gtcgtactaa 2400cacatgatgt gatagtttat gctagctagc
tataacataa gctgtctctg agtgtgttgt 2460atattaataa agatcatcac tggtgaatgg
tgatcgtgta cgtaccctac ttagtaggca 2520atggaagcac ttagagtgtg ctttgtgcat
ggccttgcct ctgttttgag acttttgtaa 2580tgttttcgag tttaaatctt tgcctttgcg
tacgtctaga ggatccccgg gtacc 2635449276DNAArtificial
Sequenceplasmid pKR970 44gtacggatcc gtcgacggcg cgcccgatca tccggatata
gttcctcctt tcagcaaaaa 60acccctcaag acccgtttag aggccccaag gggttatgct
agttattgct cagcggtggc 120agcagccaac tcagcttcct ttcgggcttt gttagcagcc
ggatcgatcc aagctgtacc 180tcactattcc tttgccctcg gacgagtgct ggggcgtcgg
tttccactat cggcgagtac 240ttctacacag ccatcggtcc agacggccgc gcttctgcgg
gcgatttgtg tacgcccgac 300agtcccggct ccggatcgga cgattgcgtc gcatcgaccc
tgcgcccaag ctgcatcatc 360gaaattgccg tcaaccaagc tctgatagag ttggtcaaga
ccaatgcgga gcatatacgc 420ccggagccgc ggcgatcctg caagctccgg atgcctccgc
tcgaagtagc gcgtctgctg 480ctccatacaa gccaaccacg gcctccagaa gaagatgttg
gcgacctcgt attgggaatc 540cccgaacatc gcctcgctcc agtcaatgac cgctgttatg
cggccattgt ccgtcaggac 600attgttggag ccgaaatccg cgtgcacgag gtgccggact
tcggggcagt cctcggccca 660aagcatcagc tcatcgagag cctgcgcgac ggacgcactg
acggtgtcgt ccatcacagt 720ttgccagtga tacacatggg gatcagcaat cgcgcatatg
aaatcacgcc atgtagtgta 780ttgaccgatt ccttgcggtc cgaatgggcc gaacccgctc
gtctggctaa gatcggccgc 840agcgatcgca tccatagcct ccgcgaccgg ctgcagaaca
gcgggcagtt cggtttcagg 900caggtcttgc aacgtgacac cctgtgcacg gcgggagatg
caataggtca ggctctcgct 960gaattcccca atgtcaagca cttccggaat cgggagcgcg
gccgatgcaa agtgccgata 1020aacataacga tctttgtaga aaccatcggc gcagctattt
acccgcagga catatccacg 1080ccctcctaca tcgaagctga aagcacgaga ttcttcgccc
tccgagagct gcatcaggtc 1140ggagacgctg tcgaactttt cgatcagaaa cttctcgaca
gacgtcgcgg tgagttcagg 1200cttttccatg ggtatatctc cttcttaaag ttaaacaaaa
ttatttctag agggaaaccg 1260ttgtggtctc cctatagtga gtcgtattaa tttcgcggga
tcgagatctg atcaacctgc 1320attaatgaat cggccaacgc gcggggagag gcggtttgcg
tattgggcgc tcttccgctt 1380cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
gcgagcggta tcagctcact 1440caaaggcggt aatacggtta tccacagaat caggggataa
cgcaggaaag aacatgtgag 1500caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
gttgctggcg tttttccata 1560ggctccgccc ccctgacgag catcacaaaa atcgacgctc
aagtcagagg tggcgaaacc 1620cgacaggact ataaagatac caggcgtttc cccctggaag
ctccctcgtg cgctctcctg 1680ttccgaccct gccgcttacc ggatacctgt ccgcctttct
cccttcggga agcgtggcgc 1740tttctcaatg ctcacgctgt aggtatctca gttcggtgta
ggtcgttcgc tccaagctgg 1800gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
cttatccggt aactatcgtc 1860ttgagtccaa cccggtaaga cacgacttat cgccactggc
agcagccact ggtaacagga 1920ttagcagagc gaggtatgta ggcggtgcta cagagttctt
gaagtggtgg cctaactacg 1980gctacactag aaggacagta tttggtatct gcgctctgct
gaagccagtt accttcggaa 2040aaagagttgg tagctcttga tccggcaaac aaaccaccgc
tggtagcggt ggtttttttg 2100tttgcaagca gcagattacg cgcagaaaaa aaggatctca
agaagatcct ttgatctttt 2160ctacggggtc tgacgctcag tggaacgaaa actcacgtta
agggattttg gtcatgacat 2220taacctataa aaataggcgt atcacgaggc cctttcgtct
cgcgcgtttc ggtgatgacg 2280gtgaaaacct ctgacacatg cagctcccgg agacggtcac
agcttgtctg taagcggatg 2340ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
tggcgggtgt cggggctggc 2400ttaactatgc ggcatcagag cagattgtac tgagagtgca
ccatatggac atattgtcgt 2460tagaacgcgg ctacaattaa tacataacct tatgtatcat
acacatacga tttaggtgac 2520actatagaac ggcgcgccaa gcttggatct cctgcaggct
agcctaagta cgtactcaaa 2580atgccaacaa ataaaaaaaa agttgcttta ataatgccaa
aacaaattaa taaaacactt 2640acaacaccgg atttttttta attaaaatgt gccatttagg
ataaatagtt aatattttta 2700ataattattt aaaaagccgt atctactaaa atgattttta
tttggttgaa aatattaata 2760tgtttaaatc aacacaatct atcaaaatta aactaaaaaa
aaaataagtg tacgtggtta 2820acattagtac agtaatataa gaggaaaatg agaaattaag
aaattgaaag cgagtctaat 2880ttttaaatta tgaacctgca tatataaaag gaaagaaaga
atccaggaag aaaagaaatg 2940aaaccatgca tggtcccctc gtcatcacga gtttctgcca
tttgcaatag aaacactgaa 3000acacctttct ctttgtcact taattgagat gccgaagcca
cctcacacca tgaacttcat 3060gaggtgtagc acccaaggct tccatagcca tgcatactga
agaatgtctc aagctcagca 3120ccctacttct gtgacgtgtc cctcattcac cttcctctct
tccctataaa taaccacgcc 3180tcaggttctc cgcttcacaa ctcaaacatt ctctccattg
gtccttaaac actcatcagt 3240catcaccgcg gccgcatggg aacggaccaa ggaaaaacct
tcacctggga agagctggcg 3300gcccataaca ccaaggacga cctactcttg gccatccgcg
gcagggtgta cgatgtcaca 3360aagttcttga gccgccatcc tggtggagtg gacactctcc
tgctcggagc tggccgagat 3420gttactccgg tctttgagat gtatcacgcg tttggggctg
cagatgccat tatgaagaag 3480tactatgtcg gtacactggt ctcgaatgag ctgcccatct
tcccggagcc aacggtgttc 3540cacaaaacca tcaagacgag agtcgagggc tactttacgg
atcggaacat tgatcccaag 3600aatagaccag agatctgggg acgatacgct cttatctttg
gatccttgat cgcttcctac 3660tacgcgcagc tctttgtgcc tttcgttgtc gaacgcacat
ggcttcaggt ggtgtttgca 3720atcatcatgg gatttgcgtg cgcacaagtc ggactcaacc
ctcttcatga tgcgtctcac 3780ttttcagtga cccacaaccc cactgtctgg aagattctgg
gagccacgca cgactttttc 3840aacggagcat cgtacctggt gtggatgtac caacatatgc
tcggccatca cccctacacc 3900aacattgctg gagcagatcc cgacgtgtcg acgtctgagc
ccgatgttcg tcgtatcaag 3960cccaaccaaa agtggtttgt caaccacatc aaccagcaca
tgtttgttcc tttcctgtac 4020ggactgctgg cgttcaaggt gcgcattcag gacatcaaca
ttttgtactt tgtcaagacc 4080aatgacgcta ttcgtgtcaa tcccatctcg acatggcaca
ctgtgatgtt ctggggcggc 4140aaggctttct ttgtctggta tcgcctgatt gttcccctgc
agtatctgcc cctgggcaag 4200gtgctgctct tgttcacggt cgcggacatg gtgtcgtctt
actggctggc gctgaccttc 4260caggcgaacc acgttgttga ggaagttcag tggccgttgc
ctgacgagaa cgggatcatc 4320caaaaggact gggcagctat gcaggtcgag actacgcagg
attacgcaca cgattcgcac 4380ctctggacca gcatcactgg cagcttgaac taccaggctg
tgcaccatct gttccccaac 4440gtgtcgcagc accattatcc cgatattctg gccatcatca
agaacacctg cagcgagtac 4500aaggttccat accttgtcaa ggatacgttt tggcaagcat
ttgcttcaca tttggagcac 4560ttgcgtgttc ttggactccg tcccaaggaa gagtaggcgg
ccgcatttcg caccaaatca 4620atgaaagtaa taatgaaaag tctgaataag aatacttagg
cttagatgcc tttgttactt 4680gtgtaaaata acttgagtca tgtacctttg gcggaaacag
aataaataaa aggtgaaatt 4740ccaatgctct atgtataagt tagtaatact taatgtgttc
tacggttgtt tcaatatcat 4800caaactctaa ttgaaacttt agaaccacaa atctcaatct
tttcttaatg aaatgaaaaa 4860tcttaattgt accatgttta tgttaaacac cttacaattg
gttggagagg aggaccaacc 4920gatgggacaa cattgggaga aagagattca atggagattt
ggataggaga acaacattct 4980ttttcacttc aatacaagat gagtgcaaca ctaaggatat
gtatgagact ttcagaagct 5040acgacaacat agatgagtga ggtggtgatt cctagcaaga
aagacattag aggaagccaa 5100aatcgaacaa ggaagacatc aagggcaaga gacaggacca
tccatctcag gaaaaggagc 5160tttgggatag tccgagaagt tgtacaagaa attttttgga
gggtgagtga tgcattgctg 5220gtgactttaa ctcaatcaaa attgagaaag aaagaaaagg
gagggggctc acatgtgaat 5280agaagggaaa cgggagaatt ttacagtttt gatctaatgg
gcatcccagc tagtggtaac 5340atattcacca tgtttaacct tcacgtaccg ggccccccct
cgaggtcgcc cgggggatcc 5400gccctaagct tcgtacgtcc tcgaagagaa gggttaataa
cacatttttt aacattttta 5460acacaaattt tagttattta aaaatttatt aaaaaattta
aaataagaag aggaactctt 5520taaataaatc taacttacaa aatttatgat ttttaataag
ttttcaccaa taaaaaatgt 5580cataaaaata tgttaaaaag tatattatca atattctctt
tatgataaat aaaaagaaaa 5640aaaaaataaa agttaagtga aaatgagatt gaagtgactt
taggtgtgta taaatatatc 5700aaccccgcca acaatttatt taatccaaat atattgaagt
atattattcc atagccttta 5760tttatttata tatttattat ataaaagctt tatttgttct
aggttgttca tgaaatattt 5820ttttggtttt atctccgttg taagaaaatc atgtgctttg
tgtcgccact cactattgca 5880gctttttcat gcattggtca gattgacggt tgattgtatt
tttgtttttt atggttttgt 5940gttatgactt aagtcttcat ctctttatct cttcatcagg
tttgatggtt acctaatatg 6000gtccatgggt acatgcatgg ttaaattagg tggccaactt
tgttgtgaac gatagaattt 6060tttttatatt aagtaaacta tttttatatt atgaaataat
aataaaaaaa atattttatc 6120attattaaca aaatcatatt agttaatttg ttaactctat
aataaaagaa atactgtaac 6180attcacatta catggtaaca tctttccacc ctttcatttg
ttttttgttt gatgactttt 6240tttcttgttt aaatttattt cccttctttt aaatttggaa
tacattatca tcatatataa 6300actaaaatac taaaaacagg attacacaaa tgataaataa
taacacaaat atttataaat 6360ctagctgcaa tatatttaaa ctagctatat cgatattgta
aaataaaact agctgcattg 6420atactgataa aaaaatatca tgtgctttct ggactgatga
tgcagtatac ttttgacatt 6480gcctttattt tatttttcag aaaagctttc ttagttctgg
gttcttcatt atttgtttcc 6540catctccatt gtgaattgaa tcatttgctt cgtgtcacaa
atacaattta gntaggtaca 6600tgcattggtc agattcacgg tttattatgt catgacttaa
gttcatggta gtacattacc 6660tgccacgcat gcattatatt ggttagattt gataggcaaa
tttggttgtc aacaatataa 6720atataaataa tgtttttata ttacgaaata acagtgatca
aaacaaacag ttttatcttt 6780attaacaaga ttttgttttt gtttgatgac gttttttaat
gtttacgctt tcccccttct 6840tttgaattta gaacacttta tcatcataaa atcaaatact
aaaaaaatta catatttcat 6900aaataataac acaaatattt ttaaaaaatc tgaaataata
atgaacaata ttacatatta 6960tcacgaaaat tcattaataa aaatattata taaataaaat
gtaatagtag ttatatgtag 7020gaaaaaagta ctgcacgcat aatatataca aaaagattaa
aatgaactat tataaataat 7080aacactaaat taatggtgaa tcatatcaaa ataatgaaaa
agtaaataaa atttgtaatt 7140aacttctata tgtattacac acacaaataa taaataatag
taaaaaaaat tatgataaat 7200atttaccatc tcataagata tttaaaataa tgataaaaat
atagattatt ttttatgcaa 7260ctagctagcc aaaaagagaa cacgggtata tataaaaaga
gtacctttaa attctactgt 7320acttccttta ttcctgacgt ttttatatca agtggacata
cgtgaagatt ttaattatca 7380gtctaaatat ttcattagca cttaatactt ttctgtttta
ttcctatcct ataagtagtc 7440ccgattctcc caacattgct tattcacaca actaactaag
aaagtcttcc atagcccccc 7500aagcggccgc accatgggca agggtggaga cggcggcgcg
caggcggtga gcgggaccga 7560cgcgtctctc gctgaggtga gctccgtcga tagcaagagc
gtgcacgtcg tgctctacgg 7620caagcgcgtg gatgtcacaa agttccagaa ggcacacccg
ggcgggagca aggtgttccg 7680catcttccag gagcgcgacg cgacggagca gttcgagtct
taccactcgc ccaaggccat 7740caagatgatg gagggcatgc tcaagaagtc ggaggatgcg
cccgcttccg tgcccctgcc 7800ctcgcggtcc accatgggca cggagttcaa ggagatgatt
gagcgccaca agagggctgg 7860tctctacgac ccttgcccgt tggacgagct gttcaagctc
accatcgtcc ttgcgcccat 7920cttcgtgggc gcctatctcg tgcggagcgg cgtctcgccc
ctcgcgggcg cgctctccat 7980gggctttggc ttctacctcg acggctggct tgctcacgac
tacctgcatc acgcagtctt 8040caagggctcg gtcaacacgc tcgtcaaggc gaacaacgcc
atgggatacg ccctcggctt 8100cctccagggc tacgacgtgg cctggtggcg cgcgcgccat
aacacgcacc acgtgtgcac 8160caacgaggat ggttcggacc cggacatcaa gacggcgccc
ctgctcatct acgtgcgaga 8220gaacccgtcc attgccaagc ggctcaactt cttccagcgc
tggcagcagt actactatgt 8280gccgaccatg gccatcctcg acctctactg gcgcctggag
tccatcgcgt acgtggctgt 8340gcgcctgcct aagatgtgga tgcaggccgc cgctcttgcc
gctcactacg cgctcctgtg 8400ctgggtcttc gcagcgcatc tcaacctcat ccctctcatg
atggttgcac gcggcttcgc 8460gacgggcatc gttgtctttg caacccacta tggtgaggac
atcctcgacc gcgagcacgt 8520cgagggcatg acgctcgtcg agcagaccgc caagacctcc
cgtaacatca cgggcggctg 8580gctagtgaac gtgctcacgg gcttcatctc cctgcagacc
gagcatcacc tcttccccat 8640gatgcccacc ggcaacctaa tgactatcca gcccgaggta
cgcgacttct tcaagaagca 8700tggcctcgag taccgcgagg gcaacctctt ccagtgcgtg
caccagaaca tcaaggctct 8760cgccttcgag cacctcctcc actgagcggc cgcgacacaa
gtgtgagagt actaaataaa 8820tgctttggtt gtacgaaatc attacactaa ataaaataat
caaagcttat atatgccttc 8880cgctaaggcc gaatgcaaag aaattggttc tttctcgtta
tcttttgcca cttttactag 8940tacgtattaa ttactactta atcatctttg tttacggctc
attatatccg gtctagagga 9000tccaaggccg cgaagttaaa agcaatgttg tcacttgtcg
tactaacaca tgatgtgata 9060gtttatgcta gctagctata acataagctg tctctgagtg
tgttgtatat taataaagat 9120catcactggt gaatggtgat cgtgtacgta ccctacttag
taggcaatgg aagcacttag 9180agtgtgcttt gtgcatggcc ttgcctctgt tttgagactt
ttgtaatgtt ttcgagttta 9240aatctttgcc tttgcgtacg tctagaggat ccccgg
92764511366DNAArtificial Sequenceplasmid pKR973
45ggagatccaa gcttggcgcg ccgttctata gtgtcaccta aatcgtatgt gtatgataca
60taaggttatg tattaattgt agccgcgttc taacgacaat atgtccatat ggtgcactct
120cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
180tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
240ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa
300gggcctcgtg atacgcctat ttttataggt taatgtcatg accaaaatcc cttaacgtga
360gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
420tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
480ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
540gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
600tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
660cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
720gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
780actgagatac ctacagcgtg agcattgaga aagcgccacg cttcccgaag ggagaaaggc
840ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
900gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
960atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
1020tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
1080tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
1140aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
1200gcctctcccc gcgcgttggc cgattcatta atgcaggttg atcagatctc gatcccgcga
1260aattaatacg actcactata gggagaccac aacggtttcc ctctagaaat aattttgttt
1320aactttaaga aggagatata cccatggaaa agcctgaact caccgcgacg tctgtcgaga
1380agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag
1440aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct
1500gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc
1560cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc
1620gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc
1680agccggtcgc ggaggctatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt
1740tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg
1800cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt
1860ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc
1920acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag
1980cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct
2040tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc
2100atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc
2160aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat
2220gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa
2280gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc
2340ccagcactcg tccgagggca aaggaatagt gaggtacagc ttggatcgat ccggctgcta
2400acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
2460cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
2520gatgatcggg cgcgccgtcg acggatccgt acccggggat cctctagacg tacgcaaagg
2580caaagattta aactcgaaaa cattacaaaa gtctcaaaac agaggcaagg ccatgcacaa
2640agcacactct aagtgcttcc attgcctact aagtagggta cgtacacgat caccattcac
2700cagtgatgat ctttattaat atacaacaca ctcagagaca gcttatgtta tagctagcta
2760gcataaacta tcacatcatg tgttagtacg acaagtgaca acattgcttt taacttcgcg
2820gccttggatc ctctagaccg gatataatga gccgtaaaca aagatgatta agtagtaatt
2880aatacgtact agtaaaagtg gcaaaagata acgagaaaga accaatttct ttgcattcgg
2940ccttagcgga aggcatatat aagctttgat tattttattt agtgtaatga tttcgtacaa
3000ccaaagcatt tatttagtac tctcacactt gtgtcgcggc cgctcagtgg aggaggtgct
3060cgaaggcgag agccttgatg ttctggtgca cgcactggaa gaggttgccc tcgcggtact
3120cgaggccatg cttcttgaag aagtcgcgta cctcgggctg gatagtcatt aggttgccgg
3180tgggcatcat ggggaagagg tgatgctcgg tctgcaggga gatgaagccc gtgagcacgt
3240tcactagcca gccgcccgtg atgttacggg aggtcttggc ggtctgctcg acgagcgtca
3300tgccctcgac gtgctcgcgg tcgaggatgt cctcaccata gtgggttgca aagacaacga
3360tgcccgtcgc gaagccgcgt gcaaccatca tgagagggat gaggttgaga tgcgctgcga
3420agacccagca caggagcgcg tagtgagcgg caagagcggc ggcctgcatc cacatcttag
3480gcaggcgcac agccacgtac gcgatggact ccaggcgcca gtagaggtcg aggatggcca
3540tggtcggcac atagtagtac tgctgccagc gctggaagaa gttgagccgc ttggcaatgg
3600acgggttctc tcgcacgtag atgagcaggg gcgccgtctt gatgtccggg tccgaaccat
3660cctcgttggt gcacacgtgg tgcgtgttat ggcgcgcgcg ccaccaggcc acgtcgtagc
3720cctggaggaa gccgagggcg tatcccatgg cgttgttcgc cttgacgagc gtgttgaccg
3780agcccttgaa gactgcgtga tgcaggtagt cgtgagcaag ccagccgtcg aggtagaagc
3840caaagcccat ggagagcgcg cccgcgaggg gcgagacgcc gctccgcacg agataggcgc
3900ccacgaagat gggcgcaagg acgatggtga gcttgaacag ctcgtccaac gggcaagggt
3960cgtagagacc agccctcttg tggcgctcaa tcatctcctt gaactccgtg cccatggtgg
4020accgcgaggg caggggcacg gaagcgggcg catcctccga cttcttgagc atgccctcca
4080tcatcttgat ggccttgggc gagtggtaag actcgaactg ctccgtcgcg tcgcgctcct
4140ggaagatgcg gaacaccttg ctcccgcccg ggtgtgcctt ctggaacttt gtgacatcca
4200cgcgcttgcc gtagagcacg acgtgcacgc tcttgctatc gacggagctc acctcagcga
4260gagacgcgtc ggtcccgctc accgcctgcg cgccgccgtc tccacccttg cccatggtgc
4320ggccgcttgg ggggctatgg aagactttct tagttagttg tgtgaataag caatgttggg
4380agaatcggga ctacttatag gataggaata aaacagaaaa gtattaagtg ctaatgaaat
4440atttagactg ataattaaaa tcttcacgta tgtccacttg atataaaaac gtcaggaata
4500aaggaagtac agtagaattt aaaggtactc tttttatata tacccgtgtt ctctttttgg
4560ctagctagtt gcataaaaaa taatctatat ttttatcatt attttaaata tcttatgaga
4620tggtaaatat ttatcataat tttttttact attatttatt atttgtgtgt gtaatacata
4680tagaagttaa ttacaaattt tatttacttt ttcattattt tgatatgatt caccattaat
4740ttagtgttat tatttataat agttcatttt aatctttttg tatatattat gcgtgcagta
4800cttttttcct acatataact actattacat tttatttata taatattttt attaatgaat
4860tttcgtgata atatgtaata ttgttcatta ttatttcaga ttttttaaaa atatttgtgt
4920tattatttat gaaatatgta atttttttag tatttgattt tatgatgata aagtgttcta
4980aattcaaaag aagggggaaa gcgtaaacat taaaaaacgt catcaaacaa aaacaaaatc
5040ttgttaataa agataaaact gtttgttttg atcactgtta tttcgtaata taaaaacatt
5100atttatattt atattgttga caaccaaatt tgcctatcaa atctaaccaa tataatgcat
5160gcgtggcagg taatgtacta ccatgaactt aagtcatgac ataataaacc gtgaatctga
5220ccaatgcatg tacctancta aattgtattt gtgacacgaa gcaaatgatt caattcacaa
5280tggagatggg aaacaaataa tgaagaaccc agaactaaga aagcttttct gaaaaataaa
5340ataaaggcaa tgtcaaaagt atactgcatc atcagtccag aaagcacatg atattttttt
5400atcagtatca atgcagctag ttttatttta caatatcgat atagctagtt taaatatatt
5460gcagctagat ttataaatat ttgtgttatt atttatcatt tgtgtaatcc tgtttttagt
5520attttagttt atatatgatg ataatgtatt ccaaatttaa aagaagggaa ataaatttaa
5580acaagaaaaa aagtcatcaa acaaaaaaca aatgaaaggg tggaaagatg ttaccatgta
5640atgtgaatgt tacagtattt cttttattat agagttaaca aattaactaa tatgattttg
5700ttaataatga taaaatattt tttttattat tatttcataa tataaaaata gtttacttaa
5760tataaaaaaa attctatcgt tcacaacaaa gttggccacc taatttaacc atgcatgtac
5820ccatggacca tattaggtaa ccatcaaacc tgatgaagag ataaagagat gaagacttaa
5880gtcataacac aaaaccataa aaaacaaaaa tacaatcaac cgtcaatctg accaatgcat
5940gaaaaagctg caatagtgag tggcgacaca aagcacatga ttttcttaca acggagataa
6000aaccaaaaaa atatttcatg aacaacctag aacaaataaa gcttttatat aataaatata
6060taaataaata aaggctatgg aataatatac ttcaatatat ttggattaaa taaattgttg
6120gcggggttga tatatttata cacacctaaa gtcacttcaa tctcattttc acttaacttt
6180tatttttttt ttctttttat ttatcataaa gagaatattg ataatatact ttttaacata
6240tttttatgac attttttatt ggtgaaaact tattaaaaat cataaatttt gtaagttaga
6300tttatttaaa gagttcctct tcttatttta aattttttaa taaattttta aataactaaa
6360atttgtgtta aaaatgttaa aaaatgtgtt attaaccctt ctcttcgagg acgtacgaag
6420cttagggcgg atcccccggg cgacctcgag ggggggcccg gtacgtgaag gttaaacatg
6480gtgaatatgt taccactagc tgggatgccc attagatcaa aactgtaaaa ttctcccgtt
6540tcccttctat tcacatgtga gccccctccc ttttctttct ttctcaattt tgattgagtt
6600aaagtcacca gcaatgcatc actcaccctc caaaaaattt cttgtacaac ttctcggact
6660atcccaaagc tccttttcct gagatggatg gtcctgtctc ttgcccttga tgtcttcctt
6720gttcgatttt ggcttcctct aatgtctttc ttgctaggaa tcaccacctc actcatctat
6780gttgtcgtag cttctgaaag tctcatacat atccttagtg ttgcactcat cttgtattga
6840agtgaaaaag aatgttgttc tcctatccaa atctccattg aatctctttc tcccaatgtt
6900gtcccatcgg ttggtcctcc tctccaacca attgtaaggt gtttaacata aacatggtac
6960aattaagatt tttcatttca ttaagaaaag attgagattt gtggttctaa agtttcaatt
7020agagtttgat gatattgaaa caaccgtaga acacattaag tattactaac ttatacatag
7080agcattggaa tttcaccttt tatttattct gtttccgcca aaggtacatg actcaagtta
7140ttttacacaa gtaacaaagg catctaagcc taagtattct tattcagact tttcattatt
7200actttcattg atttggtgcg aaatgcggcc gcctactctt ccttgggacg gagtccaaga
7260acacgcaagt gctccaaatg tgaagcaaat gcttgccaaa acgtatcctt gacaaggtat
7320ggaaccttgt actcgctgca ggtgttcttg atgatggcca gaatatcggg ataatggtgc
7380tgcgacacgt tggggaacag atggtgcaca gcctggtagt tcaagctgcc agtgatgctg
7440gtccagaggt gcgaatcgtg tgcgtaatcc tgcgtagtct cgacctgcat agctgcccag
7500tccttttgga tgatcccgtt ctcgtcaggc aacggccact gaacttcctc aacaacgtgg
7560ttcgcctgga aggtcagcgc cagccagtaa gacgacacca tgtccgcgac cgtgaacaag
7620agcagcacct tgcccagggg cagatactgc aggggaacaa tcaggcgata ccagacaaag
7680aaagccttgc cgccccagaa catcacagtg tgccatgtcg agatgggatt gacacgaata
7740gcgtcattgg tcttgacaaa gtacaaaatg ttgatgtcct gaatgcgcac cttgaacgcc
7800agcagtccgt acaggaaagg aacaaacatg tgctggttga tgtggttgac aaaccacttt
7860tggttgggct tgatacgacg aacatcgggc tcagacgtcg acacgtcggg atctgctcca
7920gcaatgttgg tgtaggggtg atggccgagc atatgttggt acatccacac caggtacgat
7980gctccgttga aaaagtcgtg cgtggctccc agaatcttcc agacagtggg gttgtgggtc
8040actgaaaagt gagacgcatc atgaagaggg ttgagtccga cttgtgcgca cgcaaatccc
8100atgatgattg caaacaccac ctgaagccat gtgcgttcga caacgaaagg cacaaagagc
8160tgcgcgtagt aggaagcgat caaggatcca aagataagag cgtatcgtcc ccagatctct
8220ggtctattct tgggatcaat gttccgatcc gtaaagtagc cctcgactct cgtcttgatg
8280gttttgtgga acaccgttgg ctccgggaag atgggcagct cattcgagac cagtgtaccg
8340acatagtact tcttcataat ggcatctgca gccccaaacg cgtgatacat ctcaaagacc
8400ggagtaacat ctcggccagc tccgagcagg agagtgtcca ctccaccagg atggcggctc
8460aagaactttg tgacatcgta caccctgccg cggatggcca agagtaggtc gtccttggtg
8520ttatgggccg ccagctcttc ccaggtgaag gtttttcctt ggtccgttcc catgcggccg
8580cggtgatgac tgatgagtgt ttaaggacca atggagagaa tgtttgagtt gtgaagcgga
8640gaacctgagg cgtggttatt tatagggaag agaggaaggt gaatgaggga cacgtcacag
8700aagtagggtg ctgagcttga gacattcttc agtatgcatg gctatggaag ccttgggtgc
8760tacacctcat gaagttcatg gtgtgaggtg gcttcggcat ctcaattaag tgacaaagag
8820aaaggtgttt cagtgtttct attgcaaatg gcagaaactc gtgatgacga ggggaccatg
8880catggtttca tttcttttct tcctggattc tttctttcct tttatatatg caggttcata
8940atttaaaaat tagactcgct ttcaatttct taatttctca ttttcctctt atattactgt
9000actaatgtta accacgtaca cttatttttt ttttagttta attttgatag attgtgttga
9060tttaaacata ttaatatttt caaccaaata aaaatcattt tagtagatac ggctttttaa
9120ataattatta aaaatattaa ctatttatcc taaatggcac attttaatta aaaaaaatcc
9180ggtgttgtaa gtgttttatt aatttgtttt ggcattatta aagcaacttt ttttttattt
9240gttggcattt tgagtacgta cttaggctag cctgcaggcc aactgcgttt ggggctccag
9300attaaacgac gccgtttcgt tcctttcgct tcacggctta acgatgtcgt ttctgtctgt
9360gcccaaaaaa taaaggcatt tgttatttgc accagatatt tactaagtgc accctagttt
9420gacaagtagg cgataattac aaatagatgc ggtgcaaata ataaattttg aaggaaataa
9480ttacaaaaga acagaactta tatttacttt attttaaaaa actaaaatga aagaacaaaa
9540aaagtaaaaa atacaaaaaa tgtgctttaa ccactttcat tatttgttac agaaagtatg
9600attctactca aattgatctg ttgtatctgg tgctgccttg tcacactggc gatttcaatc
9660ccctaaagat atggtgcaaa ctgcgaagtg atcaatatct gctcggttaa tttagattaa
9720ttaataatat tcaacgtgat gtaccaaaaa aagacaattt tttgctccat tgacaaatta
9780aacctcatca aggtaatttc caaacctata agcaaaaaaa tttcacatta attggcccgc
9840aatcctatta gtcttattat actagagtag gaaaaaaaac aattacacaa cttgtcttat
9900tattctctat gctaatgaat atttttccct tttgttagaa atcagtgttt cctaatttat
9960tgagtattaa ttccactcac cgcatatatt taccgttgaa taagaaaatt ttacacataa
10020ttctttttaa gataaataat ttttttatac tagatcttat atgattacgt gaagccaagt
10080gggttatact aatgatatat aatgtttgat agtaatcagt ttataaacca aatgcatgga
10140aatgttacgt ggaagcacgt aaattaacaa gcattgaagc aaatgcagcc accgcaccaa
10200aaccacccca cttcacttcc acgtaccata ttccatgcaa ctacaacacc ctaaaacttc
10260aataaatgcc cccaccttca cttcacttca cccatcaata gcaagcggcc gcaccatgga
10320ggtggtgaat gaaatagtct caattgggca ggaagtttta cccaaagttg attatgccca
10380actctggagt gatgccagtc actgtgaggt gctttacttg tccatcgcat ttgtcatctt
10440gaagttcact cttggccccc ttggtccaaa aggtcagtct cgtatgaagt ttgttttcac
10500caattacaac cttctcatgt ccatttattc gttgggatca ttcctctcaa tggcatatgc
10560catgtacacc atcggtgtta tgtctgacaa ctgcgagaag gcttttgaca acaacgtctt
10620caggatcacc acgcagttgt tctatttgag caagttcctg gagtatattg actccttcta
10680tttgccactg atgggcaagc ctctgacctg gttgcaattc ttccatcatt tgggggcacc
10740gatggatatg tggctgttct ataattaccg aaatgaagct gtttggattt ttgtgctgtt
10800gaatggtttc atccactgga tcatgtacgg ttattattgg accagattga tcaagctgaa
10860gttccccatg ccaaaatccc tgattacatc aatgcagatc attcaattca atgttggttt
10920ctacattgtc tggaagtaca ggaacattcc ctgttatcgc caagatggga tgaggatgtt
10980tggctggttc ttcaattact tttatgttgg cacagtcttg tgtttgttct tgaatttcta
11040tgtgcaaacg tatatcgtca ggaagcacaa gggagccaaa aagattcagt gagcggccgc
11100gaagttaaaa gcaatgttgt cacttgtcgt actaacacat gatgtgatag tttatgctag
11160ctagctataa cataagctgt ctctgagtgt gttgtatatt aataaagatc atcactggtg
11220aatggtgatc gtgtacgtac cctacttagt aggcaatgga agcacttaga gtgtgctttg
11280tgcatggcct tgcctctgtt ttgagacttt tgtaatgttt tcgagtttaa atctttgcct
11340ttgcgtacgt ctagagtcga cctgca
11366467085DNAArtificial Sequenceplasmid pKR72 46gtacggatcc gtcgacggcg
cgcccgatca tccggatata gttcctcctt tcagcaaaaa 60acccctcaag acccgtttag
aggccccaag gggttatgct agttattgct cagcggtggc 120agcagccaac tcagcttcct
ttcgggcttt gttagcagcc ggatcgatcc aagctgtacc 180tcactattcc tttgccctcg
gacgagtgct ggggcgtcgg tttccactat cggcgagtac 240ttctacacag ccatcggtcc
agacggccgc gcttctgcgg gcgatttgtg tacgcccgac 300agtcccggct ccggatcgga
cgattgcgtc gcatcgaccc tgcgcccaag ctgcatcatc 360gaaattgccg tcaaccaagc
tctgatagag ttggtcaaga ccaatgcgga gcatatacgc 420ccggagccgc ggcgatcctg
caagctccgg atgcctccgc tcgaagtagc gcgtctgctg 480ctccatacaa gccaaccacg
gcctccagaa gaagatgttg gcgacctcgt attgggaatc 540cccgaacatc gcctcgctcc
agtcaatgac cgctgttatg cggccattgt ccgtcaggac 600attgttggag ccgaaatccg
cgtgcacgag gtgccggact tcggggcagt cctcggccca 660aagcatcagc tcatcgagag
cctgcgcgac ggacgcactg acggtgtcgt ccatcacagt 720ttgccagtga tacacatggg
gatcagcaat cgcgcatatg aaatcacgcc atgtagtgta 780ttgaccgatt ccttgcggtc
cgaatgggcc gaacccgctc gtctggctaa gatcggccgc 840agcgatcgca tccatagcct
ccgcgaccgg ctgcagaaca gcgggcagtt cggtttcagg 900caggtcttgc aacgtgacac
cctgtgcacg gcgggagatg caataggtca ggctctcgct 960gaattcccca atgtcaagca
cttccggaat cgggagcgcg gccgatgcaa agtgccgata 1020aacataacga tctttgtaga
aaccatcggc gcagctattt acccgcagga catatccacg 1080ccctcctaca tcgaagctga
aagcacgaga ttcttcgccc tccgagagct gcatcaggtc 1140ggagacgctg tcgaactttt
cgatcagaaa cttctcgaca gacgtcgcgg tgagttcagg 1200cttttccatg ggtatatctc
cttcttaaag ttaaacaaaa ttatttctag agggaaaccg 1260ttgtggtctc cctatagtga
gtcgtattaa tttcgcggga tcgagatcga tccaattcca 1320atcccacaaa aatctgagct
taacagcaca gttgctcctc tcagagcaga atcgggtatt 1380caacaccctc atatcaacta
ctacgttgtg tataacggtc cacatgccgg tatatacgat 1440gactggggtt gtacaaaggc
ggcaacaaac ggcgttcccg gagttgcaca caagaaattt 1500gccactatta cagaggcaag
agcagcagct gacgcgtaca caacaagtca gcaaacagac 1560aggttgaact tcatccccaa
aggagaagct caactcaagc ccaagagctt tgctaaggcc 1620ctaacaagcc caccaaagca
aaaagcccac tggctcacgc taggaaccaa aaggcccagc 1680agtgatccag ccccaaaaga
gatctccttt gccccggaga ttacaatgga cgatttcctc 1740tatctttacg atctaggaag
gaagttcgaa ggtgaaggtg acgacactat gttcaccact 1800gataatgaga aggttagcct
cttcaatttc agaaagaatg ctgacccaca gatggttaga 1860gaggcctacg cagcaggtct
catcaagacg atctacccga gtaacaatct ccaggagatc 1920aaataccttc ccaagaaggt
taaagatgca gtcaaaagat tcaggactaa ttgcatcaag 1980aacacagaga aagacatatt
tctcaagatc agaagtacta ttccagtatg gacgattcaa 2040ggcttgcttc ataaaccaag
gcaagtaata gagattggag tctctaaaaa ggtagttcct 2100actgaatcta aggccatgca
tggagtctaa gattcaaatc gaggatctaa cagaactcgc 2160cgtgaagact ggcgaacagt
tcatacagag tcttttacga ctcaatgaca agaagaaaat 2220cttcgtcaac atggtggagc
acgacactct ggtctactcc aaaaatgtca aagatacagt 2280ctcagaagac caaagggcta
ttgagacttt tcaacaaagg ataatttcgg gaaacctcct 2340cggattccat tgcccagcta
tctgtcactt catcgaaagg acagtagaaa aggaaggtgg 2400ctcctacaaa tgccatcatt
gcgataaagg aaaggctatc attcaagatg cctctgccga 2460cagtggtccc aaagatggac
ccccacccac gaggagcatc gtggaaaaag aagacgttcc 2520aaccacgtct tcaaagcaag
tggattgatg tgacatctcc actgacgtaa gggatgacgc 2580acaatcccac tatccttcgc
aagacccttc ctctatataa ggaagttcat ttcatttgga 2640gaggacacgc tcgagctcat
ttctctatta cttcagccat aacaaaagaa ctcttttctc 2700ttcttattaa accatgaaaa
agcctgaact caccgcgacg tctgtcgaga agtttctgat 2760cgaaaagttc gacagcgtct
ccgacctgat gcagctctcg gagggcgaag aatctcgtgc 2820tttcagcttc gatgtaggag
ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg 2880tttctacaaa gatcgttatg
tttatcggca ctttgcatcg gccgcgctcc cgattccgga 2940agtgcttgac attggggaat
tcagcgagag cctgacctat tgcatctccc gccgtgcaca 3000gggtgtcacg ttgcaagacc
tgcctgaaac cgaactgccc gctgttctgc agccggtcgc 3060ggaggccatg gatgcgatcg
ctgcggccga tcttagccag acgagcgggt tcggcccatt 3120cggaccgcaa ggaatcggtc
aatacactac atggcgtgat ttcatatgcg cgattgctga 3180tccccatgtg tatcactggc
aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca 3240ggctctcgat gagctgatgc
tttgggccga ggactgcccc gaagtccggc acctcgtgca 3300cgcggatttc ggctccaaca
atgtcctgac ggacaatggc cgcataacag cggtcattga 3360ctggagcgag gcgatgttcg
gggattccca atacgaggtc gccaacatct tcttctggag 3420gccgtggttg gcttgtatgg
agcagcagac gcgctacttc gagcggaggc atccggagct 3480tgcaggatcg ccgcggctcc
gggcgtatat gctccgcatt ggtcttgacc aactctatca 3540gagcttggtt gacggcaatt
tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat 3600cgtccgatcc ggagccggga
ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt 3660ctggaccgat ggctgtgtag
aagtactcgc cgatagtgga aaccgacgcc ccagcactcg 3720tccgagggca aaggaatagt
gaggtaccta aagaaggagt gcgtcgaagc agatcgttca 3780aacatttggc aataaagttt
cttaagattg aatcctgttg ccggtcttgc gatgattatc 3840atataatttc tgttgaatta
cgttaagcat gtaataatta acatgtaatg catgacgtta 3900tttatgagat gggtttttat
gattagagtc ccgcaattat acatttaata cgcgatagaa 3960aacaaaatat agcgcgcaaa
ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta 4020gatcgatgtc gaatcgatca
acctgcatta atgaatcggc caacgcgcgg ggagaggcgg 4080tttgcgtatt gggcgctctt
ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 4140gctgcggcga gcggtatcag
ctcactcaaa ggcggtaata cggttatcca cagaatcagg 4200ggataacgca ggaaagaaca
tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4260ggccgcgttg ctggcgtttt
tccataggct ccgcccccct gacgagcatc acaaaaatcg 4320acgctcaagt cagaggtggc
gaaacccgac aggactataa agataccagg cgtttccccc 4380tggaagctcc ctcgtgcgct
ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4440ctttctccct tcgggaagcg
tggcgctttc tcaatgctca cgctgtaggt atctcagttc 4500ggtgtaggtc gttcgctcca
agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4560ctgcgcctta tccggtaact
atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4620actggcagca gccactggta
acaggattag cagagcgagg tatgtaggcg gtgctacaga 4680gttcttgaag tggtggccta
actacggcta cactagaagg acagtatttg gtatctgcgc 4740tctgctgaag ccagttacct
tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4800caccgctggt agcggtggtt
tttttgtttg caagcagcag attacgcgca gaaaaaaagg 4860atctcaagaa gatcctttga
tcttttctac ggggtctgac gctcagtgga acgaaaactc 4920acgttaaggg attttggtca
tgacattaac ctataaaaat aggcgtatca cgaggccctt 4980tcgtctcgcg cgtttcggtg
atgacggtga aaacctctga cacatgcagc tcccggagac 5040ggtcacagct tgtctgtaag
cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc 5100gggtgttggc gggtgtcggg
gctggcttaa ctatgcggca tcagagcaga ttgtactgag 5160agtgcaccat atggacatat
tgtcgttaga acgcggctac aattaataca taaccttatg 5220tatcatacac atacgattta
ggtgacacta tagaacggcg cgccaagctt gttgaaacat 5280ccctgaagtg tctcatttta
ttttatttat tctttgctga taaaaaaata aaataaaaga 5340agctaagcac acggtcaacc
attgctctac tgctaaaagg gttatgtgta gtgttttact 5400gcataaatta tgcagcaaac
aagacaactc aaattaaaaa atttcctttg cttgtttttt 5460tgttgtctct gacttgactt
tcttgtggaa gttggttgta taaggattgg gacaccattg 5520tccttcttaa tttaatttta
ttctttgctg ataaaaaaaa aaatttcata tagtgttaaa 5580taataatttg ttaaataacc
aaaaagtcaa atatgtttac tctcgtttaa ataattgaga 5640ttcgtccagc aaggctaaac
gattgtatag atttatgaca atatttactt ttttatagat 5700aaatgttata ttataataaa
tttatataca tatattatat gttatttatt attattttaa 5760atccttcaat attttatcaa
accaactcat aatttttttt ttatctgtaa gaagcaataa 5820aattaaatag acccacttta
aggatgatcc aacctttata cagagtaaga gagttcaaat 5880agtacccttt catatacata
tcaactaaaa tattagaaat atcatggatc aaaccttata 5940aagacattaa ataagtggat
aagtataata tataaatggg tagtatataa tatataaatg 6000gatacaaact tctctcttta
taattgttat gtctccttaa catcctaata taatacataa 6060gtgggtaata tataatatat
aaatggagac aaacttcttc cattataatt gttatgtctt 6120cttaacactt atgtctcgtt
cacaatgcta aggttagaat tgtttagaaa gtcttatagt 6180acacatttgt ttttgtacta
tttgaagcat tccataagcc gtcacgattc agatgattta 6240taataataag aggaaattta
tcatagaaca ataaggtgca tagatagagt gttaatatat 6300cataacatcc tttgtttatt
catagaagaa gtgagatgga gctcagttat tatactgtta 6360catggtcgga tacaatattc
catgctctcc atgagctctt acacctacat gcattttagt 6420tcatacttgc ggccgcagta
tatcttaaat tctttaatac ggtgtactag gatattgaac 6480tggttcttga tgatgaaaac
ctgggccgag attgcagcta tttatagtca taggtcttgt 6540taacatgcat ggacatttgg
ccacggggtg gcatgcagtt tgacgggtgt tgaaataaac 6600aaaaatgagg tggcggaaga
gaatacgagt ttgaggttgg gttagaaaca acaaatgtga 6660gggctcatga tgggttgagt
tggtgaatgt tttgggctgc tcgattgaca cctttgtgag 6720tacgtgttgt tgtgcatggc
ttttggggtc cagttttttt ttcttgacgc ggcgatcctg 6780atcagctagt ggataagtga
tgtccactgt gtgtgattgc gtttttgttt gaattttatg 6840aacttagaca ttgctatgca
aaggatactc tcattgtgtt ttgtcttctt ttgttccttg 6900gctttttctt atgatccaag
agactagtca gtgttgtggc attcgagact accaagatta 6960attatgatgg gggaaggata
agtaactgat tagtacggac tgttaccaaa ttaattaata 7020agcggcaaat gaagggcatg
gatcaaaagc ttggatctcc tgcaggatct ggccggccgg 7080atctc
7085477873DNAArtificial
Sequenceplasmid pKR912 47ggccgcaagt atgaactaaa atgcatgtag gtgtaagagc
tcatggagag catggaatat 60tgtatccgac catgtaacag tataataact gagctccatc
tcacttcttc tatgaataaa 120caaaggatgt tatgatatat taacactcta tctatgcacc
ttattgttct atgataaatt 180tcctcttatt attataaatc atctgaatcg tgacggctta
tggaatgctt caaatagtac 240aaaaacaaat gtgtactata agactttcta aacaattcta
accttagcat tgtgaacgag 300acataagtgt taagaagaca taacaattat aatggaagaa
gtttgtctcc atttatatat 360tatatattac ccacttatgt attatattag gatgttaagg
agacataaca attataaaga 420gagaagtttg tatccattta tatattatat actacccatt
tatatattat acttatccac 480ttatttaatg tctttataag gtttgatcca tgatatttct
aatattttag ttgatatgta 540tatgaaaggg tactatttga actctcttac tctgtataaa
ggttggatca tccttaaagt 600gggtctattt aattttattg cttcttacag ataaaaaaaa
aattatgagt tggtttgata 660aaatattgaa ggatttaaaa taataataaa taacatataa
tatatgtata taaatttatt 720ataatataac atttatctat aaaaaagtaa atattgtcat
aaatctatac aatcgtttag 780ccttgctgga cgaatctcaa ttatttaaac gagagtaaac
atatttgact ttttggttat 840ttaacaaatt attatttaac actatatgaa attttttttt
ttatcagcaa agaataaaat 900taaattaaga aggacaatgg tgtcccaatc cttatacaac
caacttccac aagaaagtca 960agtcagagac aacaaaaaaa caagcaaagg aaatttttta
atttgagttg tcttgtttgc 1020tgcataattt atgcagtaaa acactacaca taaccctttt
agcagtagag caatggttga 1080ccgtgtgctt agcttctttt attttatttt tttatcagca
aagaataaat aaaataaaat 1140gagacacttc agggatgttt caacaagctt ggcgcgccgt
tctatagtgt cacctaaatc 1200gtatgtgtat gatacataag gttatgtatt aattgtagcc
gcgttctaac gacaatatgt 1260ccatatggtg cactctcagt acaatctgct ctgatgccgc
atagttaagc cagccccgac 1320acccgccaac acccgctgac gcgccctgac gggcttgtct
gctcccggca tccgcttaca 1380gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
gttttcaccg tcatcaccga 1440aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
ataggttaat gtcatgacca 1500aaatccctta acgtgagttt tcgttccact gagcgtcaga
ccccgtagaa aagatcaaag 1560gatcttcttg agatcctttt tttctgcgcg taatctgctg
cttgcaaaca aaaaaaccac 1620cgctaccagc ggtggtttgt ttgccggatc aagagctacc
aactcttttt ccgaaggtaa 1680ctggcttcag cagagcgcag ataccaaata ctgtccttct
agtgtagccg tagttaggcc 1740accacttcaa gaactctgta gcaccgccta catacctcgc
tctgctaatc ctgttaccag 1800tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
ggactcaaga cgatagttac 1860cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
cacacagccc agcttggagc 1920gaacgaccta caccgaactg agatacctac agcgtgagca
ttgagaaagc gccacgcttc 1980ccgaagggag aaaggcggac aggtatccgg taagcggcag
ggtcggaaca ggagagcgca 2040cgagggagct tccaggggga aacgcctggt atctttatag
tcctgtcggg tttcgccacc 2100tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
gcggagccta tggaaaaacg 2160ccagcaacgc ggccttttta cggttcctgg ccttttgctg
gccttttgct cacatgttct 2220ttcctgcgtt atcccctgat tctgtggata accgtattac
cgcctttgag tgagctgata 2280ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
gagcgaggaa gcggaagagc 2340gcccaatacg caaaccgcct ctccccgcgc gttggccgat
tcattaatgc aggttgatcg 2400attcgacatc gatctagtaa catagatgac accgcgcgcg
ataatttatc ctagtttgcg 2460cgctatattt tgttttctat cgcgtattaa atgtataatt
gcgggactct aatcataaaa 2520acccatctca taaataacgt catgcattac atgttaatta
ttacatgctt aacgtaattc 2580aacagaaatt atatgataat catcgcaaga ccggcaacag
gattcaatct taagaaactt 2640tattgccaaa tgtttgaacg atctgcttcg acgcactcct
tctttaggta cctcactatt 2700cctttgccct cggacgagtg ctggggcgtc ggtttccact
atcggcgagt acttctacac 2760agccatcggt ccagacggcc gcgcttctgc gggcgatttg
tgtacgcccg acagtcccgg 2820ctccggatcg gacgattgcg tcgcatcgac cctgcgccca
agctgcatca tcgaaattgc 2880cgtcaaccaa gctctgatag agttggtcaa gaccaatgcg
gagcatatac gcccggagcc 2940gcggcgatcc tgcaagctcc ggatgcctcc gctcgaagta
gcgcgtctgc tgctccatac 3000aagccaacca cggcctccag aagaagatgt tggcgacctc
gtattgggaa tccccgaaca 3060tcgcctcgct ccagtcaatg accgctgtta tgcggccatt
gtccgtcagg acattgttgg 3120agccgaaatc cgcgtgcacg aggtgccgga cttcggggca
gtcctcggcc caaagcatca 3180gctcatcgag agcctgcgcg acggacgcac tgacggtgtc
gtccatcaca gtttgccagt 3240gatacacatg gggatcagca atcgcgcata tgaaatcacg
ccatgtagtg tattgaccga 3300ttccttgcgg tccgaatggg ccgaacccgc tcgtctggct
aagatcggcc gcagcgatcg 3360catccatggc ctccgcgacc ggctgcagaa cagcgggcag
ttcggtttca ggcaggtctt 3420gcaacgtgac accctgtgca cggcgggaga tgcaataggt
caggctctcg ctgaattccc 3480caatgtcaag cacttccgga atcgggagcg cggccgatgc
aaagtgccga taaacataac 3540gatctttgta gaaaccatcg gcgcagctat ttacccgcag
gacatatcca cgccctccta 3600catcgaagct gaaagcacga gattcttcgc cctccgagag
ctgcatcagg tcggagacgc 3660tgtcgaactt ttcgatcaga aacttctcga cagacgtcgc
ggtgagttca ggctttttca 3720tggtttaata agaagagaaa agagttcttt tgttatggct
gaagtaatag agaaatgagc 3780tcgagcgtgt cctctccaaa tgaaatgaac ttccttatat
agaggaaggg tcttgcgaag 3840gatagtggga ttgtgcgtca tcccttacgt cagtggagat
gtcacatcaa tccacttgct 3900ttgaagacgt ggttggaacg tcttcttttt ccacgatgct
cctcgtgggt gggggtccat 3960ctttgggacc actgtcggca gaggcatctt gaatgatagc
ctttccttta tcgcaatgat 4020ggcatttgta ggagccacct tccttttcta ctgtcctttc
gatgaagtga cagatagctg 4080ggcaatggaa tccgaggagg tttcccgaaa ttatcctttg
ttgaaaagtc tcaatagccc 4140tttggtcttc tgagactgta tctttgacat ttttggagta
gaccagagtg tcgtgctcca 4200ccatgttgac gaagattttc ttcttgtcat tgagtcgtaa
aagactctgt atgaactgtt 4260cgccagtctt cacggcgagt tctgttagat cctcgatttg
aatcttagac tccatgcatg 4320gccttagatt cagtaggaac taccttttta gagactccaa
tctctattac ttgccttggt 4380ttatgaagca agccttgaat cgtccatact ggaatagtac
ttctgatctt gagaaatatg 4440tctttctctg tgttcttgat gcaattagtc ctgaatcttt
tgactgcatc tttaaccttc 4500ttgggaaggt atttgatctc ctggagattg ttactcgggt
agatcgtctt gatgagacct 4560gctgcgtagg cctctctaac catctgtggg tcagcattct
ttctgaaatt gaagaggcta 4620accttctcat tatcagtggt gaacatagtg tcgtcacctt
caccttcgaa cttccttcct 4680agatcgtaaa gatagaggaa atcgtccatt gtaatctccg
gggcaaagga gatctctttt 4740ggggctggat cactgctggg ccttttggtt cctagcgtga
gccagtgggc tttttgcttt 4800ggtgggcttg ttagggcctt agcaaagctc ttgggcttga
gttgagcttc tcctttgggg 4860atgaagttca acctgtctgt ttgctgactt gttgtgtacg
cgtcagctgc tgctcttgcc 4920tctgtaatag tggcaaattt cttgtgtgca actccgggaa
cgccgtttgt tgccgccttt 4980gtacaacccc agtcatcgta tataccggca tgtggaccgt
tatacacaac gtagtagttg 5040atatgagggt gttgaatacc cgattctgct ctgagaggag
caactgtgct gttaagctca 5100gatttttgtg ggattggaat tggatcgatc tcgatcccgc
gaaattaata cgactcacta 5160tagggagacc acaacggttt ccctctagaa ataattttgt
ttaactttaa gaaggagata 5220tacccatgga aaagcctgaa ctcaccgcga cgtctgtcga
gaagtttctg atcgaaaagt 5280tcgacagcgt ctccgacctg atgcagctct cggagggcga
agaatctcgt gctttcagct 5340tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag
ctgcgccgat ggtttctaca 5400aagatcgtta tgtttatcgg cactttgcat cggccgcgct
cccgattccg gaagtgcttg 5460acattgggga attcagcgag agcctgacct attgcatctc
ccgccgtgca cagggtgtca 5520cgttgcaaga cctgcctgaa accgaactgc ccgctgttct
gcagccggtc gcggaggcta 5580tggatgcgat cgctgcggcc gatcttagcc agacgagcgg
gttcggccca ttcggaccgc 5640aaggaatcgg tcaatacact acatggcgtg atttcatatg
cgcgattgct gatccccatg 5700tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc
gtccgtcgcg caggctctcg 5760atgagctgat gctttgggcc gaggactgcc ccgaagtccg
gcacctcgtg cacgcggatt 5820tcggctccaa caatgtcctg acggacaatg gccgcataac
agcggtcatt gactggagcg 5880aggcgatgtt cggggattcc caatacgagg tcgccaacat
cttcttctgg aggccgtggt 5940tggcttgtat ggagcagcag acgcgctact tcgagcggag
gcatccggag cttgcaggat 6000cgccgcggct ccgggcgtat atgctccgca ttggtcttga
ccaactctat cagagcttgg 6060ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg
atgcgacgca atcgtccgat 6120ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag
aagcgcggcc gtctggaccg 6180atggctgtgt agaagtactc gccgatagtg gaaaccgacg
ccccagcact cgtccgaggg 6240caaaggaata gtgaggtaca gcttggatcg atccggctgc
taacaaagcc cgaaaggaag 6300ctgagttggc tgctgccacc gctgagcaat aactagcata
accccttggg gcctctaaac 6360gggtcttgag gggttttttg ctgaaaggag gaactatatc
cggatgatcg ggcgcgccgt 6420cgacggatcc gtacgagatc cggccggcca gatcctgcag
gagatccaag cttttgatcc 6480atgcccttca tttgccgctt attaattaat ttggtaacag
tccgtactaa tcagttactt 6540atccttcccc catcataatt aatcttggta gtctcgaatg
ccacaacact gactagtctc 6600ttggatcata agaaaaagcc aaggaacaaa agaagacaaa
acacaatgag agtatccttt 6660gcatagcaat gtctaagttc ataaaattca aacaaaaacg
caatcacaca cagtggacat 6720cacttatcca ctagctgatc aggatcgccg cgtcaagaaa
aaaaaactgg accccaaaag 6780ccatgcacaa caacacgtac tcacaaaggt gtcaatcgag
cagcccaaaa cattcaccaa 6840ctcaacccat catgagccct cacatttgtt gtttctaacc
caacctcaaa ctcgtattct 6900cttccgccac ctcatttttg tttatttcaa cacccgtcaa
actgcatgcc accccgtggc 6960caaatgtcca tgcatgttaa caagacctat gactataaat
agctgcaatc tcggcccagg 7020ttttcatcat caagaaccag ttcaatatcc tagtacaccg
tattaaagaa tttaagatat 7080actgcggccg caccatggag gtggtgaatg aaatagtctc
aattgggcag gaagttttac 7140ccaaagttga ttatgcccaa ctctggagtg atgccagtca
ctgtgaggtg ctttacttgt 7200ccatcgcatt tgtcatcttg aagttcactc ttggccccct
tggtccaaaa ggtcagtctc 7260gtatgaagtt tgttttcacc aattacaacc ttctcatgtc
catttattcg ttgggatcat 7320tcctctcaat ggcatatgcc atgtacacca tcggtgttat
gtctgacaac tgcgagaagg 7380cttttgacaa caacgtcttc aggatcacca cgcagttgtt
ctatttgagc aagttcctgg 7440agtatattga ctccttctat ttgccactga tgggcaagcc
tctgacctgg ttgcaattct 7500tccatcattt gggggcaccg atggatatgt ggctgttcta
taattaccga aatgaagctg 7560tttggatttt tgtgctgttg aatggtttca tccactggat
catgtacggt tattattgga 7620ccagattgat caagctgaag ttccccatgc caaaatccct
gattacatca atgcagatca 7680ttcaattcaa tgttggtttc tacattgtct ggaagtacag
gaacattccc tgttatcgcc 7740aagatgggat gaggatgttt ggctggttct tcaattactt
ttatgttggc acagtcttgt 7800gtttgttctt gaatttctat gtgcaaacgt atatcgtcag
gaagcacaag ggagccaaaa 7860agattcagtg agc
7873489892DNAArtificial Sequenceplasmid pKR886r
48ggtcgactcg acgtacgtct agaggatccg tcgacggcgc gcccgatcat ccggatatag
60ttcctccttt cagcaaaaaa cccctcaaga cccgtttaga ggccccaagg ggttatgcta
120gttattgctc agcggtggca gcagccaact cagcttcctt tcgggctttg ttagcagccg
180gatcgatcca agctgtacct cactattcct ttgccctcgg acgagtgctg gggcgtcggt
240ttccactatc ggcgagtact tctacacagc catcggtcca gacggccgcg cttctgcggg
300cgatttgtgt acgcccgaca gtcccggctc cggatcggac gattgcgtcg catcgaccct
360gcgcccaagc tgcatcatcg aaattgccgt caaccaagct ctgatagagt tggtcaagac
420caatgcggag catatacgcc cggagccgcg gcgatcctgc aagctccgga tgcctccgct
480cgaagtagcg cgtctgctgc tccatacaag ccaaccacgg cctccagaag aagatgttgg
540cgacctcgta ttgggaatcc ccgaacatcg cctcgctcca gtcaatgacc gctgttatgc
600ggccattgtc cgtcaggaca ttgttggagc cgaaatccgc gtgcacgagg tgccggactt
660cggggcagtc ctcggcccaa agcatcagct catcgagagc ctgcgcgacg gacgcactga
720cggtgtcgtc catcacagtt tgccagtgat acacatgggg atcagcaatc gcgcatatga
780aatcacgcca tgtagtgtat tgaccgattc cttgcggtcc gaatgggccg aacccgctcg
840tctggctaag atcggccgca gcgatcgcat ccatagcctc cgcgaccggc tgcagaacag
900cgggcagttc ggtttcaggc aggtcttgca acgtgacacc ctgtgcacgg cgggagatgc
960aataggtcag gctctcgctg aattccccaa tgtcaagcac ttccggaatc gggagcgcgg
1020ccgatgcaaa gtgccgataa acataacgat ctttgtagaa accatcggcg cagctattta
1080cccgcaggac atatccacgc cctcctacat cgaagctgaa agcacgagat tcttcgccct
1140ccgagagctg catcaggtcg gagacgctgt cgaacttttc gatcagaaac ttctcgacag
1200acgtcgcggt gagttcaggc ttttccatgg gtatatctcc ttcttaaagt taaacaaaat
1260tatttctaga gggaaaccgt tgtggtctcc ctatagtgag tcgtattaat ttcgcgggat
1320cgagatctga tcaacctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
1380attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
1440cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
1500gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
1560ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
1620agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
1680tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
1740ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag
1800gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
1860ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
1920gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
1980aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
2040aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
2100ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
2160gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
2220gggattttgg tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc
2280gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca
2340gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
2400ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac
2460catatggaca tattgtcgtt agaacgcggc tacaattaat acataacctt atgtatcata
2520cacatacgat ttaggtgaca ctatagaacg gcgcgccaag ctgggtctag aactagaaac
2580gtgatgccac ttgttattga agtcgattac agcatctatt ctgttttact atttataact
2640ttgccatttc tgacttttga aaactatctc tggatttcgg tatcgctttg tgaagatcga
2700gcaaaagaga cgttttgtgg acgcaatggt ccaaatccgt tctacatgaa caaattggtc
2760acaatttcca ctaaaagtaa ataaatggca agttaaaaaa ggaatatgca ttttactgat
2820tgcctaggtg agctccaaga gaagttgaat ctacacgtct accaaccgct aaaaaaagaa
2880aaacattgat atgtaacctg attccattag cttttgactt cttcaacaga ttctctactt
2940agatttctaa cagaaatatt attactagca catcattttc agtctcacta cagcaaaaaa
3000tccaacggca caatacagac aacaggagat atcagactac agagatagat agatgctact
3060gcatgtagta agttaaataa aaggaaaata aaatgtcttg ctaccaaaac tactacagac
3120tatgatgctc accacaggcc aaatcctgca actaggacag cattatctta tatatattgt
3180acaaaacaag catcaaggaa catttggtct aggcaatcag tacctcgttc taccatcacc
3240ctcagttatc acatccttga aggatccatt actgggaatc atcggcaaca catgctcctg
3300atggggcaca atgacatcaa gaaggtaggg gccaggggtg tccaacattc tctgaattgc
3360cgctctaagc tcttccttct tcgtcactcg cgctgccggt atcccacaag catcagcaaa
3420cttgagcatg tttgggaata tctcgctctc gctagacgga tctccaagat aggtgtgagc
3480tctattggac ttgtagaacc tatcctccaa ctgaaccacc atacccaaat gctgattgtt
3540caacaacaat atcttaactg ggagattctc cactcttata gtggccaact cctgaacatt
3600catgatgaaa ctaccatccc catcaatgtc aaccacaaca gccccagggt tagcaacagc
3660agcaccaata gccgcaggca atccaaaacc catggctcca agaccccctg aggtcaacca
3720ctgcctcggt ctcttgtact tgtaaaactg cgcagcccac atttgatgct gcccaacccc
3780agtactaaca atagcatctc cattagtcaa ctcatcaaga acctcgatag catgctgcgg
3840agaaatcgcg tcctggaatg tcttgtaacc caatggaaac ttgtgtttct gcacattaat
3900ctcttctctc caacctccaa gatcaaactt accctccact cctttctcct ccaaaatcat
3960attaattccc ttcaaggcca acttcaaatc cgcgcaaacc gacacgtgcg cctgcttgtt
4020cttcccaatc tcggcagaat caatatcaat gtgaacaatc ttagccctac tagcaaaagc
4080ctcaagcttc ccagtaacac ggtcatcaaa ccttacccca aaggcaagca acaaatcact
4140attgtcaaca gcatagttag cataaacagt accatgcata cccagcatct gaagggaata
4200ttcatcacca ataggaaaag ttccaagacc cattaaagtg ctagcaacgg gaataccagt
4260gagttcaaca aagcgcctca attcagcact ggaattcaaa ctgccaccgc cgacgtagag
4320aacgggcttt tgggcctcca tgatgagtct gacaatgtgt tccaattggg cctcggcggg
4380gggcctgggc agcctggcga ggtaaccggg gaggttaacg ggctcgtccc aattaggcac
4440ggcgagttgc tgctgaacgt ctttgggaat gtcgatgagg accggaccgg ggcggccgga
4500ggtggcgacg aagaaagcct cggcgacgac gcgggggatg tcgtcgacgt cgaggatgag
4560gtagttgtgc ttcgtgatgg atctgctcac ctccacgatc ggggtttctt ggaaggcgtc
4620ggtgccgatc atccggcggg cgacctggcc ggtgatggcg acgactggga cgctgtccat
4680taaagcgtcg gcgaggccgc tcacgaggtt ggtggcgccg gggccggagg tggcaatgca
4740gacgccgggg aggccggagg aacgcgcgta gccttcggcg gcgaagacgc cgccctgctc
4800gtggcgcggg agcacgttgc ggatggcggc ggagcgcgtg agcgcctggt ggatctccat
4860cgacgcaccg ccggggtacg cgaacaccgt cgtcacgccc tgcctctcca gcgcctccac
4920aaggatgtcc gcgcccttgc gaggttcgcc ggaggcgaac cgtgacacga agggctccgt
4980ggtcggcgct tccttggtga agggcgccgc cgtggggggt ttggagatgg aacatttgat
5040tttgagagcg tggttgggtt tggtgagggt ttgatgagag agagggaggg tggatctagt
5100aatgcgtttg gggaaggtgg ggtgtgaaga ggaagaagag aatcgggtgg ttctggaagc
5160ggtggccgcc attgtgttgt gtggcatggt tatacttcaa aaactgcaca acaagcctag
5220agttagtacc taaacagtaa atttacaaca gagagcaaag acacatgcaa aaatttcagc
5280cataaaaaaa gttataatag aatttaaagc aaaagtttca ttttttaaac atatatacaa
5340acaaactgga tttgaaggaa gggattaatt cccctgctca aagtttgaat tcctattgtg
5400acctatactc gaataaaatt gaagcctaag gaatgtatga gaaacaagaa aacaaaacaa
5460aactacagac aaacaagtac aattacaaaa ttcgctaaaa ttctgtaatc accaaacccc
5520atctcagtca gcacaaggcc caaggtttat tttgaaataa aaaaaaagtg attttatttc
5580tcataagcta aaagaaagaa aggcaattat gaaatgattt cgactagatc tgaaagtcca
5640acgcgtattc cgcagatatt aaagaaagag tagagtttca catggatcct agatggaccc
5700agttgaggaa aaagcaaggc aaagcaaacc agaagtgcaa gatccgaaat tgaaccacgg
5760aatctaggat ttggtagagg gagaagaaaa gtaccttgag aggtagaaga gaagagaaga
5820gcagagagat atatgaacga gtgtgtcttg gtctcaactc tgaagcgata cgagtttaga
5880ggggagcatt gagttccaat ttatagggaa accgggtggc aggggtgagt taatgacgga
5940aaagccccta agtaacgaga ttggattgtg ggttagattc aaccgtttgc atccgcggct
6000tagattgggg aagtcagagt gaatctcaac cgttgactga gttgaaaatt gaatgtagca
6060accaattgag ccaaccccag cctttgccct ttgattttga tttgtttgtt gcatactttt
6120tatttgtctt ctggttctga ctctctttct ctcgtttcaa tgccaggttg cctactccca
6180caccactcac aagaagattc tactgttagt attaaatatt ttttaatgta ttaaatgatg
6240aatgcttttg taaacagaac aagactatgt ctaataagtg tcttgcaaca ttttttaaga
6300aattaaaaaa aatatattta ttatcaaaat caaatgtatg aaaaatcatg aataatataa
6360ttttatacat ttttttaaaa aatcttttaa tttcttaatt aatatcttaa aaataatgat
6420taatatttaa cccaaaataa ttagtatgat tggtaaggaa gatatccatg ttatgtttgg
6480atgtgagttt gatctagagc aaagcttact agagtcgacc tgcagcccgg gggatccgcc
6540cacgtacggt accatctgct aatattttaa atcacatgca agagaggagg catggttcca
6600ttttctacct tcacattatt tgagaaaaac gaacttgttc tgtgttttat ttttgccctt
6660cacattagta caacgtggaa gactcatggt tacacagaat catacataag tacaatgctt
6720gtccctaaga aaacaagcac tcgttgtatt gaacctttac ggctcatgcg gccgcgaatt
6780cactagtgat tgaattcgcg gccgcttagt ccgacttggc cttggcggcc gcggccgact
6840ctttgagcgt gaagatctgc gccgtctcgg gcacagcgcc gtagttgaca aagaggtgcg
6900cggtcttgaa gaaggccgtg atgatgggct cgtcgttcct gcgcacgagg tgcgggtacg
6960cggccgcaaa gtgcttggtg gcttcgttga gcttgtagtg cggaatgatc gggaacaagt
7020ggtggacctg gtgcgtgcca atgtggtggc tcaggttgtc cacgaacgcg ccgtacgagc
7080ggtcgacgct cgagaggttg cccttgacgt acgtccactc cgagtcgccg taccacggcg
7140tcgcttcgtc gttgtggtgc aagaaggtcg taatgacgag gaacgaagca aagacaaaga
7200gcggcgcata gtagtagagg cccatgacgg caaagccgag cgagtatgtg aggtacgcgt
7260acgcggcgaa gaaggcggcc cagacgccga gcgacacgat gacggccgac gcgcggcgaa
7320ggaggagcgg gtcccacggg tcaaagtggc tcatcgtgcg cggggcatac ccgaccttca
7380agtagacaaa ccacgcaccg ccgagcgtgt agacccattg gcgcacgtcc tggaggtcct
7440tgaccgaccg gtgcgggtaa aagatctcgt ccttatcaat gttgcccgtg ttcttgtggt
7500ggtggcggtg cgtcacgcgc cagctctcga acggcgtcaa aatcgcagag tgcatgatgc
7560agccgatgat aaagttgacg ctgtggtagc gcgagaaggc cgagtggccg cagtcgtggc
7620cgaccgtgaa gaagccccag aagatgacgc cctgcacgta gatgtaggtg gcgcaaacga
7680gcgcgtggag cagaacgtta tcggcaatga acggcgtcga gcgcgccgcg tagagcagcg
7740ccgccgaggc cgacgcgttg aagatcgcgc gggccgtgta gtagagcgag aggccgaggt
7800tcgactcaaa gcacgcgttc gggatcgagt gcttgagctc cgtgagcgtc gggaactcga
7860ccttcgtctt atcctcagtc atgcggccgc tgaagtattg cttcttagtt aacctttcct
7920ttctctctca gctatgtgaa ttcattttgc tttcgtcaca atttatatag tgaaattgga
7980tctttggagt taacgccttc acaggattat cgtgttagaa caatgctttt tcatgttcta
8040attagtagta cattacaaat gtgcactcta ttcaataagc atcttttggc acgttaataa
8100atcatgtgaa aaaaaaatac tactatttca aagaaagtgt tgtaaaaaga aacggaaaga
8160gagctggctt cagttgttga gacttgtttg ctagtaaaaa tggtgtgaag agtgattcat
8220ggtgaggtgg tttttcgtcc ctttctgttt gcatgaaaaa caaatggcaa gagatgacgt
8280aggattcctt cccttaacga ttatctgttt ttaatttcaa atatacatat aggaatttat
8340gaattactaa ggttgtaaaa tatgctggtc atttatttat ggctaaaata tttttttttc
8400tcgtaaatat aaaaatattt aaaatttatt tttatcatat tttttatcct tataaaatta
8460tgtgtacaac ctatataaaa aaatatcata tttaatattg attatatgtt taatcaatat
8520aaaaaatcat tatcatatat ttagatttat tcgaatatac atctaaacaa aaaataacat
8580attttaattt tatgaagaaa aaaaaatatt ttatccttta tttatttaag attaattaat
8640agttatgtat tgtggaaaga cttttacaca tgcaatagat atactgaatc aattagatgc
8700caatgctgag ttggaaatca cttgaggagg ggaggagact tgccaatgct tttcagtttc
8760atttaaatga tttagtggag gagatagagt agtgataaag gcatgcccca attttggagt
8820gtatatatga gtggaaataa gagagggata gagagaaaaa ataaagagag taaaaataat
8880taatgtgaaa tgatatgata aaaaaataaa gaaagagata aagagaaaaa tgaaatgaga
8940gatagatgaa atagagagta gatacatgtt tgtttaggtt ttttttagga aataacacat
9000ttttttctca tcacttatta ctcactgtca atttcctctc tttcaatcat aatgatatga
9060tttgtttaac aaaaatgtga aaaaacatat aaagtaaaat atttttataa attgataaat
9120aaaaatttac aaaatttatt tcttattaaa ttgaatagaa aatgaaagaa aagaaaagaa
9180aaagtatata taaaatgata tagctttaaa aagaataaat ttttcatatc agtctttttt
9240taataattta gaaatattta agtatatagc aaaaatataa tgtactttac atatgcataa
9300ataataattt gaaaatagaa ctaatagaat agagaaaaaa gtaatataat aattaactat
9360atgaaaattt agaagggaca atatttttaa ttaagaatat aaacaatatt tcttttcatg
9420taatgaggga cggatgtacg gggccagtgt tggagtcaaa gccaaaatag tcacggggaa
9480attaatgcac tgcatgacta ttcgaaaaaa ttcactagcc ttacttagat gttagattaa
9540tagctagggg gtgcagataa ttttgaaagg catgaaaaac attaatttgt acattgcaag
9600cttttgatga caagctttgc aattgttcac actaccttat gccatttata aatagagtga
9660ttggcatatg aaggaaatca tgagagtcga agcgaaaaac aaagcttgag agtgtaggaa
9720aaatacagtt tttttggtaa aaatacagta tttgaatagg agcgaaaaat atcctttcaa
9780aatgatcctt ttcttttttt ttttttttct tgttgttctt ggtcagttat tcaaaggaaa
9840agggattgaa ataaaaactt gcatgtggga tcgtacgtcg agtcgacctg ca
9892496021DNAArtificial Sequenceplasmid pKR271 49ggccgcgaat tcaatcacta
gtgaattcgc ggccgcatga gccgtaaagg ttcaatacaa 60cgagtgcttg ttttcttagg
gacaagcatt gtacttatgt atgattctgt gtaaccatga 120gtcttccacg ttgtactaat
gtgaagggca aaaataaaac acagaacaag ttcgtttttc 180tcaaataatg tgaaggtaga
aaatggaacc atgcctcctc tcttgcatgt gatttaaaat 240attagcagat ggtaccgtac
gtgggcggat cccccgggct gcaggaattc actggccgtc 300gttttacaac gtcgtgactg
ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca 360catccccctt tcgccagctg
gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa 420cagttgcgca gcctgaatgg
cgaatggcgc ctgatgcggt attttctcct tacgcatctg 480tgcggtattt cacaccgcat
atggtgcact ctcagtacaa tctgctctga tgccgcatag 540ttaagccagc cccgacaccc
gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc 600ccggcatccg cttacagaca
agctgtgacc gtctccggga gctgcatgtg tcagaggttt 660tcaccgtcat caccgaaacg
cgcgagacga aagggcctcg tgatacgcct atttttatag 720gttaatgtca tgataataat
ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg 780cgcggaaccc ctatttgttt
atttttctaa atacattcaa atatgtatcc gctcatgaga 840caataaccct gataaatgct
tcaataatat tgaaaaagga agagtatgag tattcaacat 900ttccgtgtcg cccttattcc
cttttttgcg gcattttgcc ttcctgtttt tgctcaccca 960gaaacgctgg tgaaagtaaa
agatgctgaa gatcagttgg gtgcacgagt gggttacatc 1020gaactggatc tcaacagcgg
taagatcctt gagagttttc gccccgaaga acgttttcca 1080atgatgagca cttttaaagt
tctgctatgt ggcgcggtat tatcccgtat tgacgccggg 1140caagagcaac tcggtcgccg
catacactat tctcagaatg acttggttga gtactcacca 1200gtcacagaaa agcatcttac
ggatggcatg acagtaagag aattatgcag tgctgccata 1260accatgagtg ataacactgc
ggccaactta cttctgacaa cgatcggagg accgaaggag 1320ctaaccgctt ttttgcacaa
catgggggat catgtaactc gccttgatcg ttgggaaccg 1380gagctgaatg aagccatacc
aaacgacgag cgtgacacca cgatgcctgt agcaatggca 1440acaacgttgc gcaaactatt
aactggcgaa ctacttactc tagcttcccg gcaacaatta 1500atagactgga tggaggcgga
taaagttgca ggaccacttc tgcgctcggc ccttccggct 1560ggctggttta ttgctgataa
atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca 1620gcactggggc cagatggtaa
gccctcccgt atcgtagtta tctacacgac ggggagtcag 1680gcaactatgg atgaacgaaa
tagacagatc gctgagatag gtgcctcact gattaagcat 1740tggtaactgt cagaccaagt
ttactcatat atactttaga ttgatttaaa acttcatttt 1800taatttaaaa ggatctaggt
gaagatcctt tttgataatc tcatgaccaa aatcccttaa 1860cgtgagtttt cgttccactg
agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1920gatccttttt ttctgcgcgt
aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1980gtggtttgtt tgccggatca
agagctacca actctttttc cgaaggtaac tggcttcagc 2040agagcgcaga taccaaatac
tgtccttcta gtgtagccgt agttaggcca ccacttcaag 2100aactctgtag caccgcctac
atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 2160agtggcgata agtcgtgtct
taccgggttg gactcaagac gatagttacc ggataaggcg 2220cagcggtcgg gctgaacggg
gggttcgtgc acacagccca gcttggagcg aacgacctac 2280accgaactga gatacctaca
gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 2340aaggcggaca ggtatccggt
aagcggcagg gtcggaacag gagagcgcac gagggagctt 2400ccagggggaa acgcctggta
tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2460cgtcgatttt tgtgatgctc
gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2520gcctttttac ggttcctggc
cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2580tcccctgatt ctgtggataa
ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2640agccgaacga ccgagcgcag
cgagtcagtg agcgaggaag cggaagagcg cccaatacgc 2700aaaccgcctc tccccgcgcg
ttggccgatt cattaatgca gctggcacga caggtttccc 2760gactggaaag cgggcagtga
gcgcaacgca attaatgtga gttagctcac tcattaggca 2820ccccaggctt tacactttat
gcttccggct cgtatgttgt gtggaattgt gagcggataa 2880caatttcaca caggaaacag
ctatgaccat gattacgcca agcttgcatg cctgcaggtc 2940gactcgacgt acgatcccac
atgcaagttt ttatttcaat cccttttcct ttgaataact 3000gaccaagaac aacaagaaaa
aaaaaaaaaa agaaaaggat cattttgaaa ggatattttt 3060cgctcctatt caaatactgt
atttttacca aaaaaactgt atttttccta cactctcaag 3120ctttgttttt cgcttcgact
ctcatgattt ccttcatatg ccaatcactc tatttataaa 3180tggcataagg tagtgtgaac
aattgcaaag cttgtcatca aaagcttgca atgtacaaat 3240taatgttttt catgcctttc
aaaattatct gcacccccta gctattaatc taacatctaa 3300gtaaggctag tgaatttttt
cgaatagtca tgcagtgcat taatttcccc gtgactattt 3360tggctttgac tccaacactg
gccccgtaca tccgtccctc attacatgaa aagaaatatt 3420gtttatattc ttaattaaaa
atattgtccc ttctaaattt tcatatagtt aattattata 3480ttactttttt ctctattcta
ttagttctat tttcaaatta ttatttatgc atatgtaaag 3540tacattatat ttttgctata
tacttaaata tttctaaatt attaaaaaaa gactgatatg 3600aaaaatttat tctttttaaa
gctatatcat tttatatata ctttttcttt tcttttcttt 3660cattttctat tcaatttaat
aagaaataaa ttttgtaaat ttttatttat caatttataa 3720aaatatttta ctttatatgt
tttttcacat ttttgttaaa caaatcatat cattatgatt 3780gaaagagagg aaattgacag
tgagtaataa gtgatgagaa aaaaatgtgt tatttcctaa 3840aaaaaaccta aacaaacatg
tatctactct ctatttcatc tatctctcat ttcatttttc 3900tctttatctc tttctttatt
tttttatcat atcatttcac attaattatt tttactctct 3960ttattttttc tctctatccc
tctcttattt ccactcatat atacactcca aaattggggc 4020atgcctttat cactactcta
tctcctccac taaatcattt aaatgaaact gaaaagcatt 4080ggcaagtctc ctcccctcct
caagtgattt ccaactcagc attggcatct aattgattca 4140gtatatctat tgcatgtgta
aaagtctttc cacaatacat aactattaat taatcttaaa 4200taaataaagg ataaaatatt
tttttttctt cataaaatta aaatatgtta ttttttgttt 4260agatgtatat tcgaataaat
ctaaatatat gataatgatt ttttatattg attaaacata 4320taatcaatat taaatatgat
atttttttat ataggttgta cacataattt tataaggata 4380aaaaatatga taaaaataaa
ttttaaatat ttttatattt acgagaaaaa aaaatatttt 4440agccataaat aaatgaccag
catattttac aaccttagta attcataaat tcctatatgt 4500atatttgaaa ttaaaaacag
ataatcgtta agggaaggaa tcctacgtca tctcttgcca 4560tttgtttttc atgcaaacag
aaagggacga aaaaccacct caccatgaat cactcttcac 4620accattttta ctagcaaaca
agtctcaaca actgaagcca gctctctttc cgtttctttt 4680tacaacactt tctttgaaat
agtagtattt ttttttcaca tgatttatta acgtgccaaa 4740agatgcttat tgaatagagt
gcacatttgt aatgtactac taattagaac atgaaaaagc 4800attgttctaa cacgataatc
ctgtgaaggc gttaactcca aagatccaat ttcactatat 4860aaattgtgac gaaagcaaaa
tgaattcaca tagctgagag agaaaggaaa ggttaactaa 4920gaagcaatac ttcagcggcc
gcatgactga ggataagacg aaggtcgagt tcccgacgct 4980cacggagctc aagcactcga
tcccgaacgc gtgctttgag tcgaacctcg gcctctcgct 5040ctactacacg gcccgcgcga
tcttcaacgc gtcggcctcg gcggcgctgc tctacgcggc 5100gcgctcgacg ccgttcattg
ccgataacgt tctgctccac gcgctcgttt gcgccaccta 5160catctacgtg cagggcgtca
tcttctgggg cttcttcacg gtcggccacg actgcggcca 5220ctcggccttc tcgcgctacc
acagcgtcaa ctttatcatc ggctgcatca tgcactctgc 5280gattttgacg ccgttcgaga
gctggcgcgt gacgcaccgc caccaccaca agaacacggg 5340caacattgat aaggacgaga
tcttttaccc gcaccggtcg gtcaaggacc tccaggacgt 5400gcgccaatgg gtctacacgc
tcggcggtgc gtggtttgtc tacttgaagg tcgggtatgc 5460cccgcgcacg atgagccact
ttgacccgtg ggacccgctc ctccttcgcc gcgcgtcggc 5520cgtcatcgtg tcgctcggcg
tctgggccgc cttcttcgcc gcgtacgcgt acctcacata 5580ctcgctcggc tttgccgtca
tgggcctcta ctactatgcg ccgctctttg tctttgcttc 5640gttcctcgtc attacgacct
tcttgcacca caacgacgaa gcgacgccgt ggtacggcga 5700ctcggagtgg acgtacgtca
agggcaacct ctcgagcgtc gaccgctcgt acggcgcgtt 5760cgtggacaac ctgagccacc
acattggcac gcaccaggtc caccacttgt tcccgatcat 5820tccgcactac aagctcaacg
aagccaccaa gcactttgcg gccgcgtacc cgcacctcgt 5880gcgcaggaac gacgagccca
tcatcacggc cttcttcaag accgcgcacc tctttgtcaa 5940ctacggcgct gtgcccgaga
cggcgcagat cttcacgctc aaagagtcgg ccgcggccgc 6000caaggccaag tcggactaag c
6021506524DNAArtificial
Sequenceplasmid pKR226 50gtacgtctag aggatccgtc gacggcgcgc ccgatcatcc
ggatatagtt cctcctttca 60gcaaaaaacc cctcaagacc cgtttagagg ccccaagggg
ttatgctagt tattgctcag 120cggtggcagc agccaactca gcttcctttc gggctttgtt
agcagccgga tcgatccaag 180ctgtacctca ctattccttt gccctcggac gagtgctggg
gcgtcggttt ccactatcgg 240cgagtacttc tacacagcca tcggtccaga cggccgcgct
tctgcgggcg atttgtgtac 300gcccgacagt cccggctccg gatcggacga ttgcgtcgca
tcgaccctgc gcccaagctg 360catcatcgaa attgccgtca accaagctct gatagagttg
gtcaagacca atgcggagca 420tatacgcccg gagccgcggc gatcctgcaa gctccggatg
cctccgctcg aagtagcgcg 480tctgctgctc catacaagcc aaccacggcc tccagaagaa
gatgttggcg acctcgtatt 540gggaatcccc gaacatcgcc tcgctccagt caatgaccgc
tgttatgcgg ccattgtccg 600tcaggacatt gttggagccg aaatccgcgt gcacgaggtg
ccggacttcg gggcagtcct 660cggcccaaag catcagctca tcgagagcct gcgcgacgga
cgcactgacg gtgtcgtcca 720tcacagtttg ccagtgatac acatggggat cagcaatcgc
gcatatgaaa tcacgccatg 780tagtgtattg accgattcct tgcggtccga atgggccgaa
cccgctcgtc tggctaagat 840cggccgcagc gatcgcatcc atagcctccg cgaccggctg
cagaacagcg ggcagttcgg 900tttcaggcag gtcttgcaac gtgacaccct gtgcacggcg
ggagatgcaa taggtcaggc 960tctcgctgaa ttccccaatg tcaagcactt ccggaatcgg
gagcgcggcc gatgcaaagt 1020gccgataaac ataacgatct ttgtagaaac catcggcgca
gctatttacc cgcaggacat 1080atccacgccc tcctacatcg aagctgaaag cacgagattc
ttcgccctcc gagagctgca 1140tcaggtcgga gacgctgtcg aacttttcga tcagaaactt
ctcgacagac gtcgcggtga 1200gttcaggctt ttccatgggt atatctcctt cttaaagtta
aacaaaatta tttctagagg 1260gaaaccgttg tggtctccct atagtgagtc gtattaattt
cgcgggatcg agatctgatc 1320aacctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
gtttgcgtat tgggcgctct 1380tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
ggctgcggcg agcggtatca 1440gctcactcaa aggcggtaat acggttatcc acagaatcag
gggataacgc aggaaagaac 1500atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
aggccgcgtt gctggcgttt 1560ttccataggc tccgcccccc tgacgagcat cacaaaaatc
gacgctcaag tcagaggtgg 1620cgaaacccga caggactata aagataccag gcgtttcccc
ctggaagctc cctcgtgcgc 1680tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
cctttctccc ttcgggaagc 1740gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt
cggtgtaggt cgttcgctcc 1800aagctgggct gtgtgcacga accccccgtt cagcccgacc
gctgcgcctt atccggtaac 1860tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
cactggcagc agccactggt 1920aacaggatta gcagagcgag gtatgtaggc ggtgctacag
agttcttgaa gtggtggcct 1980aactacggct acactagaag gacagtattt ggtatctgcg
ctctgctgaa gccagttacc 2040ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
ccaccgctgg tagcggtggt 2100ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
gatctcaaga agatcctttg 2160atcttttcta cggggtctga cgctcagtgg aacgaaaact
cacgttaagg gattttggtc 2220atgacattaa cctataaaaa taggcgtatc acgaggccct
ttcgtctcgc gcgtttcggt 2280gatgacggtg aaaacctctg acacatgcag ctcccggaga
cggtcacagc ttgtctgtaa 2340gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
cgggtgttgg cgggtgtcgg 2400ggctggctta actatgcggc atcagagcag attgtactga
gagtgcacca tatggacata 2460ttgtcgttag aacgcggcta caattaatac ataaccttat
gtatcataca catacgattt 2520aggtgacact atagaacggc gcgccaagct gggtctagaa
ctagaaacgt gatgccactt 2580gttattgaag tcgattacag catctattct gttttactat
ttataacttt gccatttctg 2640acttttgaaa actatctctg gatttcggta tcgctttgtg
aagatcgagc aaaagagacg 2700ttttgtggac gcaatggtcc aaatccgttc tacatgaaca
aattggtcac aatttccact 2760aaaagtaaat aaatggcaag ttaaaaaagg aatatgcatt
ttactgattg cctaggtgag 2820ctccaagaga agttgaatct acacgtctac caaccgctaa
aaaaagaaaa acattgatat 2880gtaacctgat tccattagct tttgacttct tcaacagatt
ctctacttag atttctaaca 2940gaaatattat tactagcaca tcattttcag tctcactaca
gcaaaaaatc caacggcaca 3000atacagacaa caggagatat cagactacag agatagatag
atgctactgc atgtagtaag 3060ttaaataaaa ggaaaataaa atgtcttgct accaaaacta
ctacagacta tgatgctcac 3120cacaggccaa atcctgcaac taggacagca ttatcttata
tatattgtac aaaacaagca 3180tcaaggaaca tttggtctag gcaatcagta cctcgttcta
ccatcaccct cagttatcac 3240atccttgaag gatccattac tgggaatcat cggcaacaca
tgctcctgat ggggcacaat 3300gacatcaaga aggtaggggc caggggtgtc caacattctc
tgaattgccg ctctaagctc 3360ttccttcttc gtcactcgcg ctgccggtat cccacaagca
tcagcaaact tgagcatgtt 3420tgggaatatc tcgctctcgc tagacggatc tccaagatag
gtgtgagctc tattggactt 3480gtagaaccta tcctccaact gaaccaccat acccaaatgc
tgattgttca acaacaatat 3540cttaactggg agattctcca ctcttatagt ggccaactcc
tgaacattca tgatgaaact 3600accatcccca tcaatgtcaa ccacaacagc cccagggtta
gcaacagcag caccaatagc 3660cgcaggcaat ccaaaaccca tggctccaag accccctgag
gtcaaccact gcctcggtct 3720cttgtacttg taaaactgcg cagcccacat ttgatgctgc
ccaaccccag tactaacaat 3780agcatctcca ttagtcaact catcaagaac ctcgatagca
tgctgcggag aaatcgcgtc 3840ctggaatgtc ttgtaaccca atggaaactt gtgtttctgc
acattaatct cttctctcca 3900acctccaaga tcaaacttac cctccactcc tttctcctcc
aaaatcatat taattccctt 3960caaggccaac ttcaaatccg cgcaaaccga cacgtgcgcc
tgcttgttct tcccaatctc 4020ggcagaatca atatcaatgt gaacaatctt agccctacta
gcaaaagcct caagcttccc 4080agtaacacgg tcatcaaacc ttaccccaaa ggcaagcaac
aaatcactat tgtcaacagc 4140atagttagca taaacagtac catgcatacc cagcatctga
agggaatatt catcaccaat 4200aggaaaagtt ccaagaccca ttaaagtgct agcaacggga
ataccagtga gttcaacaaa 4260gcgcctcaat tcagcactgg aattcaaact gccaccgccg
acgtagagaa cgggcttttg 4320ggcctccatg atgagtctga caatgtgttc caattgggcc
tcggcggggg gcctgggcag 4380cctggcgagg taaccgggga ggttaacggg ctcgtcccaa
ttaggcacgg cgagttgctg 4440ctgaacgtct ttgggaatgt cgatgaggac cggaccgggg
cggccggagg tggcgacgaa 4500gaaagcctcg gcgacgacgc gggggatgtc gtcgacgtcg
aggatgaggt agttgtgctt 4560cgtgatggat ctgctcacct ccacgatcgg ggtttcttgg
aaggcgtcgg tgccgatcat 4620ccggcgggcg acctggccgg tgatggcgac gactgggacg
ctgtccatta aagcgtcggc 4680gaggccgctc acgaggttgg tggcgccggg gccggaggtg
gcaatgcaga cgccggggag 4740gccggaggaa cgcgcgtagc cttcggcggc gaagacgccg
ccctgctcgt ggcgcgggag 4800cacgttgcgg atggcggcgg agcgcgtgag cgcctggtgg
atctccatcg acgcaccgcc 4860ggggtacgcg aacaccgtcg tcacgccctg cctctccagc
gcctccacaa ggatgtccgc 4920gcccttgcga ggttcgccgg aggcgaaccg tgacacgaag
ggctccgtgg tcggcgcttc 4980cttggtgaag ggcgccgccg tggggggttt ggagatggaa
catttgattt tgagagcgtg 5040gttgggtttg gtgagggttt gatgagagag agggagggtg
gatctagtaa tgcgtttggg 5100gaaggtgggg tgtgaagagg aagaagagaa tcgggtggtt
ctggaagcgg tggccgccat 5160tgtgttgtgt ggcatggtta tacttcaaaa actgcacaac
aagcctagag ttagtaccta 5220aacagtaaat ttacaacaga gagcaaagac acatgcaaaa
atttcagcca taaaaaaagt 5280tataatagaa tttaaagcaa aagtttcatt ttttaaacat
atatacaaac aaactggatt 5340tgaaggaagg gattaattcc cctgctcaaa gtttgaattc
ctattgtgac ctatactcga 5400ataaaattga agcctaagga atgtatgaga aacaagaaaa
caaaacaaaa ctacagacaa 5460acaagtacaa ttacaaaatt cgctaaaatt ctgtaatcac
caaaccccat ctcagtcagc 5520acaaggccca aggtttattt tgaaataaaa aaaaagtgat
tttatttctc ataagctaaa 5580agaaagaaag gcaattatga aatgatttcg actagatctg
aaagtccaac gcgtattccg 5640cagatattaa agaaagagta gagtttcaca tggatcctag
atggacccag ttgaggaaaa 5700agcaaggcaa agcaaaccag aagtgcaaga tccgaaattg
aaccacggaa tctaggattt 5760ggtagaggga gaagaaaagt accttgagag gtagaagaga
agagaagagc agagagatat 5820atgaacgagt gtgtcttggt ctcaactctg aagcgatacg
agtttagagg ggagcattga 5880gttccaattt atagggaaac cgggtggcag gggtgagtta
atgacggaaa agcccctaag 5940taacgagatt ggattgtggg ttagattcaa ccgtttgcat
ccgcggctta gattggggaa 6000gtcagagtga atctcaaccg ttgactgagt tgaaaattga
atgtagcaac caattgagcc 6060aaccccagcc tttgcccttt gattttgatt tgtttgttgc
atacttttta tttgtcttct 6120ggttctgact ctctttctct cgtttcaatg ccaggttgcc
tactcccaca ccactcacaa 6180gaagattcta ctgttagtat taaatatttt ttaatgtatt
aaatgatgaa tgcttttgta 6240aacagaacaa gactatgtct aataagtgtc ttgcaacatt
ttttaagaaa ttaaaaaaaa 6300tatatttatt atcaaaatca aatgtatgaa aaatcatgaa
taatataatt ttatacattt 6360ttttaaaaaa tcttttaatt tcttaattaa tatcttaaaa
ataatgatta atatttaacc 6420caaaataatt agtatgattg gtaaggaaga tatccatgtt
atgtttggat gtgagtttga 6480tctagagcaa agcttactag agtcgacctg caggtcgact
cgac 65245132DNAArtificial SequenceoCon-1 primer
51aatctagacc tgcaggatcc atgcccttca tt
325233DNAArtificial SequenceoCon-2 primer 52tttctagacc tgcaggttga
aacatccctg aag 33534480DNAArtificial
Sequenceplasmid pKR179 53ctagacctgc aggatccatg cccttcattt gccgcttatt
aattaatttg gtaacagtcc 60gtactaatca gttacttatc cttcccccat cataattaat
cttggtagtc tcgaatgcca 120caacactgac tagtctcttg gatcataaga aaaagccaag
gaacaaaaga agacaaaaca 180caatgagagt atcctttgca tagcaatgtc taagttcata
aaattcaaac aaaaacgcaa 240tcacacacag tggacatcac ttatccacta gctgatcagg
atcgccgcgt caagaaaaaa 300aaactggacc ccaaaagcca tgcacaacaa cacgtactca
caaaggtgtc aatcgagcag 360cccaaaacat tcaccaactc aacccatcat gagccctcac
atttgttgtt tctaacccaa 420cctcaaactc gtattctctt ccgccacctc atttttgttt
atttcaacac ccgtcaaact 480gcatgccacc ccgtggccaa atgtccatgc atgttaacaa
gacctatgac tataaatagc 540tgcaatctcg gcccaggttt tcatcatcaa gaaccagttc
aatatcctag tacaccgtat 600taaagaattt aagatatact gcggccgcaa gtatgaacta
aaatgcatgt aggtgtaaga 660gctcatggag agcatggaat attgtatccg accatgtaac
agtataataa ctgagctcca 720tctcacttct tctatgaata aacaaaggat gttatgatat
attaacactc tatctatgca 780ccttattgtt ctatgataaa tttcctctta ttattataaa
tcatctgaat cgtgacggct 840tatggaatgc ttcaaatagt acaaaaacaa atgtgtacta
taagactttc taaacaattc 900taaccttagc attgtgaacg agacataagt gttaagaaga
cataacaatt ataatggaag 960aagtttgtct ccatttatat attatatatt acccacttat
gtattatatt aggatgttaa 1020ggagacataa caattataaa gagagaagtt tgtatccatt
tatatattat atactaccca 1080tttatatatt atacttatcc acttatttaa tgtctttata
aggtttgatc catgatattt 1140ctaatatttt agttgatatg tatatgaaag ggtactattt
gaactctctt actctgtata 1200aaggttggat catccttaaa gtgggtctat ttaattttat
tgcttcttac agataaaaaa 1260aaaattatga gttggtttga taaaatattg aaggatttaa
aataataata aataacatat 1320aatatatgta tataaattta ttataatata acatttatct
ataaaaaagt aaatattgtc 1380ataaatctat acaatcgttt agccttgctg gacgaatctc
aattatttaa acgagagtaa 1440acatatttga ctttttggtt atttaacaaa ttattattta
acactatatg aaattttttt 1500ttttatcagc aaagaataaa attaaattaa gaaggacaat
ggtgtcccaa tccttataca 1560accaacttcc acaagaaagt caagtcagag acaacaaaaa
aacaagcaaa ggaaattttt 1620taatttgagt tgtcttgttt gctgcataat ttatgcagta
aaacactaca cataaccctt 1680ttagcagtag agcaatggtt gaccgtgtgc ttagcttctt
ttattttatt tttttatcag 1740caaagaataa ataaaataaa atgagacact tcagggatgt
ttcaacctgc aggtctagag 1800gatccccggg taccgagctc gaattcactg gccgtcgttt
tacaacgtcg tgactgggaa 1860aaccctggcg ttacccaact taatcgcctt gcagcacatc
cccctttcgc cagctggcgt 1920aatagcgaag aggcccgcac cgatcgccct tcccaacagt
tgcgcagcct gaatggcgaa 1980tggcgcctga tgcggtattt tctccttacg catctgtgcg
gtatttcaca ccgcatatgg 2040tgcactctca gtacaatctg ctctgatgcc gcatagttaa
gccagccccg acacccgcca 2100acacccgctg acgcgccctg acgggcttgt ctgctcccgg
catccgctta cagacaagct 2160gtgaccgtct ccgggagctg catgtgtcag aggttttcac
cgtcatcacc gaaacgcgcg 2220agacgaaagg gcctcgtgat acgcctattt ttataggtta
atgtcatgat aataatggtt 2280tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
gaacccctat ttgtttattt 2340ttctaaatac attcaaatat gtatccgctc atgagacaat
aaccctgata aatgcttcaa 2400taatattgaa aaaggaagag tatgagtatt caacatttcc
gtgtcgccct tattcccttt 2460tttgcggcat tttgccttcc tgtttttgct cacccagaaa
cgctggtgaa agtaaaagat 2520gctgaagatc agttgggtgc acgagtgggt tacatcgaac
tggatctcaa cagcggtaag 2580atccttgaga gttttcgccc cgaagaacgt tttccaatga
tgagcacttt taaagttctg 2640ctatgtggcg cggtattatc ccgtattgac gccgggcaag
agcaactcgg tcgccgcata 2700cactattctc agaatgactt ggttgagtac tcaccagtca
cagaaaagca tcttacggat 2760ggcatgacag taagagaatt atgcagtgct gccataacca
tgagtgataa cactgcggcc 2820aacttacttc tgacaacgat cggaggaccg aaggagctaa
ccgctttttt gcacaacatg 2880ggggatcatg taactcgcct tgatcgttgg gaaccggagc
tgaatgaagc cataccaaac 2940gacgagcgtg acaccacgat gcctgtagca atggcaacaa
cgttgcgcaa actattaact 3000ggcgaactac ttactctagc ttcccggcaa caattaatag
actggatgga ggcggataaa 3060gttgcaggac cacttctgcg ctcggccctt ccggctggct
ggtttattgc tgataaatct 3120ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
tggggccaga tggtaagccc 3180tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
ctatggatga acgaaataga 3240cagatcgctg agataggtgc ctcactgatt aagcattggt
aactgtcaga ccaagtttac 3300tcatatatac tttagattga tttaaaactt catttttaat
ttaaaaggat ctaggtgaag 3360atcctttttg ataatctcat gaccaaaatc ccttaacgtg
agttttcgtt ccactgagcg 3420tcagaccccg tagaaaagat caaaggatct tcttgagatc
ctttttttct gcgcgtaatc 3480tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
tttgtttgcc ggatcaagag 3540ctaccaactc tttttccgaa ggtaactggc ttcagcagag
cgcagatacc aaatactgtc 3600cttctagtgt agccgtagtt aggccaccac ttcaagaact
ctgtagcacc gcctacatac 3660ctcgctctgc taatcctgtt accagtggct gctgccagtg
gcgataagtc gtgtcttacc 3720gggttggact caagacgata gttaccggat aaggcgcagc
ggtcgggctg aacggggggt 3780tcgtgcacac agcccagctt ggagcgaacg acctacaccg
aactgagata cctacagcgt 3840gagctatgag aaagcgccac gcttcccgaa gggagaaagg
cggacaggta tccggtaagc 3900ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
ggggaaacgc ctggtatctt 3960tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
gatttttgtg atgctcgtca 4020ggggggcgga gcctatggaa aaacgccagc aacgcggcct
ttttacggtt cctggccttt 4080tgctggcctt ttgctcacat gttctttcct gcgttatccc
ctgattctgt ggataaccgt 4140attaccgcct ttgagtgagc tgataccgct cgccgcagcc
gaacgaccga gcgcagcgag 4200tcagtgagcg aggaagcgga agagcgccca atacgcaaac
cgcctctccc cgcgcgttgg 4260ccgattcatt aatgcagctg gcacgacagg tttcccgact
ggaaagcggg cagtgagcgc 4320aacgcaatta atgtgagtta gctcactcat taggcacccc
aggctttaca ctttatgctt 4380ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
ttcacacagg aaacagctat 4440gaccatgatt acgccaagct tgcatgcctg caggtcgact
4480549088DNAArtificial Sequenceplasmid pKR578
54ggccgcgaca caagtgtgag agtactaaat aaatgctttg gttgtacgaa atcattacac
60taaataaaat aatcaaagct tatatatgcc ttccgctaag gccgaatgca aagaaattgg
120ttctttctcg ttatcttttg ccacttttac tagtacgtat taattactac ttaatcatct
180ttgtttacgg ctcattatat ccggtctaga ggatccaagg ccgcgaagtt aaaagcaatg
240ttgtcacttg tcgtactaac acatgatgtg atagtttatg ctagctagct ataacataag
300ctgtctctga gtgtgttgta tattaataaa gatcatcact ggtgaatggt gatcgtgtac
360gtaccctact tagtaggcaa tggaagcact tagagtgtgc tttgtgcatg gccttgcctc
420tgttttgaga cttttgtaat gttttcgagt ttaaatcttt gcctttgcgt acggatccgt
480cgacggcgcg cccgatcatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac
540ccgtttagag gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc
600agcttccttt cgggctttgt tagcagccgg atcgatccaa gctgtacctc actattcctt
660tgccctcgga cgagtgctgg ggcgtcggtt tccactatcg gcgagtactt ctacacagcc
720atcggtccag acggccgcgc ttctgcgggc gatttgtgta cgcccgacag tcccggctcc
780ggatcggacg attgcgtcgc atcgaccctg cgcccaagct gcatcatcga aattgccgtc
840aaccaagctc tgatagagtt ggtcaagacc aatgcggagc atatacgccc ggagccgcgg
900cgatcctgca agctccggat gcctccgctc gaagtagcgc gtctgctgct ccatacaagc
960caaccacggc ctccagaaga agatgttggc gacctcgtat tgggaatccc cgaacatcgc
1020ctcgctccag tcaatgaccg ctgttatgcg gccattgtcc gtcaggacat tgttggagcc
1080gaaatccgcg tgcacgaggt gccggacttc ggggcagtcc tcggcccaaa gcatcagctc
1140atcgagagcc tgcgcgacgg acgcactgac ggtgtcgtcc atcacagttt gccagtgata
1200cacatgggga tcagcaatcg cgcatatgaa atcacgccat gtagtgtatt gaccgattcc
1260ttgcggtccg aatgggccga acccgctcgt ctggctaaga tcggccgcag cgatcgcatc
1320catagcctcc gcgaccggct gcagaacagc gggcagttcg gtttcaggca ggtcttgcaa
1380cgtgacaccc tgtgcacggc gggagatgca ataggtcagg ctctcgctga attccccaat
1440gtcaagcact tccggaatcg ggagcgcggc cgatgcaaag tgccgataaa cataacgatc
1500tttgtagaaa ccatcggcgc agctatttac ccgcaggaca tatccacgcc ctcctacatc
1560gaagctgaaa gcacgagatt cttcgccctc cgagagctgc atcaggtcgg agacgctgtc
1620gaacttttcg atcagaaact tctcgacaga cgtcgcggtg agttcaggct tttccatggg
1680tatatctcct tcttaaagtt aaacaaaatt atttctagag ggaaaccgtt gtggtctccc
1740tatagtgagt cgtattaatt tcgcgggatc gagatcgatc caattccaat cccacaaaaa
1800tctgagctta acagcacagt tgctcctctc agagcagaat cgggtattca acaccctcat
1860atcaactact acgttgtgta taacggtcca catgccggta tatacgatga ctggggttgt
1920acaaaggcgg caacaaacgg cgttcccgga gttgcacaca agaaatttgc cactattaca
1980gaggcaagag cagcagctga cgcgtacaca acaagtcagc aaacagacag gttgaacttc
2040atccccaaag gagaagctca actcaagccc aagagctttg ctaaggccct aacaagccca
2100ccaaagcaaa aagcccactg gctcacgcta ggaaccaaaa ggcccagcag tgatccagcc
2160ccaaaagaga tctcctttgc cccggagatt acaatggacg atttcctcta tctttacgat
2220ctaggaagga agttcgaagg tgaaggtgac gacactatgt tcaccactga taatgagaag
2280gttagcctct tcaatttcag aaagaatgct gacccacaga tggttagaga ggcctacgca
2340gcaggtctca tcaagacgat ctacccgagt aacaatctcc aggagatcaa ataccttccc
2400aagaaggtta aagatgcagt caaaagattc aggactaatt gcatcaagaa cacagagaaa
2460gacatatttc tcaagatcag aagtactatt ccagtatgga cgattcaagg cttgcttcat
2520aaaccaaggc aagtaataga gattggagtc tctaaaaagg tagttcctac tgaatctaag
2580gccatgcatg gagtctaaga ttcaaatcga ggatctaaca gaactcgccg tgaagactgg
2640cgaacagttc atacagagtc ttttacgact caatgacaag aagaaaatct tcgtcaacat
2700ggtggagcac gacactctgg tctactccaa aaatgtcaaa gatacagtct cagaagacca
2760aagggctatt gagacttttc aacaaaggat aatttcggga aacctcctcg gattccattg
2820cccagctatc tgtcacttca tcgaaaggac agtagaaaag gaaggtggct cctacaaatg
2880ccatcattgc gataaaggaa aggctatcat tcaagatgcc tctgccgaca gtggtcccaa
2940agatggaccc ccacccacga ggagcatcgt ggaaaaagaa gacgttccaa ccacgtcttc
3000aaagcaagtg gattgatgtg acatctccac tgacgtaagg gatgacgcac aatcccacta
3060tccttcgcaa gacccttcct ctatataagg aagttcattt catttggaga ggacacgctc
3120gagctcattt ctctattact tcagccataa caaaagaact cttttctctt cttattaaac
3180catgaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga
3240cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga
3300tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga
3360tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag tgcttgacat
3420tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt
3480gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg aggccatgga
3540tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg
3600aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta
3660tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga
3720gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg
3780ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc
3840gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc
3900ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc
3960gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga
4020cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg
4080agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg
4140ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa
4200ggaatagtga ggtacctaaa gaaggagtgc gtcgaagcag atcgttcaaa catttggcaa
4260taaagtttct taagattgaa tcctgttgcc ggtcttgcga tgattatcat ataatttctg
4320ttgaattacg ttaagcatgt aataattaac atgtaatgca tgacgttatt tatgagatgg
4380gtttttatga ttagagtccc gcaattatac atttaatacg cgatagaaaa caaaatatag
4440cgcgcaaact aggataaatt atcgcgcgcg gtgtcatcta tgttactaga tcgatgtcga
4500atcgatcaac ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
4560gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
4620ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
4680aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
4740ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
4800gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
4860cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
4920gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
4980tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
5040cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
5100cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
5160gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
5220agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
5280cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
5340tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
5400tttggtcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg
5460tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
5520tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
5580gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat
5640ggacatattg tcgttagaac gcggctacaa ttaatacata accttatgta tcatacacat
5700acgatttagg tgacactata gaacggcgcg ccaagcttgg atctcctgca ggatctggcc
5760ggccggatct cgtacgtcct cgaagagaag ggttaataac acatttttta acatttttaa
5820cacaaatttt agttatttaa aaatttatta aaaaatttaa aataagaaga ggaactcttt
5880aaataaatct aacttacaaa atttatgatt tttaataagt tttcaccaat aaaaaatgtc
5940ataaaaatat gttaaaaagt atattatcaa tattctcttt atgataaata aaaagaaaaa
6000aaaaataaaa gttaagtgaa aatgagattg aagtgacttt aggtgtgtat aaatatatca
6060accccgccaa caatttattt aatccaaata tattgaagta tattattcca tagcctttat
6120ttatttatat atttattata taaaagcttt atttgttcta ggttgttcat gaaatatttt
6180tttggtttta tctccgttgt aagaaaatca tgtgctttgt gtcgccactc actattgcag
6240ctttttcatg cattggtcag attgacggtt gattgtattt ttgtttttta tggttttgtg
6300ttatgactta agtcttcatc tctttatctc ttcatcaggt ttgatggtta cctaatatgg
6360tccatgggta catgcatggt taaattaggt ggccaacttt gttgtgaacg atagaatttt
6420ttttatatta agtaaactat ttttatatta tgaaataata ataaaaaaaa tattttatca
6480ttattaacaa aatcatatta gttaatttgt taactctata ataaaagaaa tactgtaaca
6540ttcacattac atggtaacat ctttccaccc tttcatttgt tttttgtttg atgacttttt
6600ttcttgttta aatttatttc ccttctttta aatttggaat acattatcat catatataaa
6660ctaaaatact aaaaacagga ttacacaaat gataaataat aacacaaata tttataaatc
6720tagctgcaat atatttaaac tagctatatc gatattgtaa aataaaacta gctgcattga
6780tactgataaa aaaatatcat gtgctttctg gactgatgat gcagtatact tttgacattg
6840cctttatttt atttttcaga aaagctttct tagttctggg ttcttcatta tttgtttccc
6900atctccattg tgaattgaat catttgcttc gtgtcacaaa tacaatttag ntaggtacat
6960gcattggtca gattcacggt ttattatgtc atgacttaag ttcatggtag tacattacct
7020gccacgcatg cattatattg gttagatttg ataggcaaat ttggttgtca acaatataaa
7080tataaataat gtttttatat tacgaaataa cagtgatcaa aacaaacagt tttatcttta
7140ttaacaagat tttgtttttg tttgatgacg ttttttaatg tttacgcttt cccccttctt
7200ttgaatttag aacactttat catcataaaa tcaaatacta aaaaaattac atatttcata
7260aataataaca caaatatttt taaaaaatct gaaataataa tgaacaatat tacatattat
7320cacgaaaatt cattaataaa aatattatat aaataaaatg taatagtagt tatatgtagg
7380aaaaaagtac tgcacgcata atatatacaa aaagattaaa atgaactatt ataaataata
7440acactaaatt aatggtgaat catatcaaaa taatgaaaaa gtaaataaaa tttgtaatta
7500acttctatat gtattacaca cacaaataat aaataatagt aaaaaaaatt atgataaata
7560tttaccatct cataagatat ttaaaataat gataaaaata tagattattt tttatgcaac
7620tagctagcca aaaagagaac acgggtatat ataaaaagag tacctttaaa ttctactgta
7680cttcctttat tcctgacgtt tttatatcaa gtggacatac gtgaagattt taattatcag
7740tctaaatatt tcattagcac ttaatacttt tctgttttat tcctatccta taagtagtcc
7800cgattctccc aacattgctt attcacacaa ctaactaaga aagtcttcca tagcccccca
7860agcggccgca caatggcgac tcgacagcga actgccacca ctgttgtggt cgaggacctt
7920cccaaggtca ctcttgaggc caagtctgaa cctgtgttcc ccgatatcaa gaccatcaag
7980gatgccattc ccgcgcactg cttccagccc tcgctcgtca cctcattcta ctacgtcttc
8040cgcgattttg ccatggtctc tgccctcgtc tgggctgctc tcacctacat ccccagcatc
8100cccgaccaga ccctccgcgt cgcagcttgg atggtctacg gcttcgtcca gggtctgttc
8160tgcaccggtg tctggattct cggccatgag tgcggccacg gtgctttctc tctccacgga
8220aaggtcaaca atgtgaccgg ctggttcctc cactcgttcc tcctcgtccc ctacttcagc
8280tggaagtact ctcaccaccg ccaccaccgc ttcaccggcc acatggatct cgacatggct
8340ttcgtcccca agactgagcc caagccctcc aagtcgctca tgattgctgg cattgacgtc
8400gccgagcttg ttgaggacac ccccgctgct cagatggtca agctcatctt ccaccagctt
8460ttcggatggc aggcgtacct cttcttcaac gctagctctg gcaagggcag caagcagtgg
8520gagcccaaga ctggcctctc caagtggttc cgagtcagtc acttcgagcc taccagcgct
8580gtcttccgcc ccaacgaggc catcttcatc ctcatctccg atatcggtct tgctctaatg
8640ggaactgctc tgtactttgc ttccaagcaa gttggtgttt cgaccattct cttcctctac
8700cttgttccct acctgtgggt tcaccactgg ctcgttgcca ttacctacct ccaccaccac
8760cacaccgagc tccctcacta caccgctgag ggctggacct acgtcaaggg agctctcgcc
8820actgtcgacc gtgagtttgg cttcatcgga aagcacctct tccacggtat cattgagaag
8880cacgttgttc accatctctt ccctaagatc cccttctaca aggctgacga ggccaccgag
8940gccatcaagc ccgtcattgg cgaccactac tgccacgacg accgaagctt cctgggccag
9000ctgtggacca tcttcggcac gctcaagtac gtcgagcacg accctgcccg acccggtgcc
9060atgcgatgga acaaggacta ggctaggc
9088555705DNAArtificial Sequenceplasmid pKR582 55ggccgcacaa tggcgactcg
acagcgaact gccaccactg ttgtggtcga ggaccttccc 60aaggtcactc ttgaggccaa
gtctgaacct gtgttccccg atatcaagac catcaaggat 120gccattcccg cgcactgctt
ccagccctcg ctcgtcacct cattctacta cgtcttccgc 180gattttgcca tggtctctgc
cctcgtctgg gctgctctca cctacatccc cagcatcccc 240gaccagaccc tccgcgtcgc
agcttggatg gtctacggct tcgtccaggg tctgttctgc 300accggtgtct ggattctcgg
ccatgagtgc ggccacggtg ctttctctct ccacggaaag 360gtcaacaatg tgaccggctg
gttcctccac tcgttcctcc tcgtccccta cttcagctgg 420aagtactctc accaccgcca
ccaccgcttc accggccaca tggatctcga catggctttc 480gtccccaaga ctgagcccaa
gccctccaag tcgctcatga ttgctggcat tgacgtcgcc 540gagcttgttg aggacacccc
cgctgctcag atggtcaagc tcatcttcca ccagcttttc 600ggatggcagg cgtacctctt
cttcaacgct agctctggca agggcagcaa gcagtgggag 660cccaagactg gcctctccaa
gtggttccga gtcagtcact tcgagcctac cagcgctgtc 720ttccgcccca acgaggccat
cttcatcctc atctccgata tcggtcttgc tctaatggga 780actgctctgt actttgcttc
caagcaagtt ggtgtttcga ccattctctt cctctacctt 840gttccctacc tgtgggttca
ccactggctc gttgccatta cctacctcca ccaccaccac 900accgagctcc ctcactacac
cgctgagggc tggacctacg tcaagggagc tctcgccact 960gtcgaccgtg agtttggctt
catcggaaag cacctcttcc acggtatcat tgagaagcac 1020gttgttcacc atctcttccc
taagatcccc ttctacaagg ctgacgaggc caccgaggcc 1080atcaagcccg tcattggcga
ccactactgc cacgacgacc gaagcttcct gggccagctg 1140tggaccatct tcggcacgct
caagtacgtc gagcacgacc ctgcccgacc cggtgccatg 1200cgatggaaca aggactaggc
taggcggccg caagtatgaa ctaaaatgca tgtaggtgta 1260agagctcatg gagagcatgg
aatattgtat ccgaccatgt aacagtataa taactgagct 1320ccatctcact tcttctatga
ataaacaaag gatgttatga tatattaaca ctctatctat 1380gcaccttatt gttctatgat
aaatttcctc ttattattat aaatcatctg aatcgtgacg 1440gcttatggaa tgcttcaaat
agtacaaaaa caaatgtgta ctataagact ttctaaacaa 1500ttctaacctt agcattgtga
acgagacata agtgttaaga agacataaca attataatgg 1560aagaagtttg tctccattta
tatattatat attacccact tatgtattat attaggatgt 1620taaggagaca taacaattat
aaagagagaa gtttgtatcc atttatatat tatatactac 1680ccatttatat attatactta
tccacttatt taatgtcttt ataaggtttg atccatgata 1740tttctaatat tttagttgat
atgtatatga aagggtacta tttgaactct cttactctgt 1800ataaaggttg gatcatcctt
aaagtgggtc tatttaattt tattgcttct tacagataaa 1860aaaaaaatta tgagttggtt
tgataaaata ttgaaggatt taaaataata ataaataaca 1920tataatatat gtatataaat
ttattataat ataacattta tctataaaaa agtaaatatt 1980gtcataaatc tatacaatcg
tttagccttg ctggacgaat ctcaattatt taaacgagag 2040taaacatatt tgactttttg
gttatttaac aaattattat ttaacactat atgaaatttt 2100tttttttatc agcaaagaat
aaaattaaat taagaaggac aatggtgtcc caatccttat 2160acaaccaact tccacaagaa
agtcaagtca gagacaacaa aaaaacaagc aaaggaaatt 2220ttttaatttg agttgtcttg
tttgctgcat aatttatgca gtaaaacact acacataacc 2280cttttagcag tagagcaatg
gttgaccgtg tgcttagctt cttttatttt atttttttat 2340cagcaaagaa taaataaaat
aaaatgagac acttcaggga tgtttcaacc tgcaggtcta 2400gaggatcccc gggtaccgag
ctcgaattca ctggccgtcg ttttacaacg tcgtgactgg 2460gaaaaccctg gcgttaccca
acttaatcgc cttgcagcac atcccccttt cgccagctgg 2520cgtaatagcg aagaggcccg
caccgatcgc ccttcccaac agttgcgcag cctgaatggc 2580gaatggcgcc tgatgcggta
ttttctcctt acgcatctgt gcggtatttc acaccgcata 2640tggtgcactc tcagtacaat
ctgctctgat gccgcatagt taagccagcc ccgacacccg 2700ccaacacccg ctgacgcgcc
ctgacgggct tgtctgctcc cggcatccgc ttacagacaa 2760gctgtgaccg tctccgggag
ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc 2820gcgagacgaa agggcctcgt
gatacgccta tttttatagg ttaatgtcat gataataatg 2880gtttcttaga cgtcaggtgg
cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 2940tttttctaaa tacattcaaa
tatgtatccg ctcatgagac aataaccctg ataaatgctt 3000caataatatt gaaaaaggaa
gagtatgagt attcaacatt tccgtgtcgc ccttattccc 3060ttttttgcgg cattttgcct
tcctgttttt gctcacccag aaacgctggt gaaagtaaaa 3120gatgctgaag atcagttggg
tgcacgagtg ggttacatcg aactggatct caacagcggt 3180aagatccttg agagttttcg
ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt 3240ctgctatgtg gcgcggtatt
atcccgtatt gacgccgggc aagagcaact cggtcgccgc 3300atacactatt ctcagaatga
cttggttgag tactcaccag tcacagaaaa gcatcttacg 3360gatggcatga cagtaagaga
attatgcagt gctgccataa ccatgagtga taacactgcg 3420gccaacttac ttctgacaac
gatcggagga ccgaaggagc taaccgcttt tttgcacaac 3480atgggggatc atgtaactcg
ccttgatcgt tgggaaccgg agctgaatga agccatacca 3540aacgacgagc gtgacaccac
gatgcctgta gcaatggcaa caacgttgcg caaactatta 3600actggcgaac tacttactct
agcttcccgg caacaattaa tagactggat ggaggcggat 3660aaagttgcag gaccacttct
gcgctcggcc cttccggctg gctggtttat tgctgataaa 3720tctggagccg gtgagcgtgg
gtctcgcggt atcattgcag cactggggcc agatggtaag 3780ccctcccgta tcgtagttat
ctacacgacg gggagtcagg caactatgga tgaacgaaat 3840agacagatcg ctgagatagg
tgcctcactg attaagcatt ggtaactgtc agaccaagtt 3900tactcatata tactttagat
tgatttaaaa cttcattttt aatttaaaag gatctaggtg 3960aagatccttt ttgataatct
catgaccaaa atcccttaac gtgagttttc gttccactga 4020gcgtcagacc ccgtagaaaa
gatcaaagga tcttcttgag atcctttttt tctgcgcgta 4080atctgctgct tgcaaacaaa
aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 4140gagctaccaa ctctttttcc
gaaggtaact ggcttcagca gagcgcagat accaaatact 4200gtccttctag tgtagccgta
gttaggccac cacttcaaga actctgtagc accgcctaca 4260tacctcgctc tgctaatcct
gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 4320accgggttgg actcaagacg
atagttaccg gataaggcgc agcggtcggg ctgaacgggg 4380ggttcgtgca cacagcccag
cttggagcga acgacctaca ccgaactgag atacctacag 4440cgtgagctat gagaaagcgc
cacgcttccc gaagggagaa aggcggacag gtatccggta 4500agcggcaggg tcggaacagg
agagcgcacg agggagcttc cagggggaaa cgcctggtat 4560ctttatagtc ctgtcgggtt
tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 4620tcaggggggc ggagcctatg
gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 4680ttttgctggc cttttgctca
catgttcttt cctgcgttat cccctgattc tgtggataac 4740cgtattaccg cctttgagtg
agctgatacc gctcgccgca gccgaacgac cgagcgcagc 4800gagtcagtga gcgaggaagc
ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt 4860tggccgattc attaatgcag
ctggcacgac aggtttcccg actggaaagc gggcagtgag 4920cgcaacgcaa ttaatgtgag
ttagctcact cattaggcac cccaggcttt acactttatg 4980cttccggctc gtatgttgtg
tggaattgtg agcggataac aatttcacac aggaaacagc 5040tatgaccatg attacgccaa
gcttgcatgc ctgcaggtcg actctagacc tgcaggatcc 5100atgcccttca tttgccgctt
attaattaat ttggtaacag tccgtactaa tcagttactt 5160atccttcccc catcataatt
aatcttggta gtctcgaatg ccacaacact gactagtctc 5220ttggatcata agaaaaagcc
aaggaacaaa agaagacaaa acacaatgag agtatccttt 5280gcatagcaat gtctaagttc
ataaaattca aacaaaaacg caatcacaca cagtggacat 5340cacttatcca ctagctgatc
aggatcgccg cgtcaagaaa aaaaaactgg accccaaaag 5400ccatgcacaa caacacgtac
tcacaaaggt gtcaatcgag cagcccaaaa cattcaccaa 5460ctcaacccat catgagccct
cacatttgtt gtttctaacc caacctcaaa ctcgtattct 5520cttccgccac ctcatttttg
tttatttcaa cacccgtcaa actgcatgcc accccgtggc 5580caaatgtcca tgcatgttaa
caagacctat gactataaat agctgcaatc tcggcccagg 5640ttttcatcat caagaaccag
ttcaatatcc tagtacaccg tattaaagaa tttaagatat 5700actgc
57055612897DNAArtificial
Sequenceplasmid pKR983 56ggtcgactcg acgtacgtct agaggatccg tcgacggcgc
gcccgatcat ccggatatag 60ttcctccttt cagcaaaaaa cccctcaaga cccgtttaga
ggccccaagg ggttatgcta 120gttattgctc agcggtggca gcagccaact cagcttcctt
tcgggctttg ttagcagccg 180gatcgatcca agctgtacct cactattcct ttgccctcgg
acgagtgctg gggcgtcggt 240ttccactatc ggcgagtact tctacacagc catcggtcca
gacggccgcg cttctgcggg 300cgatttgtgt acgcccgaca gtcccggctc cggatcggac
gattgcgtcg catcgaccct 360gcgcccaagc tgcatcatcg aaattgccgt caaccaagct
ctgatagagt tggtcaagac 420caatgcggag catatacgcc cggagccgcg gcgatcctgc
aagctccgga tgcctccgct 480cgaagtagcg cgtctgctgc tccatacaag ccaaccacgg
cctccagaag aagatgttgg 540cgacctcgta ttgggaatcc ccgaacatcg cctcgctcca
gtcaatgacc gctgttatgc 600ggccattgtc cgtcaggaca ttgttggagc cgaaatccgc
gtgcacgagg tgccggactt 660cggggcagtc ctcggcccaa agcatcagct catcgagagc
ctgcgcgacg gacgcactga 720cggtgtcgtc catcacagtt tgccagtgat acacatgggg
atcagcaatc gcgcatatga 780aatcacgcca tgtagtgtat tgaccgattc cttgcggtcc
gaatgggccg aacccgctcg 840tctggctaag atcggccgca gcgatcgcat ccatagcctc
cgcgaccggc tgcagaacag 900cgggcagttc ggtttcaggc aggtcttgca acgtgacacc
ctgtgcacgg cgggagatgc 960aataggtcag gctctcgctg aattccccaa tgtcaagcac
ttccggaatc gggagcgcgg 1020ccgatgcaaa gtgccgataa acataacgat ctttgtagaa
accatcggcg cagctattta 1080cccgcaggac atatccacgc cctcctacat cgaagctgaa
agcacgagat tcttcgccct 1140ccgagagctg catcaggtcg gagacgctgt cgaacttttc
gatcagaaac ttctcgacag 1200acgtcgcggt gagttcaggc ttttccatgg gtatatctcc
ttcttaaagt taaacaaaat 1260tatttctaga gggaaaccgt tgtggtctcc ctatagtgag
tcgtattaat ttcgcgggat 1320cgagatctga tcaacctgca ttaatgaatc ggccaacgcg
cggggagagg cggtttgcgt 1380attgggcgct cttccgcttc ctcgctcact gactcgctgc
gctcggtcgt tcggctgcgg 1440cgagcggtat cagctcactc aaaggcggta atacggttat
ccacagaatc aggggataac 1500gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
ggaaccgtaa aaaggccgcg 1560ttgctggcgt ttttccatag gctccgcccc cctgacgagc
atcacaaaaa tcgacgctca 1620agtcagaggt ggcgaaaccc gacaggacta taaagatacc
aggcgtttcc ccctggaagc 1680tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
gatacctgtc cgcctttctc 1740ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta
ggtatctcag ttcggtgtag 1800gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
ttcagcccga ccgctgcgcc 1860ttatccggta actatcgtct tgagtccaac ccggtaagac
acgacttatc gccactggca 1920gcagccactg gtaacaggat tagcagagcg aggtatgtag
gcggtgctac agagttcttg 1980aagtggtggc ctaactacgg ctacactaga aggacagtat
ttggtatctg cgctctgctg 2040aagccagtta ccttcggaaa aagagttggt agctcttgat
ccggcaaaca aaccaccgct 2100ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
gcagaaaaaa aggatctcaa 2160gaagatcctt tgatcttttc tacggggtct gacgctcagt
ggaacgaaaa ctcacgttaa 2220gggattttgg tcatgacatt aacctataaa aataggcgta
tcacgaggcc ctttcgtctc 2280gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
agctcccgga gacggtcaca 2340gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
agggcgcgtc agcgggtgtt 2400ggcgggtgtc ggggctggct taactatgcg gcatcagagc
agattgtact gagagtgcac 2460catatggaca tattgtcgtt agaacgcggc tacaattaat
acataacctt atgtatcata 2520cacatacgat ttaggtgaca ctatagaacg gcgcgccaag
ctgggtctag aactagaaac 2580gtgatgccac ttgttattga agtcgattac agcatctatt
ctgttttact atttataact 2640ttgccatttc tgacttttga aaactatctc tggatttcgg
tatcgctttg tgaagatcga 2700gcaaaagaga cgttttgtgg acgcaatggt ccaaatccgt
tctacatgaa caaattggtc 2760acaatttcca ctaaaagtaa ataaatggca agttaaaaaa
ggaatatgca ttttactgat 2820tgcctaggtg agctccaaga gaagttgaat ctacacgtct
accaaccgct aaaaaaagaa 2880aaacattgat atgtaacctg attccattag cttttgactt
cttcaacaga ttctctactt 2940agatttctaa cagaaatatt attactagca catcattttc
agtctcacta cagcaaaaaa 3000tccaacggca caatacagac aacaggagat atcagactac
agagatagat agatgctact 3060gcatgtagta agttaaataa aaggaaaata aaatgtcttg
ctaccaaaac tactacagac 3120tatgatgctc accacaggcc aaatcctgca actaggacag
cattatctta tatatattgt 3180acaaaacaag catcaaggaa catttggtct aggcaatcag
tacctcgttc taccatcacc 3240ctcagttatc acatccttga aggatccatt actgggaatc
atcggcaaca catgctcctg 3300atggggcaca atgacatcaa gaaggtaggg gccaggggtg
tccaacattc tctgaattgc 3360cgctctaagc tcttccttct tcgtcactcg cgctgccggt
atcccacaag catcagcaaa 3420cttgagcatg tttgggaata tctcgctctc gctagacgga
tctccaagat aggtgtgagc 3480tctattggac ttgtagaacc tatcctccaa ctgaaccacc
atacccaaat gctgattgtt 3540caacaacaat atcttaactg ggagattctc cactcttata
gtggccaact cctgaacatt 3600catgatgaaa ctaccatccc catcaatgtc aaccacaaca
gccccagggt tagcaacagc 3660agcaccaata gccgcaggca atccaaaacc catggctcca
agaccccctg aggtcaacca 3720ctgcctcggt ctcttgtact tgtaaaactg cgcagcccac
atttgatgct gcccaacccc 3780agtactaaca atagcatctc cattagtcaa ctcatcaaga
acctcgatag catgctgcgg 3840agaaatcgcg tcctggaatg tcttgtaacc caatggaaac
ttgtgtttct gcacattaat 3900ctcttctctc caacctccaa gatcaaactt accctccact
cctttctcct ccaaaatcat 3960attaattccc ttcaaggcca acttcaaatc cgcgcaaacc
gacacgtgcg cctgcttgtt 4020cttcccaatc tcggcagaat caatatcaat gtgaacaatc
ttagccctac tagcaaaagc 4080ctcaagcttc ccagtaacac ggtcatcaaa ccttacccca
aaggcaagca acaaatcact 4140attgtcaaca gcatagttag cataaacagt accatgcata
cccagcatct gaagggaata 4200ttcatcacca ataggaaaag ttccaagacc cattaaagtg
ctagcaacgg gaataccagt 4260gagttcaaca aagcgcctca attcagcact ggaattcaaa
ctgccaccgc cgacgtagag 4320aacgggcttt tgggcctcca tgatgagtct gacaatgtgt
tccaattggg cctcggcggg 4380gggcctgggc agcctggcga ggtaaccggg gaggttaacg
ggctcgtccc aattaggcac 4440ggcgagttgc tgctgaacgt ctttgggaat gtcgatgagg
accggaccgg ggcggccgga 4500ggtggcgacg aagaaagcct cggcgacgac gcgggggatg
tcgtcgacgt cgaggatgag 4560gtagttgtgc ttcgtgatgg atctgctcac ctccacgatc
ggggtttctt ggaaggcgtc 4620ggtgccgatc atccggcggg cgacctggcc ggtgatggcg
acgactggga cgctgtccat 4680taaagcgtcg gcgaggccgc tcacgaggtt ggtggcgccg
gggccggagg tggcaatgca 4740gacgccgggg aggccggagg aacgcgcgta gccttcggcg
gcgaagacgc cgccctgctc 4800gtggcgcggg agcacgttgc ggatggcggc ggagcgcgtg
agcgcctggt ggatctccat 4860cgacgcaccg ccggggtacg cgaacaccgt cgtcacgccc
tgcctctcca gcgcctccac 4920aaggatgtcc gcgcccttgc gaggttcgcc ggaggcgaac
cgtgacacga agggctccgt 4980ggtcggcgct tccttggtga agggcgccgc cgtggggggt
ttggagatgg aacatttgat 5040tttgagagcg tggttgggtt tggtgagggt ttgatgagag
agagggaggg tggatctagt 5100aatgcgtttg gggaaggtgg ggtgtgaaga ggaagaagag
aatcgggtgg ttctggaagc 5160ggtggccgcc attgtgttgt gtggcatggt tatacttcaa
aaactgcaca acaagcctag 5220agttagtacc taaacagtaa atttacaaca gagagcaaag
acacatgcaa aaatttcagc 5280cataaaaaaa gttataatag aatttaaagc aaaagtttca
ttttttaaac atatatacaa 5340acaaactgga tttgaaggaa gggattaatt cccctgctca
aagtttgaat tcctattgtg 5400acctatactc gaataaaatt gaagcctaag gaatgtatga
gaaacaagaa aacaaaacaa 5460aactacagac aaacaagtac aattacaaaa ttcgctaaaa
ttctgtaatc accaaacccc 5520atctcagtca gcacaaggcc caaggtttat tttgaaataa
aaaaaaagtg attttatttc 5580tcataagcta aaagaaagaa aggcaattat gaaatgattt
cgactagatc tgaaagtcca 5640acgcgtattc cgcagatatt aaagaaagag tagagtttca
catggatcct agatggaccc 5700agttgaggaa aaagcaaggc aaagcaaacc agaagtgcaa
gatccgaaat tgaaccacgg 5760aatctaggat ttggtagagg gagaagaaaa gtaccttgag
aggtagaaga gaagagaaga 5820gcagagagat atatgaacga gtgtgtcttg gtctcaactc
tgaagcgata cgagtttaga 5880ggggagcatt gagttccaat ttatagggaa accgggtggc
aggggtgagt taatgacgga 5940aaagccccta agtaacgaga ttggattgtg ggttagattc
aaccgtttgc atccgcggct 6000tagattgggg aagtcagagt gaatctcaac cgttgactga
gttgaaaatt gaatgtagca 6060accaattgag ccaaccccag cctttgccct ttgattttga
tttgtttgtt gcatactttt 6120tatttgtctt ctggttctga ctctctttct ctcgtttcaa
tgccaggttg cctactccca 6180caccactcac aagaagattc tactgttagt attaaatatt
ttttaatgta ttaaatgatg 6240aatgcttttg taaacagaac aagactatgt ctaataagtg
tcttgcaaca ttttttaaga 6300aattaaaaaa aatatattta ttatcaaaat caaatgtatg
aaaaatcatg aataatataa 6360ttttatacat ttttttaaaa aatcttttaa tttcttaatt
aatatcttaa aaataatgat 6420taatatttaa cccaaaataa ttagtatgat tggtaaggaa
gatatccatg ttatgtttgg 6480atgtgagttt gatctagagc aaagcttact agagtcgacc
tgcagcccgg gggatccgcc 6540cacgtacggt accatctgct aatattttaa atcacatgca
agagaggagg catggttcca 6600ttttctacct tcacattatt tgagaaaaac gaacttgttc
tgtgttttat ttttgccctt 6660cacattagta caacgtggaa gactcatggt tacacagaat
catacataag tacaatgctt 6720gtccctaaga aaacaagcac tcgttgtatt gaacctttac
ggctcatgcg gccgcgaatt 6780cactagtgat tgaattcgcg gccgcttagt ccgacttggc
cttggcggcc gcggccgact 6840ctttgagcgt gaagatctgc gccgtctcgg gcacagcgcc
gtagttgaca aagaggtgcg 6900cggtcttgaa gaaggccgtg atgatgggct cgtcgttcct
gcgcacgagg tgcgggtacg 6960cggccgcaaa gtgcttggtg gcttcgttga gcttgtagtg
cggaatgatc gggaacaagt 7020ggtggacctg gtgcgtgcca atgtggtggc tcaggttgtc
cacgaacgcg ccgtacgagc 7080ggtcgacgct cgagaggttg cccttgacgt acgtccactc
cgagtcgccg taccacggcg 7140tcgcttcgtc gttgtggtgc aagaaggtcg taatgacgag
gaacgaagca aagacaaaga 7200gcggcgcata gtagtagagg cccatgacgg caaagccgag
cgagtatgtg aggtacgcgt 7260acgcggcgaa gaaggcggcc cagacgccga gcgacacgat
gacggccgac gcgcggcgaa 7320ggaggagcgg gtcccacggg tcaaagtggc tcatcgtgcg
cggggcatac ccgaccttca 7380agtagacaaa ccacgcaccg ccgagcgtgt agacccattg
gcgcacgtcc tggaggtcct 7440tgaccgaccg gtgcgggtaa aagatctcgt ccttatcaat
gttgcccgtg ttcttgtggt 7500ggtggcggtg cgtcacgcgc cagctctcga acggcgtcaa
aatcgcagag tgcatgatgc 7560agccgatgat aaagttgacg ctgtggtagc gcgagaaggc
cgagtggccg cagtcgtggc 7620cgaccgtgaa gaagccccag aagatgacgc cctgcacgta
gatgtaggtg gcgcaaacga 7680gcgcgtggag cagaacgtta tcggcaatga acggcgtcga
gcgcgccgcg tagagcagcg 7740ccgccgaggc cgacgcgttg aagatcgcgc gggccgtgta
gtagagcgag aggccgaggt 7800tcgactcaaa gcacgcgttc gggatcgagt gcttgagctc
cgtgagcgtc gggaactcga 7860ccttcgtctt atcctcagtc atgcggccgc tgaagtattg
cttcttagtt aacctttcct 7920ttctctctca gctatgtgaa ttcattttgc tttcgtcaca
atttatatag tgaaattgga 7980tctttggagt taacgccttc acaggattat cgtgttagaa
caatgctttt tcatgttcta 8040attagtagta cattacaaat gtgcactcta ttcaataagc
atcttttggc acgttaataa 8100atcatgtgaa aaaaaaatac tactatttca aagaaagtgt
tgtaaaaaga aacggaaaga 8160gagctggctt cagttgttga gacttgtttg ctagtaaaaa
tggtgtgaag agtgattcat 8220ggtgaggtgg tttttcgtcc ctttctgttt gcatgaaaaa
caaatggcaa gagatgacgt 8280aggattcctt cccttaacga ttatctgttt ttaatttcaa
atatacatat aggaatttat 8340gaattactaa ggttgtaaaa tatgctggtc atttatttat
ggctaaaata tttttttttc 8400tcgtaaatat aaaaatattt aaaatttatt tttatcatat
tttttatcct tataaaatta 8460tgtgtacaac ctatataaaa aaatatcata tttaatattg
attatatgtt taatcaatat 8520aaaaaatcat tatcatatat ttagatttat tcgaatatac
atctaaacaa aaaataacat 8580attttaattt tatgaagaaa aaaaaatatt ttatccttta
tttatttaag attaattaat 8640agttatgtat tgtggaaaga cttttacaca tgcaatagat
atactgaatc aattagatgc 8700caatgctgag ttggaaatca cttgaggagg ggaggagact
tgccaatgct tttcagtttc 8760atttaaatga tttagtggag gagatagagt agtgataaag
gcatgcccca attttggagt 8820gtatatatga gtggaaataa gagagggata gagagaaaaa
ataaagagag taaaaataat 8880taatgtgaaa tgatatgata aaaaaataaa gaaagagata
aagagaaaaa tgaaatgaga 8940gatagatgaa atagagagta gatacatgtt tgtttaggtt
ttttttagga aataacacat 9000ttttttctca tcacttatta ctcactgtca atttcctctc
tttcaatcat aatgatatga 9060tttgtttaac aaaaatgtga aaaaacatat aaagtaaaat
atttttataa attgataaat 9120aaaaatttac aaaatttatt tcttattaaa ttgaatagaa
aatgaaagaa aagaaaagaa 9180aaagtatata taaaatgata tagctttaaa aagaataaat
ttttcatatc agtctttttt 9240taataattta gaaatattta agtatatagc aaaaatataa
tgtactttac atatgcataa 9300ataataattt gaaaatagaa ctaatagaat agagaaaaaa
gtaatataat aattaactat 9360atgaaaattt agaagggaca atatttttaa ttaagaatat
aaacaatatt tcttttcatg 9420taatgaggga cggatgtacg gggccagtgt tggagtcaaa
gccaaaatag tcacggggaa 9480attaatgcac tgcatgacta ttcgaaaaaa ttcactagcc
ttacttagat gttagattaa 9540tagctagggg gtgcagataa ttttgaaagg catgaaaaac
attaatttgt acattgcaag 9600cttttgatga caagctttgc aattgttcac actaccttat
gccatttata aatagagtga 9660ttggcatatg aaggaaatca tgagagtcga agcgaaaaac
aaagcttgag agtgtaggaa 9720aaatacagtt tttttggtaa aaatacagta tttgaatagg
agcgaaaaat atcctttcaa 9780aatgatcctt ttcttttttt ttttttttct tgttgttctt
ggtcagttat tcaaaggaaa 9840agggattgaa ataaaaactt gcatgtggga tcgtacgtcg
agtcgacctg caggatccat 9900gcccttcatt tgccgcttat taattaattt ggtaacagtc
cgtactaatc agttacttat 9960ccttccccca tcataattaa tcttggtagt ctcgaatgcc
acaacactga ctagtctctt 10020ggatcataag aaaaagccaa ggaacaaaag aagacaaaac
acaatgagag tatcctttgc 10080atagcaatgt ctaagttcat aaaattcaaa caaaaacgca
atcacacaca gtggacatca 10140cttatccact agctgatcag gatcgccgcg tcaagaaaaa
aaaactggac cccaaaagcc 10200atgcacaaca acacgtactc acaaaggtgt caatcgagca
gcccaaaaca ttcaccaact 10260caacccatca tgagccctca catttgttgt ttctaaccca
acctcaaact cgtattctct 10320tccgccacct catttttgtt tatttcaaca cccgtcaaac
tgcatgccac cccgtggcca 10380aatgtccatg catgttaaca agacctatga ctataaatag
ctgcaatctc ggcccaggtt 10440ttcatcatca agaaccagtt caatatccta gtacaccgta
ttaaagaatt taagatatac 10500tgcggccgca caatggcgac tcgacagcga actgccacca
ctgttgtggt cgaggacctt 10560cccaaggtca ctcttgaggc caagtctgaa cctgtgttcc
ccgatatcaa gaccatcaag 10620gatgccattc ccgcgcactg cttccagccc tcgctcgtca
cctcattcta ctacgtcttc 10680cgcgattttg ccatggtctc tgccctcgtc tgggctgctc
tcacctacat ccccagcatc 10740cccgaccaga ccctccgcgt cgcagcttgg atggtctacg
gcttcgtcca gggtctgttc 10800tgcaccggtg tctggattct cggccatgag tgcggccacg
gtgctttctc tctccacgga 10860aaggtcaaca atgtgaccgg ctggttcctc cactcgttcc
tcctcgtccc ctacttcagc 10920tggaagtact ctcaccaccg ccaccaccgc ttcaccggcc
acatggatct cgacatggct 10980ttcgtcccca agactgagcc caagccctcc aagtcgctca
tgattgctgg cattgacgtc 11040gccgagcttg ttgaggacac ccccgctgct cagatggtca
agctcatctt ccaccagctt 11100ttcggatggc aggcgtacct cttcttcaac gctagctctg
gcaagggcag caagcagtgg 11160gagcccaaga ctggcctctc caagtggttc cgagtcagtc
acttcgagcc taccagcgct 11220gtcttccgcc ccaacgaggc catcttcatc ctcatctccg
atatcggtct tgctctaatg 11280ggaactgctc tgtactttgc ttccaagcaa gttggtgttt
cgaccattct cttcctctac 11340cttgttccct acctgtgggt tcaccactgg ctcgttgcca
ttacctacct ccaccaccac 11400cacaccgagc tccctcacta caccgctgag ggctggacct
acgtcaaggg agctctcgcc 11460actgtcgacc gtgagtttgg cttcatcgga aagcacctct
tccacggtat cattgagaag 11520cacgttgttc accatctctt ccctaagatc cccttctaca
aggctgacga ggccaccgag 11580gccatcaagc ccgtcattgg cgaccactac tgccacgacg
accgaagctt cctgggccag 11640ctgtggacca tcttcggcac gctcaagtac gtcgagcacg
accctgcccg acccggtgcc 11700atgcgatgga acaaggacta ggctaggcgg ccgcaagtat
gaactaaaat gcatgtaggt 11760gtaagagctc atggagagca tggaatattg tatccgacca
tgtaacagta taataactga 11820gctccatctc acttcttcta tgaataaaca aaggatgtta
tgatatatta acactctatc 11880tatgcacctt attgttctat gataaatttc ctcttattat
tataaatcat ctgaatcgtg 11940acggcttatg gaatgcttca aatagtacaa aaacaaatgt
gtactataag actttctaaa 12000caattctaac cttagcattg tgaacgagac ataagtgtta
agaagacata acaattataa 12060tggaagaagt ttgtctccat ttatatatta tatattaccc
acttatgtat tatattagga 12120tgttaaggag acataacaat tataaagaga gaagtttgta
tccatttata tattatatac 12180tacccattta tatattatac ttatccactt atttaatgtc
tttataaggt ttgatccatg 12240atatttctaa tattttagtt gatatgtata tgaaagggta
ctatttgaac tctcttactc 12300tgtataaagg ttggatcatc cttaaagtgg gtctatttaa
ttttattgct tcttacagat 12360aaaaaaaaaa ttatgagttg gtttgataaa atattgaagg
atttaaaata ataataaata 12420acatataata tatgtatata aatttattat aatataacat
ttatctataa aaaagtaaat 12480attgtcataa atctatacaa tcgtttagcc ttgctggacg
aatctcaatt atttaaacga 12540gagtaaacat atttgacttt ttggttattt aacaaattat
tatttaacac tatatgaaat 12600tttttttttt atcagcaaag aataaaatta aattaagaag
gacaatggtg tcccaatcct 12660tatacaacca acttccacaa gaaagtcaag tcagagacaa
caaaaaaaca agcaaaggaa 12720attttttaat ttgagttgtc ttgtttgctg cataatttat
gcagtaaaac actacacata 12780acccttttag cagtagagca atggttgacc gtgtgcttag
cttcttttat tttatttttt 12840tatcagcaaa gaataaataa aataaaatga gacacttcag
ggatgtttca acctgca 12897571272DNAArtificial Sequencesynthetic delta-8
desaturase (codon-optimized for Yarrowia lipolytica) 57atgggcaagg
gtggagacgg tggagcacag gctgtgtctg gcaccgatgc ctccctcgct 60gaggtctcct
ctgtggacag caagtccgtc cacgtggttc tgtacggcaa gcgagtggat 120gtcaccaagt
tccagaaggc tcaccctgga ggttcgaagg tgttccgaat ctttcaggag 180cgagacgcca
cagaacagtt cgagtcctac cactctccca aggccatcaa gatgatggaa 240ggtatgctca
aaaagtcgga ggatgctccc gcttccgtgc ctcttccctc tcgatccact 300atgggcaccg
agttcaagga gatgatcgaa cgacacaaga gagccggtct ctacgaccct 360tgtcccttgg
acgagctgtt caagctcacc attgtccttg ctcctatctt tgtgggagcc 420tatctcgttc
gatccggtgt ctctcctctt gctggagccc tgtcgatggg cttcggattc 480tacctcgacg
gctggcttgc tcacgactac ctgcatcacg cagtgttcaa gggctccgtc 540aacacactcg
tcaaggccaa caacgctatg ggatacgcct tgggcttcct ccagggttac 600gacgttgctt
ggtggcgagc cagacacaac actcatcacg tgtgcaccaa cgaggacggc 660tccgatcccg
acatcaagac ggctcctctg ctcatttacg tgcgagagaa tccctccatt 720gccaagcggc
tcaacttctt tcagcgatgg caacagtact actatgttcc tactatggcc 780attctggatc
tctactggcg actggagtct atcgcatacg tcgctgtgcg actgcccaag 840atgtggatgc
aggctgccgc tcttgccgct cactacgcac tcctgtgttg ggtcttcgct 900gcccatctca
acctgattcc tctcatgatg gttgcacgag gtttcgcgac cggaatcgtg 960gtctttgcca
cccactatgg cgaggacatt ctcgaccgag agcacgtcga aggcatgact 1020ctggtcgagc
agaccgccaa gacctcccga aacatcactg gtggatggct tgtcaacgtg 1080ctcaccggct
tcatttctct gcagaccgag catcatctgt ttcccatgat gcctactgga 1140aacctcatga
ccattcaacc cgaggttcga gactttttca aaaagcacgg tctcgagtac 1200cgagaaggaa
acctgtttca gtgcgtgcat cagaacatca aggctctcgc cttcgagcac 1260ctgcttcact
aa
1272583992DNAArtificial Sequenceplasmid pPiD8S 58tcgcgcgttt cggtgatgac
ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat
gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg
cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata
ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240attcgccatt caggctgcgc
aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300tacgccagct ggcgaaaggg
ggatgtgctg caaggcgatt aagttgggta acgccagggt 360tttcccagtc acgacgttgt
aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa 420tgcatctaga tccatgggca
agggtggaga cggtggagca caggctgtgt ctggcaccga 480tgcctccctc gctgaggtct
cctctgtgga cagcaagtcc gtccacgtgg ttctgtacgg 540caagcgagtg gatgtcacca
agttccagaa ggctcaccct ggaggttcga aggtgttccg 600aatctttcag gagcgagacg
ccacagaaca gttcgagtcc taccactctc ccaaggccat 660caagatgatg gaaggtatgc
tcaaaaagtc ggaggatgct cccgcttccg tgcctcttcc 720ctctcgatcc actatgggca
ccgagttcaa ggagatgatc gaacgacaca agagagccgg 780tctctacgac ccttgtccct
tggacgagct gttcaagctc accattgtcc ttgctcctat 840ctttgtggga gcctatctcg
ttcgatccgg tgtctctcct cttgctggag ccctgtcgat 900gggcttcgga ttctacctcg
acggctggct tgctcacgac tacctgcatc acgcagtgtt 960caagggctcc gtcaacacac
tcgtcaaggc caacaacgct atgggatacg ccttgggctt 1020cctccagggt tacgacgttg
cttggtggcg agccagacac aacactcatc acgtgtgcac 1080caacgaggac ggctccgatc
ccgacatcaa gacggctcct ctgctcattt acgtgcgaga 1140gaatccctcc attgccaagc
ggctcaactt ctttcagcga tggcaacagt actactatgt 1200tcctactatg gccattctgg
atctctactg gcgactggag tctatcgcat acgtcgctgt 1260gcgactgccc aagatgtgga
tgcaggctgc cgctcttgcc gctcactacg cactcctgtg 1320ttgggtcttc gctgcccatc
tcaacctgat tcctctcatg atggttgcac gaggtttcgc 1380gaccggaatc gtggtctttg
ccacccacta tggcgaggac attctcgacc gagagcacgt 1440cgaaggcatg actctggtcg
agcagaccgc caagacctcc cgaaacatca ctggtggatg 1500gcttgtcaac gtgctcaccg
gcttcatttc tctgcagacc gagcatcatc tgtttcccat 1560gatgcctact ggaaacctca
tgaccattca acccgaggtt cgagactttt tcaaaaagca 1620cggtctcgag taccgagaag
gaaacctgtt tcagtgcgtg catcagaaca tcaaggctct 1680cgccttcgag cacctgcttc
actaagcggc cgcatcggat cccgggcccg tcgactgcag 1740aggcctgcat gcaagcttgg
cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta 1800tccgctcaca attccacaca
acatacgagc cggaagcata aagtgtaaag cctggggtgc 1860ctaatgagtg agctaactca
cattaattgc gttgcgctca ctgcccgctt tccagtcggg 1920aaacctgtcg tgccagctgc
attaatgaat cggccaacgc gcggggagag gcggtttgcg 1980tattgggcgc tcttccgctt
cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 2040gcgagcggta tcagctcact
caaaggcggt aatacggtta tccacagaat caggggataa 2100cgcaggaaag aacatgtgag
caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 2160gttgctggcg tttttccata
ggctccgccc ccctgacgag catcacaaaa atcgacgctc 2220aagtcagagg tggcgaaacc
cgacaggact ataaagatac caggcgtttc cccctggaag 2280ctccctcgtg cgctctcctg
ttccgaccct gccgcttacc ggatacctgt ccgcctttct 2340cccttcggga agcgtggcgc
tttctcatag ctcacgctgt aggtatctca gttcggtgta 2400ggtcgttcgc tccaagctgg
gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 2460cttatccggt aactatcgtc
ttgagtccaa cccggtaaga cacgacttat cgccactggc 2520agcagccact ggtaacagga
ttagcagagc gaggtatgta ggcggtgcta cagagttctt 2580gaagtggtgg cctaactacg
gctacactag aagaacagta tttggtatct gcgctctgct 2640gaagccagtt accttcggaa
aaagagttgg tagctcttga tccggcaaac aaaccaccgc 2700tggtagcggt ggtttttttg
tttgcaagca gcagattacg cgcagaaaaa aaggatctca 2760agaagatcct ttgatctttt
ctacggggtc tgacgctcag tggaacgaaa actcacgtta 2820agggattttg gtcatgagat
tatcaaaaag gatcttcacc tagatccttt taaattaaaa 2880atgaagtttt aaatcaatct
aaagtatata tgagtaaact tggtctgaca gttaccaatg 2940cttaatcagt gaggcaccta
tctcagcgat ctgtctattt cgttcatcca tagttgcctg 3000actccccgtc gtgtagataa
ctacgatacg ggagggctta ccatctggcc ccagtgctgc 3060aatgataccg cgagacccac
gctcaccggc tccagattta tcagcaataa accagccagc 3120cggaagggcc gagcgcagaa
gtggtcctgc aactttatcc gcctccatcc agtctattaa 3180ttgttgccgg gaagctagag
taagtagttc gccagttaat agtttgcgca acgttgttgc 3240cattgctaca ggcatcgtgg
tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 3300ttcccaacga tcaaggcgag
ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 3360cttcggtcct ccgatcgttg
tcagaagtaa gttggccgca gtgttatcac tcatggttat 3420ggcagcactg cataattctc
ttactgtcat gccatccgta agatgctttt ctgtgactgg 3480tgagtactca accaagtcat
tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 3540ggcgtcaata cgggataata
ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 3600aaaacgttct tcggggcgaa
aactctcaag gatcttaccg ctgttgagat ccagttcgat 3660gtaacccact cgtgcaccca
actgatcttc agcatctttt actttcacca gcgtttctgg 3720gtgagcaaaa acaggaaggc
aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 3780ttgaatactc atactcttcc
tttttcaata ttattgaagc atttatcagg gttattgtct 3840catgagcgga tacatatttg
aatgtattta gaaaaataaa caaatagggg ttccgcgcac 3900atttccccga aaagtgccac
ctgacgtcta agaaaccatt attatcatga cattaaccta 3960taaaaatagg cgtatcacga
ggccctttcg tc 399259792DNAIsochrysis
galbana 59atggccctcg caaacgacgc gggagagcgc atctgggcgg ctgtgaccga
cccggaaatc 60ctcattggca ccttctcgta cttgctactc aaaccgctgc tccgcaattc
cgggctggtg 120gatgagaaga agggcgcata caggacgtcc atgatctggt acaacgttct
gctggcgctc 180ttctctgcgc tgagcttcta cgtgacggcg accgccctcg gctgggacta
tggtacgggc 240gcgtggctgc gcaggcaaac cggcgacaca ccgcagccgc tcttccagtg
cccgtccccg 300gtttgggact cgaagctctt cacatggacc gccaaggcat tctattactc
caagtacgtg 360gagtacctcg acacggcctg gctggtgctc aagggcaaga gggtctcctt
tctccaggcc 420ttccaccact ttggcgcgcc gtgggatgtg tacctcggca ttcggctgca
caacgagggc 480gtatggatct tcatgttttt caactcgttc attcacacca tcatgtacac
ctactacggc 540ctcaccgccg ccgggtataa gttcaaggcc aagccgctca tcaccgcgat
gcagatctgc 600cagttcgtgg gcggcttcct gttggtctgg gactacatca acgtcccctg
cttcaactcg 660gacaaaggga agttgttcag ctgggctttc aactatgcat acgtcggctc
ggtcttcttg 720ctcttctgcc actttttcta ccaggacaac ttggcaacga agaaatcggc
caaggcgggc 780aagcagctct ag
79260757DNAEuglena gracilismisc_feature(677)..(677)n is a, c,
g, or t 60ttttttttcg aacacttaat ggaggtggtg aatgaaatag tctcaattgg
gcaggaagtt 60ttacccaaag ttgattatgc ccaactctgg agtgatgcca gtcactgtga
ggtgctttac 120ttgtccatcg catttgtcat cttgaagttc actcttggcc cccttggtcc
aaaaggtcag 180tctcgtatga agtttgtttt caccaattac aaccttctca tgtccattta
ttcgttggga 240tcattcctct caatggcata tgccatgtac accatcggtg ttatgtctga
caactgcgag 300aaggcttttg acaacaacgt cttcaggatc accacgcagt tgttctattt
gagcaagttc 360ctggagtata ttgactcctt ctatttgcca ctgatgggca agcctctgac
ctggttgcaa 420ttcttccatc atttgggggc accgatggat atgtggctgt tctataatta
ccgaaatgaa 480gctgtttgga tttttgtgct gttgaatggt ttcatccact ggatcatgta
cggttattat 540tggaccagat tgatcaagct gaagttcccc atgccaaaat ccctgattac
atcaatgcag 600atcattcaat tcaatgttgg tttctacatt gtctggaagt acaggaacat
tccctgttat 660cgccaagatg ggatgangat gtttggctgg ttcttcaatt acttttatgt
tggcacagtc 720ttgtgtttgt tcttgaattt ctatgtgcaa acgtata
75761774DNAEuglena gracilismisc_feature(34)..(34)n is a, c,
g, or t 61tcaggatcac cacgcagttg ttctatttga gcangttcct ggagtatatt
gactccttct 60atttgccant gatgggcaag cntctgacct ggttgcaatt cttccatcat
tngggggcac 120cgatggatat gtggctgttc tataattacc gaaatgaagc tgtttggatt
tttgtgctgt 180tgaatggttt catccactgg atcatgtacg gttattannn gaccagattg
atcaagctga 240agttccccat gccaaaatcc ctgattacat caatgcagat cattcaattc
aatgttggtt 300tctacattgt ctggaagtac aggaacattc cctgttatcg ccaagatggg
atgaggatgt 360ttggctggtt cttcaattac ttttatgttg gcacagtctt gtgtttgttc
ttgaatttct 420atgtgcaaac gtatatcgtc aggaagcaca agggagccaa aaagattcag
tgatatttcc 480tcctctgcgg tggcctcttt tgacctcccc ttgacaccta taatgtggag
gtgtcgggct 540ctctccgtct caccagcact tgactctgca ggtgctcact tttatttttt
acccatcttt 600gcttgttgac cattcacctc tcccacttcc acatagtcca ttctaactgt
tgcagactgc 660ggtccatttt ttccagagct cccaatgacc atacgcgaca ccttgtnnnc
ncccagccca 720ttgtgcacaa ttcatagtgg catcgttttg ccttgatacg tgtgcatcca
gcgg 774621201DNAEuglena gracilismisc_feature(1134)..(1136)n is
a, c, g, or t 62gacatggcaa ctatgatttt attttgactg atagtgacct gttcgttgca
acaaattgat 60gagcaatgct tttttataat gccaactttg tacaaaaaag ttggattttt
tttcgaacac 120ttaatggagg tggtgaatga aatagtctca attgggcagg aagttttacc
caaagttgat 180tatgcccaac tctggagtga tgccagtcac tgtgaggtgc tttacttgtc
catcgcattt 240gtcatcttga agttcactct tggccccctt ggtccaaaag gtcagtctcg
tatgaagttt 300gttttcacca attacaacct tctcatgtcc atttattcgt tgggatcatt
cctctcaatg 360gcatatgcca tgtacaccat cggtgttatg tctgacaact gcgagaaggc
ttttgacaac 420aacgtcttca ggatcaccac gcagttgttc tatttgagca agttcctgga
gtatattgac 480tccttctatt tgccactgat gggcaagcct ctgacctggt tgcaattctt
ccatcatttg 540ggggcaccga tggatatgtg gctgttctat aattaccgaa atgaagctgt
ttggattttt 600gtgctgttga atggtttcat ccactggatc atgtacggtt attattggac
cagattgatc 660aagctgaagt tccccatgcc aaaatccctg attacatcaa tgcagatcat
tcaattcaat 720gttggtttct acattgtctg gaagtacagg aacattccct gttatcgcca
agatgggatg 780aggatgtttg gctggttctt caattacttt tatgttggca cagtcttgtg
tttgttcttg 840aatttctatg tgcaaacgta tatcgtcagg aagcacaagg gagccaaaaa
gattcagtga 900tatttcctcc tctgcggtgg cctcttttga cctccccttg acacctataa
tgtggaggtg 960tcgggctctc tccgtctcac cagcacttga ctctgcaggt gctcactttt
attttttacc 1020catctttgct tgttgaccat tcacctctcc cacttccaca tagtccattc
taactgttgc 1080agactgcggt ccattttttc cagagctccc aatgaccata cgcgacacct
tgtnnncncc 1140cagcccattg tgcacaattc atagtggcat cgttttgcct tgatacgtgt
gcatccagcg 1200g
12016318DNAArtificial SequenceM13F universal primer
63tgtaaaacga cggccagt
1864258PRTEuglena gracilis 64Met Glu Val Val Asn Glu Ile Val Ser Ile Gly
Gln Glu Val Leu Pro1 5 10
15Lys Val Asp Tyr Ala Gln Leu Trp Ser Asp Ala Ser His Cys Glu Val
20 25 30Leu Tyr Leu Ser Ile Ala Phe
Val Ile Leu Lys Phe Thr Leu Gly Pro 35 40
45Leu Gly Pro Lys Gly Gln Ser Arg Met Lys Phe Val Phe Thr Asn
Tyr 50 55 60Asn Leu Leu Met Ser Ile
Tyr Ser Leu Gly Ser Phe Leu Ser Met Ala65 70
75 80Tyr Ala Met Tyr Thr Ile Gly Val Met Ser Asp
Asn Cys Glu Lys Ala 85 90
95Phe Asp Asn Asn Val Phe Arg Ile Thr Thr Gln Leu Phe Tyr Leu Ser
100 105 110Lys Phe Leu Glu Tyr Ile
Asp Ser Phe Tyr Leu Pro Leu Met Gly Lys 115 120
125Pro Leu Thr Trp Leu Gln Phe Phe His His Leu Gly Ala Pro
Met Asp 130 135 140Met Trp Leu Phe Tyr
Asn Tyr Arg Asn Glu Ala Val Trp Ile Phe Val145 150
155 160Leu Leu Asn Gly Phe Ile His Trp Ile Met
Tyr Gly Tyr Tyr Trp Thr 165 170
175Arg Leu Ile Lys Leu Lys Phe Pro Met Pro Lys Ser Leu Ile Thr Ser
180 185 190Met Gln Ile Ile Gln
Phe Asn Val Gly Phe Tyr Ile Val Trp Lys Tyr 195
200 205Arg Asn Ile Pro Cys Tyr Arg Gln Asp Gly Met Arg
Met Phe Gly Trp 210 215 220Phe Phe Asn
Tyr Phe Tyr Val Gly Thr Val Leu Cys Leu Phe Leu Asn225
230 235 240Phe Tyr Val Gln Thr Tyr Ile
Val Arg Lys His Lys Gly Ala Lys Lys 245
250 255Ile Gln65263PRTIsochrysis galbana 65Met Ala Leu
Ala Asn Asp Ala Gly Glu Arg Ile Trp Ala Ala Val Thr1 5
10 15Asp Pro Glu Ile Leu Ile Gly Thr Phe
Ser Tyr Leu Leu Leu Lys Pro 20 25
30Leu Leu Arg Asn Ser Gly Leu Val Asp Glu Lys Lys Gly Ala Tyr Arg
35 40 45Thr Ser Met Ile Trp Tyr Asn
Val Leu Leu Ala Leu Phe Ser Ala Leu 50 55
60Ser Phe Tyr Val Thr Ala Thr Ala Leu Gly Trp Asp Tyr Gly Thr Gly65
70 75 80Ala Trp Leu Arg
Arg Gln Thr Gly Asp Thr Pro Gln Pro Leu Phe Gln 85
90 95Cys Pro Ser Pro Val Trp Asp Ser Lys Leu
Phe Thr Trp Thr Ala Lys 100 105
110Ala Phe Tyr Tyr Ser Lys Tyr Val Glu Tyr Leu Asp Thr Ala Trp Leu
115 120 125Val Leu Lys Gly Lys Arg Val
Ser Phe Leu Gln Ala Phe His His Phe 130 135
140Gly Ala Pro Trp Asp Val Tyr Leu Gly Ile Arg Leu His Asn Glu
Gly145 150 155 160Val Trp
Ile Phe Met Phe Phe Asn Ser Phe Ile His Thr Ile Met Tyr
165 170 175Thr Tyr Tyr Gly Leu Thr Ala
Ala Gly Tyr Lys Phe Lys Ala Lys Pro 180 185
190Leu Ile Thr Ala Met Gln Ile Cys Gln Phe Val Gly Gly Phe
Leu Leu 195 200 205Val Trp Asp Tyr
Ile Asn Val Pro Cys Phe Asn Ser Asp Lys Gly Lys 210
215 220Leu Phe Ser Trp Ala Phe Asn Tyr Ala Tyr Val Gly
Ser Val Phe Leu225 230 235
240Leu Phe Cys His Phe Phe Tyr Gln Asp Asn Leu Ala Thr Lys Lys Ser
245 250 255Ala Lys Ala Gly Lys
Gln Leu 26066777DNAArtificial Sequencesynthetic
(codon-optimized) delta-9 elongase derived from Isochrysis galbana
codon-optimized for expression in Yarrowia lipolytica 66atggaggtcg
tgaacgaaat cgtctccatt ggccaggagg ttcttcccaa ggtcgactat 60gctcagctct
ggtctgatgc ctcgcactgc gaggtgctgt acctctccat cgccttcgtc 120atcctgaagt
tcacccttgg tcctctcgga cccaagggtc agtctcgaat gaagtttgtg 180ttcaccaact
acaacctgct catgtccatc tactcgctgg gctccttcct ctctatggcc 240tacgccatgt
acaccattgg tgtcatgtcc gacaactgcg agaaggcttt cgacaacaat 300gtcttccgaa
tcaccactca gctgttctac ctcagcaagt tcctcgagta cattgactcc 360ttctatctgc
ccctcatggg caagcctctg acctggttgc agttctttca ccatctcgga 420gctcctatgg
acatgtggct gttctacaac taccgaaacg aagccgtttg gatctttgtg 480ctgctcaacg
gcttcattca ctggatcatg tacggctact attggacccg actgatcaag 540ctcaagttcc
ctatgcccaa gtccctgatt acttctatgc agatcattca gttcaacgtt 600ggcttctaca
tcgtctggaa gtaccggaac attccctgct accgacaaga tggaatgaga 660atgtttggct
ggtttttcaa ctacttctac gttggtactg tcctgtgtct gttcctcaac 720ttctacgtgc
agacctacat cgtccgaaag cacaagggag ccaaaaagat tcagtga
777673497DNAArtificial Sequenceplasmid pEgD9ES 67tcgcgcgttt cggtgatgac
ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat
gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg
cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata
ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240attcgccatt caggctgcgc
aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300tacgccagct ggcgaaaggg
ggatgtgctg caaggcgatt aagttgggta acgccagggt 360tttcccagtc acgacgttgt
aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa 420tgcatctaga tccatggagg
tcgtgaacga aatcgtctcc attggccagg aggttcttcc 480caaggtcgac tatgctcagc
tctggtctga tgcctcgcac tgcgaggtgc tgtacctctc 540catcgccttc gtcatcctga
agttcaccct tggtcctctc ggacccaagg gtcagtctcg 600aatgaagttt gtgttcacca
actacaacct gctcatgtcc atctactcgc tgggctcctt 660cctctctatg gcctacgcca
tgtacaccat tggtgtcatg tccgacaact gcgagaaggc 720tttcgacaac aatgtcttcc
gaatcaccac tcagctgttc tacctcagca agttcctcga 780gtacattgac tccttctatc
tgcccctcat gggcaagcct ctgacctggt tgcagttctt 840tcaccatctc ggagctccta
tggacatgtg gctgttctac aactaccgaa acgaagccgt 900ttggatcttt gtgctgctca
acggcttcat tcactggatc atgtacggct actattggac 960ccgactgatc aagctcaagt
tccctatgcc caagtccctg attacttcta tgcagatcat 1020tcagttcaac gttggcttct
acatcgtctg gaagtaccgg aacattccct gctaccgaca 1080agatggaatg agaatgtttg
gctggttttt caactacttc tacgttggta ctgtcctgtg 1140tctgttcctc aacttctacg
tgcagaccta catcgtccga aagcacaagg gagccaaaaa 1200gattcagtga gcggccgcat
cggatcccgg gcccgtcgac tgcagaggcc tgcatgcaag 1260cttggcgtaa tcatggtcat
agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc 1320acacaacata cgagccggaa
gcataaagtg taaagcctgg ggtgcctaat gagtgagcta 1380actcacatta attgcgttgc
gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca 1440gctgcattaa tgaatcggcc
aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc 1500cgcttcctcg ctcactgact
cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 1560tcactcaaag gcggtaatac
ggttatccac agaatcaggg gataacgcag gaaagaacat 1620gtgagcaaaa ggccagcaaa
aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 1680ccataggctc cgcccccctg
acgagcatca caaaaatcga cgctcaagtc agaggtggcg 1740aaacccgaca ggactataaa
gataccaggc gtttccccct ggaagctccc tcgtgcgctc 1800tcctgttccg accctgccgc
ttaccggata cctgtccgcc tttctccctt cgggaagcgt 1860ggcgctttct catagctcac
gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 1920gctgggctgt gtgcacgaac
cccccgttca gcccgaccgc tgcgccttat ccggtaacta 1980tcgtcttgag tccaacccgg
taagacacga cttatcgcca ctggcagcag ccactggtaa 2040caggattagc agagcgaggt
atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 2100ctacggctac actagaagaa
cagtatttgg tatctgcgct ctgctgaagc cagttacctt 2160cggaaaaaga gttggtagct
cttgatccgg caaacaaacc accgctggta gcggtggttt 2220ttttgtttgc aagcagcaga
ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 2280cttttctacg gggtctgacg
ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 2340gagattatca aaaaggatct
tcacctagat ccttttaaat taaaaatgaa gttttaaatc 2400aatctaaagt atatatgagt
aaacttggtc tgacagttac caatgcttaa tcagtgaggc 2460acctatctca gcgatctgtc
tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 2520gataactacg atacgggagg
gcttaccatc tggccccagt gctgcaatga taccgcgaga 2580cccacgctca ccggctccag
atttatcagc aataaaccag ccagccggaa gggccgagcg 2640cagaagtggt cctgcaactt
tatccgcctc catccagtct attaattgtt gccgggaagc 2700tagagtaagt agttcgccag
ttaatagttt gcgcaacgtt gttgccattg ctacaggcat 2760cgtggtgtca cgctcgtcgt
ttggtatggc ttcattcagc tccggttccc aacgatcaag 2820gcgagttaca tgatccccca
tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat 2880cgttgtcaga agtaagttgg
ccgcagtgtt atcactcatg gttatggcag cactgcataa 2940ttctcttact gtcatgccat
ccgtaagatg cttttctgtg actggtgagt actcaaccaa 3000gtcattctga gaatagtgta
tgcggcgacc gagttgctct tgcccggcgt caatacggga 3060taataccgcg ccacatagca
gaactttaaa agtgctcatc attggaaaac gttcttcggg 3120gcgaaaactc tcaaggatct
taccgctgtt gagatccagt tcgatgtaac ccactcgtgc 3180acccaactga tcttcagcat
cttttacttt caccagcgtt tctgggtgag caaaaacagg 3240aaggcaaaat gccgcaaaaa
agggaataag ggcgacacgg aaatgttgaa tactcatact 3300cttccttttt caatattatt
gaagcattta tcagggttat tgtctcatga gcggatacat 3360atttgaatgt atttagaaaa
ataaacaaat aggggttccg cgcacatttc cccgaaaagt 3420gccacctgac gtctaagaaa
ccattattat catgacatta acctataaaa ataggcgtat 3480cacgaggccc tttcgtc
3497689472DNAArtificial
Sequenceplasmid pDMW263 68catggcatgg atggtacgtc ctgtagaaac cccaacccgt
gaaatcaaaa aactcgacgg 60cctgtgggca ttcagtctgg atcgcgaaaa ctgtggaatt
gatcagcgtt ggtgggaaag 120cgcgttacaa gaaagccggg caattgctgt gccaggcagt
tttaacgatc agttcgccga 180tgcagatatt cgtaattatg cgggcaacgt ctggtatcag
cgcgaagtct ttataccgaa 240aggttgggca ggccagcgta tcgtgctgcg tttcgatgcg
gtcactcatt acggcaaagt 300gtgggtcaat aatcaggaag tgatggagca tcagggcggc
tatacgccat ttgaagccga 360tgtcacgccg tatgttattg ccgggaaaag tgtacgtatc
accgtttgtg tgaacaacga 420actgaactgg cagactatcc cgccgggaat ggtgattacc
gacgaaaacg gcaagaaaaa 480gcagtcttac ttccatgatt tctttaacta tgccgggatc
catcgcagcg taatgctcta 540caccacgccg aacacctggg tggacgatat caccgtggtg
acgcatgtcg cgcaagactg 600taaccacgcg tctgttgact ggcaggtggt ggccaatggt
gatgtcagcg ttgaactgcg 660tgatgcggat caacaggtgg ttgcaactgg acaaggcact
agcgggactt tgcaagtggt 720gaatccgcac ctctggcaac cgggtgaagg ttatctctat
gaactgtgcg tcacagccaa 780aagccagaca gagtgtgata tctacccgct tcgcgtcggc
atccggtcag tggcagtgaa 840gggcgaacag ttcctgatta accacaaacc gttctacttt
actggctttg gtcgtcatga 900agatgcggac ttacgtggca aaggattcga taacgtgctg
atggtgcacg accacgcatt 960aatggactgg attggggcca actcctaccg tacctcgcat
tacccttacg ctgaagagat 1020gctcgactgg gcagatgaac atggcatcgt ggtgattgat
gaaactgctg ctgtcggctt 1080taacctctct ttaggcattg gtttcgaagc gggcaacaag
ccgaaagaac tgtacagcga 1140agaggcagtc aacggggaaa ctcagcaagc gcacttacag
gcgattaaag agctgatagc 1200gcgtgacaaa aaccacccaa gcgtggtgat gtggagtatt
gccaacgaac cggatacccg 1260tccgcaagtg cacgggaata tttcgccact ggcggaagca
acgcgtaaac tcgacccgac 1320gcgtccgatc acctgcgtca atgtaatgtt ctgcgacgct
cacaccgata ccatcagcga 1380tctctttgat gtgctgtgcc tgaaccgtta ttacggatgg
tatgtccaaa gcggcgattt 1440ggaaacggca gagaaggtac tggaaaaaga acttctggcc
tggcaggaga aactgcatca 1500gccgattatc atcaccgaat acggcgtgga tacgttagcc
gggctgcact caatgtacac 1560cgacatgtgg agtgaagagt atcagtgtgc atggctggat
atgtatcacc gcgtctttga 1620tcgcgtcagc gccgtcgtcg gtgaacaggt atggaatttc
gccgattttg cgacctcgca 1680aggcatattg cgcgttggcg gtaacaagaa agggatcttc
actcgcgacc gcaaaccgaa 1740gtcggcggct tttctgctgc aaaaacgctg gactggcatg
aacttcggtg aaaaaccgca 1800gcagggaggc aaacaatgat taattaacta gagcggccgc
caccgcggcc cgagattccg 1860gcctcttcgg ccgccaagcg acccgggtgg acgtctagag
gtacctagca attaacagat 1920agtttgccgg tgataattct cttaacctcc cacactcctt
tgacataacg atttatgtaa 1980cgaaactgaa atttgaccag atattgtgtc cgcggtggag
ctccagcttt tgttcccttt 2040agtgagggtt aatttcgagc ttggcgtaat catggtcata
gctgtttcct gtgtgaaatt 2100gttatccgct cacaattcca cacaacatac gagccggaag
cataaagtgt aaagcctggg 2160gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
ctcactgccc gctttccagt 2220cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
acgcgcgggg agaggcggtt 2280tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
gctgcgctcg gtcgttcggc 2340tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
gttatccaca gaatcagggg 2400ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
ggccaggaac cgtaaaaagg 2460ccgcgttgct ggcgtttttc cataggctcc gcccccctga
cgagcatcac aaaaatcgac 2520gctcaagtca gaggtggcga aacccgacag gactataaag
ataccaggcg tttccccctg 2580gaagctccct cgtgcgctct cctgttccga ccctgccgct
taccggatac ctgtccgcct 2640ttctcccttc gggaagcgtg gcgctttctc atagctcacg
ctgtaggtat ctcagttcgg 2700tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
ccccgttcag cccgaccgct 2760gcgccttatc cggtaactat cgtcttgagt ccaacccggt
aagacacgac ttatcgccac 2820tggcagcagc cactggtaac aggattagca gagcgaggta
tgtaggcggt gctacagagt 2880tcttgaagtg gtggcctaac tacggctaca ctagaaggac
agtatttggt atctgcgctc 2940tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
ttgatccggc aaacaaacca 3000ccgctggtag cggtggtttt tttgtttgca agcagcagat
tacgcgcaga aaaaaaggat 3060ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
tcagtggaac gaaaactcac 3120gttaagggat tttggtcatg agattatcaa aaaggatctt
cacctagatc cttttaaatt 3180aaaaatgaag ttttaaatca atctaaagta tatatgagta
aacttggtct gacagttacc 3240aatgcttaat cagtgaggca cctatctcag cgatctgtct
atttcgttca tccatagttg 3300cctgactccc cgtcgtgtag ataactacga tacgggaggg
cttaccatct ggccccagtg 3360ctgcaatgat accgcgagac ccacgctcac cggctccaga
tttatcagca ataaaccagc 3420cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
atccgcctcc atccagtcta 3480ttaattgttg ccgggaagct agagtaagta gttcgccagt
taatagtttg cgcaacgttg 3540ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
tggtatggct tcattcagct 3600ccggttccca acgatcaagg cgagttacat gatcccccat
gttgtgcaaa aaagcggtta 3660gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
cgcagtgtta tcactcatgg 3720ttatggcagc actgcataat tctcttactg tcatgccatc
cgtaagatgc ttttctgtga 3780ctggtgagta ctcaaccaag tcattctgag aatagtgtat
gcggcgaccg agttgctctt 3840gcccggcgtc aatacgggat aataccgcgc cacatagcag
aactttaaaa gtgctcatca 3900ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
accgctgttg agatccagtt 3960cgatgtaacc cactcgtgca cccaactgat cttcagcatc
ttttactttc accagcgttt 4020ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
gggaataagg gcgacacgga 4080aatgttgaat actcatactc ttcctttttc aatattattg
aagcatttat cagggttatt 4140gtctcatgag cggatacata tttgaatgta tttagaaaaa
taaacaaata ggggttccgc 4200gcacatttcc ccgaaaagtg ccacctgacg cgccctgtag
cggcgcatta agcgcggcgg 4260gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
cgccctagcg cccgctcctt 4320tcgctttctt cccttccttt ctcgccacgt tcgccggctt
tccccgtcaa gctctaaatc 4380gggggctccc tttagggttc cgatttagtg ctttacggca
cctcgacccc aaaaaacttg 4440attagggtga tggttcacgt agtgggccat cgccctgata
gacggttttt cgccctttga 4500cgttggagtc cacgttcttt aatagtggac tcttgttcca
aactggaaca acactcaacc 4560ctatctcggt ctattctttt gatttataag ggattttgcc
gatttcggcc tattggttaa 4620aaaatgagct gatttaacaa aaatttaacg cgaattttaa
caaaatatta acgcttacaa 4680tttccattcg ccattcaggc tgcgcaactg ttgggaaggg
cgatcggtgc gggcctcttc 4740gctattacgc cagctggcga aagggggatg tgctgcaagg
cgattaagtt gggtaacgcc 4800agggttttcc cagtcacgac gttgtaaaac gacggccagt
gaattgtaat acgactcact 4860atagggcgaa ttgggtaccg ggccccccct cgaggtcgat
ggtgtcgata agcttgatat 4920cgaattcatg tcacacaaac cgatcttcgc ctcaaggaaa
cctaattcta catccgagag 4980actgccgaga tccagtctac actgattaat tttcgggcca
ataatttaaa aaaatcgtgt 5040tatataatat tatatgtatt atatatatac atcatgatga
tactgacagt catgtcccat 5100tgctaaatag acagactcca tctgccgcct ccaactgatg
ttctcaatat ttaaggggtc 5160atctcgcatt gtttaataat aaacagactc catctaccgc
ctccaaatga tgttctcaaa 5220atatattgta tgaacttatt tttattactt agtattatta
gacaacttac ttgctttatg 5280aaaaacactt cctatttagg aaacaattta taatggcagt
tcgttcattt aacaatttat 5340gtagaataaa tgttataaat gcgtatggga aatcttaaat
atggatagca taaatgatat 5400ctgcattgcc taattcgaaa tcaacagcaa cgaaaaaaat
cccttgtaca acataaatag 5460tcatcgagaa atatcaacta tcaaagaaca gctattcaca
cgttactatt gagattatta 5520ttggacgaga atcacacact caactgtctt tctctcttct
agaaatacag gtacaagtat 5580gtactattct cattgttcat acttctagtc atttcatccc
acatattcct tggatttctc 5640tccaatgaat gacattctat cttgcaaatt caacaattat
aataagatat accaaagtag 5700cggtatagtg gcaatcaaaa agcttctctg gtgtgcttct
cgtatttatt tttattctaa 5760tgatccatta aaggtatata tttatttctt gttatataat
ccttttgttt attacatggg 5820ctggatacat aaaggtattt tgatttaatt ttttgcttaa
attcaatccc ccctcgttca 5880gtgtcaactg taatggtagg aaattaccat acttttgaag
aagcaaaaaa aatgaaagaa 5940aaaaaaaatc gtatttccag gttagacgtt ccgcagaatc
tagaatgcgg tatgcggtac 6000attgttcttc gaacgtaaaa gttgcgctcc ctgagatatt
gtacattttt gcttttacaa 6060gtacaagtac atcgtacaac tatgtactac tgttgatgca
tccacaacag tttgttttgt 6120ttttttttgt tttttttttt tctaatgatt cattaccgct
atgtatacct acttgtactt 6180gtagtaagcc gggttattgg cgttcaatta atcatagact
tatgaatctg cacggtgtgc 6240gctgcgagtt acttttagct tatgcatgct acttgggtgt
aatattggga tctgttcgga 6300aatcaacgga tgctcaaccg atttcgacag taataatttg
aatcgaatcg gagcctaaaa 6360tgaacccgag tatatctcat aaaattctcg gtgagaggtc
tgtgactgtc agtacaaggt 6420gccttcatta tgccctcaac cttaccatac ctcactgaat
gtagtgtacc tctaaaaatg 6480aaatacagtg ccaaaagcca aggcactgag ctcgtctaac
ggacttgata tacaaccaat 6540taaaacaaat gaaaagaaat acagttcttt gtatcatttg
taacaattac cctgtacaaa 6600ctaaggtatt gaaatcccac aatattccca aagtccaccc
ctttccaaat tgtcatgcct 6660acaactcata taccaagcac taacctacca aacaccacta
aaaccccaca aaatatatct 6720taccgaatat acagtaacaa gctaccacca cactcgttgg
gtgcagtcgc cagcttaaag 6780atatctatcc acatcagcca caactccctt cctttaataa
accgactaca cccttggcta 6840ttgaggttat gagtgaatat actgtagaca agacactttc
aagaagactg tttccaaaac 6900gtaccactgt cctccactac aaacacaccc aatctgcttc
ttctagtcaa ggttgctaca 6960ccggtaaatt ataaatcatc atttcattag cagggcaggg
ccctttttat agagtcttat 7020acactagcgg accctgccgg tagaccaacc cgcaggcgcg
tcagtttgct ccttccatca 7080atgcgtcgta gaaacgactt actccttctt gagcagctcc
ttgaccttgt tggcaacaag 7140tctccgacct cggaggtgga ggaagagcct ccgatatcgg
cggtagtgat accagcctcg 7200acggactcct tgacggcagc ctcaacagcg tcaccggcgg
gcttcatgtt aagagagaac 7260ttgagcatca tggcggcaga cagaatggtg gcaatggggt
tgaccttctg cttgccgaga 7320tcgggggcag atccgtgaca gggctcgtac agaccgaacg
cctcgttggt gtcgggcaga 7380gaagccagag aggcggaggg cagcagaccc agagaaccgg
ggatgacgga ggcctcgtcg 7440gagatgatat cgccaaacat gttggtggtg atgatgatac
cattcatctt ggagggctgc 7500ttgatgagga tcatggcggc cgagtcgatc agctggtggt
tgagctcgag ctgggggaat 7560tcgtccttga ggactcgagt gacagtcttt cgccaaagtc
gagaggaggc cagcacgttg 7620gccttgtcaa gagaccacac gggaagaggg gggttgtgct
gaagggccag gaaggcggcc 7680attcgggcaa ttcgctcaac ctcaggaacg gagtaggtct
cggtgtcgga agcgacgcca 7740gatccgtcat cctcctttcg ctctccaaag tagatacctc
cgacgagctc tcggacaatg 7800atgaagtcgg tgccctcaac gtttcggatg ggggagagat
cggcgagctt gggcgacagc 7860agctggcagg gtcgcaggtt ggcgtacagg ttcaggtcct
ttcgcagctt gaggagaccc 7920tgctcgggtc gcacgtcggt tcgtccgtcg ggagtggtcc
atacggtgtt ggcagcgcct 7980ccgacagcac cgagcataat agagtcagcc tttcggcaga
tgtcgagagt agcgtcggtg 8040atgggctcgc cctccttctc aatggcagct cctccaatga
gtcggtcctc aaacacaaac 8100tcggtgccgg aggcctcagc aacagacttg agcaccttga
cggcctcggc aatcacctcg 8160gggccacaga agtcgccgcc gagaagaaca atcttcttgg
agtcagtctt ggtcttctta 8220gtttcgggtt ccattgtgga tgtgtgtggt tgtatgtgtg
atgtggtgtg tggagtgaaa 8280atctgtggct ggcaaacgct cttgtatata tacgcacttt
tgcccgtgct atgtggaaga 8340ctaaacctcc gaagattgtg actcaggtag tgcggtatcg
gctagggacc caaaccttgt 8400cgatgccgat agcgctatcg aacgtacccc agccggccgg
gagtatgtcg gaggggacat 8460acgagatcgt caagggtttg tggccaactg gtaaataaat
gatgtcgacg tttaaacagt 8520gtacgcagat ctactataga ggaacattta aattgccccg
gagaagacgg ccaggccgcc 8580tagatgacaa attcaacaac tcacagctga ctttctgcca
ttgccactag gggggggcct 8640ttttatatgg ccaagccaag ctctccacgt cggttgggct
gcacccaaca ataaatgggt 8700agggttgcac caacaaaggg atgggatggg gggtagaaga
tacgaggata acggggctca 8760atggcacaaa taagaacgaa tactgccatt aagactcgtg
atccagcgac tgacaccatt 8820gcatcatcta agggcctcaa aactacctcg gaactgctgc
gctgatctgg acaccacaga 8880ggttccgagc actttaggtt gcaccaaatg tcccaccagg
tgcaggcaga aaacgctgga 8940acagcgtgta cagtttgtct taacaaaaag tgagggcgct
gaggtcgagc agggtggtgt 9000gacttgttat agcctttaga gctgcgaaag cgcgtatgga
tttggctcat caggccagat 9060tgagggtctg tggacacatg tcatgttagt gtacttcaat
cgccccctgg atatagcccc 9120gacaataggc cgtggcctca tttttttgcc ttccgcacat
ttccattgct cgatacccac 9180accttgcttc tcctgcactt gccaacctta atactggttt
acattgacca acatcttaca 9240agcggggggc ttgtctaggg tatatataaa cagtggctct
cccaatcggt tgccagtctc 9300ttttttcctt tctttcccca cagattcgaa atctaaacta
cacatcacag aattccgagc 9360cgtgagtatc cacgacaaga tcagtgtcga gacgacgcgt
tttgtgtaat gacacaatcc 9420gaaagtcgct agcaacacac actctctaca caaactaacc
cagctctggt ac 9472698165DNAArtificial Sequenceplasmid pZUF17
69gtacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
60ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
120taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
180tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
240aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
300aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
360ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
420acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
480ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
540tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
600tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
660gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
720agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
780tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
840agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
900tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
960acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
1020tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
1080agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
1140tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
1200acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
1260tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
1320ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
1380agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
1440tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
1500acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
1560agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
1620actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
1680tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
1740gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
1800ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
1860tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
1920aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
1980tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
2040tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
2100gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
2160gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
2220acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
2280agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
2340ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
2400ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
2460taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
2520aacgcgaatt ttaacaaaat attaacgctt acaatttcca ttcgccattc aggctgcgca
2580actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
2640gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
2700aaacgacggc cagtgaattg taatacgact cactataggg cgaattgggt accgggcccc
2760ccctcgaggt cgatggtgtc gataagcttg atatcgaatt catgtcacac aaaccgatct
2820tcgcctcaag gaaacctaat tctacatccg agagactgcc gagatccagt ctacactgat
2880taattttcgg gccaataatt taaaaaaatc gtgttatata atattatatg tattatatat
2940atacatcatg atgatactga cagtcatgtc ccattgctaa atagacagac tccatctgcc
3000gcctccaact gatgttctca atatttaagg ggtcatctcg cattgtttaa taataaacag
3060actccatcta ccgcctccaa atgatgttct caaaatatat tgtatgaact tatttttatt
3120acttagtatt attagacaac ttacttgctt tatgaaaaac acttcctatt taggaaacaa
3180tttataatgg cagttcgttc atttaacaat ttatgtagaa taaatgttat aaatgcgtat
3240gggaaatctt aaatatggat agcataaatg atatctgcat tgcctaattc gaaatcaaca
3300gcaacgaaaa aaatcccttg tacaacataa atagtcatcg agaaatatca actatcaaag
3360aacagctatt cacacgttac tattgagatt attattggac gagaatcaca cactcaactg
3420tctttctctc ttctagaaat acaggtacaa gtatgtacta ttctcattgt tcatacttct
3480agtcatttca tcccacatat tccttggatt tctctccaat gaatgacatt ctatcttgca
3540aattcaacaa ttataataag atataccaaa gtagcggtat agtggcaatc aaaaagcttc
3600tctggtgtgc ttctcgtatt tatttttatt ctaatgatcc attaaaggta tatatttatt
3660tcttgttata taatcctttt gtttattaca tgggctggat acataaaggt attttgattt
3720aattttttgc ttaaattcaa tcccccctcg ttcagtgtca actgtaatgg taggaaatta
3780ccatactttt gaagaagcaa aaaaaatgaa agaaaaaaaa aatcgtattt ccaggttaga
3840cgttccgcag aatctagaat gcggtatgcg gtacattgtt cttcgaacgt aaaagttgcg
3900ctccctgaga tattgtacat ttttgctttt acaagtacaa gtacatcgta caactatgta
3960ctactgttga tgcatccaca acagtttgtt ttgttttttt ttgttttttt tttttctaat
4020gattcattac cgctatgtat acctacttgt acttgtagta agccgggtta ttggcgttca
4080attaatcata gacttatgaa tctgcacggt gtgcgctgcg agttactttt agcttatgca
4140tgctacttgg gtgtaatatt gggatctgtt cggaaatcaa cggatgctca atcgatttcg
4200acagtaatta attaagtcat acacaagtca gctttcttcg agcctcatat aagtataagt
4260agttcaacgt attagcactg tacccagcat ctccgtatcg agaaacacaa caacatgccc
4320cattggacag atcatgcgga tacacaggtt gtgcagtatc atacatactc gatcagacag
4380gtcgtctgac catcatacaa gctgaacaag cgctccatac ttgcacgctc tctatataca
4440cagttaaatt acatatccat agtctaacct ctaacagtta atcttctggt aagcctccca
4500gccagccttc tggtatcgct tggcctcctc aataggatct cggttctggc cgtacagacc
4560tcggccgaca attatgatat ccgttccggt agacatgaca tcctcaacag ttcggtactg
4620ctgtccgaga gcgtctccct tgtcgtcaag acccaccccg ggggtcagaa taagccagtc
4680ctcagagtcg cccttaggtc ggttctgggc aatgaagcca accacaaact cggggtcgga
4740tcgggcaagc tcaatggtct gcttggagta ctcgccagtg gccagagagc ccttgcaaga
4800cagctcggcc agcatgagca gacctctggc cagcttctcg ttgggagagg ggactaggaa
4860ctccttgtac tgggagttct cgtagtcaga gacgtcctcc ttcttctgtt cagagacagt
4920ttcctcggca ccagctcgca ggccagcaat gattccggtt ccgggtacac cgtgggcgtt
4980ggtgatatcg gaccactcgg cgattcggtg acaccggtac tggtgcttga cagtgttgcc
5040aatatctgcg aactttctgt cctcgaacag gaagaaaccg tgcttaagag caagttcctt
5100gagggggagc acagtgccgg cgtaggtgaa gtcgtcaatg atgtcgatat gggttttgat
5160catgcacaca taaggtccga ccttatcggc aagctcaatg agctccttgg tggtggtaac
5220atccagagaa gcacacaggt tggttttctt ggctgccacg agcttgagca ctcgagcggc
5280aaaggcggac ttgtggacgt tagctcgagc ttcgtaggag ggcattttgg tggtgaagag
5340gagactgaaa taaatttagt ctgcagaact ttttatcgga accttatctg gggcagtgaa
5400gtatatgtta tggtaatagt tacgagttag ttgaacttat agatagactg gactatacgg
5460ctatcggtcc aaattagaaa gaacgtcaat ggctctctgg gcgtcgcctt tgccgacaaa
5520aatgtgatca tgatgaaagc cagcaatgac gttgcagctg atattgttgt cggccaaccg
5580cgccgaaaac gcagctgtca gacccacagc ctccaacgaa gaatgtatcg tcaaagtgat
5640ccaagcacac tcatagttgg agtcgtactc caaaggcggc aatgacgagt cagacagata
5700ctcgtcgact caggcgacga cggaattcct gcagcccatc tgcagaattc aggagagacc
5760gggttggcgg cgtatttgtg tcccaaaaaa cagccccaat tgccccggag aagacggcca
5820ggccgcctag atgacaaatt caacaactca cagctgactt tctgccattg ccactagggg
5880ggggcctttt tatatggcca agccaagctc tccacgtcgg ttgggctgca cccaacaata
5940aatgggtagg gttgcaccaa caaagggatg ggatgggggg tagaagatac gaggataacg
6000gggctcaatg gcacaaataa gaacgaatac tgccattaag actcgtgatc cagcgactga
6060caccattgca tcatctaagg gcctcaaaac tacctcggaa ctgctgcgct gatctggaca
6120ccacagaggt tccgagcact ttaggttgca ccaaatgtcc caccaggtgc aggcagaaaa
6180cgctggaaca gcgtgtacag tttgtcttaa caaaaagtga gggcgctgag gtcgagcagg
6240gtggtgtgac ttgttatagc ctttagagct gcgaaagcgc gtatggattt ggctcatcag
6300gccagattga gggtctgtgg acacatgtca tgttagtgta cttcaatcgc cccctggata
6360tagccccgac aataggccgt ggcctcattt ttttgccttc cgcacatttc cattgctcgg
6420tacccacacc ttgcttctcc tgcacttgcc aaccttaata ctggtttaca ttgaccaaca
6480tcttacaagc ggggggcttg tctagggtat atataaacag tggctctccc aatcggttgc
6540cagtctcttt tttcctttct ttccccacag attcgaaatc taaactacac atcacacaat
6600gcctgttact gacgtcctta agcgaaagtc cggtgtcatc gtcggcgacg atgtccgagc
6660cgtgagtatc cacgacaaga tcagtgtcga gacgacgcgt tttgtgtaat gacacaatcc
6720gaaagtcgct agcaacacac actctctaca caaactaacc cagctctcca tggctgagga
6780taagaccaag gtcgagttcc ctaccctgac tgagctgaag cactctatcc ctaacgcttg
6840ctttgagtcc aacctcggac tctcgctcta ctacactgcc cgagcgatct tcaacgcatc
6900tgcctctgct gctctgctct acgctgcccg atctactccc ttcattgccg ataacgttct
6960gctccacgct ctggtttgcg ccacctacat ctacgtgcag ggtgtcatct tctggggttt
7020ctttaccgtc ggtcacgact gtggtcactc tgccttctcc cgataccact ccgtcaactt
7080catcattggc tgcatcatgc actctgccat tctgactccc ttcgagtcct ggcgagtgac
7140ccaccgacac catcacaaga acactggcaa cattgataag gacgagatct tctaccctca
7200tcggtccgtc aaggacctcc aggacgtgcg acaatgggtc tacaccctcg gaggtgcttg
7260gtttgtctac ctgaaggtcg gatatgctcc tcgaaccatg tcccactttg acccctggga
7320ccctctcctg cttcgacgag cctccgctgt catcgtgtcc ctcggagtct gggctgcctt
7380cttcgctgcc tacgcctacc tcacatactc gctcggcttt gccgtcatgg gcctctacta
7440ctatgctcct ctctttgtct ttgcttcgtt cctcgtcatt actaccttct tgcatcacaa
7500cgacgaagct actccctggt acggtgactc ggagtggacc tacgtcaagg gcaacctgag
7560ctccgtcgac cgatcgtacg gagctttcgt ggacaacctg tctcaccaca ttggcaccca
7620ccaggtccat cacttgttcc ctatcattcc ccactacaag ctcaacgaag ccaccaagca
7680ctttgctgcc gcttaccctc acctcgtgag acgtaacgac gagcccatca ttactgcctt
7740cttcaagacc gctcacctct ttgtcaacta cggagctgtg cccgagactg ctcagatttt
7800caccctcaaa gagtctgccg ctgcagccaa ggccaagagc gactaagcgg ccgcaagtgt
7860ggatggggaa gtgagtgccc ggttctgtgt gcacaattgg caatccaaga tggatggatt
7920caacacaggg atatagcgag ctacgtggtg gtgcgaggat atagcaacgg atatttatgt
7980ttgacacttg agaatgtacg atacaagcac tgtccaagta caatactaaa catactgtac
8040atactcatac tcgtacccgg gcaacggttt cacttgagtg cagtggctag tgctcttact
8100cgtacagtgt gcaatactgc gtatcatagt ctttgatgta tatcgtattc attcatgtta
8160gttgc
8165707769DNAArtificial Sequenceplasmid pZUFmEgD9ES 70ggccgcaagt
gtggatgggg aagtgagtgc ccggttctgt gtgcacaatt ggcaatccaa 60gatggatgga
ttcaacacag ggatatagcg agctacgtgg tggtgcgagg atatagcaac 120ggatatttat
gtttgacact tgagaatgta cgatacaagc actgtccaag tacaatacta 180aacatactgt
acatactcat actcgtaccc gggcaacggt ttcacttgag tgcagtggct 240agtgctctta
ctcgtacagt gtgcaatact gcgtatcata gtctttgatg tatatcgtat 300tcattcatgt
tagttgcgta cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 360gagtgagcta
actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 420tgtcgtgcca
gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 480ggcgctcttc
cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 540cggtatcagc
tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 600gaaagaacat
gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 660tggcgttttt
ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 720agaggtggcg
aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 780tcgtgcgctc
tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 840cgggaagcgt
ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 900ttcgctccaa
gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 960ccggtaacta
tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 1020ccactggtaa
caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 1080ggtggcctaa
ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 1140cagttacctt
cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 1200gcggtggttt
ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 1260atcctttgat
cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 1320ttttggtcat
gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 1380gttttaaatc
aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 1440tcagtgaggc
acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 1500ccgtcgtgta
gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 1560taccgcgaga
cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 1620gggccgagcg
cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 1680gccgggaagc
tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 1740ctacaggcat
cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 1800aacgatcaag
gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 1860gtcctccgat
cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 1920cactgcataa
ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 1980actcaaccaa
gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 2040caatacggga
taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 2100gttcttcggg
gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 2160ccactcgtgc
acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 2220caaaaacagg
aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 2280tactcatact
cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 2340gcggatacat
atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 2400cccgaaaagt
gccacctgac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg 2460ttacgcgcag
cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct 2520tcccttcctt
tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc 2580ctttagggtt
ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg 2640atggttcacg
tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt 2700ccacgttctt
taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg 2760tctattcttt
tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc 2820tgatttaaca
aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttccattc 2880gccattcagg
ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg 2940ccagctggcg
aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc 3000ccagtcacga
cgttgtaaaa cgacggccag tgaattgtaa tacgactcac tatagggcga 3060attgggtacc
gggccccccc tcgaggtcga tggtgtcgat aagcttgata tcgaattcat 3120gtcacacaaa
ccgatcttcg cctcaaggaa acctaattct acatccgaga gactgccgag 3180atccagtcta
cactgattaa ttttcgggcc aataatttaa aaaaatcgtg ttatataata 3240ttatatgtat
tatatatata catcatgatg atactgacag tcatgtccca ttgctaaata 3300gacagactcc
atctgccgcc tccaactgat gttctcaata tttaaggggt catctcgcat 3360tgtttaataa
taaacagact ccatctaccg cctccaaatg atgttctcaa aatatattgt 3420atgaacttat
ttttattact tagtattatt agacaactta cttgctttat gaaaaacact 3480tcctatttag
gaaacaattt ataatggcag ttcgttcatt taacaattta tgtagaataa 3540atgttataaa
tgcgtatggg aaatcttaaa tatggatagc ataaatgata tctgcattgc 3600ctaattcgaa
atcaacagca acgaaaaaaa tcccttgtac aacataaata gtcatcgaga 3660aatatcaact
atcaaagaac agctattcac acgttactat tgagattatt attggacgag 3720aatcacacac
tcaactgtct ttctctcttc tagaaataca ggtacaagta tgtactattc 3780tcattgttca
tacttctagt catttcatcc cacatattcc ttggatttct ctccaatgaa 3840tgacattcta
tcttgcaaat tcaacaatta taataagata taccaaagta gcggtatagt 3900ggcaatcaaa
aagcttctct ggtgtgcttc tcgtatttat ttttattcta atgatccatt 3960aaaggtatat
atttatttct tgttatataa tccttttgtt tattacatgg gctggataca 4020taaaggtatt
ttgatttaat tttttgctta aattcaatcc cccctcgttc agtgtcaact 4080gtaatggtag
gaaattacca tacttttgaa gaagcaaaaa aaatgaaaga aaaaaaaaat 4140cgtatttcca
ggttagacgt tccgcagaat ctagaatgcg gtatgcggta cattgttctt 4200cgaacgtaaa
agttgcgctc cctgagatat tgtacatttt tgcttttaca agtacaagta 4260catcgtacaa
ctatgtacta ctgttgatgc atccacaaca gtttgttttg tttttttttg 4320tttttttttt
ttctaatgat tcattaccgc tatgtatacc tacttgtact tgtagtaagc 4380cgggttattg
gcgttcaatt aatcatagac ttatgaatct gcacggtgtg cgctgcgagt 4440tacttttagc
ttatgcatgc tacttgggtg taatattggg atctgttcgg aaatcaacgg 4500atgctcaatc
gatttcgaca gtaattaatt aagtcataca caagtcagct ttcttcgagc 4560ctcatataag
tataagtagt tcaacgtatt agcactgtac ccagcatctc cgtatcgaga 4620aacacaacaa
catgccccat tggacagatc atgcggatac acaggttgtg cagtatcata 4680catactcgat
cagacaggtc gtctgaccat catacaagct gaacaagcgc tccatacttg 4740cacgctctct
atatacacag ttaaattaca tatccatagt ctaacctcta acagttaatc 4800ttctggtaag
cctcccagcc agccttctgg tatcgcttgg cctcctcaat aggatctcgg 4860ttctggccgt
acagacctcg gccgacaatt atgatatccg ttccggtaga catgacatcc 4920tcaacagttc
ggtactgctg tccgagagcg tctcccttgt cgtcaagacc caccccgggg 4980gtcagaataa
gccagtcctc agagtcgccc ttaggtcggt tctgggcaat gaagccaacc 5040acaaactcgg
ggtcggatcg ggcaagctca atggtctgct tggagtactc gccagtggcc 5100agagagccct
tgcaagacag ctcggccagc atgagcagac ctctggccag cttctcgttg 5160ggagagggga
ctaggaactc cttgtactgg gagttctcgt agtcagagac gtcctccttc 5220ttctgttcag
agacagtttc ctcggcacca gctcgcaggc cagcaatgat tccggttccg 5280ggtacaccgt
gggcgttggt gatatcggac cactcggcga ttcggtgaca ccggtactgg 5340tgcttgacag
tgttgccaat atctgcgaac tttctgtcct cgaacaggaa gaaaccgtgc 5400ttaagagcaa
gttccttgag ggggagcaca gtgccggcgt aggtgaagtc gtcaatgatg 5460tcgatatggg
ttttgatcat gcacacataa ggtccgacct tatcggcaag ctcaatgagc 5520tccttggtgg
tggtaacatc cagagaagca cacaggttgg ttttcttggc tgccacgagc 5580ttgagcactc
gagcggcaaa ggcggacttg tggacgttag ctcgagcttc gtaggagggc 5640attttggtgg
tgaagaggag actgaaataa atttagtctg cagaactttt tatcggaacc 5700ttatctgggg
cagtgaagta tatgttatgg taatagttac gagttagttg aacttataga 5760tagactggac
tatacggcta tcggtccaaa ttagaaagaa cgtcaatggc tctctgggcg 5820tcgcctttgc
cgacaaaaat gtgatcatga tgaaagccag caatgacgtt gcagctgata 5880ttgttgtcgg
ccaaccgcgc cgaaaacgca gctgtcagac ccacagcctc caacgaagaa 5940tgtatcgtca
aagtgatcca agcacactca tagttggagt cgtactccaa aggcggcaat 6000gacgagtcag
acagatactc gtcgacgttt aaacagtgta cgcagatcta ctatagagga 6060acatttaaat
tgccccggag aagacggcca ggccgcctag atgacaaatt caacaactca 6120cagctgactt
tctgccattg ccactagggg ggggcctttt tatatggcca agccaagctc 6180tccacgtcgg
ttgggctgca cccaacaata aatgggtagg gttgcaccaa caaagggatg 6240ggatgggggg
tagaagatac gaggataacg gggctcaatg gcacaaataa gaacgaatac 6300tgccattaag
actcgtgatc cagcgactga caccattgca tcatctaagg gcctcaaaac 6360tacctcggaa
ctgctgcgct gatctggaca ccacagaggt tccgagcact ttaggttgca 6420ccaaatgtcc
caccaggtgc aggcagaaaa cgctggaaca gcgtgtacag tttgtcttaa 6480caaaaagtga
gggcgctgag gtcgagcagg gtggtgtgac ttgttatagc ctttagagct 6540gcgaaagcgc
gtatggattt ggctcatcag gccagattga gggtctgtgg acacatgtca 6600tgttagtgta
cttcaatcgc cccctggata tagccccgac aataggccgt ggcctcattt 6660ttttgccttc
cgcacatttc cattgctcga tacccacacc ttgcttctcc tgcacttgcc 6720aaccttaata
ctggtttaca ttgaccaaca tcttacaagc ggggggcttg tctagggtat 6780atataaacag
tggctctccc aatcggttgc cagtctcttt tttcctttct ttccccacag 6840attcgaaatc
taaactacac atcacagaat tccgagccgt gagtatccac gacaagatca 6900gtgtcgagac
gacgcgtttt gtgtaatgac acaatccgaa agtcgctagc aacacacact 6960ctctacacaa
actaacccag ctctggtacc atggaggtcg tgaacgaaat cgtctccatt 7020ggccaggagg
ttcttcccaa ggtcgactat gctcagctct ggtctgatgc ctcgcactgc 7080gaggtgctgt
acctctccat cgccttcgtc atcctgaagt tcacccttgg tcctctcgga 7140cccaagggtc
agtctcgaat gaagtttgtg ttcaccaact acaacctgct catgtccatc 7200tactcgctgg
gctccttcct ctctatggcc tacgccatgt acaccattgg tgtcatgtcc 7260gacaactgcg
agaaggcttt cgacaacaat gtcttccgaa tcaccactca gctgttctac 7320ctcagcaagt
tcctcgagta cattgactcc ttctatctgc ccctcatggg caagcctctg 7380acctggttgc
agttctttca ccatctcgga gctcctatgg acatgtggct gttctacaac 7440taccgaaacg
aagccgtttg gatctttgtg ctgctcaacg gcttcattca ctggatcatg 7500tacggctact
attggacccg actgatcaag ctcaagttcc ctatgcccaa gtccctgatt 7560acttctatgc
agatcattca gttcaacgtt ggcttctaca tcgtctggaa gtaccggaac 7620attccctgct
accgacaaga tggaatgaga atgtttggct ggtttttcaa ctacttctac 7680gttggtactg
tcctgtgtct gttcctcaac ttctacgtgc agacctacat cgtccgaaag 7740cacaagggag
ccaaaaagat tcagtgagc
77697110206DNAArtificial Sequenceplasmid pZUFmE9SP8S 71taagtcatac
acaagtcagc tttcttcgag cctcatataa gtataagtag ttcaacgtat 60tagcactgta
cccagcatct ccgtatcgag aaacacaaca acatgcccca ttggacagat 120catgcggata
cacaggttgt gcagtatcat acatactcga tcagacaggt cgtctgacca 180tcatacaagc
tgaacaagcg ctccatactt gcacgctctc tatatacaca gttaaattac 240atatccatag
tctaacctct aacagttaat cttctggtaa gcctcccagc cagccttctg 300gtatcgcttg
gcctcctcaa taggatctcg gttctggccg tacagacctc ggccgacaat 360tatgatatcc
gttccggtag acatgacatc ctcaacagtt cggtactgct gtccgagagc 420gtctcccttg
tcgtcaagac ccaccccggg ggtcagaata agccagtcct cagagtcgcc 480cttaggtcgg
ttctgggcaa tgaagccaac cacaaactcg gggtcggatc gggcaagctc 540aatggtctgc
ttggagtact cgccagtggc cagagagccc ttgcaagaca gctcggccag 600catgagcaga
cctctggcca gcttctcgtt gggagagggg actaggaact ccttgtactg 660ggagttctcg
tagtcagaga cgtcctcctt cttctgttca gagacagttt cctcggcacc 720agctcgcagg
ccagcaatga ttccggttcc gggtacaccg tgggcgttgg tgatatcgga 780ccactcggcg
attcggtgac accggtactg gtgcttgaca gtgttgccaa tatctgcgaa 840ctttctgtcc
tcgaacagga agaaaccgtg cttaagagca agttccttga gggggagcac 900agtgccggcg
taggtgaagt cgtcaatgat gtcgatatgg gttttgatca tgcacacata 960aggtccgacc
ttatcggcaa gctcaatgag ctccttggtg gtggtaacat ccagagaagc 1020acacaggttg
gttttcttgg ctgccacgag cttgagcact cgagcggcaa aggcggactt 1080gtggacgtta
gctcgagctt cgtaggaggg cattttggtg gtgaagagga gactgaaata 1140aatttagtct
gcagaacttt ttatcggaac cttatctggg gcagtgaagt atatgttatg 1200gtaatagtta
cgagttagtt gaacttatag atagactgga ctatacggct atcggtccaa 1260attagaaaga
acgtcaatgg ctctctgggc gtcgcctttg ccgacaaaaa tgtgatcatg 1320atgaaagcca
gcaatgacgt tgcagctgat attgttgtcg gccaaccgcg ccgaaaacgc 1380agctgtcaga
cccacagcct ccaacgaaga atgtatcgtc aaagtgatcc aagcacactc 1440atagttggag
tcgtactcca aaggcggcaa tgacgagtca gacagatact cgtcgacgtt 1500taaacagtgt
acgcagatct actatagagg aacatttaaa ttgccccgga gaagacggcc 1560aggccgccta
gatgacaaat tcaacaactc acagctgact ttctgccatt gccactaggg 1620gggggccttt
ttatatggcc aagccaagct ctccacgtcg gttgggctgc acccaacaat 1680aaatgggtag
ggttgcacca acaaagggat gggatggggg gtagaagata cgaggataac 1740ggggctcaat
ggcacaaata agaacgaata ctgccattaa gactcgtgat ccagcgactg 1800acaccattgc
atcatctaag ggcctcaaaa ctacctcgga actgctgcgc tgatctggac 1860accacagagg
ttccgagcac tttaggttgc accaaatgtc ccaccaggtg caggcagaaa 1920acgctggaac
agcgtgtaca gtttgtctta acaaaaagtg agggcgctga ggtcgagcag 1980ggtggtgtga
cttgttatag cctttagagc tgcgaaagcg cgtatggatt tggctcatca 2040ggccagattg
agggtctgtg gacacatgtc atgttagtgt acttcaatcg ccccctggat 2100atagccccga
caataggccg tggcctcatt tttttgcctt ccgcacattt ccattgctcg 2160atacccacac
cttgcttctc ctgcacttgc caaccttaat actggtttac attgaccaac 2220atcttacaag
cggggggctt gtctagggta tatataaaca gtggctctcc caatcggttg 2280ccagtctctt
ttttcctttc tttccccaca gattcgaaat ctaaactaca catcacagaa 2340ttccgagccg
tgagtatcca cgacaagatc agtgtcgaga cgacgcgttt tgtgtaatga 2400cacaatccga
aagtcgctag caacacacac tctctacaca aactaaccca gctctggtac 2460catggaggtc
gtgaacgaaa tcgtctccat tggccaggag gttcttccca aggtcgacta 2520tgctcagctc
tggtctgatg cctcgcactg cgaggtgctg tacctctcca tcgccttcgt 2580catcctgaag
ttcacccttg gtcctctcgg acccaagggt cagtctcgaa tgaagtttgt 2640gttcaccaac
tacaacctgc tcatgtccat ctactcgctg ggctccttcc tctctatggc 2700ctacgccatg
tacaccattg gtgtcatgtc cgacaactgc gagaaggctt tcgacaacaa 2760tgtcttccga
atcaccactc agctgttcta cctcagcaag ttcctcgagt acattgactc 2820cttctatctg
cccctcatgg gcaagcctct gacctggttg cagttctttc accatctcgg 2880agctcctatg
gacatgtggc tgttctacaa ctaccgaaac gaagccgttt ggatctttgt 2940gctgctcaac
ggcttcattc actggatcat gtacggctac tattggaccc gactgatcaa 3000gctcaagttc
cctatgccca agtccctgat tacttctatg cagatcattc agttcaacgt 3060tggcttctac
atcgtctgga agtaccggaa cattccctgc taccgacaag atggaatgag 3120aatgtttggc
tggtttttca actacttcta cgttggtact gtcctgtgtc tgttcctcaa 3180cttctacgtg
cagacctaca tcgtccgaaa gcacaaggga gccaaaaaga ttcagtgagc 3240ggccgcaagt
gtggatgggg aagtgagtgc ccggttctgt gtgcacaatt ggcaatccaa 3300gatggatgga
ttcaacacag ggatatagcg agctacgtgg tggtgcgagg atatagcaac 3360ggatatttat
gtttgacact tgagaatgta cgatacaagc actgtccaag tacaatacta 3420aacatactgt
acatactcat actcgtaccc gggcaacggt ttcacttgag tgcagtggct 3480agtgctctta
ctcgtacagt gtgcaatact gcgtatcata gtctttgatg tatatcgtat 3540tcattcatgt
tagttgcgta cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 3600gagtgagcta
actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 3660tgtcgtgcca
gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 3720ggcgctcttc
cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 3780cggtatcagc
tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 3840gaaagaacat
gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 3900tggcgttttt
ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 3960agaggtggcg
aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 4020tcgtgcgctc
tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 4080cgggaagcgt
ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 4140ttcgctccaa
gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 4200ccggtaacta
tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 4260ccactggtaa
caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 4320ggtggcctaa
ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 4380cagttacctt
cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 4440gcggtggttt
ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 4500atcctttgat
cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 4560ttttggtcat
gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 4620gttttaaatc
aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 4680tcagtgaggc
acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 4740ccgtcgtgta
gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 4800taccgcgaga
cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 4860gggccgagcg
cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 4920gccgggaagc
tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 4980ctacaggcat
cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 5040aacgatcaag
gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 5100gtcctccgat
cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 5160cactgcataa
ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 5220actcaaccaa
gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 5280caatacggga
taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 5340gttcttcggg
gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 5400ccactcgtgc
acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 5460caaaaacagg
aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 5520tactcatact
cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 5580gcggatacat
atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 5640cccgaaaagt
gccacctgac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg 5700ttacgcgcag
cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct 5760tcccttcctt
tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc 5820ctttagggtt
ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg 5880atggttcacg
tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt 5940ccacgttctt
taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg 6000tctattcttt
tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc 6060tgatttaaca
aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttccattc 6120gccattcagg
ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg 6180ccagctggcg
aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc 6240ccagtcacga
cgttgtaaaa cgacggccag tgaattgtaa tacgactcac tatagggcga 6300attgggtacc
gggccccccc tcgaggtcga tggtgtcgat aagcttgata tcgaattcat 6360gtcacacaaa
ccgatcttcg cctcaaggaa acctaattct acatccgaga gactgccgag 6420atccagtcta
cactgattaa ttttcgggcc aataatttaa aaaaatcgtg ttatataata 6480ttatatgtat
tatatatata catcatgatg atactgacag tcatgtccca ttgctaaata 6540gacagactcc
atctgccgcc tccaactgat gttctcaata tttaaggggt catctcgcat 6600tgtttaataa
taaacagact ccatctaccg cctccaaatg atgttctcaa aatatattgt 6660atgaacttat
ttttattact tagtattatt agacaactta cttgctttat gaaaaacact 6720tcctatttag
gaaacaattt ataatggcag ttcgttcatt taacaattta tgtagaataa 6780atgttataaa
tgcgtatggg aaatcttaaa tatggatagc ataaatgata tctgcattgc 6840ctaattcgaa
atcaacagca acgaaaaaaa tcccttgtac aacataaata gtcatcgaga 6900aatatcaact
atcaaagaac agctattcac acgttactat tgagattatt attggacgag 6960aatcacacac
tcaactgtct ttctctcttc tagaaataca ggtacaagta tgtactattc 7020tcattgttca
tacttctagt catttcatcc cacatattcc ttggatttct ctccaatgaa 7080tgacattcta
tcttgcaaat tcaacaatta taataagata taccaaagta gcggtatagt 7140ggcaatcaaa
aagcttctct ggtgtgcttc tcgtatttat ttttattcta atgatccatt 7200aaaggtatat
atttatttct tgttatataa tccttttgtt tattacatgg gctggataca 7260taaaggtatt
ttgatttaat tttttgctta aattcaatcc cccctcgttc agtgtcaact 7320gtaatggtag
gaaattacca tacttttgaa gaagcaaaaa aaatgaaaga aaaaaaaaat 7380cgtatttcca
ggttagacgt tccgcagaat ctagaatgcg gtatgcggta cattgttctt 7440cgaacgtaaa
agttgcgctc cctgagatat tgtacatttt tgcttttaca agtacaagta 7500catcgtacaa
ctatgtacta ctgttgatgc atccacaaca gtttgttttg tttttttttg 7560tttttttttt
ttctaatgat tcattaccgc tatgtatacc tacttgtact tgtagtaagc 7620cgggttattg
gcgttcaatt aatcatagac ttatgaatct gcacggtgtg cgctgcgagt 7680tacttttagc
ttatgcatgc tacttgggtg taatattggg atctgttcgg aaatcaacgg 7740atgctcaatc
gatggttaat gctgctgtgt gctgtgtgtg tgtgttgttt ggcgctcatt 7800gttgcgttat
gcagcgtaca ccacaatatt ggaagcttat tagcctttct attttttcgt 7860ttgcaaggct
taacaacatt gctgtggaga gggatgggga tatggaggcc gctggaggga 7920gtcggagagg
cgttttggag cggcttggcc tggcgcccag ctcgcgaaac gcacctagga 7980ccctttggca
cgccgaaatg tgccactttt cagtctagta acgccttacc tacgtcattc 8040catgcgtgca
tgtttgcgcc ttttttccct tgcccttgat cgccacacag tacagtgcac 8100tgtacagtgg
aggttttggg ggggtcttag atgggagcta aaagcggcct agcggtacac 8160tagtgggatt
gtatggagtg gcatggagcc taggtggagc ctgacaggac gcacgaccgg 8220ctagcccgtg
acagacgatg ggtggctcct gttgtccacc gcgtacaaat gtttgggcca 8280aagtcttgtc
agccttgctt gcgaacctaa ttcccaattt tgtcacttcg cacccccatt 8340gatcgagccc
taacccctgc ccatcaggca atccaattaa gctcgcattg tctgccttgt 8400ttagtttggc
tcctgcccgt ttcggcgtcc acttgcacaa acacaaacaa gcattatata 8460taaggctcgt
ctctccctcc caaccacact cacttttttg cccgtcttcc cttgctaaca 8520caaaagtcaa
gaacacaaac aaccacccca acccccttac acacaagaca tatctacagc 8580aatggccatg
ggcaagggtg gagacggtgg agcacaggct gtgtctggca ccgatgcctc 8640cctcgctgag
gtctcctctg tggacagcaa gtccgtccac gtggttctgt acggcaagcg 8700agtggatgtc
accaagttcc agaaggctca ccctggaggt tcgaaggtgt tccgaatctt 8760tcaggagcga
gacgccacag aacagttcga gtcctaccac tctcccaagg ccatcaagat 8820gatggaaggt
atgctcaaaa agtcggagga tgctcccgct tccgtgcctc ttccctctcg 8880atccactatg
ggcaccgagt tcaaggagat gatcgaacga cacaagagag ccggtctcta 8940cgacccttgt
cccttggacg agctgttcaa gctcaccatt gtccttgctc ctatctttgt 9000gggagcctat
ctcgttcgat ccggtgtctc tcctcttgct ggagccctgt cgatgggctt 9060cggattctac
ctcgacggct ggcttgctca cgactacctg catcacgcag tgttcaaggg 9120ctccgtcaac
acactcgtca aggccaacaa cgctatggga tacgccttgg gcttcctcca 9180gggttacgac
gttgcttggt ggcgagccag acacaacact catcacgtgt gcaccaacga 9240ggacggctcc
gatcccgaca tcaagacggc tcctctgctc atttacgtgc gagagaatcc 9300ctccattgcc
aagcggctca acttctttca gcgatggcaa cagtactact atgttcctac 9360tatggccatt
ctggatctct actggcgact ggagtctatc gcatacgtcg ctgtgcgact 9420gcccaagatg
tggatgcagg ctgccgctct tgccgctcac tacgcactcc tgtgttgggt 9480cttcgctgcc
catctcaacc tgattcctct catgatggtt gcacgaggtt tcgcgaccgg 9540aatcgtggtc
tttgccaccc actatggcga ggacattctc gaccgagagc acgtcgaagg 9600catgactctg
gtcgagcaga ccgccaagac ctcccgaaac atcactggtg gatggcttgt 9660caacgtgctc
accggcttca tttctctgca gaccgagcat catctgtttc ccatgatgcc 9720tactggaaac
ctcatgacca ttcaacccga ggttcgagac tttttcaaaa agcacggtct 9780cgagtaccga
gaaggaaacc tgtttcagtg cgtgcatcag aacatcaagg ctctcgcctt 9840cgagcacctg
cttcactaag cggccgcatt gatgattgga aacacacaca tgggttatat 9900ctaggtgaga
gttagttgga cagttatata ttaaatcagc tatgccaacg gtaacttcat 9960tcatgtcaac
gaggaaccag tgactgcaag taatatagaa tttgaccacc ttgccattct 10020cttgcactcc
tttactatat ctcatttatt tcttatatac aaatcacttc ttcttcccag 10080catcgagctc
ggaaacctca tgagcaataa catcgtggat ctcgtcaata gagggctttt 10140tggactcctt
gctgttggcc accttgtcct tgctgtctgg ctcattctgt ttcaacgcct 10200tttaat
10206726871DNAArtificial Sequenceplasmid pEXPGUS1-C 72aattcgtcct
tgaggactcg agtgacagtc tttcgccaaa gtcgagagga ggccagcacg 60ttggccttgt
caagagacca cacgggaaga ggggggttgt gctgaagggc caggaaggcg 120gccattcggg
caattcgctc aacctcagga acggagtagg tctcggtgtc ggaagcgacg 180ccagatccgt
catcctcctt tcgctctcca aagtagatac ctccgacgag ctctcggaca 240atgatgaagt
cggtgccctc aacgtttcgg atgggggaga gatcggcgag cttgggcgac 300agcagctggc
agggtcgcag gttggcgtac aggttcaggt cctttcgcag cttgaggaga 360ccctgctcgg
gtcgcacgtc ggttcgtccg tcgggagtgg tccatacggt gttggcagcg 420cctccgacag
caccgagcat aatagagtca gcctttcggc agatgtcgag agtagcgtcg 480gtgatgggct
cgccctcctt ctcaatggca gctcctccaa tgagtcggtc ctcaaacaca 540aactcggtgc
cggaggcctc agcaacagac ttgagcacct tgacggcctc ggcaatcacc 600tcggggccac
agaagtcgcc gccgagaaga acaatcttct tggagtcagt cttggtcttc 660ttagtttcgg
gttccattgt ggatgtgtgt ggttgtatgt gtgatgtggt gtgtggagtg 720aaaatctgtg
gctggcaaac gctcttgtat atatacgcac ttttgcccgt gctatgtgga 780agactaaacc
tccgaagatt gtgactcagg tagtgcggta tcggctaggg acccaaacct 840tgtcgatgcc
gatagcgcta tcgaacgtac cccagccggc cgggagtatg tcggagggga 900catacgagat
cgtcaagggt ttgtggccaa ctggtaaata aatgatgtcg agggagtttg 960gcgcccgttt
tttcgagccc cacacgtttc ggtgagtatg agcggcggca gattcgagcg 1020tttccggttt
ccgcggctgg acgagagccc atgatggggg ctcccaccac cagcaatcag 1080ggccctgatt
acacacccac ctgtaatgtc atgctgttca tcgatggtta atgctgctgt 1140gtgctgtgtg
tgtgtgttgt ttggcgctca ttgttgcgtt atgcagcgta caccacaata 1200ttggaagctt
attagccttt ctattttttc gtttgcaagg cttaacaaca ttgctgtgga 1260gagggatggg
gatatggagg ccgctggagg gagtcggaga ggcgttttgg agcggcttgg 1320cctggcgccc
agctcgcgaa acgcacctag gaccctttgg cacgccgaaa tgtgccactt 1380ttcagtctag
taacgcctta cctacgtcat tccatgcgtg catgtttgcg ccttttttcc 1440cttgcccttg
atcgccacac agtacagtgc actgtacagt ggaggttttg ggggggtctt 1500agatgggagc
taaaagcggc ctagcggtac actagtggga ttgtatggag tggcatggag 1560cctaggtgga
gcctgacagg acgcacgacc ggctagcccg tgacagacga tgggtggctc 1620ctgttgtcca
ccgcgtacaa atgtttgggc caaagtcttg tcagccttgc ttgcgaacct 1680aattcccaat
tttgtcactt cgcaccccca ttgatcgagc cctaacccct gcccatcagg 1740caatccaatt
aagctcgcat tgtctgcctt gtttagtttg gctcctgccc gtttcggcgt 1800ccacttgcac
aaacacaaac aagcattata tataaggctc gtctctccct cccaaccaca 1860ctcacttttt
tgcccgtctt cccttgctaa cacaaaagtc aagaacacaa acaaccaccc 1920caaccccctt
acacacaaga catatctaca gcaatggcca tggtacgtcc tgtagaaacc 1980ccaacccgtg
aaatcaaaaa actcgacggc ctgtgggcat tcagtctgga tcgcgaaaac 2040tgtggaattg
atcagcgttg gtgggaaagc gcgttacaag aaagccgggc aattgctgtg 2100ccaggcagtt
ttaacgatca gttcgccgat gcagatattc gtaattatgc gggcaacgtc 2160tggtatcagc
gcgaagtctt tataccgaaa ggttgggcag gccagcgtat cgtgctgcgt 2220ttcgatgcgg
tcactcatta cggcaaagtg tgggtcaata atcaggaagt gatggagcat 2280cagggcggct
atacgccatt tgaagccgat gtcacgccgt atgttattgc cgggaaaagt 2340gtacgtatca
ccgtttgtgt gaacaacgaa ctgaactggc agactatccc gccgggaatg 2400gtgattaccg
acgaaaacgg caagaaaaag cagtcttact tccatgattt ctttaactat 2460gccgggatcc
atcgcagcgt aatgctctac accacgccga acacctgggt ggacgatatc 2520accgtggtga
cgcatgtcgc gcaagactgt aaccacgcgt ctgttgactg gcaggtggtg 2580gccaatggtg
atgtcagcgt tgaactgcgt gatgcggatc aacaggtggt tgcaactgga 2640caaggcacta
gcgggacttt gcaagtggtg aatccgcacc tctggcaacc gggtgaaggt 2700tatctctatg
aactgtgcgt cacagccaaa agccagacag agtgtgatat ctacccgctt 2760cgcgtcggca
tccggtcagt ggcagtgaag ggcgaacagt tcctgattaa ccacaaaccg 2820ttctacttta
ctggctttgg tcgtcatgaa gatgcggact tacgtggcaa aggattcgat 2880aacgtgctga
tggtgcacga ccacgcatta atggactgga ttggggccaa ctcctaccgt 2940acctcgcatt
acccttacgc tgaagagatg ctcgactggg cagatgaaca tggcatcgtg 3000gtgattgatg
aaactgctgc tgtcggcttt aacctctctt taggcattgg tttcgaagcg 3060ggcaacaagc
cgaaagaact gtacagcgaa gaggcagtca acggggaaac tcagcaagcg 3120cacttacagg
cgattaaaga gctgatagcg cgtgacaaaa accacccaag cgtggtgatg 3180tggagtattg
ccaacgaacc ggatacccgt ccgcaagtgc acgggaatat ttcgccactg 3240gcggaagcaa
cgcgtaaact cgacccgacg cgtccgatca cctgcgtcaa tgtaatgttc 3300tgcgacgctc
acaccgatac catcagcgat ctctttgatg tgctgtgcct gaaccgttat 3360tacggatggt
atgtccaaag cggcgatttg gaaacggcag agaaggtact ggaaaaagaa 3420cttctggcct
ggcaggagaa actgcatcag ccgattatca tcaccgaata cggcgtggat 3480acgttagccg
ggctgcactc aatgtacacc gacatgtgga gtgaagagta tcagtgtgca 3540tggctggata
tgtatcaccg cgtctttgat cgcgtcagcg ccgtcgtcgg tgaacaggta 3600tggaatttcg
ccgattttgc gacctcgcaa ggcatattgc gcgttggcgg taacaagaaa 3660gggatcttca
ctcgcgaccg caaaccgaag tcggcggctt ttctgctgca aaaacgctgg 3720actggcatga
acttcggtga aaaaccgcag cagggaggca aacaatgatt aattaactag 3780agcggccgcc
accgcggccc gagattccgg cctcttcggc cgccaagcga cccgggtgga 3840cgtctagagg
tacctagcaa ttaacagata gtttgccggt gataattctc ttaacctccc 3900acactccttt
gacataacga tttatgtaac gaaactgaaa tttgaccaga tattgtgtcc 3960gcggtggagc
tccagctttt gttcccttta gtgagggtta atttcgagct tggcgtaatc 4020atggtcatag
ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg 4080agccggaagc
ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat 4140tgcgttgcgc
tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg 4200aatcggccaa
cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct 4260cactgactcg
ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 4320ggtaatacgg
ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 4380ccagcaaaag
gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 4440cccccctgac
gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 4500actataaaga
taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 4560cctgccgctt
accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 4620tagctcacgc
tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 4680gcacgaaccc
cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 4740caacccggta
agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 4800agcgaggtat
gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 4860tagaaggaca
gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 4920tggtagctct
tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 4980gcagcagatt
acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 5040gtctgacgct
cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 5100aaggatcttc
acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat 5160atatgagtaa
acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc 5220gatctgtcta
tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat 5280acgggagggc
ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc 5340ggctccagat
ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc 5400tgcaacttta
tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag 5460ttcgccagtt
aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg 5520ctcgtcgttt
ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg 5580atcccccatg
ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag 5640taagttggcc
gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt 5700catgccatcc
gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga 5760atagtgtatg
cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc 5820acatagcaga
actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc 5880aaggatctta
ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc 5940ttcagcatct
tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc 6000cgcaaaaaag
ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca 6060atattattga
agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat 6120ttagaaaaat
aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgc 6180gccctgtagc
ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac 6240acttgccagc
gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt 6300cgccggcttt
ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc 6360tttacggcac
ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc 6420gccctgatag
acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact 6480cttgttccaa
actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg 6540gattttgccg
atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc 6600gaattttaac
aaaatattaa cgcttacaat ttccattcgc cattcaggct gcgcaactgt 6660tgggaagggc
gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt 6720gctgcaaggc
gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg 6780acggccagtg
aattgtaata cgactcacta tagggcgaat tgggtaccgg gccccccctc 6840gaggtcgatg
gtgtcgataa gcttgatatc g
6871735460DNAArtificial Sequenceplasmid pZGD5T-CP 73ggccgcattg atgattggaa
acacacacat gggttatatc taggtgagag ttagttggac 60agttatatat taaatcagct
atgccaacgg taacttcatt catgtcaacg aggaaccagt 120gactgcaagt aatatagaat
ttgaccacct tgccattctc ttgcactcct ttactatatc 180tcatttattt cttatataca
aatcacttct tcttcccagc atcgagctcg gaaacctcat 240gagcaataac atcgtggatc
tcgtcaatag agggcttttt ggactccttg ctgttggcca 300ccttgtcctt gctgtctggc
tcattctgtt tcaacgcctt ttaattaatc gagcttggcg 360taatcatggt catagctgtt
tcctgtgtga aattgttatc cgctcacaat tccacacaac 420atacgagccg gaagcataaa
gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 480ttaattgcgt tgcgctcact
gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 540taatgaatcg gccaacgcgc
ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 600tcgctcactg actcgctgcg
ctcggtcgtt cggctgcggc gagcggtatc agctcactca 660aaggcggtaa tacggttatc
cacagaatca ggggataacg caggaaagaa catgtgagca 720aaaggccagc aaaaggccag
gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 780ctccgccccc ctgacgagca
tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 840acaggactat aaagatacca
ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 900ccgaccctgc cgcttaccgg
atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 960tctcatagct cacgctgtag
gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 1020tgtgtgcacg aaccccccgt
tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 1080gagtccaacc cggtaagaca
cgacttatcg ccactggcag cagccactgg taacaggatt 1140agcagagcga ggtatgtagg
cggtgctaca gagttcttga agtggtggcc taactacggc 1200tacactagaa ggacagtatt
tggtatctgc gctctgctga agccagttac cttcggaaaa 1260agagttggta gctcttgatc
cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 1320tgcaagcagc agattacgcg
cagaaaaaaa ggatctcaag aagatccttt gatcttttct 1380acggggtctg acgctcagtg
gaacgaaaac tcacgttaag ggattttggt catgagatta 1440tcaaaaagga tcttcaccta
gatcctttta aattaaaaat gaagttttaa atcaatctaa 1500agtatatatg agtaaacttg
gtctgacagt taccaatgct taatcagtga ggcacctatc 1560tcagcgatct gtctatttcg
ttcatccata gttgcctgac tccccgtcgt gtagataact 1620acgatacggg agggcttacc
atctggcccc agtgctgcaa tgataccgcg agacccacgc 1680tcaccggctc cagatttatc
agcaataaac cagccagccg gaagggccga gcgcagaagt 1740ggtcctgcaa ctttatccgc
ctccatccag tctattaatt gttgccggga agctagagta 1800agtagttcgc cagttaatag
tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 1860tcacgctcgt cgtttggtat
ggcttcattc agctccggtt cccaacgatc aaggcgagtt 1920acatgatccc ccatgttgtg
caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 1980agaagtaagt tggccgcagt
gttatcactc atggttatgg cagcactgca taattctctt 2040actgtcatgc catccgtaag
atgcttttct gtgactggtg agtactcaac caagtcattc 2100tgagaatagt gtatgcggcg
accgagttgc tcttgcccgg cgtcaatacg ggataatacc 2160gcgccacata gcagaacttt
aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 2220ctctcaagga tcttaccgct
gttgagatcc agttcgatgt aacccactcg tgcacccaac 2280tgatcttcag catcttttac
tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 2340aatgccgcaa aaaagggaat
aagggcgaca cggaaatgtt gaatactcat actcttcctt 2400tttcaatatt attgaagcat
ttatcagggt tattgtctca tgagcggata catatttgaa 2460tgtatttaga aaaataaaca
aataggggtt ccgcgcacat ttccccgaaa agtgccacct 2520gacgcgccct gtagcggcgc
attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 2580gctacacttg ccagcgccct
agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 2640acgttcgccg gctttccccg
tcaagctcta aatcgggggc tccctttagg gttccgattt 2700agtgctttac ggcacctcga
ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 2760ccatcgccct gatagacggt
ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 2820ggactcttgt tccaaactgg
aacaacactc aaccctatct cggtctattc ttttgattta 2880taagggattt tgccgatttc
ggcctattgg ttaaaaaatg agctgattta acaaaaattt 2940aacgcgaatt ttaacaaaat
attaacgctt acaatttcca ttcgccattc aggctgcgca 3000actgttggga agggcgatcg
gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 3060gatgtgctgc aaggcgatta
agttgggtaa cgccagggtt ttcccagtca cgacgttgta 3120aaacgacggc cagtgaattg
taatacgact cactataggg cgaattgggt accgggcccc 3180ccctcgaggt cgacggtatc
gatggaagcc ggtagaaccg ggctgcttgt gcttggagat 3240ggaagccggt agaaccgggc
tgcttggggg gatttggggc cgctgggctc caaagagggg 3300taggcatttc gttggggtta
cgtaattgcg gcatttgggt cctgcgcgca tgtcccattg 3360gtcagaatta gtccggatag
gagacttatc agccaatcac agcgccggat ccacctgtag 3420gttgggttgg gtgggagcac
ccctccacag agtagagtca aacagcagca gcaacatgat 3480agttgggggt gtgcgtgtta
aaggaaaaaa aagaagcttg ggttatattc ccgctctatt 3540tagaggttgc gggatagacg
ccgacggagg gcaatggcgc tatggaacct tgcggatatc 3600catacgccgc ggcggactgc
gtccgaacca gctccagcag cgttttttcc gggccattga 3660gccgactgcg accccgccaa
cgtgtcttgg cccacgcact catgtcatgt tggtgttggg 3720aggccacttt ttaagtagca
caaggcacct agctcgcagc aaggtgtccg aaccaaagaa 3780gcggctgcag tggtgcaaac
ggggcggaaa cggcgggaaa aagccacggg ggcacgaatt 3840gaggcacgcc ctcgaatttg
agacgagtca cggccccatt cgcccgcgca atggctcgcc 3900aacgcccggt cttttgcacc
acatcaggtt accccaagcc aaacctttgt gttaaaaagc 3960ttaacatatt ataccgaacg
taggtttggg cgggcttgct ccgtctgtcc aaggcaacat 4020ttatataagg gtctgcatcg
ccggctcaat tgaatctttt ttcttcttct cttctctata 4080ttcattcttg aattaaacac
acatcaacca tgggaacgga ccaaggaaaa accttcacct 4140gggaagagct ggcggcccat
aacaccaagg acgacctact cttggccatc cgcggcaggg 4200tgtacgatgt cacaaagttc
ttgagccgcc atcctggtgg agtggacact ctcctgctcg 4260gagctggccg agatgttact
ccggtctttg agatgtatca cgcgtttggg gctgcagatg 4320ccattatgaa gaagtactat
gtcggtacac tggtctcgaa tgagctgccc atcttcccgg 4380agccaacggt gttccacaaa
accatcaaga cgagagtcga gggctacttt acggatcgga 4440acattgatcc caagaataga
ccagagatct ggggacgata cgctcttatc tttggatcct 4500tgatcgcttc ctactacgcg
cagctctttg tgcctttcgt tgtcgaacgc acatggcttc 4560aggtggtgtt tgcaatcatc
atgggatttg cgtgcgcaca agtcggactc aaccctcttc 4620atgatgcgtc tcacttttca
gtgacccaca accccactgt ctggaagatt ctgggagcca 4680cgcacgactt tttcaacgga
gcatcgtacc tggtgtggat gtaccaacat atgctcggcc 4740atcaccccta caccaacatt
gctggagcag atcccgacgt gtcgacgtct gagcccgatg 4800ttcgtcgtat caagcccaac
caaaagtggt ttgtcaacca catcaaccag cacatgtttg 4860ttcctttcct gtacggactg
ctggcgttca aggtgcgcat tcaggacatc aacattttgt 4920actttgtcaa gaccaatgac
gctattcgtg tcaatcccat ctcgacatgg cacactgtga 4980tgttctgggg cggcaaggct
ttctttgtct ggtatcgcct gattgttccc ctgcagtatc 5040tgcccctggg caaggtgctg
ctcttgttca cggtcgcgga catggtgtcg tcttactggc 5100tggcgctgac cttccaggcg
aaccacgttg ttgaggaagt tcagtggccg ttgcctgacg 5160agaacgggat catccaaaag
gactgggcag ctatgcaggt cgagactacg caggattacg 5220cacacgattc gcacctctgg
accagcatca ctggcagctt gaactaccag gctgtgcacc 5280atctgttccc caacgtgtcg
cagcaccatt atcccgatat tctggccatc atcaagaaca 5340cctgcagcga gtacaaggtt
ccataccttg tcaaggatac gttttggcaa gcatttgctt 5400cacatttgga gcacttgcgt
gttcttggac tccgtcccaa ggaagagtag gcagctaagc 5460748630DNAArtificial
Sequenceplasmid pYZDE2-S 74ggtggagctc cagcttttgt tccctttagt gagggttaat
ttcgagcttg gcgtaatcat 60ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
aattccacac aacgtacgag 120ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
gagctaactc acattaattg 180cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
gtgccagctg cattaatgaa 240tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
ctcttccgct tcctcgctca 300ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
atcagctcac tcaaaggcgg 360taatacggtt atccacagaa tcaggggata acgcaggaaa
gaacatgtga gcaaaaggcc 420agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
gtttttccat aggctccgcc 480cccctgacga gcatcacaaa aatcgacgct caagtcagag
gtggcgaaac ccgacaggac 540tataaagata ccaggcgttt ccccctggaa gctccctcgt
gcgctctcct gttccgaccc 600tgccgcttac cggatacctg tccgcctttc tcccttcggg
aagcgtggcg ctttctcata 660gctcacgctg taggtatctc agttcggtgt aggtcgttcg
ctccaagctg ggctgtgtgc 720acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
taactatcgt cttgagtcca 780acccggtaag acacgactta tcgccactgg cagcagccac
tggtaacagg attagcagag 840cgaggtatgt aggcggtgct acagagttct tgaagtggtg
gcctaactac ggctacacta 900gaaggacagt atttggtatc tgcgctctgc tgaagccagt
taccttcgga aaaagagttg 960gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
tggttttttt gtttgcaagc 1020agcagattac gcgcagaaaa aaaggatctc aagaagatcc
tttgatcttt tctacggggt 1080ctgacgctca gtggaacgaa aactcacgtt aagggatttt
ggtcatgaga ttatcaaaaa 1140ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
taaatcaatc taaagtatat 1200atgagtaaac ttggtctgac agttaccaat gcttaatcag
tgaggcacct atctcagcga 1260tctgtctatt tcgttcatcc atagttgcct gactccccgt
cgtgtagata actacgatac 1320gggagggctt accatctggc cccagtgctg caatgatacc
gcgagaccca cgctcaccgg 1380ctccagattt atcagcaata aaccagccag ccggaagggc
cgagcgcaga agtggtcctg 1440caactttatc cgcctccatc cagtctatta attgttgccg
ggaagctaga gtaagtagtt 1500cgccagttaa tagtttgcgc aacgttgttg ccattgctac
aggcatcgtg gtgtcacgct 1560cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
atcaaggcga gttacatgat 1620cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
tccgatcgtt gtcagaagta 1680agttggccgc agtgttatca ctcatggtta tggcagcact
gcataattct cttactgtca 1740tgccatccgt aagatgcttt tctgtgactg gtgagtactc
aaccaagtca ttctgagaat 1800agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
acgggataat accgcgccac 1860atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
ttcggggcga aaactctcaa 1920ggatcttacc gctgttgaga tccagttcga tgtaacccac
tcgtgcaccc aactgatctt 1980cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
aacaggaagg caaaatgccg 2040caaaaaaggg aataagggcg acacggaaat gttgaatact
catactcttc ctttttcaat 2100attattgaag catttatcag ggttattgtc tcatgagcgg
atacatattt gaatgtattt 2160agaaaaataa acaaataggg gttccgcgca catttccccg
aaaagtgcca cctgacgcgc 2220cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
gcgcagcgtg accgctacac 2280ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
ttcctttctc gccacgttcg 2340ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
agggttccga tttagtgctt 2400tacggcacct cgaccccaaa aaacttgatt agggtgatgg
ttcacgtagt gggccatcgc 2460cctgatagac ggtttttcgc cctttgacgt tggagtccac
gttctttaat agtggactct 2520tgttccaaac tggaacaaca ctcaacccta tctcggtcta
ttcttttgat ttataaggga 2580ttttgccgat ttcggcctat tggttaaaaa atgagctgat
ttaacaaaaa tttaacgcga 2640attttaacaa aatattaacg cttacaattt ccattcgcca
ttcaggctgc gcaactgttg 2700ggaagggcga tcggtgcggg cctcttcgct attacgccag
ctggcgaaag ggggatgtgc 2760tgcaaggcga ttaagttggg taacgccagg gttttcccag
tcacgacgtt gtaaaacgac 2820ggccagtgaa ttgtaatacg actcactata gggcgaattg
ggtaccgggc cccccctcga 2880ggtcgatggt gtcgataagc ttgatatcga attcatgtca
cacaaaccga tcttcgcctc 2940aaggaaacct aattctacat ccgagagact gccgagatcc
agtctacact gattaatttt 3000cgggccaata atttaaaaaa atcgtgttat ataatattat
atgtattata tatatacatc 3060atgatgatac tgacagtcat gtcccattgc taaatagaca
gactccatct gccgcctcca 3120actgatgttc tcaatattta aggggtcatc tcgcattgtt
taataataaa cagactccat 3180ctaccgcctc caaatgatgt tctcaaaata tattgtatga
acttattttt attacttagt 3240attattagac aacttacttg ctttatgaaa aacacttcct
atttaggaaa caatttataa 3300tggcagttcg ttcatttaac aatttatgta gaataaatgt
tataaatgcg tatgggaaat 3360cttaaatatg gatagcataa atgatatctg cattgcctaa
ttcgaaatca acagcaacga 3420aaaaaatccc ttgtacaaca taaatagtca tcgagaaata
tcaactatca aagaacagct 3480attcacacgt tactattgag attattattg gacgagaatc
acacactcaa ctgtctttct 3540ctcttctaga aatacaggta caagtatgta ctattctcat
tgttcatact tctagtcatt 3600tcatcccaca tattccttgg atttctctcc aatgaatgac
attctatctt gcaaattcaa 3660caattataat aagatatacc aaagtagcgg tatagtggca
atcaaaaagc ttctctggtg 3720tgcttctcgt atttattttt attctaatga tccattaaag
gtatatattt atttcttgtt 3780atataatcct tttgtttatt acatgggctg gatacataaa
ggtattttga tttaattttt 3840tgcttaaatt caatcccccc tcgttcagtg tcaactgtaa
tggtaggaaa ttaccatact 3900tttgaagaag caaaaaaaat gaaagaaaaa aaaaatcgta
tttccaggtt agacgttccg 3960cagaatctag aatgcggtat gcggtacatt gttcttcgaa
cgtaaaagtt gcgctccctg 4020agatattgta catttttgct tttacaagta caagtacatc
gtacaactat gtactactgt 4080tgatgcatcc acaacagttt gttttgtttt tttttgtttt
ttttttttct aatgattcat 4140taccgctatg tatacctact tgtacttgta gtaagccggg
ttattggcgt tcaattaatc 4200atagacttat gaatctgcac ggtgtgcgct gcgagttact
tttagcttat gcatgctact 4260tgggtgtaat attgggatct gttcggaaat caacggatgc
tcaaccgatt tcgacagtaa 4320taatttgaat cgaatcggag cctaaaatga acccgagtat
atctcataaa attctcggtg 4380agaggtctgt gactgtcagt acaaggtgcc ttcattatgc
cctcaacctt accatacctc 4440actgaatgta gtgtacctct aaaaatgaaa tacagtgcca
aaagccaagg cactgagctc 4500gtctaacgga cttgatatac aaccaattaa aacaaatgaa
aagaaataca gttctttgta 4560tcatttgtaa caattaccct gtacaaacta aggtattgaa
atcccacaat attcccaaag 4620tccacccctt tccaaattgt catgcctaca actcatatac
caagcactaa cctaccaaac 4680accactaaaa ccccacaaaa tatatcttac cgaatataca
gtaacaagct accaccacac 4740tcgttgggtg cagtcgccag cttaaagata tctatccaca
tcagccacaa ctcccttcct 4800ttaataaacc gactacaccc ttggctattg aggttatgag
tgaatatact gtagacaaga 4860cactttcaag aagactgttt ccaaaacgta ccactgtcct
ccactacaaa cacacccaat 4920ctgcttcttc tagtcaaggt tgctacaccg gtaaattata
aatcatcatt tcattagcag 4980ggcagggccc tttttataga gtcttataca ctagcggacc
ctgccggtag accaacccgc 5040aggcgcgtca gtttgctcct tccatcaatg cgtcgtagaa
acgacttact ccttcttgag 5100cagctccttg accttgttgg caacaagtct ccgacctcgg
aggtggagga agagcctccg 5160atatcggcgg tagtgatacc agcctcgacg gactccttga
cggcagcctc aacagcgtca 5220ccggcgggct tcatgttaag agagaacttg agcatcatgg
cggcagacag aatggtggca 5280atggggttga ccttctgctt gccgagatcg ggggcagatc
cgtgacaggg ctcgtacaga 5340ccgaacgcct cgttggtgtc gggcagagaa gccagagagg
cggagggcag cagacccaga 5400gaaccgggga tgacggaggc ctcgtcggag atgatatcgc
caaacatgtt ggtggtgatg 5460atgataccat tcatcttgga gggctgcttg atgaggatca
tggcggccga gtcgatcagc 5520tggtggttga gctcgagctg ggggaattcg tccttgagga
ctcgagtgac agtctttcgc 5580caaagtcgag aggaggccag cacgttggcc ttgtcaagag
accacacggg aagagggggg 5640ttgtgctgaa gggccaggaa ggcggccatt cgggcaattc
gctcaacctc aggaacggag 5700taggtctcgg tgtcggaagc gacgccagat ccgtcatcct
cctttcgctc tccaaagtag 5760atacctccga cgagctctcg gacaatgatg aagtcggtgc
cctcaacgtt tcggatgggg 5820gagagatcgg cgagcttggg cgacagcagc tggcagggtc
gcaggttggc gtacaggttc 5880aggtcctttc gcagcttgag gagaccctgc tcgggtcgca
cgtcggttcg tccgtcggga 5940gtggtccata cggtgttggc agcgcctccg acagcaccga
gcataataga gtcagccttt 6000cggcagatgt cgagagtagc gtcggtgatg ggctcgccct
ccttctcaat ggcagctcct 6060ccaatgagtc ggtcctcaaa cacaaactcg gtgccggagg
cctcagcaac agacttgagc 6120accttgacgg cctcggcaat cacctcgggg ccacagaagt
cgccgccgag aagaacaatc 6180ttcttggagt cagtcttggt cttcttagtt tcgggttcca
ttgtggatgt gtgtggttgt 6240atgtgtgatg tggtgtgtgg agtgaaaatc tgtggctggc
aaacgctctt gtatatatac 6300gcacttttgc ccgtgctatg tggaagacta aacctccgaa
gattgtgact caggtagtgc 6360ggtatcggct agggacccaa accttgtcga tgccgatagc
gctatcgaac gtaccccagc 6420cggccgggag tatgtcggag gggacatacg agatcgtcaa
gggtttgtgg ccaactggta 6480aatttaaatg atgtcgacgc agtaggatgt cctgcacggg
tctttttgtg gggtgtggag 6540aaaggggtgc ttggagatgg aagccggtag aaccgggctg
cttgtgcttg gagatggaag 6600ccggtagaac cgggctgctt ggggggattt ggggccgctg
ggctccaaag aggggtaggc 6660atttcgttgg ggttacgtaa ttgcggcatt tgggtcctgc
gcgcatgtcc cattggtcag 6720aattagtccg gataggagac ttatcagcca atcacagcgc
cggatccacc tgtaggttgg 6780gttgggtggg agcacccctc cacagagtag agtcaaacag
cagcagcaac atgatagttg 6840ggggtgtgcg tgttaaagga aaaaaaagaa gcttgggtta
tattcccgct ctatttagag 6900gttgcgggat agacgccgac ggagggcaat ggcgccatgg
aaccttgcgg atatcgatac 6960gccgcggcgg actgcgtccg aaccagctcc agcagcgttt
tttccgggcc attgagccga 7020ctgcgacccc gccaacgtgt cttggcccac gcactcatgt
catgttggtg ttgggaggcc 7080actttttaag tagcacaagg cacctagctc gcagcaaggt
gtccgaacca aagaagcggc 7140tgcagtggtg caaacggggc ggaaacggcg ggaaaaagcc
acgggggcac gaattgaggc 7200acgccctcga atttgagacg agtcacggcc ccattcgccc
gcgcaatggc tcgccaacgc 7260ccggtctttt gcaccacatc aggttacccc aagccaaacc
tttgtgttaa aaagcttaac 7320atattatacc gaacgtaggt ttgggcgggc ttgctccgtc
tgtccaaggc aacatttata 7380taagggtctg catcgccggc tcaattgaat cttttttctt
cttctcttct ctatattcat 7440tcttgaatta aacacacatc aatccatggc aaacagcagc
gtgtgggatg atgtggtggg 7500ccgcgtggag accggcgtgg accagtggat ggatggcgcc
aagccgtacg cactcaccga 7560tgggctcccg atgatggacg tgtccaccat gctggcattc
gaggtgggat acatggccat 7620gctgctcttc ggcatcccga tcatgaagca gatggagaag
ccttttgagc tcaagaccat 7680caagctcttg cacaacttgt ttctcttcgg actttccttg
tacatgtgcg tggagaccat 7740ccgccaggct atcctcggag gctacaaagt gtttggaaac
gacatggaga agggcaacga 7800gtctcatgct cagggcatgt ctcgcatcgt gtacgtgttc
tacgtgtcca aggcatacga 7860gttcttggat accgccatca tgatcctttg caagaagttc
aaccaggttt ccttcttgca 7920tgtgtaccac catgccactc atttttgcca tctggtgggc
tatccgccaa gtacgctcca 7980ggaggtgatg cgtacttttt cagtgatcct caactctttc
gtgcacaccg tcatgtacgg 8040catactactt cttctcctcc caagggttcg ggttcgtgaa
gccaatcaag ccgtacatca 8100ccacccttca gatgacccag ttcatggcaa tgcttgtgca
gtccttgtac gactacctct 8160tcccatgcga ctacccacag gctcttgtgc agctccttgg
agtgtacatg atcaccttgc 8220ttgccctctt cggcaacttt tttgtgcaga gctatcttaa
aaagccaaaa aagagcaaga 8280ccaactaaaa ctgcctgcat gatatgccgc tcgccggcgt
tcgaattgac tcagaaagcg 8340agttaaggcg acacgcaaac tctatatttt ttcaaacgtg
ttgccgtcac tcattcgcca 8400tctgtttact acgtgtctgt tcaatgagca tgttcttgaa
tctaaagaat ctcgaatgtt 8460ttttaaaaaa agaattcgat atcaagctta cgcgtcgacc
cgggtggacg tctagaggta 8520cctagcaatt aacagatagt ttgccggtga taattctctt
aacctcccac actcctttga 8580cataacgatt tatgtaacga aactgaaatt tgaccagata
ttgtgtccgc 8630756473DNAArtificial Sequenceplasmid pY5-22
75ggtggagctc cagcttttgt tccctttagt gagggttaat ttcgagcttg gcgtaatcat
60ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacgtacgag
120ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
180cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
240tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
300ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
360taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
420agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
480cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
540tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
600tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
660gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
720acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
780acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
840cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
900gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
960gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
1020agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
1080ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
1140ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
1200atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
1260tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
1320gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
1380ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
1440caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
1500cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
1560cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
1620cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
1680agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
1740tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
1800agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
1860atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
1920ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
1980cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
2040caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
2100attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
2160agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgcgc
2220cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
2280ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
2340ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
2400tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
2460cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
2520tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
2580ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
2640attttaacaa aatattaacg cttacaattt ccattcgcca ttcaggctgc gcaactgttg
2700ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
2760tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
2820ggccagtgaa ttgtaatacg actcactata gggcgaattg ggtaccgggc cccccctcga
2880ggtcgatggt gtcgataagc ttgatatcga attcatgtca cacaaaccga tcttcgcctc
2940aaggaaacct aattctacat ccgagagact gccgagatcc agtctacact gattaatttt
3000cgggccaata atttaaaaaa atcgtgttat ataatattat atgtattata tatatacatc
3060atgatgatac tgacagtcat gtcccattgc taaatagaca gactccatct gccgcctcca
3120actgatgttc tcaatattta aggggtcatc tcgcattgtt taataataaa cagactccat
3180ctaccgcctc caaatgatgt tctcaaaata tattgtatga acttattttt attacttagt
3240attattagac aacttacttg ctttatgaaa aacacttcct atttaggaaa caatttataa
3300tggcagttcg ttcatttaac aatttatgta gaataaatgt tataaatgcg tatgggaaat
3360cttaaatatg gatagcataa atgatatctg cattgcctaa ttcgaaatca acagcaacga
3420aaaaaatccc ttgtacaaca taaatagtca tcgagaaata tcaactatca aagaacagct
3480attcacacgt tactattgag attattattg gacgagaatc acacactcaa ctgtctttct
3540ctcttctaga aatacaggta caagtatgta ctattctcat tgttcatact tctagtcatt
3600tcatcccaca tattccttgg atttctctcc aatgaatgac attctatctt gcaaattcaa
3660caattataat aagatatacc aaagtagcgg tatagtggca atcaaaaagc ttctctggtg
3720tgcttctcgt atttattttt attctaatga tccattaaag gtatatattt atttcttgtt
3780atataatcct tttgtttatt acatgggctg gatacataaa ggtattttga tttaattttt
3840tgcttaaatt caatcccccc tcgttcagtg tcaactgtaa tggtaggaaa ttaccatact
3900tttgaagaag caaaaaaaat gaaagaaaaa aaaaatcgta tttccaggtt agacgttccg
3960cagaatctag aatgcggtat gcggtacatt gttcttcgaa cgtaaaagtt gcgctccctg
4020agatattgta catttttgct tttacaagta caagtacatc gtacaactat gtactactgt
4080tgatgcatcc acaacagttt gttttgtttt tttttgtttt ttttttttct aatgattcat
4140taccgctatg tatacctact tgtacttgta gtaagccggg ttattggcgt tcaattaatc
4200atagacttat gaatctgcac ggtgtgcgct gcgagttact tttagcttat gcatgctact
4260tgggtgtaat attgggatct gttcggaaat caacggatgc tcaaccgatt tcgacagtaa
4320ttaattaagt catacacaag tcagctttct tcgagcctca tataagtata agtagttcaa
4380cgtattagca ctgtacccag catctccgta tcgagaaaca caacaacatg ccccattgga
4440cagatcatgc ggatacacag gttgtgcagt atcatacata ctcgatcaga caggtcgtct
4500gaccatcata caagctgaac aagcgctcca tacttgcacg ctctctatat acacagttaa
4560attacatatc catagtctaa cctctaacag ttaatcttct ggtaagcctc ccagccagcc
4620ttctggtatc gcttggcctc ctcaatagga tctcggttct ggccgtacag acctcggccg
4680acaattatga tatccgttcc ggtagacatg acatcctcaa cagttcggta ctgctgtccg
4740agagcgtctc ccttgtcgtc aagacccacc ccgggggtca gaataagcca gtcctcagag
4800tcgcccttag gtcggttctg ggcaatgaag ccaaccacaa actcggggtc ggatcgggca
4860agctcaatgg tctgcttgga gtactcgcca gtggccagag agcccttgca agacagctcg
4920gccagcatga gcagacctct ggccagcttc tcgttgggag aggggactag gaactccttg
4980tactgggagt tctcgtagtc agagacgtcc tccttcttct gttcagagac agtttcctcg
5040gcaccagctc gcaggccagc aatgattccg gttccgggta caccgtgggc gttggtgata
5100tcggaccact cggcgattcg gtgacaccgg tactggtgct tgacagtgtt gccaatatct
5160gcgaactttc tgtcctcgaa caggaagaaa ccgtgcttaa gagcaagttc cttgaggggg
5220agcacagtgc cggcgtaggt gaagtcgtca atgatgtcga tatgggtttt gatcatgcac
5280acataaggtc cgaccttatc ggcaagctca atgagctcct tggtggtggt aacatccaga
5340gaagcacaca ggttggtttt cttggctgcc acgagcttga gcactcgagc ggcaaaggcg
5400gacttgtgga cgttagctcg agcttcgtag gagggcattt tggtggtgaa gaggagactg
5460aaataaattt agtctgcaga actttttatc ggaaccttat ctggggcagt gaagtatatg
5520ttatggtaat agttacgagt tagttgaact tatagataga ctggactata cggctatcgg
5580tccaaattag aaagaacgtc aatggctctc tgggcgtcgc ctttgccgac aaaaatgtga
5640tcatgatgaa agccagcaat gacgttgcag ctgatattgt tgtcggccaa ccgcgccgaa
5700aacgcagctg tcagacccac agcctccaac gaagaatgta tcgtcaaagt gatccaagca
5760cactcatagt tggagtcgta ctccaaaggc ggcaatgacg agtcagacag atactcgtcg
5820actcaggcga cgacggaatt cctgcagccc atctgcagaa ttcaggagag accgggttgg
5880cggcgtattt gtgtcccaaa aaacagcccc aattgcccca attgacccca aattgaccca
5940gtagcgggcc caaccccggc gagagccccc ttcaccccac atatcaaacc tcccccggtt
6000cccacacttg ccgttaaggg cgtagggtac tgcagtctgg aatctacgct tgttcagact
6060ttgtactagt ttctttgtct ggccatccgg gtaacccatg ccggacgcaa aatagactac
6120tgaaaatttt tttgctttgt ggttgggact ttagccaagg gtataaaaga ccaccgtccc
6180cgaattacct ttcctcttct tttctctctc tccttgtcaa ctcacacccg aaatcgttaa
6240gcatttcctt ctgagtataa gaatcattca ccatggatcc actagttcta gagcggccgc
6300caccgcggcc cgagattccg gcctcttcgg ccgccaagcg acccgggtgg acgtctagag
6360gtacctagca attaacagat agtttgccgg tgataattct cttaacctcc cacactcctt
6420tgacataacg atttatgtaa cgaaactgaa atttgaccag atattgtgtc cgc
647376427PRTPavlova salina 76Met Gly Arg Gly Gly Asp Ser Ser Gly Gln Ala
His Pro Ala Ala Glu1 5 10
15Leu Ala Val Pro Ser Asp Arg Ala Glu Val Ser Asn Ala Asp Ser Lys
20 25 30Ala Leu His Ile Val Leu Tyr
Gly Lys Arg Val Asp Val Thr Lys Phe 35 40
45Gln Arg Thr His Pro Gly Gly Ser Lys Val Phe Arg Ile Phe Gln
Asp 50 55 60Arg Asp Ala Thr Glu Gln
Phe Glu Ser Tyr His Ser Lys Arg Ala Ile65 70
75 80Lys Met Met Glu Gly Met Leu Lys Lys Ser Glu
Asp Ala Pro Ala Asp 85 90
95Thr Pro Leu Pro Ser Gln Ser Pro Met Gly Lys Asp Phe Lys Ala Met
100 105 110Ile Glu Arg His Val Ala
Ala Gly Tyr Tyr Asp Pro Cys Pro Leu Asp 115 120
125Glu Leu Phe Lys Leu Ser Leu Val Leu Leu Pro Thr Phe Ala
Gly Met 130 135 140Tyr Met Leu Lys Ala
Gly Val Gly Ser Pro Leu Cys Gly Ala Leu Met145 150
155 160Val Ser Phe Gly Trp Tyr Leu Asp Gly Trp
Leu Ala His Asp Tyr Leu 165 170
175His His Ser Val Phe Lys Gly Ser Val Ala Arg Thr Val Gly Trp Asn
180 185 190Asn Ala Ala Gly Tyr
Phe Leu Gly Phe Val Gln Gly Tyr Ala Val Glu 195
200 205Trp Trp Arg Ala Arg His Asn Thr His His Val Cys
Thr Asn Glu Asp 210 215 220Gly Ser Asp
Pro Asp Ile Lys Thr Ala Pro Leu Leu Ile Tyr Val Arg225
230 235 240Asn Lys Pro Ser Ile Ala Lys
Arg Leu Asn Ala Phe Gln Arg Tyr Gln 245
250 255Gln Tyr Tyr Tyr Val Pro Val Met Ala Ile Leu Asp
Leu Tyr Trp Arg 260 265 270Leu
Glu Ser Ile Ala Tyr Val Ala Met Arg Leu Pro Lys Met Leu Pro 275
280 285Gln Ala Leu Ala Leu Val Ala His Tyr
Ala Ile Val Ala Trp Val Phe 290 295
300Ala Gly Asn Tyr His Leu Leu Pro Leu Val Thr Val Leu Arg Gly Phe305
310 315 320Gly Thr Gly Ile
Thr Val Phe Ala Thr His Tyr Gly Glu Asp Ile Leu 325
330 335Asp Ala Asp Gln Val Arg His Met Thr Leu
Val Glu Gln Thr Ala Leu 340 345
350Thr Ser Arg Asn Ile Ser Gly Gly Trp Leu Val Asn Val Leu Thr Gly
355 360 365Phe Ile Ser Leu Gln Thr Glu
His His Leu Phe Pro Met Met Pro Thr 370 375
380Gly Asn Leu Met Thr Ile Gln Pro Glu Val Arg Ala Phe Phe Lys
Lys385 390 395 400His Gly
Leu Glu Tyr Arg Glu Gly Asn Leu Ile Glu Cys Val Arg Gln
405 410 415Asn Ile Arg Ala Leu Ala Phe
Glu His Leu Leu 420 42577421PRTEuglena
gracilis 77Met Lys Ser Lys Arg Gln Ala Leu Pro Leu Thr Ile Asp Gly Thr
Thr1 5 10 15Tyr Asp Val
Ser Ala Trp Val Asn Phe His Pro Gly Gly Ala Glu Ile 20
25 30Ile Glu Asn Tyr Gln Gly Arg Asp Ala Thr
Asp Ala Phe Met Val Met 35 40
45His Ser Gln Glu Ala Phe Asp Lys Leu Lys Arg Met Pro Lys Ile Asn 50
55 60Pro Ser Ser Glu Leu Pro Pro Gln Ala
Ala Val Asn Glu Ala Gln Glu65 70 75
80Asp Phe Arg Lys Leu Arg Glu Glu Leu Ile Ala Thr Gly Met
Phe Asp 85 90 95Ala Ser
Pro Leu Trp Tyr Ser Tyr Lys Ile Ser Thr Thr Leu Gly Leu 100
105 110Gly Val Leu Gly Tyr Phe Leu Met Val
Gln Tyr Gln Met Tyr Phe Ile 115 120
125Gly Ala Val Leu Leu Gly Met His Tyr Gln Gln Met Gly Trp Leu Ser
130 135 140His Asp Ile Cys His His Gln
Thr Phe Lys Asn Arg Asn Trp Asn Asn145 150
155 160Leu Val Gly Leu Val Phe Gly Asn Gly Leu Gln Gly
Phe Ser Val Thr 165 170
175Trp Trp Lys Asp Arg His Asn Ala His His Ser Ala Thr Asn Val Gln
180 185 190Gly His Asp Pro Asp Ile
Asp Asn Leu Pro Leu Leu Ala Trp Ser Glu 195 200
205Asp Asp Val Thr Arg Ala Ser Pro Ile Ser Arg Lys Leu Ile
Gln Phe 210 215 220Gln Gln Tyr Tyr Phe
Leu Val Ile Cys Ile Leu Leu Arg Phe Ile Trp225 230
235 240Cys Phe Gln Ser Val Leu Thr Val Arg Ser
Leu Lys Asp Arg Asp Asn 245 250
255Gln Phe Tyr Arg Ser Gln Tyr Lys Lys Glu Ala Ile Gly Leu Ala Leu
260 265 270His Trp Thr Leu Lys
Thr Leu Phe His Leu Phe Phe Met Pro Ser Ile 275
280 285Leu Thr Ser Leu Leu Val Phe Phe Val Ser Glu Leu
Val Gly Gly Phe 290 295 300Gly Ile Ala
Ile Val Val Phe Met Asn His Tyr Pro Leu Glu Lys Ile305
310 315 320Gly Asp Ser Val Trp Asp Gly
His Gly Phe Ser Val Gly Gln Ile His 325
330 335Glu Thr Met Asn Ile Arg Arg Gly Ile Ile Thr Asp
Trp Phe Phe Gly 340 345 350Gly
Leu Asn Tyr Gln Ile Glu His His Leu Trp Pro Thr Leu Pro Arg 355
360 365His Asn Leu Thr Ala Val Ser Tyr Gln
Val Glu Gln Leu Cys Gln Lys 370 375
380His Asn Leu Pro Tyr Arg Asn Pro Leu Pro His Glu Gly Leu Val Ile385
390 395 400Leu Leu Arg Tyr
Leu Ala Val Phe Ala Arg Met Ala Glu Lys Gln Pro 405
410 415Ala Gly Lys Ala Leu
420788953DNAArtificial Sequenceplasmid pY5-30 78ggtggagctc cagcttttgt
tccctttagt gagggttaat ttcgagcttg gcgtaatcat 60ggtcatagct gtttcctgtg
tgaaattgtt atccgctcac aattccacac aacatacgag 120ccggaagcat aaagtgtaaa
gcctggggtg cctaatgagt gagctaactc acattaattg 180cgttgcgctc actgcccgct
ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 240tcggccaacg cgcggggaga
ggcggtttgc gtattgggcg ctcttccgct tcctcgctca 300ctgactcgct gcgctcggtc
gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 360taatacggtt atccacagaa
tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 420agcaaaaggc caggaaccgt
aaaaaggccg cgttgctggc gtttttccat aggctccgcc 480cccctgacga gcatcacaaa
aatcgacgct caagtcagag gtggcgaaac ccgacaggac 540tataaagata ccaggcgttt
ccccctggaa gctccctcgt gcgctctcct gttccgaccc 600tgccgcttac cggatacctg
tccgcctttc tcccttcggg aagcgtggcg ctttctcata 660gctcacgctg taggtatctc
agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 720acgaaccccc cgttcagccc
gaccgctgcg ccttatccgg taactatcgt cttgagtcca 780acccggtaag acacgactta
tcgccactgg cagcagccac tggtaacagg attagcagag 840cgaggtatgt aggcggtgct
acagagttct tgaagtggtg gcctaactac ggctacacta 900gaaggacagt atttggtatc
tgcgctctgc tgaagccagt taccttcgga aaaagagttg 960gtagctcttg atccggcaaa
caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 1020agcagattac gcgcagaaaa
aaaggatctc aagaagatcc tttgatcttt tctacggggt 1080ctgacgctca gtggaacgaa
aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 1140ggatcttcac ctagatcctt
ttaaattaaa aatgaagttt taaatcaatc taaagtatat 1200atgagtaaac ttggtctgac
agttaccaat gcttaatcag tgaggcacct atctcagcga 1260tctgtctatt tcgttcatcc
atagttgcct gactccccgt cgtgtagata actacgatac 1320gggagggctt accatctggc
cccagtgctg caatgatacc gcgagaccca cgctcaccgg 1380ctccagattt atcagcaata
aaccagccag ccggaagggc cgagcgcaga agtggtcctg 1440caactttatc cgcctccatc
cagtctatta attgttgccg ggaagctaga gtaagtagtt 1500cgccagttaa tagtttgcgc
aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 1560cgtcgtttgg tatggcttca
ttcagctccg gttcccaacg atcaaggcga gttacatgat 1620cccccatgtt gtgcaaaaaa
gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 1680agttggccgc agtgttatca
ctcatggtta tggcagcact gcataattct cttactgtca 1740tgccatccgt aagatgcttt
tctgtgactg gtgagtactc aaccaagtca ttctgagaat 1800agtgtatgcg gcgaccgagt
tgctcttgcc cggcgtcaat acgggataat accgcgccac 1860atagcagaac tttaaaagtg
ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 1920ggatcttacc gctgttgaga
tccagttcga tgtaacccac tcgtgcaccc aactgatctt 1980cagcatcttt tactttcacc
agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 2040caaaaaaggg aataagggcg
acacggaaat gttgaatact catactcttc ctttttcaat 2100attattgaag catttatcag
ggttattgtc tcatgagcgg atacatattt gaatgtattt 2160agaaaaataa acaaataggg
gttccgcgca catttccccg aaaagtgcca cctgacgcgc 2220cctgtagcgg cgcattaagc
gcggcgggtg tggtggttac gcgcagcgtg accgctacac 2280ttgccagcgc cctagcgccc
gctcctttcg ctttcttccc ttcctttctc gccacgttcg 2340ccggctttcc ccgtcaagct
ctaaatcggg ggctcccttt agggttccga tttagtgctt 2400tacggcacct cgaccccaaa
aaacttgatt agggtgatgg ttcacgtagt gggccatcgc 2460cctgatagac ggtttttcgc
cctttgacgt tggagtccac gttctttaat agtggactct 2520tgttccaaac tggaacaaca
ctcaacccta tctcggtcta ttcttttgat ttataaggga 2580ttttgccgat ttcggcctat
tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 2640attttaacaa aatattaacg
cttacaattt ccattcgcca ttcaggctgc gcaactgttg 2700ggaagggcga tcggtgcggg
cctcttcgct attacgccag ctggcgaaag ggggatgtgc 2760tgcaaggcga ttaagttggg
taacgccagg gttttcccag tcacgacgtt gtaaaacgac 2820ggccagtgaa ttgtaatacg
actcactata gggcgaattg ggtaccgggc cccccctcga 2880ggtcgatggt gtcgataagc
ttgatatcga attcatgtca cacaaaccga tcttcgcctc 2940aaggaaacct aattctacat
ccgagagact gccgagatcc agtctacact gattaatttt 3000cgggccaata atttaaaaaa
atcgtgttat ataatattat atgtattata tatatacatc 3060atgatgatac tgacagtcat
gtcccattgc taaatagaca gactccatct gccgcctcca 3120actgatgttc tcaatattta
aggggtcatc tcgcattgtt taataataaa cagactccat 3180ctaccgcctc caaatgatgt
tctcaaaata tattgtatga acttattttt attacttagt 3240attattagac aacttacttg
ctttatgaaa aacacttcct atttaggaaa caatttataa 3300tggcagttcg ttcatttaac
aatttatgta gaataaatgt tataaatgcg tatgggaaat 3360cttaaatatg gatagcataa
atgatatctg cattgcctaa ttcgaaatca acagcaacga 3420aaaaaatccc ttgtacaaca
taaatagtca tcgagaaata tcaactatca aagaacagct 3480attcacacgt tactattgag
attattattg gacgagaatc acacactcaa ctgtctttct 3540ctcttctaga aatacaggta
caagtatgta ctattctcat tgttcatact tctagtcatt 3600tcatcccaca tattccttgg
atttctctcc aatgaatgac attctatctt gcaaattcaa 3660caattataat aagatatacc
aaagtagcgg tatagtggca atcaaaaagc ttctctggtg 3720tgcttctcgt atttattttt
attctaatga tccattaaag gtatatattt atttcttgtt 3780atataatcct tttgtttatt
acatgggctg gatacataaa ggtattttga tttaattttt 3840tgcttaaatt caatcccccc
tcgttcagtg tcaactgtaa tggtaggaaa ttaccatact 3900tttgaagaag caaaaaaaat
gaaagaaaaa aaaaatcgta tttccaggtt agacgttccg 3960cagaatctag aatgcggtat
gcggtacatt gttcttcgaa cgtaaaagtt gcgctccctg 4020agatattgta catttttgct
tttacaagta caagtacatc gtacaactat gtactactgt 4080tgatgcatcc acaacagttt
gttttgtttt tttttgtttt ttttttttct aatgattcat 4140taccgctatg tatacctact
tgtacttgta gtaagccggg ttattggcgt tcaattaatc 4200atagacttat gaatctgcac
ggtgtgcgct gcgagttact tttagcttat gcatgctact 4260tgggtgtaat attgggatct
gttcggaaat caacggatgc tcaaccgatt tcgacagtaa 4320taatttgaat cgaatcggag
cctaaaatga acccgagtat atctcataaa attctcggtg 4380agaggtctgt gactgtcagt
acaaggtgcc ttcattatgc cctcaacctt accatacctc 4440actgaatgta gtgtacctct
aaaaatgaaa tacagtgcca aaagccaagg cactgagctc 4500gtctaacgga cttgatatac
aaccaattaa aacaaatgaa aagaaataca gttctttgta 4560tcatttgtaa caattaccct
gtacaaacta aggtattgaa atcccacaat attcccaaag 4620tccacccctt tccaaattgt
catgcctaca actcatatac caagcactaa cctaccaaac 4680accactaaaa ccccacaaaa
tatatcttac cgaatataca gtaacaagct accaccacac 4740tcgttgggtg cagtcgccag
cttaaagata tctatccaca tcagccacaa ctcccttcct 4800ttaataaacc gactacaccc
ttggctattg aggttatgag tgaatatact gtagacaaga 4860cactttcaag aagactgttt
ccaaaacgta ccactgtcct ccactacaaa cacacccaat 4920ctgcttcttc tagtcaaggt
tgctacaccg gtaaattata aatcatcatt tcattagcag 4980ggcagggccc tttttataga
gtcttataca ctagcggacc ctgccggtag accaacccgc 5040aggcgcgtca gtttgctcct
tccatcaatg cgtcgtagaa acgacttact ccttcttgag 5100cagctccttg accttgttgg
caacaagtct ccgacctcgg aggtggagga agagcctccg 5160atatcggcgg tagtgatacc
agcctcgacg gactccttga cggcagcctc aacagcgtca 5220ccggcgggct tcatgttaag
agagaacttg agcatcatgg cggcagacag aatggtggca 5280atggggttga ccttctgctt
gccgagatcg ggggcagatc cgtgacaggg ctcgtacaga 5340ccgaacgcct cgttggtgtc
gggcagagaa gccagagagg cggagggcag cagacccaga 5400gaaccgggga tgacggaggc
ctcgtcggag atgatatcgc caaacatgtt ggtggtgatg 5460atgataccat tcatcttgga
gggctgcttg atgaggatca tggcggccga gtcgatcagc 5520tggtggttga gctcgagctg
ggggaattcg tccttgagga ctcgagtgac agtctttcgc 5580caaagtcgag aggaggccag
cacgttggcc ttgtcaagag accacacggg aagagggggg 5640ttgtgctgaa gggccaggaa
ggcggccatt cgggcaattc gctcaacctc aggaacggag 5700taggtctcgg tgtcggaagc
gacgccagat ccgtcatcct cctttcgctc tccaaagtag 5760atacctccga cgagctctcg
gacaatgatg aagtcggtgc cctcaacgtt tcggatgggg 5820gagagatcgg cgagcttggg
cgacagcagc tggcagggtc gcaggttggc gtacaggttc 5880aggtcctttc gcagcttgag
gagaccctgc tcgggtcgca cgtcggttcg tccgtcggga 5940gtggtccata cggtgttggc
agcgcctccg acagcaccga gcataataga gtcagccttt 6000cggcagatgt cgagagtagc
gtcggtgatg ggctcgccct ccttctcaat ggcagctcct 6060ccaatgagtc ggtcctcaaa
cacaaactcg gtgccggagg cctcagcaac agacttgagc 6120accttgacgg cctcggcaat
cacctcgggg ccacagaagt cgccgccgag aagaacaatc 6180ttcttggagt cagtcttggt
cttcttagtt tcgggttcca ttgtggatgt gtgtggttgt 6240atgtgtgatg tggtgtgtgg
agtgaaaatc tgtggctggc aaacgctctt gtatatatac 6300gcacttttgc ccgtgctatg
tggaagacta aacctccgaa gattgtgact caggtagtgc 6360ggtatcggct agggacccaa
accttgtcga tgccgatagc gctatcgaac gtaccccagc 6420cggccgggag tatgtcggag
gggacatacg agatcgtcaa gggtttgtgg ccaactggta 6480aataaatgat gtcgactcag
gcgacgacgg aattcctgca gcccatctgc agaattcagg 6540agagaccggg ttggcggcgt
atttgtgtcc caaaaaacag ccccaattgc cccaattgac 6600cccaaattga cccagtagcg
ggcccaaccc cggcgagagc ccccttcacc ccacatatca 6660aacctccccc ggttcccaca
cttgccgtta agggcgtagg gtactgcagt ctggaatcta 6720cgcttgttca gactttgtac
tagtttcttt gtctggccat ccgggtaacc catgccggac 6780gcaaaataga ctactgaaaa
tttttttgct ttgtggttgg gactttagcc aagggtataa 6840aagaccaccg tccccgaatt
acctttcctc ttcttttctc tctctccttg tcaactcaca 6900cccgaaatcg ttaagcattt
ccttctgagt ataagaatca ttcaccatgg atggtacgtc 6960ctgtagaaac cccaacccgt
gaaatcaaaa aactcgacgg cctgtgggca ttcagtctgg 7020atcgcgaaaa ctgtggaatt
gatcagcgtt ggtgggaaag cgcgttacaa gaaagccggg 7080caattgctgt gccaggcagt
tttaacgatc agttcgccga tgcagatatt cgtaattatg 7140cgggcaacgt ctggtatcag
cgcgaagtct ttataccgaa aggttgggca ggccagcgta 7200tcgtgctgcg tttcgatgcg
gtcactcatt acggcaaagt gtgggtcaat aatcaggaag 7260tgatggagca tcagggcggc
tatacgccat ttgaagccga tgtcacgccg tatgttattg 7320ccgggaaaag tgtacgtatc
accgtttgtg tgaacaacga actgaactgg cagactatcc 7380cgccgggaat ggtgattacc
gacgaaaacg gcaagaaaaa gcagtcttac ttccatgatt 7440tctttaacta tgccgggatc
catcgcagcg taatgctcta caccacgccg aacacctggg 7500tggacgatat caccgtggtg
acgcatgtcg cgcaagactg taaccacgcg tctgttgact 7560ggcaggtggt ggccaatggt
gatgtcagcg ttgaactgcg tgatgcggat caacaggtgg 7620ttgcaactgg acaaggcact
agcgggactt tgcaagtggt gaatccgcac ctctggcaac 7680cgggtgaagg ttatctctat
gaactgtgcg tcacagccaa aagccagaca gagtgtgata 7740tctacccgct tcgcgtcggc
atccggtcag tggcagtgaa gggcgaacag ttcctgatta 7800accacaaacc gttctacttt
actggctttg gtcgtcatga agatgcggac ttacgtggca 7860aaggattcga taacgtgctg
atggtgcacg accacgcatt aatggactgg attggggcca 7920actcctaccg tacctcgcat
tacccttacg ctgaagagat gctcgactgg gcagatgaac 7980atggcatcgt ggtgattgat
gaaactgctg ctgtcggctt taacctctct ttaggcattg 8040gtttcgaagc gggcaacaag
ccgaaagaac tgtacagcga agaggcagtc aacggggaaa 8100ctcagcaagc gcacttacag
gcgattaaag agctgatagc gcgtgacaaa aaccacccaa 8160gcgtggtgat gtggagtatt
gccaacgaac cggatacccg tccgcaagtg cacgggaata 8220tttcgccact ggcggaagca
acgcgtaaac tcgacccgac gcgtccgatc acctgcgtca 8280atgtaatgtt ctgcgacgct
cacaccgata ccatcagcga tctctttgat gtgctgtgcc 8340tgaaccgtta ttacggatgg
tatgtccaaa gcggcgattt ggaaacggca gagaaggtac 8400tggaaaaaga acttctggcc
tggcaggaga aactgcatca gccgattatc atcaccgaat 8460acggcgtgga tacgttagcc
gggctgcact caatgtacac cgacatgtgg agtgaagagt 8520atcagtgtgc atggctggat
atgtatcacc gcgtctttga tcgcgtcagc gccgtcgtcg 8580gtgaacaggt atggaatttc
gccgattttg cgacctcgca aggcatattg cgcgttggcg 8640gtaacaagaa agggatcttc
actcgcgacc gcaaaccgaa gtcggcggct tttctgctgc 8700aaaaacgctg gactggcatg
aacttcggtg aaaaaccgca gcagggaggc aaacaatgat 8760taattaacta gagcggccgc
caccgcggcc cgagattccg gcctcttcgg ccgccaagcg 8820acccgggtgg acgtctagag
gtacctagca attaacagat agtttgccgg tgataattct 8880cttaacctcc cacactcctt
tgacataacg atttatgtaa cgaaactgaa atttgaccag 8940atattgtgtc cgc
8953
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