CN113151340B - Genetic engineering bacterium for increasing yield of beta-carotene and application thereof - Google Patents

Genetic engineering bacterium for increasing yield of beta-carotene and application thereof Download PDF

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CN113151340B
CN113151340B CN202011341160.4A CN202011341160A CN113151340B CN 113151340 B CN113151340 B CN 113151340B CN 202011341160 A CN202011341160 A CN 202011341160A CN 113151340 B CN113151340 B CN 113151340B
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carotene
beta
plasmid
gene
dna
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CN113151340A (en
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吴世林
***
祝重阳
江华峰
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Guangzhou Wisdom Bio Technology Co ltd
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Abstract

The invention discloses a genetic engineering bacterium for improving the yield of beta-carotene and application thereof, wherein the genetic engineering bacterium comprises a beta-carotene synthesis pathway related gene, a phosphoketolase pathway related gene and a lipid droplet synthesis pathway related gene, and covers the synthesis pathway of the beta-carotene; and increases the phosphoketolase approach, reduces the carbon loss, and leads the carbon source to generate more precursor substance acetyl coenzyme A for synthesizing the beta-carotene; the supply of NADPH as a reducing substance and ATP as an energy substance is increased; and lipid drop synthesis paths are increased, and the storage of beta-carotene can be increased by increasing the content and volume of intracellular lipid drops. The highest yield of the beta-carotene of the genetic engineering bacteria reaches 7.8g/L, and the yield of the beta-carotene is obviously improved.

Description

Genetic engineering bacterium for increasing yield of beta-carotene and application thereof
Technical Field
The invention relates to the field of genetic engineering and fermentation engineering, in particular to a genetic engineering bacterium capable of improving the biosynthesis yield of beta-carotene and a construction method and application thereof.
Background
Beta-carotene is a tetraterpene compound formed by connecting 8 isoprene units through unsaturated carbon bonds, has excellent physiological functions, and is widely applied to the industries of medical health products, food additives, cosmetics, feed additives and the like.
The production of beta-carotene mainly relies on chemical synthesis. Different from natural beta-carotene which is a mixture of trans-configuration and cis-configuration, the chemically synthesized beta-carotene is almost all in the trans-configuration, the structure greatly reduces the absorption rate of human bodies to the beta-carotene, and in addition, the chemical synthesis method has the advantages of complex process flow, high energy consumption and great pollution. The microbial fermentation method is a method for obtaining beta-carotene by fermenting and culturing a microbial strain for producing the beta-carotene and then separating and purifying the beta-carotene. In contrast, the microbial fermentation method is not limited by raw materials, has mild production conditions and green and environment-friendly production process, and has great advantages. The main synthetic route of the microorganism is that isoprene pyrophosphate (IPP) is firstly synthesized through a deoxyxylulose phosphate pathway (MEP) or a mevalonate pathway (MVA), then dimethylallyl pyrophosphate (DAMPP) is generated through the action of isomerase (idi), the IPP and the DAMPP are condensed into geranyl pyrophosphate (GPP) under the catalysis of geranyl pyrophosphate synthase, the latter combines one molecule of IPP under the catalysis of the farnesyl pyrophosphate synthase to form farnesyl pyrophosphate (FPP), and the FPP combines one molecule of IPP under the catalysis of the digeranyl pyrophosphate synthase (crtE) to form digeranyl pyrophosphate (GGPP). Finally, the beta-carotene is formed by the lycopene and the lycopene under the catalysis of lycopene synthase (crtB), lycopene dehydrogenase (crtI) and lycopene cyclase (crtY).
The biggest bottleneck in the fermentative production of beta-carotene by microorganisms lies in how to realize industrialization, and the necessary condition of industrialization is that the yield of the beta-carotene must meet the level of industrialization. Although there are many reports of synthesizing beta-carotene by genetically modified engineering bacteria, these modifications only enhance the gene expression of a metabolic pathway from acetyl coenzyme A to beta-carotene via mevalonate, but do not consider short-plate problems of carbon source utilization, reducing power supply and final product storage, and thus, the yield cannot be further broken through to meet the requirement of industrial production.
Disclosure of Invention
The first object of the present invention is to provide a nucleic acid molecule which addresses the technical deficiencies of the prior art in the utilization of a β -carotene carbon source, the supply of reducing power, and the storage of the final product.
The second object of the present invention is to provide an expression system comprising the above-mentioned nucleic acid molecule.
The third object of the present invention is to provide a cell containing the above expression system.
The fourth purpose of the invention is to provide a recombinant engineering bacterium, the preservation number is CCTCC NO: m2020606, named ZTQ5320, deposited in CCTCC-China center for type culture Collection in 10 months and 19 days in 2020; address: wuhan, wuhan university (eight-way Lojia mountain in Wuchang region, wuhan city, hubei, post: 430072); the preservation number is CCTCC NO: m2020606, classified and named as Yarrowia lipolytica ZTQ5320 (Yarrowia lipolytica ZTQ 5320).
The fifth object of the present invention is to provide a method for producing beta-carotene, which comprises culturing the above-mentioned cells by fermentation to extract beta-carotene from the cells.
The technical scheme adopted by the invention is as follows:
in a first aspect of the invention, there is provided a nucleic acid molecule comprising the nucleic acid sequence of:
(I) a gene related to the beta-carotene synthesis pathway;
(II) phosphoketolase pathway-related genes;
(III) genes involved in the lipid droplet synthesis pathway.
Further, according to the nucleic acid molecule of the first aspect of the present invention, the lipid droplet synthesis pathway-associated gene includes at least one of the following genes:
(1) Acetyl-coa acidifying enzyme gene ACC1;
(2) Diacylglycerol acyltransferase gene DAG1;
(3) Delta-9 stearoyl-coa desaturase gene SCD;
(4) Fat storage mediated lipid droplet fusion induces the transmembrane protein gene FIT.
Preferably, the nucleic acid molecule according to the first aspect of the present invention, wherein the lipid droplet pathway-associated gene is at least one of ACC1, DGA1, SCD and FIT derived from yarrowia lipolytica W29.
More preferably, the nucleic acid molecule according to the first aspect of the present invention, wherein said lipid droplet pathway-associated genes are ACC1, DGA1, SCD and FIT derived from yarrowia lipolytica W29.
More specifically, the lipid droplet pathway-associated genes are preferably ACC1 (CP 028450.1: 1555944-1562432), DGA1 (CP 028452.1: 3883639-3885183), SCD (CP 028450.1: 745375-746823) and FIT (CP 028450.1: 3104019-3105197) derived from yarrowia lipolytica W29.
In some embodiments of the present invention, the recombinant engineering bacteria selected are yarrowia lipolytica, which is an ester-producing yeast, and which does not produce large lipid droplets in cells under natural conditions, and the genes ACC1, DGA1, SCD and FIT from yarrowia lipolytica are selected to maximize the lipid droplet content and β -carotene storage capacity in yarrowia lipolytica.
The nucleic acid molecule according to the first aspect of the present invention, wherein the beta-carotene synthesis pathway-related gene comprises at least one of the following genes:
(1) A gene AtoB catalyzing the condensation of acetyl coenzyme A into acetoacetyl coenzyme A;
(2) A gene HMGS catalyzing the condensation of acetoacetyl-CoA and acetyl-CoA into HMG-CoA;
(3) A gene tHMGR catalyzing the reduction of HMG-CoA to mevalonate;
(4) Gene ERG12 catalyzing the phosphorylation of mevalonate into mevalonate-5-phosphate;
(5) Gene ERG8 catalyzing mevalonate-5-phosphate to produce mevalonate-5-pyrophosphate;
(6) Gene ERG19 for catalyzing the decarboxylation of mevalonate-5-pyrophosphate to generate isoprene pyrophosphate;
(7) Gene IDI catalyzing the isomerization of isoprene pyrophosphate into dimethylallyl pyrophosphate;
(8) Gene ERG20 for catalyzing isopentenyl pyrophosphate and dimethylallyl pyrophosphate to generate farnesyl pyrophosphate;
(9) A gene GGS1 for catalyzing farnesyl pyrophosphate to combine with one molecule of isoprene pyrophosphate to generate geranyl pyrophosphate;
(10) Gene CarB for catalyzing the dehydrogenation of lycopene to lycopene;
(11) A gene CarRP which catalyzes two molecules of digeranyl pyrophosphate to generate lycopene and catalyzes lycopene cyclization to generate beta-carotene.
Preferably, the beta-carotene pathway-associated gene is at least one of atoB derived from Escherichia coli BL21 (DE 3), HMGS derived from yarrowia lipolytica W29, tHMGR, ERG12, ERG8, ERG19, IDI, ERG20, GGS1, carB derived from Mucor circinelloides, and CarRP.
More preferably, the beta-carotene pathway-associated genes are atoB derived from Escherichia coli BL21 (DE 3), HMGS, tHMGR, ERG12, ERG8, ERG19, IDI, ERG20, GGS1, carB and CarRP derived from Mucor circinelloides yarrowia.
More specifically, the beta-carotene pathway-related genes are atoB (CP 001509.3: 2216470-2217654) derived from Escherichia coli BL21 (DE 3), HMGS (CP 028459.1: 3780129-3781469) derived from yarrowia lipolytica W29, tHMGR (CP 028452.1: 571078-572577), ERG12 (CP 028449.1: 2082357-2083706), ERG8 (CP 028452.1: 748052-746796), ERG19 (CP 453 0281: 818770-819933), IDI (CP 028453.1: 5862-584850), ERG20 (028452.1: 673063063063063063068-672034), GGS1 (CP 451.1: 2085775-2084792) and Carb (K9717.J) derived from Rhizomucor circinella (K028971, 9918).
In previous studies by the team of the inventors, it was found that CarB and CarRP derived from Mucor circinelloides were more catalytically efficient than CarB and CarRA derived from Blakeslea trispora by comparing the yield of beta-carotene by shake flask culture, and thus CarB and CarRP derived from Mucor circinelloides were selected. .
The nucleic acid molecule according to the first aspect of the present invention, wherein the phosphoketolase pathway-associated gene comprises at least one of:
(1) Gene PK catalyzing fructose 6 phosphate or xylulose 5 phosphate to generate acetyl phosphate;
(2) PTA gene catalyzing acetyl phosphate to generate acetyl coenzyme A.
Preferably, the phosphoketolase pathway-associated gene is at least one of PK derived from leuconostoc mesenteroides and PTA derived from clostridium sp.
More preferably, the phosphoketolase pathway-associated genes are PK derived from leuconostoc mesenteroides and PTA derived from clostridium sp.
More specifically, the phosphoketolase pathway-associated gene is preferably selected from PK (CP 000414.1: 1936622-1934181) derived from Leuconostoc mesenteroides (Leuconostoc mesenteroides) and PTA (CP 018335.1: 1695414-1696415) derived from Clostridium kluyveri (Clostridium kluyveri).
The inventors' group found in previous studies that the phosphoketolase pathway of the combination could maximally increase beta-carrot yield by comparing beta-carrot yield in shake flask culture, and therefore selected the above combination.
Preferably, the nucleic acid molecule according to the first aspect of the present invention, wherein a promoter sequence is linked upstream of each gene in order to ensure that the gene can be expressed efficiently in an expression system or recombinant cell;
promoter sequences selected include, but are not limited to: pTEF, pEXP, pFBA and pGPD.
More preferably, a terminator sequence is added downstream of each gene.
In a second aspect of the invention, there is provided an expression system comprising a nucleic acid molecule according to the first aspect of the invention.
Preferably, the genes of the nucleic acid molecules are split into several groups, which are inserted into different expression vectors.
More preferably, to ensure that the individual genes can be expressed efficiently in an expression system or recombinant cell, a promoter sequence is added upstream and a terminator sequence is added downstream of each gene.
Promoter sequences selected include, but are not limited to: pTEF, pEXP, pFBA and pGPD.
In one embodiment of the present invention, the genes of the nucleic acid molecules are split into 6 groups, which are inserted into 6 different expression vectors.
More specifically, plasmid pZ1 uses pUC19 as a backbone vector and contains CarB, carRP and GGS1 genes;
plasmid pZ2 uses pUC19 as a skeleton vector and comprises AtoB, HMGS, HMGR and ERG12 genes;
plasmid pZ3 uses pUC19 as a skeleton vector and comprises ERG8, ERG19, ERG20 and IDI genes;
plasmid pZ4 takes pUC19 as a skeleton vector and contains PK and PTA genes;
plasmid pZ5 uses pUC19 as a skeleton vector and contains ACC1 gene;
plasmid pZ6 uses pUC19 as a backbone vector and contains DGA1, SCD and FIT genes.
Further, the plasmid pZ1, the plasmid pZ2, the plasmid pZ3, the plasmid pZ4, the plasmid pZ5, and the plasmid pZ6 each contain a URA3 uracil deficiency screening gene.
Those skilled in the art will appreciate that other groupings can be used to accomplish the objectives of the present invention.
In a third aspect, the invention provides a cell comprising a nucleic acid molecule according to the first aspect of the invention or an expression system according to the second aspect of the invention.
Further, the nucleic acid molecule is inserted into the genome of the cell.
Still further, the nucleic acid molecule is inserted into the genome of the cell by non-homologous recombinant biological means.
It will be appreciated by those skilled in the art that the insertion into the genome of said cells by means of non-homologous recombinant organisms is intended to obtain more strains which are likely to be stably inherited, and that the object of the invention can equally well be achieved by transforming only the above-mentioned expression systems.
In addition, compared with a fixed-point embedding mode, non-homologous recombination has the possibility of obtaining a recombinant cell or a recombinant bacterium which is embedded for multiple times and has higher efficiency.
Preferably, the cell according to the third aspect of the present invention is a yeast cell.
More preferably, the cell is yarrowia lipolytica.
In one embodiment of the present invention, the construction method of the recombinant engineering bacteria is as follows: plasmid pZ1, plasmid pZ2, plasmid pZ3, plasmid pZ4, plasmid pZ5, and plasmid pZ6 were randomly integrated into the yarrowia lipolytica genome by non-homologous recombination.
More specifically, the gene integration sequence of the above recombinant engineered bacteria is a bacterium obtained by sequentially performing the following steps of plasmid pZ1, plasmid pZ2, plasmid pZ3, plasmid pZ4, plasmid pZ5, and plasmid pZ6.
Further, the method comprises the following steps:
s01, introducing the plasmid pZ1 into a yarrowia lipolytica strain Polf with uracil synthesis defect, and screening a strain with the highest beta-carotene yield to complete the first round of strain recombination iteration;
s02, cutting out URA3 gene by introducing circular plasmid Y-Cre for expressing Cre enzyme to recover the screening marker of the first generation of beta-carotene-producing engineering strain, then introducing linearized pZ2 plasmid again and screening high-yield beta-carotene to complete the second round of recombination iteration;
s03, introducing linearized plasmids pZ3, pZ4, pZ5 and pZ6 in sequence according to the same method in S02 to complete the third, fourth, fifth and sixth rounds of recombination iteration to obtain a recombinant strain integrating pZ1, pZ2, pZ3, pZ4, pZ5 and pZ6.
In a fourth aspect of the present invention, there is provided a recombinant engineering bacterium, having a preservation number of CCTCC NO: m2020606, named ZTQ5320, deposited in CCTCC-China center for type culture Collection in 10 months and 19 days in 2020; address: wuhan, wuhan university (eight-way Lojia mountain in Wuchang region, wuhan city, hubei, post: 430072); the preservation number is CCTCC NO: m2020606, classified and named as Yarrowia lipolytica ZTQ5320 (Yarrowia lipolytica ZTQ 5320).
In a fifth aspect of the present invention, there is provided a method for producing beta-carotene by culturing the cell of the third aspect of the present invention to extract beta-carotene;
or, the beta-carotene is extracted by fermenting the recombinant engineering bacteria of the fourth aspect of the invention.
Specifically, the fermentation method of the recombinant engineering bacteria comprises the following steps:
s1, culturing a primary seed solution;
s2, culturing a secondary seed solution;
and S3, culturing and fermenting by a secondary seed liquid method.
More specifically, the fermentation method of the recombinant engineering bacteria comprises the following steps:
s1, primary seed liquid culture: the recombinant strain F5 single colony on the solid YPD plate is inoculated into a 100ml conical flask containing 20ml YPD liquid culture medium, and cultured for 24-48 hours at 28-32 ℃ and the rotating speed of a shaking table of 180-200 rpm.
S2, culturing a secondary seed solution: transferring the primary seed solution obtained in the step S1 into a 300ml conical flask containing 100ml YPD liquid culture medium according to the inoculation amount of 1% (wt), and culturing at 28-32 ℃ and the rotating speed of a shaking table of 180-200 rpm until the OD600 of the cells reaches 10 to obtain a secondary seed solution.
S3, transferring the secondary seed liquid obtained in the step S2 into a 3L fermentation tank containing 1L of fermentation medium according to the inoculation amount of 10% (wt) for fermentation, wherein the initial medium consists of 30g/L of yeast extract, 60g/L of peptone and 50g/L of glucose, the fermentation temperature is 28-32 ℃, the stirring speed is 300-800rpm, the ventilation volume is more than 2L/min, the pH value is 6.8, and the dissolved oxygen is more than 25%, and the fed-batch fermentation is continued by using 60% of glucose stock solution.
The invention has the beneficial effects that:
the invention provides a nucleic acid molecule comprising a gene related to the beta-carotene synthesis pathway; phosphoketolase pathway-related genes and lipid droplet synthesis pathway-related genes, which cover the synthesis pathway of beta-carotene; and increases the phosphoketolase approach, reduces the carbon loss, and leads the carbon source to generate more precursor substance acetyl coenzyme A for synthesizing the beta-carotene; the supply of NADPH as a reducing substance and ATP as an energy substance is increased; and the lipid drop synthesis path is increased, and the storage of beta-carotene can be increased by increasing the content and volume of intracellular lipid drops.
The invention provides an expression system, which comprises a beta-carotene synthesis pathway related gene; the nucleic acid molecules of the phosphoketolase pathway related gene and the lipid droplet synthesis pathway related gene cover the synthesis pathway of beta-carotene; and increases the phosphoketolase approach, reduces the carbon loss, and leads the carbon source to generate more precursor substance acetyl coenzyme A for synthesizing the beta-carotene; the supply of NADPH as a reducing substance and ATP as an energy substance is increased; and lipid drop synthesis paths are increased, and the storage of beta-carotene can be increased by increasing the content and volume of intracellular lipid drops.
The present invention provides a cell, in particular a yeast cell, comprising a gene involved in the beta-carotene synthesis pathway; the expression system of the phosphoketolase pathway related gene and the lipid droplet synthesis pathway related gene can complete the synthesis of beta-carotene in cells, and in addition, the increased phosphoketolase pathway can reduce the carbon loss and enable a carbon source to generate precursor material acetyl coenzyme A for synthesizing the beta-carotene more; the supply of NADPH as a reducing substance and ATP as an energy substance is also increased; and the lipid drop synthesis way is increased, and the intracellular storage of the beta-carotene can be improved by improving the content and the volume of intracellular lipid drops.
The invention provides a recombinant engineering bacterium, the preservation number is CCTCC NO: m2020606, named ZTQ5320, deposited in CCTCC-China center for type culture Collection in 10 months and 19 days in 2020; address: wuhan, wuhan university (eight-way Lojia mountain in Wuchang region, wuhan city, hubei, post: 430072); the preservation number is CCTCC NO: m2020606, classified and named Yarrowia lipolytica ZTQ5320 (Yarrowia lipolytica ZTQ 5320). Contains genes related to the synthesis pathway of beta-carotene; an expression system of phosphoketolase pathway-related genes and lipid droplet synthesis pathway-related genes, which can improve the yield of beta-carotene by various methods from raw material supply, synthesis pathway and storage pathway.
The present invention also provides a method for producing beta-carotene by culturing/fermenting a microorganism containing a gene related to the synthetic pathway of beta-carotene; the cells or recombinant engineering bacteria of the expression system of the phosphoketolase pathway related gene and the lipid droplet synthesis pathway related gene obviously improve the fermentation yield of the beta-carotene stream supplemented material to 7.8g/L, and have good industrial application prospect.
Drawings
FIG. 1 is a schematic diagram of the phosphoketolase pathway.
FIG. 2 is a schematic diagram of the intracellular lipid droplet synthesis pathway.
FIG. 3 schematic representation of plasmid pZ1.
FIG. 4 is a schematic diagram of plasmid pZ2.
FIG. 5 is a schematic diagram of plasmid pZ3.
FIG. 6 schematic representation of plasmid pZ4.
FIG. 7 is a schematic diagram of plasmid pZ5.
FIG. 8 is a schematic diagram of plasmid pZ6.
FIG. 9 is a graph of the productivity of the strains with the highest beta-carotene productivity per iteration.
FIG. 10 is a graph of beta-carotene fermentation yield for strains that incorporate the beta-carotene pathway and the phosphoketolase pathway as well as the phosphoketolase pathway plus lipid drop pathway.
Detailed Description
The present invention will be described in further detail with reference to the following specific embodiments and accompanying drawings. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.
Yarrowia lipolytica strain was Polf (ATCC accession number MAY-2613; genotype MATA ura3-302 leu2-270 XPR-322axp2-deltaNU49 XPR2:: SUC2, available from ATCC), restriction enzymes used were purchased from ThermoFisher, kits for plasmid extraction and gel recovery were purchased from OMEGA, USA, and reagents for PCR and seamless cloning, E.coli for molecular cloning (DH 5. Alpha.) were purchased from Nanjing Novowed Biotech, inc., and the procedures were performed according to the product instructions.
The method for measuring the beta-carotene comprises the following steps:
(1) 1mL of the suspension was centrifuged to remove the supernatant, and the pellet was resuspended in 0.7mL of DMSO.
(2) After incubation at 55 ℃ for 10min, an equal volume of 0.7mL of acetone was added.
(3) Incubate at 45 ℃ for 15min, and centrifuge at 12000rpm for 5min.
(4) The supernatant was transferred to a new EP tube and the carotene production was measured by HPLC at 450nm (standard curve of acetone dissolved standards).
Example 1 Gene selection and vector construction
acetyl-CoA is an important precursor substance for the microbial synthesis of fatty acids, organic acids and terpenes, but most microbial strains naturally synthesize acetyl-CoA in a way that results in the loss of carbon. Phosphoketolase (PK) and Phosphoacetyltransferase (PTA) of the phosphoketolase pathway (as shown in FIG. 1) catalyze the formation of acetyl-CoA from fructose-6-phosphate (F6P) or xylulose-5-phosphate (X5P), without carbon dioxide formation, carbon loss, ATP production and NADPH production, providing energy and reducing power for cellular anabolism.
Lipid Droplets (LDs) are an organelle that are widely present in cells and are surrounded by a single phospholipid membrane that coats neutral lipids. The lipid droplets are formed by that malonyl-CoA is generated under the catalysis of ACC1 by acetyl-CoA, the malonyl-CoA is generated under the action of FAS by the malonyl-CoA, palmitoyl-CoA and stearoyl-CoA are generated under the action of SCD by the two saturated fatty acids, triglyceride is generated by acyl conversion of DGA1 and enters the lipid droplets, and the lipid droplets are fused to generate large lipid droplets, as shown in figure 2. In order to reduce the damage of hydrophobic carotenoids such as lycopene, beta-carotene and astaxanthin to cells, the microorganism strains store the hydrophobic carotenoids in lipid droplets of the cells,
therefore, the inventor thinks of increasing the content and volume of lipid droplets to improve the intracellular storage of hydrophobic compounds, thereby achieving the purpose of increasing the yield of beta-carotene.
The inventor screens out genes related to a beta-carotene synthesis pathway, a phosphoketolase pathway and a lipid droplet synthesis pathway.
The beta-carotene pathway related genes comprise (1) a gene AtoB catalyzing condensation of acetyl coenzyme A to acetyl coenzyme A, (2) a gene HMGS catalyzing condensation of acetyl coenzyme A and acetyl coenzyme A to HMG-CoA, (3) a gene tHMGR catalyzing reduction of HMG-CoA to mevalonate, (4) a gene ERG12 catalyzing phosphorylation of mevalonate to mevalonate-5-phosphate, (5) a gene ERG8 catalyzing formation of mevalonate-5-phosphate to mevalonate-5-pyrophosphate, (6) a gene ERG19 catalyzing decarboxylation of mevalonate-5-pyrophosphate to isoprene pyrophosphate, (7) a gene IDI catalyzing isomerization of isoprene pyrophosphate to dimethylallyl pyrophosphate, (8) a gene ERG20 catalyzing isomerization of isopentenyl pyrophosphate and dimethylallyl pyrophosphate to farnesyl pyrophosphate, (9) a gene ERP catalyzing combination of farnesyl pyrophosphate and a molecule of prenyl pyrophosphate to geranyl pyrophosphate, GGS1, (10) a gene CarB catalyzing dehydrogenation of lycopene to lycopene, and (11) a gene CarB catalyzing formation of diglygenin to lycopene and cyclization of lycopene.
The beta-carotene pathway-related genes are preferably atoB derived from Escherichia coli BL21 (DE 3) (CP 001509.3: 2216470-2217654), HMGS derived from yarrowia lipolytica W29 (CP 028459.1: 3780129-3781469), tHMGR (CP 028452.1: 571078-572577), ERG12 (CP 028449.1: 2082357-2083706), ERG8 (CP 028452.1: 748052-746796), ERG19 (CP 028453.1: 818770-819933), IDI (CP 453.1: 585662-584850), ERG20 (CP 028452.1: 673068-672034), GGS1 (CP 451.1: 2085775-2084720392), and CarB (K02817.0289717) and 0289718) derived from Mucor circinelloides.
In previous studies by the team of the inventors, it was found that CarB and CarRP derived from Mucor circinelloides were more catalytically efficient than CarB and CarRA derived from Blakeslea trispora by comparing the yield of beta-carotene by shake flask culture, and thus CarB and CarRP derived from Mucor circinelloides were selected.
The phosphoketolase pathway related genes include (1) gene PK catalyzing fructose 6 phosphate or xylulose 5 phosphate to generate acetyl phosphate and (2) gene PTA catalyzing acetyl phosphate to generate acetyl coenzyme A.
The phosphoketolase pathway-related gene is preferably selected from PK (CP 000414.1: 1936622-1934181) derived from Leuconostoc mesenteroides and PTA (CP 018335.1: 1695414-1696415) derived from Clostridium kluyveri.
The inventors' group found in previous studies that the phosphoketolase pathway of the combination could maximally increase beta-carrot yield by comparing beta-carrot yield in shake flask culture, and therefore selected the above combination.
The genes related to the lipid droplet synthesis pathway include (1) acetyl-CoA acidifying enzyme gene ACC1, (2) diacylglycerol acyltransferase gene DAG1, (3) delta-9 stearoyl-CoA desaturase gene SCD, and (4) fat storage-inducing transmembrane protein gene FIT mediating lipid droplet fusion.
The lipid droplet pathway-associated genes are preferably ACC1 (CP 028450.1: 1555944-1562432), DGA1 (CP 028452.1: 3883639-3885183), SCD (CP 028450.1: 745375-746823) and FIT (CP 028450.1: 3104019-3105197) derived from yarrowia lipolytica W29.
The genes from yarrowia lipolytica ACC1, DGA1, SCD and FIT are selected, so that the content of lipid droplets and the storage amount of beta-carotene in yarrowia lipolytica can be increased to the maximum.
Primers were designed to clone the genes described above, and the primer sequences are shown in Table 1.
TABLE 1 primer List
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Plasmids pZ1, pZ2, pZ3, pZ4, pZ5, and pZ6 were constructed.
To ensure that the individual genes can be expressed efficiently in vectors or recombinant cells, a promoter sequence is added upstream and a terminator sequence is added downstream of each gene. Promoter sequences selected for use include, but are not limited to: pTEF, pEXP, pFBA and pGPD.
Plasmid pZ1 takes pUC19 as a skeleton vector and contains CarB, carRP and GGS1 genes, promoters are pTEF, pEXP and pFBA respectively, and terminators are CYC1, tICL and tLIP respectively; the sequence of pZ1 without a skeleton vector is shown in SEQ ID NO.55, and the plasmid map is shown in figure 3.
Plasmid pZ2 takes pUC19 as a skeleton vector, contains AtoB, HMGS, HMGR and ERG12 genes, the promoters are pTEF, pGPD, pFBA and pEXP respectively, and the terminators are CYC1, tXPR2, tLIP and tICL respectively; the sequence of pZ2 without the framework vector is shown in SEQ ID NO. 56; the plasmid map is shown in FIG. 4.
Plasmid pZ3 takes pUC19 as a skeleton vector and contains ERG8, ERG19, ERG20 and IDI genes, promoters are pGPD, pTEF, pFBBA and pEXP respectively, and terminators are tXPR2, CYC1, tLIP and tICL respectively; the pZ3 does not contain a framework vector has a sequence shown in SEQ ID NO. 57; the plasmid map is shown in FIG. 5.
Plasmid pZ4 takes pUC19 as a skeleton vector and contains PK and PTA genes, promoters are pTEF and pEXP respectively, and terminators are CYC1 and tXPR2 respectively; the sequence of pZ4 without the framework vector is shown as SEQ ID NO. 58; the plasmid map is shown in FIG. 6.
Plasmid pZ5 uses pUC19 as a skeleton vector, contains ACC1 gene, a promoter is pFBA, and a terminator is CYC1; the pZ5 does not contain a framework vector and has a sequence shown as SEQ ID NO. 59; the plasmid map is shown in FIG. 7.
Plasmid pZ6 takes pUC19 as a skeleton vector and contains DGA1, SCD and FIT genes, promoters are pTEF, pEXP and pFBA respectively, and terminators are CYC1, tICL and tLIP respectively; the pZ6 does not contain a framework vector and has a sequence shown as SEQ ID NO. 60; the plasmid map is shown in FIG. 8.
Plasmid pZ1, plasmid pZ2, plasmid pZ3, plasmid pZ4, plasmid pZ5 and plasmid pZ6 each contain a URA3 uracil deficiency selection gene (SEQ ID NO. 61), and the upstream and downstream sides of the URA3 gene are inserted with a loxP sequence in the same direction, wherein the upstream loxP base motif is 5' ATCGTTCGGATAACTCCTGCATATACGAAGTTAT-.
Specifically, plasmid backbone pUC19URA3 construction
Amplifying URA3 gene by using a primer loxP-URA3-F/loxP-URA3-R and yarrowia lipolytica Polf genome DNA as a template, and amplifying URA3 gene fragment by using a primer pUC19-URA3-F/pUC19-URA3-R and a PCR product as a template; plasmid pUC19 was digested simultaneously with Nde I and Kpn I, and the URA3 gene fragment was cloned into pUC19 between Nde I and Kpn I by using a seamless Cloning (Novozan Clonexpress Ultra One Step Cloning Kit) to construct plasmid pUC19URA3.
Plasmid pZ1 construction
Respectively amplifying pTEF, pEXP, pFBA, tICL and tLIP fragments by using primers pUC19-pTEF-F/pTEF-R, pEXP-F/pEXP-R, pFBA-F/pFBA-tICL-R, tLIP-F/pUC19-tLIP-R and using yarrowia lipolytica Polf genome DNA as a template; and amplifying the CYC1 fragment by using the primer CYC1-F/pEXP-CYC1-R and the Saccharomyces cerevisiae CEN. PK2-1C genomic DNA as a template. The CarB gene and the CarRP gene from mucor circinelloides (Rhizomucor circinelloides) are synthesized by codon optimization of Nanjing Kinshire technology GmbH, and the primers pTEF-CarB-F/tCYC1-CarB-R and pEXP-CarRP-F/tICL-CarRP-R are respectively used for amplifying the CarB and CarRP fragments by taking the synthesized gene as a template; GGS1 fragments were amplified using primers pFBA-GGS1-F/tLIP-GGS1-R and yarrowia lipolytica genomic DNA as a template.
Splicing a promoter, a gene and a terminator into pTEF-CarB-CYC1, pEXP-CarRP-tICL and pFBA-GGS1-tLIP expression frames by using overlap PCR, carrying out double enzyme digestion, purification and recovery on a plasmid framework pUC19URA3 by using BamHI and Hind III, and assembling the enzyme digestion plasmid framework and the three expression frames by using seamless Cloning (Novozan Clonexpress Ultra One Step Cloning Kit) to construct a plasmid pZ1.
Plasmid pZ2 construction
Amplifying pEXP, pFBA, pGPD, pTEF, tICL, tLIP, tXPR 2-F/tXPR, pXP-ERG 12-F/tICL-ERG12-R, pFBA-HMGR-F/tLIP-R and pGPD-HMGS-F/tXPR 2-R fragments with the genomic DNA of yarrowia lipolytica Polf as a template, pFBA, pGPD, pTEF, tICL, tLIP, tXPR2, tXPG 12, HMGR and HMGS fragments with primers pUC19-pEXP-F/pEXP-R, pTEF-F/pEXP-R, pFBBA-F/pFBA-R, pFBBA-T-F/tICL-R, tLIP-F/pFBA-R, pGPD-R, pGGS-R12, HMGR and HMGS respectively; amplifying a CYC1 fragment by using a primer CYC1-F/pUC19-CYC1-R and saccharomyces cerevisiae CEN. PK2-1C genome DNA as a template; the primer pTEF-AtoB-F/CYC1-AtoB-R is used for amplifying the AtoB fragment by taking the genome DNA of escherichia coli BL21 (DE 3) as a template.
Splicing a promoter, a gene and a terminator into four expression frames of pEXP-ERG12-tICL, pFBA-HMGR-tLIP, pGPD-HMGS-tXPR2 and pTEF-AtoB-CYC1 by overlap PCR, carrying out double enzyme digestion purification and recovery on a plasmid framework pUC19URA3 by using BamHI and Hind III, and assembling the enzyme digestion plasmid framework and the four expression frames by using seamless Cloning (Xinnop ClonExpress Ultra Step Cloning Kit) to construct a plasmid pZ2.
Plasmid pZ3 construction
Amplifying fragments of pGPD-F/pGPD-R, pGPD-ERG8-F/tXPR2-ERG8-R, tXPR2-F/pTEF-tXPR2-R, pTEF-F/pTEF-R, pTEF-ERG19-F/CYC1-ERG19-R, pEXP-F/pEXP-R, pEXP-IDI-F/tICL-IDI-R, tICL-F/pFBA-tICL-R, pFBA-F/pFBA-R, pFBA-ERG20-F/tLIP-ERG20-R and tLIP-F/pUCp 19-tLIP-R with yara yarrowia lipolytica Polf genomic DNA as a template, ERPD, ERG8, tXPR2, pEEF, pEXP, ERG19, XP, tICL, pFLA, and tLIP-20, respectively; and amplifying the CYC1 fragment by using the primer CYC1-F/pEXP-CYC1-R and the Saccharomyces cerevisiae CEN. PK2-1C genomic DNA as a template.
The promoter, the gene and the terminator are spliced into four expression frames of pGPD-ERG8-tXPR2, pTEF-ERG19-CYC1, pEXP-IDI-tICL and pFBA-ERG20-tLIP by overlap PCR, the plasmid framework pUC19URA3 is purified and recovered by double enzyme digestion of BamHI and Hind III, and then the enzyme digestion plasmid framework and the four expression frames are assembled by seamless Cloning (Novozan ClonExpress Ultra One Step Cloning Kit) to construct a plasmid pZ3.
Plasmid pZ4 construction
Respectively using primers pUC19-pTEF-F/pTEF-R, pEXP-F/pEXP-R and tXPR2-F/pUC19-tXPR2-R to amplify pTEF, pEXP and tXPR2 fragments by using yarrowia lipolytica Polf genome DNA as a template; amplifying a CYC1 fragment by using a primer CYC1-F/pEXP-CYC1-R and saccharomyces cerevisiae CEN. PK2-1C genomic DNA as a template; PKT derived from Leuconostoc mesenteroides (Leuconostoc mesenteroides) and PTA derived from Clostridium (Clostridium kluyveri) are synthesized by codon optimization of Nanjing Kinsys scientific and technology Co., ltd, and PK and PTA fragments are amplified by using primers pTEF-PK-F/CYC1-PK-R and pEXP-PTA-F/tXPR2-PTA-R respectively and using the synthesized genes as templates.
Splicing the promoter, the gene and the terminator into two expression frames of pTEF-PK-CYC1 and pEXP-PTA-tXPR2 by overlap PCR, carrying out double enzyme digestion and purification on a plasmid skeleton pUC19URA3 by using BamHI and Hind III, recovering, and assembling the enzyme digestion plasmid skeleton and the two expression frames by utilizing seamless Cloning (Novozan Clonexpress Ultra One Step Cloning Kit) to construct a plasmid pZ4.
Plasmid pZ5 construction
The promoter pFBA and ACC1 gene fragment are respectively amplified by using primers pUC 19-pFBA-F/pFBBA-R and pFBA-ACC1-F/CYC1-ACC1-R and yarrowia lipolytica Polf genome DNA as a template, and the terminator CYC1 is amplified by using primers ACC1-CYC1-F/pUC19-CYC1-R and saccharomyces cerevisiae CEN.PK2-1C genome DNA as a template.
Plasmid backbone pUC19URA3 is purified and recovered by double enzyme digestion with BamHI and Hind III, and then the plasmid backbone, promoter pFBA, ACC1 gene CDS region and terminator CYC1 are assembled to construct plasmid pZ5 by using seamless Cloning (Novozan Clonexpress Ultra One Step Cloning Kit).
Plasmid pZ6 construction
Respectively amplifying promoters pTEF, pEXP and pFBA by using primers pUC19-pTEF-F/pTEF-R, pEXP-F/pEXP-R and pFBA-F/pFBA-R and using yarrowia lipolytica Polf genome DNA as a template; amplifying a terminator CYC1 by using a primer CYC1-F/pEXP-CYC1-R and saccharomyces cerevisiae CEN. PK2-1C genomic DNA as a template; the terminators tICL and tLIP were amplified using the primers tICL-F/pFBA-tICL-R and tLIP-F/pUC19-tLIP-R using yarrowia lipolytica Polf genomic DNA as a template; respectively using primers pTEF-DGA1-F/CYC1-DGA1-R, pEXP-SCD-F/tICL-SCD-R and pFBA-FIT-F/tLIP-FIT-R to amplify three gene segments of DGA1, SCD and FIT by using yarrowia lipolytica Polf genome DNA as a template;
the promoter, the gene and the terminator are spliced into pTEF-DGA1-CYC1, pEXP-SCD-tICL and pFBA-FIT-tLIP expression frames by overlap PCR, the plasmid framework pUC19URA3 is purified and recovered by double enzyme digestion of BamHI and Hind III, and the enzyme digested plasmid framework and the three expression frames are assembled to construct a plasmid pZ6 by utilizing seamless Cloning (Novonopsis Clonexpress One Step Cloning Kit).
It will be appreciated by those skilled in the art that the insertion into the genome of said cells by means of non-homologous recombinant organisms is intended to obtain more strains which are likely to be stably inherited, and that the object of the invention can equally well be achieved by transforming only the above-mentioned expression systems.
In addition, compared with a fixed-point embedding mode, non-homologous recombination has the possibility of obtaining a recombinant cell or a recombinant bacterium which is embedded for multiple times and has higher efficiency, so that the production efficiency of the recombinant cell or the recombinant bacterium is further improved.
EXAMPLE 2 construction of high-yield beta-carotene integrating Strain
The recombinant engineering bacteria randomly integrate the plasmid pZ1, the plasmid pZ2, the plasmid pZ3, the plasmid pZ4, the plasmid pZ5 and the plasmid pZ6 into the genome of the yarrowia lipolytica in a non-homologous recombination mode; the method comprises the following steps:
(1) And (3) cutting the original pUC19 skeleton part of the constructed recombinant plasmid pZ1 by using Not I, linearizing, introducing into a uracil synthesis-deficient yarrowia lipolytica strain Polf, and detecting the strain with the highest beta-carotene yield to complete the first round of strain recombination iteration.
(2) And (3) cutting out URA3 gene by introducing circular plasmid Y-Cre for expressing Cre enzyme to recover the selection marker of the first generation of beta-carotene-producing engineering strain, then introducing linearized pZ2 plasmid again and screening high-yield beta-carotene to complete the second round of recombination iteration.
(3) And (3) introducing linearized plasmids pZ3, pZ4, pZ5 and pZ6 in sequence according to the same method in the step (2) to complete the third, fourth, fifth and sixth rounds of recombination iteration to obtain a recombinant bacterium integrating pZ1, pZ2, pZ3, pZ4, pZ5 and pZ6.
Specifically, the method comprises the following steps: plasmid pZ1 is cut by Not I enzyme digestion glue, a fragment containing a beta-carotene synthesis gene is recovered, yarrowia lipolytica Polf is transformed by a lithium acetate method, an SD-URA defect culture medium is coated, after 2-3 days, 60 transformants with the deepest orange red color are selected and inoculated into 10ml of liquid YPD40 culture medium, the culture is carried out for 72 hours at 200rpm and 30 ℃, three samples with the deepest orange red color are screened out from 60 transformants which are subjected to secondary culture, the beta-carotene yield is detected, and the strain ZTQ01 with the highest yield is obtained.
Plasmid pZ2 is cut by Not I enzyme to recover a fragment containing a beta-carotene synthesis gene, yarrowia lipolytica ZTQ01 is transformed by a lithium acetate method, an SD-URA defect culture medium is coated, 60 transformants with the deepest orange red color are selected after 2-3 days and inoculated into 10ml of liquid YPD40 culture medium, the culture is carried out for 72 hours at 200rpm and 30 ℃, three samples with the deepest orange red color are screened out from 60 transformants which are subjected to secondary culture to detect the yield of the beta-carotene, and a strain ZTQ21 with the highest yield is obtained.
Plasmid pZ3 is cut by Not I enzyme to recover a fragment containing a beta-carotene synthesis gene, yarrowia lipolytica ZTQ21 is transformed by a lithium acetate method, an SD-URA defect culture medium is coated, 60 transformants with the deepest orange red color are selected after 2-3 days and inoculated into 10ml of liquid YPD40 culture medium, the culture is carried out for 72 hours at 200rpm and 30 ℃, three samples with the deepest orange red color are screened out from 60 transformants which are subjected to secondary culture to detect the yield of the beta-carotene, and a strain ZTQ211 with the highest yield is obtained.
Plasmid pZ4 is cut by Not I enzyme to recover a fragment containing a beta-carotene synthesis gene, yarrowia lipolytica ZTQ211 is transformed by a lithium acetate method, an SD-URA defect culture medium is coated, 60 transformants with the deepest orange red color are selected after 2-3 days and inoculated into 10ml of liquid YPD40 culture medium, the culture is carried out for 72 hours at 200rpm and 30 ℃, three samples with the deepest orange red color are screened out from 60 transformants which are subjected to secondary culture to detect the yield of the beta-carotene, and a strain ZTQ30 with the highest yield is obtained.
Plasmid pZ5 is cut by Not I enzyme to recover a fragment containing a beta-carotene synthesis gene, yarrowia lipolytica ZTQ30 is transformed by a lithium acetate method, an SD-URA defect culture medium is coated, 60 transformants with the deepest orange red color are selected after 2-3 days and inoculated into 10ml of liquid YPD40 culture medium, the culture is carried out for 72 hours at 200rpm and 30 ℃, three samples with the deepest orange red color are screened out from 60 transformants which are subjected to secondary culture to detect the yield of the beta-carotene, and the strain ZTQ320 with the highest yield is obtained.
Plasmid pZ6 is cut by Not I enzyme to recover a fragment containing a beta-carotene synthesis gene, yarrowia lipolytica ZTQ320 is transformed by a lithium acetate method, an SD-URA defect culture medium is coated, 60 transformants with the deepest orange red color are selected after 2-3 days and inoculated into 10ml of liquid YPD40 culture medium, the culture is carried out for 72 hours at 200rpm and 30 ℃, three samples with the deepest orange red color are screened out from 60 transformants which are subjected to secondary culture to detect the yield of the beta-carotene, and a strain ZTQ5320 with the highest yield is obtained.
The yields of the three strains with the highest yield of beta-carotene obtained by each gene integration iteration are shown in FIG. 9, and finally the strain ZTQ5320 with the highest yield is obtained by screening.
ZTQ5320 is preserved in CCTCC-China center for type culture Collection in 2020, 10 months and 19 days; address: wuhan, wuhan university (eight-way Lojia mountain in Wuchang region, wuhan city, hubei, post: 430072); the preservation number is CCTCC NO: m2020606, classified and named as Yarrowia lipolytica ZTQ5320 (Yarrowia lipolytica ZTQ 5320).
Example 3 Industrial fermentation test
The genetically engineered bacteria ZTQ211, ZTQ30 and ZTQ5320 constructed in example 2 were subjected to high density fermentation in a 3L fermentation tank, and then the yield per unit of beta-carotene was examined.
The fermentation method comprises the following steps:
s1, primary seed liquid culture: a single colony of the recombinant strain F5 on a solid YPD plate was inoculated into a 100ml Erlenmeyer flask containing 20ml of YPD liquid medium, and cultured at 30 ℃ for 48 hours with a shaker rotation speed of 200 rpm.
S2, transferring the primary seed solution obtained in the step S1 into a 300ml conical flask containing 100ml YPD liquid culture medium according to the inoculation amount of 1% (wt), and culturing for 10 hours at the temperature of 30 ℃ at the rotating speed of a shaking table of 200rpm until the cell OD600 reaches 10 to obtain a secondary seed solution.
S3, transferring the secondary seed liquid obtained in the step S2 into a 3L fermentation tank containing 1L of fermentation medium for fermentation according to the inoculation amount of 10 percent (wt), wherein the initial medium consists of 30g/L of yeast extract, 60g/L of peptone and 50g/L of glucose, the fermentation temperature is 30 ℃, the stirring speed is 300-800rpm, the ventilation volume is 2L/min, the pH value is 6.8, and the dissolved oxygen content is more than 25 percent, and the secondary seed liquid is cultured for 12 hours and then fed-batch fermentation is continued for 264 hours by using 60 percent of glucose stock solution.
The results of the unit yield of beta-carotene in 3 genetic engineering bacteria ZTQ211, ZTQ30 and ZTQ5320 are shown in FIG. 10, the yield of the strain ZTQ5320 introduced into a phosphoketolase pathway and a lipid droplet strengthening pathway is increased by 69 percent compared with that of the ZTQ211, the highest yield of the beta-carotene reaches 7.8g/L, and the yield of the beta-carotene is remarkably improved.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> Guangzhou Zhiteqi Biotechnology GmbH
<120> genetic engineering bacterium for improving yield of beta-carotene and application thereof
<130>
<160> 63
<170> PatentIn version 3.5
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atcgcttcgg ataactcctg ctatacgaag ttatacgaat tcgcgcccag agagccattg 60
<210> 2
<211> 63
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ataacttcgt atagcataca tcatacgaag ttattctgaa ttccgagaaa cacaacaaca 60
tgc 63
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<211> 48
<212> DNA
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attgtactga gagtgcacca gcggccgcat cgcttcggat aactcctg 48
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aggtcgactc tagaggatcc ccggataact tcgtatagca tacat 45
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atgtatgcta tacgaagtta tccgggagag accgggttgg cggcg 45
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ctgcggttag tactgcaaaa 20
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actttttgca gtactaaccg cagatttatg tccaagaagc acattgt 47
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gcgtgacata actaattaca tgaattttta gatgacgtta gagttg 46
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tcatgtaatt agttatgtca cgc 23
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cgggcgccaa actccgcgat gcaaattaaa gccttcgagc 40
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ggagtttggc gcccgttttt 20
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tgctgtagat atgtcttgtg 20
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cacaagacat atctacagca tttatgctgc tgacctacat gg 42
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aatatatttt gctaaacaaa ctgcattttt agatggtgtt caggtttcgc 50
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gcagtttgtt tagcaaaata 20
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ccatttattg ttgggtgcag tttaaactgt atgattgatg ttactac 47
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tgcacccaac aataaatggg 20
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ctgggttagt ttgtgtagag 20
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<211> 42
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ctctctacac aaactaaccc agatggatta taacagcgcg ga 42
<210> 20
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gttgtaaaga gtgataaata gcgtcactgc gcatcctcaa agt 43
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gctatttatc actctttaca 20
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ctttttgcag tactaaccgc agatggctga cttcgactct aa 42
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gacataacta attacatgat tacttcaatg gagaccaagt c 41
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cacacaagac atatctacag caatgaagtt gatggaaaac atc 43
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gggacaggcc atggaggtac ttaaccttga gcttgagctt g 41
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tgaccatgat tacgccagcg gccgccatct cacttgcgta tgtat 45
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ctatgaccat gattacgcca gcggccgcga cccttcgtgg gtctcaat 48
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tcggccgatt catttcaacc gcgatgaccc ttcgtgggtc tcaat 45
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tgttgatgtg tgtttaattc 20
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cttgaattaa acacacatca acagatgtcg caaccccaga ac 42
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ggggacaggc catggaggta cgctactgct tgatctcgta ct 42
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gtacctccat ggcctgtccc 20
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ccgccaaccc ggtctctgtt taaaccatct cacttgcgta tgtatgg 47
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gtttaaacag agaccgggtt ggcgg 25
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ctttttgcag tactaaccgc agatttatga agaactgtgt catcgtg 47
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cataactaat tacatgaatt tttagttcag tcgctcaatg acc 43
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tcatgtaatt agttatgtca cgc 23
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tatgaccatg attacgccag cggccgcaaa ttaaagcctt cgagc 45
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<400> 39
tgtatgctat acgaagttat ccgggggttg aaatgaatcg gccga 45
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<400> 40
gaattaaaca cacatcaaca gatgaccacc tattcggctc c 41
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gggacaggcc atggaggtac gctacttgaa ccccttctcg 40
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tgcagtacta accgcagatt tatgatccac caggcctcca c 41
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<400> 43
gacataacta attacatgaa tttctacttg ctgttcttca gag 43
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caagacatat ctacagcatt tatgacgacg tcttacagcg a 41
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<400> 45
tttgctaaac aaactgcatt tctacttgat ccaccgccg 39
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<400> 46
ctctacacaa actaacccag atgtccaagg cgaaattcg 39
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gtaaagagtg ataaatagcg ctacttctgt cgcttgtaaa 40
<210> 48
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<400> 48
gaattaaaca cacatcaaca atgactatcg actcacaata c 41
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<400> 49
gtaaagagtg ataaatagct tactcaatca ttcggaactc 40
<210> 50
<211> 45
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<400> 50
ccgccaaccc ggtctctgtt taaacgaccc ttcgtgggtc tcaat 45
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ctttttgcag tactaaccgc agatggtgaa aaacgtggac ca 42
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cgtgacataa ctaattacat gactaagcag ccatgccaga ca 42
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<211> 40
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ctacacaaac taacccagat gtctagcaaa tggtttaatg 40
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<400> 54
gtaaagagtg ataaatagcg tcatactgga cgtagcgcg 39
<210> 55
<211> 9527
<212> DNA
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<400> 55
ggccgcatcg cttcggataa ctcctgctat acgaagttat acgcgcccag agagccattg 60
acgttctttc taatttggac cgatagccgt atagtccagt ctatctataa gttcaactaa 120
ctcgtaacta ttaccataac atatacttca ctgccccaga taaggttccg ataaaaagtt 180
ctgcagacta aatttatttc agtctcctct tcaccaccaa aatgccctcc tacgaagctc 240
gagctaacgt ccacaagtcc gcctttgccg ctcgagtgct caagctcgtg gcagccaaga 300
aaaccaacct gtgtgcttct ctggatgtta ccaccaccaa ggagctcatt gagcttgccg 360
ataaggtcgg accttatgtg tgcatgatca agacccatat cgacatcatt gacgacttca 420
cctacgccag cactgtgctc cccctcaagg aacttgctct taagcacggt ttcttcctgt 480
tcgaggacag aaagttcgca gatattggca acactgtcaa gcaccagtac aagaacggtg 540
tctaccgaat cgccgagtgg tccgatatca ccaacgccca cggtgtaccc ggaaccggaa 600
tcattgctgg cctgcgagct ggtgccgagg aaactgtctc tgaacagaag aaggaggacg 660
tctctgacta cgagaactcc cagtacaagg agttcctggt cccctctccc aacgagaagc 720
tggccagagg tctgctcatg ctggccgagc tgtcttgcaa gggctctctg gccactggcg 780
agtactccaa gcagaccatt gagcttgccc gatccgaccc cgagtttgtg gttggcttcg 840
ttgcccagaa ccgacctaag ggcgactctg aggactggct tattctgacc cccggggtgg 900
gtcttgacga caagggagac gctctcggac agcagtaccg aactgttgag gatgtcatgt 960
ctaccggaac ggatatcata attgtcggcc gaggtctgta cggccagaac cgagatccta 1020
tcgaggaggc caagcgatac cagaaggctg gctgggaggc ttaccagaag attaactgtt 1080
agaggttaga ctatggatat gtaatttaac tgtgtatata gagagcgtgc aagtatggag 1140
cgcttgttca gcttgtatga tggtcagacg acctgtctga tcgagtatgt atgatactgc 1200
acaacctgtg tatccgcatg atctgtccaa tggggcatgt tgttgtgttt ctcgagaata 1260
acttcgtatg atgtatgcta tacgaagtta tgcgatcgca gagaccgggt tggcggcgta 1320
tttgtgtccc aaaaaacagc cccaattgcc ccaattgacc ccaaattgac ccagtagcgg 1380
gcccaacccc ggcgagagcc cccttcaccc cacatatcaa acctcccccg gttcccacac 1440
ttgccgttaa gggcgtaggg tactgcagtc tggaatctac gcttgttcag actttgtact 1500
agtttctttg tctggccatc cgggtaaccc atgccggacg caaaatagac tactgaaaat 1560
ttttttgctt tgtggttggg actttagcca agggtataaa agaccaccgt ccccgaatta 1620
cctttcctct tcttttctct ctctccttgt caactcacac ccgaaatcgt taagcatttc 1680
cttctgagta taagaatcat tcaaaatggt gagtttcaga ggcagcagca attgccacgg 1740
gctttgagca cacggccggg tgtggtccca ttcccatcga cacaagacgc cacgtcatcc 1800
gaccagcact ttttgcagta ctaaccgcag atttatgtcc aagaagcaca ttgtgatcat 1860
tggcgccggt gtcggcggca ccgctactgc tgctcgactg gctcgagagg gcttcaaggt 1920
gaccgtggtg gagaagaacg acttcggtgg tggtcgatgc tctctgattc accaccaggg 1980
ccaccgattc gaccagggcc cttccctgta cctgatgccc aagtacttcg aggacgcctt 2040
cgccgacctg gacgagcgaa ttcaggacca cctggagctg ctgcgatgcg acaacaacta 2100
caaggtccac ttcgacgacg gtgagtccat tcagctgtcc tctgacctga cccgaatgaa 2160
ggccgagctg gaccgagtcg agggccctct tggcttcggc cgattcctgg acttcatgaa 2220
ggagacccac atccactacg agtccggtac cctgatcgcc ctgaagaaga acttcgagtc 2280
tatctgggac ctgatccgaa tcaagtacgc ccccgagatt ttccgactgc acctgttcgg 2340
caagatctac gaccgagcct ccaagtactt caagaccaag aagatgcgaa tggccttcac 2400
cttccagacc atgtacatgg gtatgtctcc ctacgacgcc cccgccgtct actctctgct 2460
gcagtacacc gagttcgccg agggcatctg gtacccccga ggtggtttca acatggtggt 2520
ccagaagctg gaggccatcg ccaagcagaa gtacgacgcc gagttcatct ataacgcccc 2580
cgtcgccaag atcaacaccg acgacgccac caagcaggtc accggtgtca ccctggagaa 2640
cggtcacatt atcgacgccg acgccgtcgt ctgcaacgcc gatctggtct acgcctacca 2700
caacctgctg cccccctgtc gatggaccca gaacaccctg gcctccaaga agctgacctc 2760
ctcctccatc tctttctact ggtccatgtc caccaaggtc ccccagctgg acgtccacaa 2820
cattttcctg gccgaggcct accaggagtc cttcgacgag atcttcaagg acttcggtct 2880
gccctccgag gcctccttct acgtcaacgt cccctctcga atcgacccct ccgccgctcc 2940
tgacggaaag gactctgtca tcgtcctggt gcccattggc cacatgaagt ctaagaccgg 3000
tgacgcctct accgagaact accccgccat ggtcgacaag gcccgaaaga tggtcctggc 3060
cgtgattgag cgacgactgg gcatgtccaa cttcgccgac ctcatcgagc acgagcaggt 3120
caacgacccc gccgtgtggc agtccaagtt caacctgtgg cgaggttcta ttctgggtct 3180
gtctcacgac gtcctgcagg tgctgtggtt ccgaccctcc accaaggact ccaccggccg 3240
atacgacaac ctgttcttcg tgggcgcctc cacccacccc ggtactggtg ttcccatcgt 3300
cctggccggc tccaagctga cctctgacca ggtggtcaag tctttcggta aaacccccaa 3360
gccccgaaag atcgagatgg agaacaccca ggcccccctg gaggagcctg acgctgagtc 3420
taccttcccc gtctggttct ggctgcgagc cgccttctgg gtcatgttca tgttcttcta 3480
cttcttcccc cagtctaacg gtcagacccc cgcctctttc atcaacaacc tgctgcctga 3540
ggtgttccga gtccacaact ctaacgtcat ctaaaaattc atgtaattag ttatgtcacg 3600
cttacattca cgccctccct ccacatccgc tctaaccgaa aaggaaggag ttagacaacc 3660
tgaagtctag gtccctattt atttttttat agttatgtta gtattaagaa cgttatttat 3720
atttcaaatt tttctttttt ttctgtacag acgcgtgtac gcatgtaaca ttatactgaa 3780
aaccttgctt gagaaggttt tgggacgctc gaaggcttta atttgcatcg cggagtttgg 3840
cgcccgtttt ttcgagcccc acacgtttcg gtgagtatga gcggcggcag attcgagcgt 3900
ttccggtttc cgcggctgga cgagagccca tgatgggggc tcccaccacc agcaatcagg 3960
gccctgatta cacacccacc tgtaatgtca tgctgttcat cgtggttaat gctgctgtgt 4020
gctgtgtgtg tgtgttgttt ggcgctcatt gttgcgttat gcagcgtaca ccacaatatt 4080
ggaagcttat tagcctttct attttttcgt ttgcaaggct taacaacatt gctgtggaga 4140
gggatgggga tatggaggcc gctggaggga gtcggagagg cgttttggag cggcttggcc 4200
tggcgcccag ctcgcgaaac gcacctagga ccctttggca cgccgaaatg tgccactttt 4260
cagtctagta acgccttacc tacgtcattc catgcatgca tgtttgcgcc ttttttccct 4320
tgcccttgat cgccacacag tacagtgcac tgtacagtgg aggttttggg ggggtcttag 4380
atgggagcta aaagcggcct agcggtacac tagtgggatt gtatggagtg gcatggagcc 4440
taggtggagc ctgacaggac gcacgaccgg ctagcccgtg acagacgatg ggtggctcct 4500
gttgtccacc gcgtacaaat gtttgggcca aagtcttgtc agccttgctt gcgaacctaa 4560
ttcccaattt tgtcacttcg cacccccatt gatcgagccc taacccctgc ccatcaggca 4620
atccaattaa gctcgcattg tctgccttgt ttagtttggc tcctgcccgt ttcggcgtcc 4680
acttgcacaa acacaaacaa gcattatata taaggctcgt ctctccctcc caaccacact 4740
cacttttttg cccgtcttcc cttgctaaca caaaagtcaa gaacacaaac aaccacccca 4800
acccccttac acacaagaca tatctacagc atttatgctg ctgacctaca tggaggtcca 4860
cctgtactac accctgcccg tcctgggcgt cctgtcttgg ctgtcccgac cctactacac 4920
cgccaccgac gccctgaagt tcaagttcct gaccctggtg gccttcacca ccgcctccgc 4980
ttgggacaac tacattgtct accacaaggc ctggtcctac tgccccacct gcgtgaccgc 5040
cgtcattggt tacgtgcccc tggaggagta catgttcttc atcattatga ccctgctgac 5100
cgtggccttc actaacctgg tcatgcgatg gcacctgcac tctttcttca tccgacccga 5160
gacccccgtg atgcagtctg tcctggtgcg actggtcccc atcaccgccc tgctgatcac 5220
cgcctacaag gcctggcacc tggccgtccc tggtaaaccc ctgttctacg gctcttgcat 5280
tctgtggtac gcctgccccg tgctggccct tctgtggttc ggcgctggcg agtacatgat 5340
gcgacgaccc ctggccgtcc tggtgtctat tgccctgccc accctgttcc tgtgctgggt 5400
cgacgtggtc gccattggcg ccggaacctg ggacatctcc ctggctacct ccaccggcaa 5460
gttcgtggtg ccccacctgc ccgtggagga gttcatgttc ttcgccctga tcaacaccgt 5520
gctggtgttc ggtacctgcg ccatcgaccg aaccatggcc attctgcacc tgttcaagaa 5580
caagtccccc taccagcgac cctaccagca ctctaagtcc ttcctgcacc agatcctgga 5640
gatgacctgg gccttctgtc tgcccgacca ggtcctgcac tctgacacct tccacgacct 5700
gtccgtctct tgggacatcc tgcgaaaggc ctccaagtct ttctacaccg cctctgccgt 5760
cttccccggc gacgttcgac aggagctggg tgtcctgtac gccttctgcc gagccaccga 5820
cgacctgtgc gacaacgagc aggtgcccgt ccagacccga aaggagcagc tgatcctgac 5880
ccaccagttc gtctccgacc tgttcggcca gaagacctcc gcccccaccg ctattgactg 5940
ggacttctac aacgaccagc tgcccgcctc ctgcatttcc gccttcaagt ccttcacccg 6000
actgcgacac gtcctggagg ccggagctat taaggagctg ctggacggtt acaagtggga 6060
cctggagcga cgatccattc gagaccagga ggacctgcga tactactccg cctgcgtggc 6120
ctcctctgtc ggcgagatgt gcacccgaat cattctggcc cacgccgaca agcccgcctc 6180
ccgacagcag actcagtgga tcatccagcg agcccgagag atgggtctgg tcctgcagta 6240
caccaacatc gcccgagaca ttgtcaccga ctccgaggag ctgggccgat gttacctgcc 6300
ccaggactgg ctgaccgaga aggaggtggc cctgatccag ggcggtctgg ctcgagagat 6360
tggcgaggag cgactgctgt ctctgtctca ccgactgatc taccaggccg acgagctgat 6420
ggtcgtcgcc aacaagggca ttgacaagct gccctcccac tgccagggtg gcgtgcgagc 6480
tgcttgcaac gtctacgcct ccatcggcac caagctgaag tcctacaagc accactaccc 6540
ctcccgagcc cacgtgggaa actctaagcg agtggagatc gccctgctgt ctgtctacaa 6600
cctgtacacc gcccccattg ccacctcttc caccacccac tgtcgacagg gtaaaatgcg 6660
aaacctgaac accatctaaa aatgcagttt gtttagcaaa atatatttaa cgagtttgat 6720
agaggcgctg gactacataa ttactgaatc acgcgtacat gtttcagctc aaattgtatc 6780
acggtttctt tgtagcaatg gagggggaga gttgacaagg cattagagaa gagagcgaga 6840
ggagaagaca agtggataga cgactgcaat catatgatct gcacaaactg cgatgttttc 6900
ctgtcagatc atgttctttt gctcatagtt aagctatcgt gactttacgg atccgccgag 6960
catcttagta gcgaggtttg cggtctgggc tgatcggctt ttgttgatcg ggttcggaac 7020
gaattagaag tgggtcgata gcaatgatca attcggggtg agtgagtgcg gttagtggga 7080
aaccggggag atactgtggg gatttagggg acagtgttaa gaagaggaag gggtgaggga 7140
ggtgaggtga gggagaggga agtatagagg tttatgtagt aacatcaatc atacagttta 7200
aactgcaccc aacaataaat gggtagggtt gcaccaacaa agggatggga tggggggtag 7260
aagatacgag gataacgggg ctcaatggca caaataagaa cgaatactgc cattaagact 7320
cgtgatccag cgactgacac cattgcatca tctaagggcc tcaaaactac ctcggaactg 7380
ctgcgctgat ctggacacca cagaggttcc gagcacttta ggttgcacca aatgtcccac 7440
caggtgcagg cagaaaacgc tggaacagcg tgtacagttt gtcttagcaa aaagtgaagg 7500
cgctgaggtc gagcagggtg gtgtgacttg ttatagcctt tagagctgcg aaagcgcgta 7560
tggatttggc tcatcaggcc agattgaggg tctgtggaca catgtcatgt tagtgtactt 7620
caatcgcccc ctggatatag ccccgacaat aggccgtggc ctcatttttt tgccttccgc 7680
acatttccat tgctcggtac ccacaccttg cttctcctgc acttgccaac cttaatactg 7740
gtttacattg accaacatct tacaagcggg gggcttgtct agggtatata taaacagtgg 7800
ctctcccaat cggttgccag tctctttttt cctttctttc cccacagatt cgaaatctaa 7860
actacacatc acacaatgcc tgttactgac gtccttaagc gaaagtccgg tgtcatcgtc 7920
ggcgacgatg tccgagccgt gagtatccac gacaagatca gtgtcgagac gacgcgtttt 7980
gtgtaatgac acaatccgaa agtcgctagc aacacacact ctctacacaa actaacccag 8040
atggattata acagcgcgga tttcaaggag atatggggca aggccgccga caccgcgctg 8100
ctgggaccgt acaactacct cgccaacaac cggggccaca acatcagaga acacttgatc 8160
gcagcgttcg gagcggttat caaggtggac aagagcgatc tcgagaccat ttcgcacatc 8220
accaagattt tgcataactc gtcgctgctt gttgatgacg tggaagacaa ctcgatgctc 8280
cgacgaggcc tgccggcagc ccattgtctg tttggagtcc cccaaaccat caactccgcc 8340
aactacatgt actttgtggc tctgcaggag gtgctcaagc tcaagtctta tgatgccgtc 8400
tccattttca ccgaggaaat gatcaacttg catagaggtc agggtatgga tctctactgg 8460
agagaaacac tcacttgccc ctcggaagac gagtatctgg agatggtggt gcacaagacc 8520
ggtggactgt ttcggctggc tctgagactt atgctgtcgg tggcatcgaa acaggaggac 8580
catgaaaaga tcaactttga tctcacacac cttaccgaca cactgggagt catttaccag 8640
attctggatg attacctcaa cctgcagtcc acggaattga ccgagaacaa gggattctgc 8700
gaagatatca gcgaaggaaa gttttcgttt ccgctgattc acagcatacg caccaacccg 8760
gataaccacg agattctcaa cattctcaaa cagcgaacaa gcgacgcttc actcaaaaag 8820
tacgccgtgg actacatgag aacagaaacc aagagtttcg actactgcct caagaggata 8880
caggccatgt cactcaaggc aagttcgtac attgatgatc tagcagcagc tggccacgat 8940
gtctccaagc tacgagccat tttgcattat tttgtgtcca cctctgactg tgaggagaga 9000
aagtactttg aggatgcgca gtgacgctat ttatcactct ttacaacttc tacctcaact 9060
atctacttta ataaatgaat atcgtttatt ctctatgatt actgtatatg cgttcctcta 9120
agacaaatcg aaaccagcat gcgatcgaat ggcatacaaa agtttcttcc gaagttgatc 9180
aatgtcctga tagtcaggca gcttgagaag attgacacag gtggaggccg tagggaaccg 9240
atcaacctgt ctaccagcgt tacgaatggc aaatgacggg ttcaaagcct tgaatccttg 9300
caatggtgcc ttggatactg atgtcacaaa cttaagaagc agccgcttgt cctcttcctc 9360
gaaactctca aacacagtcc agaggtcctt tatagcttga tctgtatcca gatagcctcc 9420
gtaattggtg tgtgtcttca aatcccagac gtccacattg gcatgtcctc cactgataag 9480
catttgaagt tcatctgcgt tgaacattga gacccacgaa gggtcgc 9527
<210> 56
<211> 11544
<212> DNA
<213> Artificial sequence
<400> 56
ggccgcatcg cttcggataa ctcctgctat acgaagttat acgaattcgc gcccagagag 60
ccattgacgt tctttctaat ttggaccgat agccgtatag tccagtctat ctataagttc 120
aactaactcg taactattac cataacatat acttcactgc cccagataag gttccgataa 180
aaagttctgc agactaaatt tatttcagtc tcctcttcac caccaaaatg ccctcctacg 240
aagctcgagc taacgtccac aagtccgcct ttgccgctcg agtgctcaag ctcgtggcag 300
ccaagaaaac caacctgtgt gcttctctgg atgttaccac caccaaggag ctcattgagc 360
ttgccgataa ggtcggacct tatgtgtgca tgatcaagac ccatatcgac atcattgacg 420
acttcaccta cgccagcact gtgctccccc tcaaggaact tgctcttaag cacggtttct 480
tcctgttcga ggacagaaag ttcgcagata ttggcaacac tgtcaagcac cagtacaaga 540
acggtgtcta ccgaatcgcc gagtggtccg atatcaccaa cgcccacggt gtacccggaa 600
ccggaatcat tgctggcctg cgagctggtg ccgaggaaac tgtctctgaa cagaagaagg 660
aggacgtctc tgactacgag aactcccagt acaaggagtt cctggtcccc tctcccaacg 720
agaagctggc cagaggtctg ctcatgctgg ccgagctgtc ttgcaagggc tctctggcca 780
ctggcgagta ctccaagcag accattgagc ttgcccgatc cgaccccgag tttgtggttg 840
gcttcgttgc ccagaaccga cctaagggcg actctgagga ctggcttatt ctgacccccg 900
gggtgggtct tgacgacaag ggagacgctc tcggacagca gtaccgaact gttgaggatg 960
tcatgtctac cggaacggat atcataattg tcggccgagg tctgtacggc cagaaccgag 1020
atcctatcga ggaggccaag cgataccaga aggctggctg ggaggcttac cagaagatta 1080
actgttagag gttagactat ggatatgtaa tttaactgtg tatatagaga gcgtgcaagt 1140
atggagcgct tgttcagctt gtatgatggt cagacgacct gtctgatcga gtatgtatga 1200
tactgcacaa cctgtgtatc cgcatgatct gtccaatggg gcatgttgtt gtgtttctcg 1260
gaattcagaa taacttcgta tgatgtatgc tatacgaagt tatccggggg agtttggcgc 1320
ccgttttttc gagccccaca cgtttcggtg agtatgagcg gcggcagatt cgagcgtttc 1380
cggtttccgc ggctggacga gagcccatga tgggggctcc caccaccagc aatcagggcc 1440
ctgattacac acccacctgt aatgtcatgc tgttcatcgt ggttaatgct gctgtgtgct 1500
gtgtgtgtgt gttgtttggc gctcattgtt gcgttatgca gcgtacacca caatattgga 1560
agcttattag cctttctatt ttttcgtttg caaggcttaa caacattgct gtggagaggg 1620
atggggatat ggaggccgct ggagggagtc ggagaggcgt tttggagcgg cttggcctgg 1680
cgcccagctc gcgaaacgca cctaggaccc tttggcacgc cgaaatgtgc cacttttcag 1740
tctagtaacg ccttacctac gtcattccat gcatgcatgt ttgcgccttt tttcccttgc 1800
ccttgatcgc cacacagtac agtgcactgt acagtggagg ttttgggggg gtcttagatg 1860
ggagctaaaa gcggcctagc ggtacactag tgggattgta tggagtggca tggagcctag 1920
gtggagcctg acaggacgca cgaccggcta gcccgtgaca gacgatgggt ggctcctgtt 1980
gtccaccgcg tacaaatgtt tgggccaaag tcttgtcagc cttgcttgcg aacctaattc 2040
ccaattttgt cacttcgcac ccccattgat cgagccctaa cccctgccca tcaggcaatc 2100
caattaagct cgcattgtct gccttgttta gtttggctcc tgcccgtttc ggcgtccact 2160
tgcacaaaca caaacaagca ttatatataa ggctcgtctc tccctcccaa ccacactcac 2220
ttttttgccc gtcttccctt gctaacacaa aagtcaagaa cacaaacaac caccccaacc 2280
cccttacaca caagacatat ctacagcatt tatggactac atcatttcgg cgccaggcaa 2340
agtgattcta tttggtgaac atgccgctgt gtttggtaag cctgcgattg cagcagccat 2400
cgacttgcga acatacctgc ttgtcgaaac cacaacatcc gacaccccga cagtcacgtt 2460
ggagtttcca gacatccact tgaacttcaa ggtccaggtg gacaagctgg catctctcac 2520
agcccagacc aaggccgacc atctcaattg gtcgactccc aaaactctgg ataagcacat 2580
tttcgacagc ttgtctagct tggcgcttct ggaagaacct gggctcacta aggtccagca 2640
ggccgctgtt gtgtcgttct tgtacctcta catccaccta tgtccccctt ctgtgtgcga 2700
agattcatca aactgggtag ttcgatcaac gctgcctatc ggcgcgggcc tgggctcttc 2760
cgcatccatt tgtgtctgtt tggctgcagg tcttctggtt ctcaacggcc agctgagcat 2820
tgaccaggca agagatttca agtccctgac cgagaagcag ctgtctctgg tggacgactg 2880
gtccttcgtc ggtgaaatgt gcattcacgg caacccgtcg ggcatcgaca atgctgtggc 2940
tactcaggga ggtgctctgt tgttccagcg acctaacaac cgagtccctc ttgttgacat 3000
tcccgagatg aagctgctgc ttaccaatac gaagcatcct cgatctaccg cagacctggt 3060
tggtggagtc ggagttctca ctaaagagtt tggctccatc atggatccca tcatgacttc 3120
agtaggcgag atttccaacc aggccatgga gatcatttct agaggcaaga agatggtgga 3180
ccagtctaac cttgagattg agcagggtat cttgcctcaa cccacctctg aggatgcctg 3240
caacgtgatg gaagatggag ctactcttca aaagttgaga gatatcggtt cggaaatgca 3300
gcatctagtg agaatcaatc acggcctgct tatcgctatg ggtgtttccc acccgaagct 3360
cgaaatcatt cgaactgcct ccattgtcca caacctgggt gagaccaagc tcactggtgc 3420
tggaggagga ggttgcgcca tcactctagt cacttctaaa gacaagactg cgacccagct 3480
ggaggaaaat gtcattgctt tcacagagga gatggctacc catggcttcg aggtgcacga 3540
gactactatt ggtgccagag gagttggtat gtgcattgac catccctctc tcaagactgt 3600
tgaagccttc aagaaggtgg agcgggcgga tctcaaaaac atcggtccct ggacccatta 3660
gaaatgcagt ttgtttagca aaatatattt aacgagtttg atagaggcgc tggactacat 3720
aattactgaa tcacgcgtac atgtttcagc tcaaattgta tcacggtttc tttgtagcaa 3780
tggaggggga gagttgacaa ggcattagag aagagagcga gaggagaaga caagtggata 3840
gacgactgca atcatatgat ctgcacaaac tgcgatgttt tcctgtcaga tcatgttctt 3900
ttgctcatag ttaagctatc gtgactttac ggatccgccg agcatcttag tagcgaggtt 3960
tgcggtctgg gctgatcggc ttttgttgat cgggttcgga acgaattaga agtgggtcga 4020
tagcaatgat caattcgggg tgagtgagtg cggttagtgg gaaaccgggg agatactgtg 4080
gggatttagg ggacagtgtt aagaagagga aggggtgagg gaggtgaggt gagggagagg 4140
gaagtataga ggtttatgta gtaacatcaa tcatacagtt taaactgcac ccaacaataa 4200
atgggtaggg ttgcaccaac aaagggatgg gatggggggt agaagatacg aggataacgg 4260
ggctcaatgg cacaaataag aacgaatact gccattaaga ctcgtgatcc agcgactgac 4320
accattgcat catctaaggg cctcaaaact acctcggaac tgctgcgctg atctggacac 4380
cacagaggtt ccgagcactt taggttgcac caaatgtccc accaggtgca ggcagaaaac 4440
gctggaacag cgtgtacagt ttgtcttagc aaaaagtgaa ggcgctgagg tcgagcaggg 4500
tggtgtgact tgttatagcc tttagagctg cgaaagcgcg tatggatttg gctcatcagg 4560
ccagattgag ggtctgtgga cacatgtcat gttagtgtac ttcaatcgcc ccctggatat 4620
agccccgaca ataggccgtg gcctcatttt tttgccttcc gcacatttcc attgctcggt 4680
acccacacct tgcttctcct gcacttgcca accttaatac tggtttacat tgaccaacat 4740
cttacaagcg gggggcttgt ctagggtata tataaacagt ggctctccca atcggttgcc 4800
agtctctttt ttcctttctt tccccacaga ttcgaaatct aaactacaca tcacacaatg 4860
cctgttactg acgtccttaa gcgaaagtcc ggtgtcatcg tcggcgacga tgtccgagcc 4920
gtgagtatcc acgacaagat cagtgtcgag acgacgcgtt ttgtgtaatg acacaatccg 4980
aaagtcgcta gcaacacaca ctctctacac aaactaaccc aggtgtctac cgacgccaag 5040
aactctcgaa tttctggttt ccacaaggac gacatcccca cccgactggc ccgagtcgcc 5100
gctttcgccg gtctggacga cgagaccgtg cagcacctgg ccaacatggg taacctggac 5160
ccccagctgg ccgaccgact gattgagaac gtggtggcca ccctgaacgt ccccattggc 5220
atcgccacca acatgaaggt cgacggtgag gacgtgctgg tccccatggc caccgaggag 5280
tcctccgtcg tggccgctgt gtgcaacgcc gcccgacagt gttacgacca gggcggtttc 5340
accacctcta tgtccggttc cctgatgatt gcccaggtcc agctggtcga cgtccccgac 5400
gccgctcacg ctcgaatgcg aattctggag cacaaggccg aggtcaaggc cctgtgcgac 5460
gactgtgacc ccctgctggt caagctgggt ggtggtctgc aggacgtgga ggtccgaatc 5520
gtcgacgccg ccggtggtcc catggtggtc acccacctga tcgtggacac ccgagacgcc 5580
atgggtgcca acgccgtcaa ctccatggcc gagaagctgg ccccccacat cgagtcctgg 5640
accggcggtc gagtgtacct gcgaatcctg tccaacctgg ccgaccgacg actggcccga 5700
gcccgagctg tctggacctg tgacgccatc ggtggcgcct ctgtgcgaga cggtattatc 5760
tccgcctacc gattcgccgc cgccgaccct taccgagccg ctactcacaa caagggtatt 5820
atgaacggcg tgtccgccgt ggtgctggcc accggtaacg acacccgagc cgtggaggcc 5880
ggcgctcatg cttacgccgc ccgaaagggt tggtactcct ctctgaccga ctgggaggtc 5940
accgccgagg gccacctggc tggaaccctg gagatgccca tggccgtggg tctggtgggc 6000
ggtgctacca agctgcaccc caccgcccga gcctgcctga agatcctggg cgtgtccacc 6060
gccgagcgac tggctcgact gatcgccgcc gtcggcctgg ctcagaactt ctctgccctg 6120
aaggccctgg ccaccaccgg cattcagaag ggtcacatgt ccctgcacgc ccagaacatc 6180
gccatgatgg ccggtgccgt gggtgacgag atcgagcccg tcgccaaggc cctggtcgcc 6240
cagggtgctg tccgagtgga cgtcgccgag gccgagctgg ctcgactccg aggtcagggc 6300
taacgctatt tatcactctt tacaacttct acctcaacta tctactttaa taaatgaata 6360
tcgtttattc tctatgatta ctgtatatgc gttcctctaa gacaaatcga aaccagcatg 6420
cgatcgaatg gcatacaaaa gtttcttccg aagttgatca atgtcctgat agtcaggcag 6480
cttgagaaga ttgacacagg tggaggccgt agggaaccga tcaacctgtc taccagcgtt 6540
acgaatggca aatgacgggt tcaaagcctt gaatccttgc aatggtgcct tggatactga 6600
tgtcacaaac ttaagaagca gccgcttgtc ctcttcctcg aaactctcaa acacagtcca 6660
gaggtccttt atagcttgat ctgtatccag atagcctccg taattggtgt gtgtcttcaa 6720
atcccagacg tccacattgg catgtcctcc actgataagc atttgaagtt catctgcgtt 6780
gaacattgag acccacgaag ggtcatcgcg gttgaaatga atcggccgac gctcggtagt 6840
cggaaagagc cgggaccggc cggcgagcat aaaccggacg cagtaggatg tcctgcacgg 6900
gtctttttgt ggggtgtgga gaaaggggtg cttggagatg gaagccggta gaaccgggct 6960
gcttgggggg atttggggcc gctgggctcc aaagaggggt aggcatttcg ttggggttac 7020
gtaattgcgg catttgggtc ctgcgcgcat gtcccattgg tcagaattag tccggatagg 7080
agacttatca gccaatcaca gcgccggatc cacctgtagg ttgggttggg tgggagcacc 7140
cctccacaga gtagagtcaa acagcagcag caacatgata gttgggggtg tgcgtgttaa 7200
aggaaaaaaa aagaagcttg ggttatattc ccgctctatt tagaggttgc gggatagacg 7260
ccgacggagg gcaatggcgc catggaacct tgcggatatc gatacgccgc ggcggactgc 7320
gtccgaacca gctccagcag cgttttttcc gggccattga gccgactgcg accccgccaa 7380
cgtgtcttgg cccacgcact catgtcatgt tggtgttggg aggccacttt ttaagtagca 7440
caaggcacct agctcgcagc aaggtgtccg aaccaaagaa gcggctgcag tggtgcaaac 7500
ggggcggaaa cggcgggaaa aagccacggg ggcacgaatt gaggcacgcc ctcgaatttg 7560
agacgagtca cggccccatt cgcccgcgca atggctcgcc aacgcccggt cttttgcacc 7620
acatcaggtt accccaagcc aaacctttgt gttaaaaagc ttaacatatt ataccgaacg 7680
taggtttggg cgggcttgct ccgtctgtcc aaggcaacat ttatataagg gtctgcatcg 7740
ccggctcaat tgaatctttt ttcttcttct cttctctata ttcattcttg aattaaacac 7800
acatcaacag atgtcgcaac cccagaacgt tggaatcaaa gccctcgaga tctacgtgcc 7860
ttctcgaatt gtcaaccagg ctgagctcga gaagcacgac ggtgtcgctg ctggcaagta 7920
caccattggt cttggtcaga ccaacatggc ctttgtcgac gacagagagg acatctattc 7980
ctttgccctg accgccgtct ctcgactgct caagaacaac aacatcgacc ctgcatctat 8040
tggtcgaatc gaggttggta ctgaaaccct tctggacaag tccaagtccg tcaagtctgt 8100
gctcatgcag ctctttggcg agaacagcaa cattgagggt gtggacaacg tcaacgcctg 8160
ctacggagga accaacgccc tgttcaacgc tatcaactgg gttgagggtc gatcttggga 8220
cggccgaaac gccatcgtcg ttgccggtga cattgccctc tacgcaaagg gcgctgcccg 8280
acccaccgga ggtgccggct gtgttgccat gctcattggc cccgacgctc ccctggttct 8340
tgacaacgtc cacggatctt acttcgagca tgcctacgat ttctacaagc ctgatctgac 8400
ctccgagtac ccctatgttg atggccacta ctccctgacc tgttacacaa aggccctcga 8460
caaggcctac gctgcctaca acgcccgagc cgagaaggtc ggtctgttca aggactccga 8520
caagaagggt gctgaccgat ttgactactc tgccttccac gtgcccacct gcaagcttgt 8580
caccaagtct tacgctcgac ttctctacaa cgactacctc aacgacaaga gcctgtacga 8640
gggccaggtc cccgaggagg ttgctgccgt ctcctacgat gcctctctca ccgacaagac 8700
cgtcgagaag accttccttg gtattgccaa ggctcagtcc gccgagcgaa tggctccttc 8760
tctccaggga cccaccaaca ccggtaacat gtacaccgcc tctgtgtacg cttctctcat 8820
ctctctgctg acttttgtcc ccgctgagca gctgcagggc aagcgaatct ctctcttctc 8880
ttacggatct ggtcttgctt ccactctttt ctctctgacc gtcaagggag acatttctcc 8940
catcgtcaag gcctgcgact tcaaggctaa gctcgatgac cgatccaccg agactcccgt 9000
cgactacgag gctgccaccg atctccgaga gaaggcccac ctcaagaaga actttgagcc 9060
ccagggagac atcaagcaca tcaagtctgg cgtctactac ctcaccaaca tcgatgacat 9120
gttccgacga aagtacgaga tcaagcagta gcgtacctcc atggcctgtc cccacgttgc 9180
cggtcttgcc tcctactacc tgtccatcaa tgacgaggtt ctcacccctg cccaggtcga 9240
ggctcttatt actgagtcca acaccggtgt tcttcccacc accaacctca agggctctcc 9300
caacgctgtt gcctacaacg gtgttggcat ttaggcaatt aacagatagt ttgccggtga 9360
taattctctt aacctcccac actcctttga cataacgatt tatgtaacga aactgaaatt 9420
tgaccagata ttgttgtaaa tagaaaatct ggcttgtagg tggcaaaatc ccgtctttgt 9480
tcatcaattc cctctgtgac tactcgtcat ccctttatgt tcgactgtcg tatttttatt 9540
ttccatacat acgcaagtga gatggtttaa acagagaccg ggttggcggc gtatttgtgt 9600
cccaaaaaac agccccaatt gccccaattg accccaaatt gacccagtag cgggcccaac 9660
cccggcgaga gcccccttca ccccacatat caaacctccc ccggttccca cacttgccgt 9720
taagggcgta gggtactgca gtctggaatc tacgcttgtt cagactttgt actagtttct 9780
ttgtctggcc atccgggtaa cccatgccgg acgcaaaata gactactgaa aatttttttg 9840
ctttgtggtt gggactttag ccaagggtat aaaagaccac cgtccccgaa ttacctttcc 9900
tcttcttttc tctctctcct tgtcaactca cacccgaaat cgttaagcat ttccttctga 9960
gtataagaat cattcaaaat ggtgagtttc agaggcagca gcaattgcca cgggctttga 10020
gcacacggcc gggtgtggtc ccattcccat cgacacaaga cgccacgtca tccgaccagc 10080
actttttgca gtactaaccg cagatttatg aagaactgtg tcatcgtgtc cgccgtgcga 10140
accgccatcg gctccttcaa cggttctctg gcctccacct ccgccattga cctgggcgcc 10200
accgtcatta aggccgccat cgagcgagcc aagatcgact cccagcacgt ggacgaggtc 10260
attatgggta acgtcctgca ggccggcctg ggtcagaacc ccgctcgaca ggccctgctg 10320
aagtccggcc tggccgagac cgtctgtggt ttcaccgtga acaaggtgtg cggctccggt 10380
ctgaagtccg tcgccctggc cgcccaggct atccaggctg gacaggccca gtccatcgtg 10440
gccggcggaa tggagaacat gtccctggcc ccctacctgc tggacgccaa ggcccgatcc 10500
ggctaccgac tgggcgacgg tcaggtgtac gacgtgattc tgcgagacgg tctgatgtgt 10560
gccacccacg gttaccacat gggcatcacc gccgagaacg tcgccaagga gtacggtatc 10620
acccgagaga tgcaggacga gctggccctg cactcccagc gaaaggccgc cgctgccatc 10680
gagtccggtg ccttcaccgc cgagattgtc cccgtcaacg tcgtgacccg aaagaagacc 10740
ttcgtcttct cccaggacga gttccccaag gccaactcta ccgccgaggc cctgggcgct 10800
ctgcgacctg ctttcgacaa ggccggtacc gtgaccgccg gtaacgcctc cggtattaac 10860
gacggcgccg ccgccctggt cattatggag gagtccgccg ccctggccgc tggtcttacc 10920
cctctggccc gaatcaagtc ttacgcctct ggtggcgtgc cccccgccct tatgggcatg 10980
ggtcccgtgc ccgccaccca gaaggccctt cagctggccg gtctgcagct ggccgacatt 11040
gacctgatcg aggccaacga ggccttcgcc gcccagttcc tggccgtcgg aaagaacctg 11100
ggtttcgact ctgagaaggt caacgtgaac ggcggtgcca tcgccctggg ccaccctatc 11160
ggcgcttccg gagcccgaat cctggtcacc ctgctgcacg ccatgcaggc ccgagacaag 11220
accctgggcc tggccaccct gtgtattggt ggcggccagg gtattgccat ggtcattgag 11280
cgactgaact aaaaattcat gtaattagtt atgtcacgct tacattcacg ccctccctcc 11340
acatccgctc taaccgaaaa ggaaggagtt agacaacctg aagtctaggt ccctatttat 11400
ttttttatag ttatgttagt attaagaacg ttatttatat ttcaaatttt tctttttttt 11460
ctgtacagac gcgtgtacgc atgtaacatt atactgaaaa ccttgcttga gaaggttttg 11520
ggacgctcga aggctttaat ttgc 11544
<210> 57
<211> 10658
<212> DNA
<213> Artificial sequence
<400> 57
ggccgcatcg cttcggataa ctcctgctat acgaagttat acgaattcgc gcccagagag 60
ccattgacgt tctttctaat ttggaccgat agccgtatag tccagtctat ctataagttc 120
aactaactcg taactattac cataacatat acttcactgc cccagataag gttccgataa 180
aaagttctgc agactaaatt tatttcagtc tcctcttcac caccaaaatg ccctcctacg 240
aagctcgagc taacgtccac aagtccgcct ttgccgctcg agtgctcaag ctcgtggcag 300
ccaagaaaac caacctgtgt gcttctctgg atgttaccac caccaaggag ctcattgagc 360
ttgccgataa ggtcggacct tatgtgtgca tgatcaagac ccatatcgac atcattgacg 420
acttcaccta cgccagcact gtgctccccc tcaaggaact tgctcttaag cacggtttct 480
tcctgttcga ggacagaaag ttcgcagata ttggcaacac tgtcaagcac cagtacaaga 540
acggtgtcta ccgaatcgcc gagtggtccg atatcaccaa cgcccacggt gtacccggaa 600
ccggaatcat tgctggcctg cgagctggtg ccgaggaaac tgtctctgaa cagaagaagg 660
aggacgtctc tgactacgag aactcccagt acaaggagtt cctggtcccc tctcccaacg 720
agaagctggc cagaggtctg ctcatgctgg ccgagctgtc ttgcaagggc tctctggcca 780
ctggcgagta ctccaagcag accattgagc ttgcccgatc cgaccccgag tttgtggttg 840
gcttcgttgc ccagaaccga cctaagggcg actctgagga ctggcttatt ctgacccccg 900
gggtgggtct tgacgacaag ggagacgctc tcggacagca gtaccgaact gttgaggatg 960
tcatgtctac cggaacggat atcataattg tcggccgagg tctgtacggc cagaaccgag 1020
atcctatcga ggaggccaag cgataccaga aggctggctg ggaggcttac cagaagatta 1080
actgttagag gttagactat ggatatgtaa tttaactgtg tatatagaga gcgtgcaagt 1140
atggagcgct tgttcagctt gtatgatggt cagacgacct gtctgatcga gtatgtatga 1200
tactgcacaa cctgtgtatc cgcatgatct gtccaatggg gcatgttgtt gtgtttctcg 1260
gaattcagaa taacttcgta tgatgtatgc tatacgaagt tatccggggg ttgaaatgaa 1320
tcggccgacg ctcggtagtc ggaaagagcc gggaccggcc ggcgagcata aaccggacgc 1380
agtaggatgt cctgcacggg tctttttgtg gggtgtggag aaaggggtgc ttggagatgg 1440
aagccggtag aaccgggctg cttgggggga tttggggccg ctgggctcca aagaggggta 1500
ggcatttcgt tggggttacg taattgcggc atttgggtcc tgcgcgcatg tcccattggt 1560
cagaattagt ccggatagga gacttatcag ccaatcacag cgccggatcc acctgtaggt 1620
tgggttgggt gggagcaccc ctccacagag tagagtcaaa cagcagcagc aacatgatag 1680
ttgggggtgt gcgtgttaaa ggaaaaaaaa agaagcttgg gttatattcc cgctctattt 1740
agaggttgcg ggatagacgc cgacggaggg caatggcgcc atggaacctt gcggatatcg 1800
atacgccgcg gcggactgcg tccgaaccag ctccagcagc gttttttccg ggccattgag 1860
ccgactgcga ccccgccaac gtgtcttggc ccacgcactc atgtcatgtt ggtgttggga 1920
ggccactttt taagtagcac aaggcaccta gctcgcagca aggtgtccga accaaagaag 1980
cggctgcagt ggtgcaaacg gggcggaaac ggcgggaaaa agccacgggg gcacgaattg 2040
aggcacgccc tcgaatttga gacgagtcac ggccccattc gcccgcgcaa tggctcgcca 2100
acgcccggtc ttttgcacca catcaggtta ccccaagcca aacctttgtg ttaaaaagct 2160
taacatatta taccgaacgt aggtttgggc gggcttgctc cgtctgtcca aggcaacatt 2220
tatataaggg tctgcatcgc cggctcaatt gaatcttttt tcttcttctc ttctctatat 2280
tcattcttga attaaacaca catcaacaga tgaccaccta ttcggctccg ggaaaggccc 2340
tcctttgcgg cggttatttg gttattgatc cggcgtattc agcatacgtc gtgggcctct 2400
cggcgcgtat ttacgcgaca gtttcggctt ccgaggcctc caccacctct gtccatgtcg 2460
tctctccgca gtttgacaag ggtgaatgga cctacaacta cacgaacggc cagctgacgg 2520
ccatcggaca caacccattt gctcacgcgg ccgtcaacac cgttctgcat tacgttcctc 2580
ctcgaaacct ccacatcaac atcagcatca aaagtgacaa cgcgtaccac tcgcaaattg 2640
acagcacgca gagaggccag tttgcatacc acaaaaaggc gatccacgag gtgcctaaaa 2700
cgggcctcgg tagctccgct gctcttacca ccgttcttgt ggcagctttg ctcaagtcat 2760
acggcattga tcccttgcat aacacccacc tcgttcacaa cctgtcccag gttgcacact 2820
gctcggcaca gaagaagatt gggtctggat ttgacgtggc ttcggccgtt tgtggctctc 2880
tagtctatag acgtttcccg gcggagtccg tgaacatggt cattgcagct gaagggacct 2940
ccgaatacgg ggctctgttg agaactaccg ttaatcaaaa gtggaaggtg actctggaac 3000
catccttctt gccgccggga atcagcctgc ttatgggaga cgtccaggga ggatctgaga 3060
ctccaggtat ggtggccaag gtgatggcat ggcgaaaagc aaagccccga gaagccgaga 3120
tggtgtggag agatctcaac gctgccaaca tgctcatggt caagttgttc aacgacctgc 3180
gcaagctctc tctcactaac aacgaggcct acgaacaact tttggccgag gctgctcctc 3240
tcaacgctct aaagatgata atgttgcaga accctctcgg agaactagca cgatgcatta 3300
tcactattcg aaagcatctc aagaagatga cacgggagac tggtgctgct attgagccgg 3360
atgagcagtc tgcattgctc aacaagtgca acacttatag tggagtcatt ggaggtgttg 3420
tgcctggagc aggaggctac gatgctattt ctcttctggt gatcagctct acggtgaaca 3480
atgtcaagcg agagagccag ggagtccaat ggatggagct caaggaggag aacgagggtc 3540
tgcggctcga gaaggggttc aagtagcgta cctccatggc ctgtccccac gttgccggtc 3600
ttgcctccta ctacctgtcc atcaatgacg aggttctcac ccctgcccag gtcgaggctc 3660
ttattactga gtccaacacc ggtgttcttc ccaccaccaa cctcaagggc tctcccaacg 3720
ctgttgccta caacggtgtt ggcatttagg caattaacag atagtttgcc ggtgataatt 3780
ctcttaacct cccacactcc tttgacataa cgatttatgt aacgaaactg aaatttgacc 3840
agatattgtt gtaaatagaa aatctggctt gtaggtggca aaatcccgtc tttgttcatc 3900
aattccctct gtgactactc gtcatccctt tatgttcgac tgtcgtattt ttattttcca 3960
tacatacgca agtgagatgg tttaaacaga gaccgggttg gcggcgtatt tgtgtcccaa 4020
aaaacagccc caattgcccc aattgacccc aaattgaccc agtagcgggc ccaaccccgg 4080
cgagagcccc cttcacccca catatcaaac ctcccccggt tcccacactt gccgttaagg 4140
gcgtagggta ctgcagtctg gaatctacgc ttgttcagac tttgtactag tttctttgtc 4200
tggccatccg ggtaacccat gccggacgca aaatagacta ctgaaaattt ttttgctttg 4260
tggttgggac tttagccaag ggtataaaag accaccgtcc ccgaattacc tttcctcttc 4320
ttttctctct ctccttgtca actcacaccc gaaatcgtta agcatttcct tctgagtata 4380
agaatcattc aaaatggtga gtttcagagg cagcagcaat tgccacgggc tttgagcaca 4440
cggccgggtg tggtcccatt cccatcgaca caagacgcca cgtcatccga ccagcacttt 4500
ttgcagtact aaccgcagat ttatgatcca ccaggcctcc accaccgctc cggtgaacat 4560
tgcgacactc aagtactggg gcaagcgaga ccctgctctc aatctgccca ctaacaactc 4620
catctccgtg actttgtcgc aggatgatct gcggaccctc accacagcct cgtgttcccc 4680
tgatttcacc caggacgagc tgtggctcaa tggcaagcag gaggacgtga gcggcaaacg 4740
tctggttgcg tgtttccgag agctgcgggc tctgcgacac aaaatggagg actccgactc 4800
ttctctgcct aagctggccg atcagaagct caagatcgtg tccgagaaca acttccccac 4860
cgccgctggt ctcgcctcat cggctgctgg ctttgccgcc ctgatccgag ccgttgcaaa 4920
tctctacgag ctccaggaga cccccgagca gctgtccatt gtggctcgac agggctctgg 4980
atccgcctgt cgatctctct acggaggcta cgtggcatgg gaaatgggca ccgagtctga 5040
cggaagcgac tcgcgagcgg tccagatcgc caccgccgac cactggcccg agatgcgagc 5100
cgccatcctc gttgtctctg ccgacaagaa ggacacgtcg tccactaccg gtatgcaggt 5160
gactgtgcac acttctcccc tcttcaagga gcgagtcacc actgtggttc ccgagcggtt 5220
tgcccagatg aagaagtcga ttctggaccg agacttcccc acctttgccg agctcaccat 5280
gcgagactca aaccagttcc acgccacctg tctggactcg tatcctccca ttttctacct 5340
caacgacgtg tcgcgagcct ccattcgggt agttgaggcc atcaacaagg ctgccggagc 5400
caccattgcc gcctacacct ttgatgctgg acccaactgt gtcatctact acgaggacaa 5460
gaacgaggag ctggttctgg gtgctctcaa ggccattctg ggccgtgtgg agggatggga 5520
gaagcaccag tctgtggacg ccaagaagat tgatgttgac gagcggtggg agtccgagct 5580
ggccaacgga attcagcggg tgatccttac caaggttgga ggagatcccg tgaagaccgc 5640
tgagtcgctt atcaacgagg atggttctct gaagaacagc aagtagaaat tcatgtaatt 5700
agttatgtca cgcttacatt cacgccctcc ctccacatcc gctctaaccg aaaaggaagg 5760
agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt tagtattaag 5820
aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt acgcatgtaa 5880
cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaaggctt taatttgcat 5940
cgcggagttt ggcgcccgtt ttttcgagcc ccacacgttt cggtgagtat gagcggcggc 6000
agattcgagc gtttccggtt tccgcggctg gacgagagcc catgatgggg gctcccacca 6060
ccagcaatca gggccctgat tacacaccca cctgtaatgt catgctgttc atcgtggtta 6120
atgctgctgt gtgctgtgtg tgtgtgttgt ttggcgctca ttgttgcgtt atgcagcgta 6180
caccacaata ttggaagctt attagccttt ctattttttc gtttgcaagg cttaacaaca 6240
ttgctgtgga gagggatggg gatatggagg ccgctggagg gagtcggaga ggcgttttgg 6300
agcggcttgg cctggcgccc agctcgcgaa acgcacctag gaccctttgg cacgccgaaa 6360
tgtgccactt ttcagtctag taacgcctta cctacgtcat tccatgcatg catgtttgcg 6420
ccttttttcc cttgcccttg atcgccacac agtacagtgc actgtacagt ggaggttttg 6480
ggggggtctt agatgggagc taaaagcggc ctagcggtac actagtggga ttgtatggag 6540
tggcatggag cctaggtgga gcctgacagg acgcacgacc ggctagcccg tgacagacga 6600
tgggtggctc ctgttgtcca ccgcgtacaa atgtttgggc caaagtcttg tcagccttgc 6660
ttgcgaacct aattcccaat tttgtcactt cgcaccccca ttgatcgagc cctaacccct 6720
gcccatcagg caatccaatt aagctcgcat tgtctgcctt gtttagtttg gctcctgccc 6780
gtttcggcgt ccacttgcac aaacacaaac aagcattata tataaggctc gtctctccct 6840
cccaaccaca ctcacttttt tgcccgtctt cccttgctaa cacaaaagtc aagaacacaa 6900
acaaccaccc caaccccctt acacacaaga catatctaca gcatttatga cgacgtctta 6960
cagcgacaaa atcaagagta tcagcgtgag ctctgtggct cagcagtttc ctgaggtggc 7020
gccgattgcg gacgtgtcca aggctagccg gcccagcacg gagtcgtcgg actcgtcggc 7080
caagctattt gatggccacg acgaggagca gatcaagctg atggacgaga tctgtgtggt 7140
gctggactgg gacgacaagc cgattggcgg cgcgtccaaa aagtgctgtc atctgatgga 7200
caacatcaac gacggactgg tgcatcgggc cttttccgtg ttcatgttca acgaccgcgg 7260
tgagctgctt ctgcagcagc gggcggcgga aaaaatcacc tttgccaaca tgtggaccaa 7320
cacgtgctgc tcgcatcctc tggcggtgcc cagcgagatg ggcgggctgg atctggagtc 7380
ccggatccag ggcgccaaaa acgccgcggt ccggaagctt gagcacgagc tgggaatcga 7440
ccccaaggcc gttccggcag acaagttcca tttcctcacc cggatccact acgccgcgcc 7500
ctcctcgggc ccctggggcg agcacgagat tgactacatt ctgtttgtcc ggggcgaccc 7560
cgagctcaag gtggtggcca acgaggtccg cgataccgtg tgggtgtcgc agcagggact 7620
caaggacatg atggccgatc ccaagctggt tttcacccct tggttccggc tcatttgtga 7680
gcaggcgctg tttccctggt gggaccagtt ggacaatctg cccgcgggcg atgacgagat 7740
tcggcggtgg atcaagtaga aatgcagttt gtttagcaaa atatatttaa cgagtttgat 7800
agaggcgctg gactacataa ttactgaatc acgcgtacat gtttcagctc aaattgtatc 7860
acggtttctt tgtagcaatg gagggggaga gttgacaagg cattagagaa gagagcgaga 7920
ggagaagaca agtggataga cgactgcaat catatgatct gcacaaactg cgatgttttc 7980
ctgtcagatc atgttctttt gctcatagtt aagctatcgt gactttacgg atccgccgag 8040
catcttagta gcgaggtttg cggtctgggc tgatcggctt ttgttgatcg ggttcggaac 8100
gaattagaag tgggtcgata gcaatgatca attcggggtg agtgagtgcg gttagtggga 8160
aaccggggag atactgtggg gatttagggg acagtgttaa gaagaggaag gggtgaggga 8220
ggtgaggtga gggagaggga agtatagagg tttatgtagt aacatcaatc atacagttta 8280
aactgcaccc aacaataaat gggtagggtt gcaccaacaa agggatggga tggggggtag 8340
aagatacgag gataacgggg ctcaatggca caaataagaa cgaatactgc cattaagact 8400
cgtgatccag cgactgacac cattgcatca tctaagggcc tcaaaactac ctcggaactg 8460
ctgcgctgat ctggacacca cagaggttcc gagcacttta ggttgcacca aatgtcccac 8520
caggtgcagg cagaaaacgc tggaacagcg tgtacagttt gtcttagcaa aaagtgaagg 8580
cgctgaggtc gagcagggtg gtgtgacttg ttatagcctt tagagctgcg aaagcgcgta 8640
tggatttggc tcatcaggcc agattgaggg tctgtggaca catgtcatgt tagtgtactt 8700
caatcgcccc ctggatatag ccccgacaat aggccgtggc ctcatttttt tgccttccgc 8760
acatttccat tgctcggtac ccacaccttg cttctcctgc acttgccaac cttaatactg 8820
gtttacattg accaacatct tacaagcggg gggcttgtct agggtatata taaacagtgg 8880
ctctcccaat cggttgccag tctctttttt cctttctttc cccacagatt cgaaatctaa 8940
actacacatc acacaatgcc tgttactgac gtccttaagc gaaagtccgg tgtcatcgtc 9000
ggcgacgatg tccgagccgt gagtatccac gacaagatca gtgtcgagac gacgcgtttt 9060
gtgtaatgac acaatccgaa agtcgctagc aacacacact ctctacacaa actaacccag 9120
atgtccaagg cgaaattcga aagcgtgttc ccccgaatct ccgaggagct ggtgcagctg 9180
ctgcgagacg agggtctgcc ccaggatgcc gtgcagtggt tttccgactc acttcagtac 9240
aactgtgtgg gtggaaagct caaccgaggc ctgtctgtgg tcgacaccta ccagctactg 9300
accggcaaga aggagctcga tgacgaggag tactaccgac tcgcgctgct cggctggctg 9360
attgagctgc tgcaggcgtt tttcctcgtg tcggacgaca ttatggatga gtccaagacc 9420
cgacgaggcc agccctgctg gtacctcaag cccaaggtcg gcatgattgc catcaacgat 9480
gctttcatgc tagagagtgg catctacatt ctgcttaaga agcatttccg acaggagaag 9540
tactacattg accttgtcga gctgttccac gacatttcgt tcaagaccga gctgggccag 9600
ctggtggatc ttctgactgc ccccgaggat gaggttgatc tcaaccggtt ctctctggac 9660
aagcactcct ttattgtgcg atacaagact gcttactact ccttctacct gcccgttgtt 9720
ctagccatgt acgtggccgg cattaccaac cccaaggacc tgcagcaggc catggatgtg 9780
ctgatccctc tcggagagta cttccaggtc caggacgact accttgacaa ctttggagac 9840
cccgagttca ttggtaagat cggcaccgac atccaggaca acaagtgctc ctggctcgtt 9900
aacaaagccc ttcagaaggc cacccccgag cagcgacaga tcctcgagga caactacggc 9960
gtcaaggaca agtccaagga gctcgtcatc aagaaactgt atgatgacat gaagattgag 10020
caggactacc ttgactacga ggaggaggtt gttggcgaca tcaagaagaa gatcgagcag 10080
gttgacgaga gccgaggctt caagaaggag gtgctcaacg ctttcctcgc caagatttac 10140
aagcgacaga agtagcgcta tttatcactc tttacaactt ctacctcaac tatctacttt 10200
aataaatgaa tatcgtttat tctctatgat tactgtatat gcgttcctct aagacaaatc 10260
gaaaccagca tgcgatcgaa tggcatacaa aagtttcttc cgaagttgat caatgtcctg 10320
atagtcaggc agcttgagaa gattgacaca ggtggaggcc gtagggaacc gatcaacctg 10380
tctaccagcg ttacgaatgg caaatgacgg gttcaaagcc ttgaatcctt gcaatggtgc 10440
cttggatact gatgtcacaa acttaagaag cagccgcttg tcctcttcct cgaaactctc 10500
aaacacagtc cagaggtcct ttatagcttg atctgtatcc agatagcctc cgtaattggt 10560
gtgtgtcttc aaatcccaga cgtccacatt ggcatgtcct ccactgataa gcatttgaag 10620
ttcatctgcg ttgaacattg agacccacga agggtcgc 10658
<210> 58
<211> 6950
<212> DNA
<213> Artificial sequence
<400> 58
ggccgcatcg cttcggataa ctcctgctat acgaagttat acgaattcgc gcccagagag 60
ccattgacgt tctttctaat ttggaccgat agccgtatag tccagtctat ctataagttc 120
aactaactcg taactattac cataacatat acttcactgc cccagataag gttccgataa 180
aaagttctgc agactaaatt tatttcagtc tcctcttcac caccaaaatg ccctcctacg 240
aagctcgagc taacgtccac aagtccgcct ttgccgctcg agtgctcaag ctcgtggcag 300
ccaagaaaac caacctgtgt gcttctctgg atgttaccac caccaaggag ctcattgagc 360
ttgccgataa ggtcggacct tatgtgtgca tgatcaagac ccatatcgac atcattgacg 420
acttcaccta cgccagcact gtgctccccc tcaaggaact tgctcttaag cacggtttct 480
tcctgttcga ggacagaaag ttcgcagata ttggcaacac tgtcaagcac cagtacaaga 540
acggtgtcta ccgaatcgcc gagtggtccg atatcaccaa cgcccacggt gtacccggaa 600
ccggaatcat tgctggcctg cgagctggtg ccgaggaaac tgtctctgaa cagaagaagg 660
aggacgtctc tgactacgag aactcccagt acaaggagtt cctggtcccc tctcccaacg 720
agaagctggc cagaggtctg ctcatgctgg ccgagctgtc ttgcaagggc tctctggcca 780
ctggcgagta ctccaagcag accattgagc ttgcccgatc cgaccccgag tttgtggttg 840
gcttcgttgc ccagaaccga cctaagggcg actctgagga ctggcttatt ctgacccccg 900
gggtgggtct tgacgacaag ggagacgctc tcggacagca gtaccgaact gttgaggatg 960
tcatgtctac cggaacggat atcataattg tcggccgagg tctgtacggc cagaaccgag 1020
atcctatcga ggaggccaag cgataccaga aggctggctg ggaggcttac cagaagatta 1080
actgttagag gttagactat ggatatgtaa tttaactgtg tatatagaga gcgtgcaagt 1140
atggagcgct tgttcagctt gtatgatggt cagacgacct gtctgatcga gtatgtatga 1200
tactgcacaa cctgtgtatc cgcatgatct gtccaatggg gcatgttgtt gtgtttctcg 1260
gaattcagaa taacttcgta tgatgtatgc tatacgaagt tatccgggag agaccgggtt 1320
ggcggcgtat ttgtgtccca aaaaacagcc ccaattgccc caattgaccc caaattgacc 1380
cagtagcggg cccaaccccg gcgagagccc ccttcacccc acatatcaaa cctcccccgg 1440
ttcccacact tgccgttaag ggcgtagggt actgcagtct ggaatctacg cttgttcaga 1500
ctttgtacta gtttctttgt ctggccatcc gggtaaccca tgccggacgc aaaatagact 1560
actgaaaatt tttttgcttt gtggttggga ctttagccaa gggtataaaa gaccaccgtc 1620
cccgaattac ctttcctctt cttttctctc tctccttgtc aactcacacc cgaaatcgtt 1680
aagcatttcc ttctgagtat aagaatcatt caaaatggtg agtttcagag gcagcagcaa 1740
ttgccacggg ctttgagcac acggccgggt gtggtcccat tcccatcgac acaagacgcc 1800
acgtcatccg accagcactt tttgcagtac taaccgcaga tggctgactt cgactctaag 1860
gaatacttgg aattggttga caagtggtgg agagctacta actacttgtc tgctggtatg 1920
atcttcttga agtctaaccc attgttctct gttactaaca ctccaatcaa ggctgaagac 1980
gttaaggtta agccaatcgg tcactggggt actatctctg gtcaaacttt cttgtacgct 2040
cacgctaaca gattgatcaa caagtacggt ttgaacatgt tctacgttgg tggtccaggt 2100
cacggtggtc aagttatggt tactaacgct tacttggacg gtgcttacac tgaagactac 2160
ccagaaatca ctcaagacat cgaaggtatg tctcacttgt tcaagagatt ctctttccca 2220
ggtggtatcg gttctcacat gactgctcaa actccaggtt ctttgcacga aggtggtgaa 2280
ttgggttact ctttgtctca cgctttcggt gctgttttgg acaacccaga ccaagttgct 2340
ttcgctgttg ttggtgacgg tgaagctgaa actggtccat ctatggcttc ttggcactct 2400
atcaagttct tgaacgctaa gaacgacggt gctgttttgc cagttttgga cttgaacggt 2460
ttcaagatct ctaacccaac tatcttctct agaatgtctg acgaagaaat cactaagttc 2520
ttcgaaggtt tgggttactc tccaagattc atcgaaaacg acgacatcca cgactacgct 2580
acttaccacc aattggctgc taacatcttg gaccaagcta tcgaagacat ccaagctatc 2640
caaaacgacg ctagagaaaa cggtaagtac caagacggtg aaatcccagc ttggccagtt 2700
atcatcgcta gattgccaaa gggttggggt ggtccaactc acgacgcttc taacaaccca 2760
atcgaaaact ctttcagagc tcaccaagtt ccattgccat tggaacaaca cgacttggct 2820
actttgccag aattcgaaga ctggatgaac tcttacaagc cagaagaatt gttcaacgct 2880
gacggttctt tgaaggacga attgaaggct atcgctccaa agggtgacaa gagaatgtct 2940
gctaacccaa tcactaacgg tggtgctgac agatctgact tgaagttgcc aaactggaga 3000
gaattcgcta acgacatcaa cgacgacact agaggtaagg aattcgctga ctctaagaga 3060
aacatggaca tggctacttt gtctaactac ttgggtgctg tttctcaatt gaacccaact 3120
agattcagat tcttcggtcc agacgaaact atgtctaaca gattgtgggg tttgttcaac 3180
gttactccaa gacaatggat ggaagaaatc aaggaaccac aagaccaatt gttgtctcca 3240
actggtagaa tcatcgactc tcaattgtct gaacaccaag ctgaaggttg gttggaaggt 3300
tacactttga ctggtagagt tggtatcttc gcttcttacg aatctttctt gagagttgtt 3360
gacactatgg ttactcaaca cttcaagtgg ttgagacacg cttctgaaca agcttggaga 3420
aacgactacc catctttgaa cttgatcgct acttctactg ctttccaaca agaccacaac 3480
ggttacactc accaagaccc aggtatgttg actcacttgg ctgaaaagaa gtctaacttc 3540
atcagagaat acttgccagc tgacggtaac tctttgttgg ctgttcaaga aagagctttc 3600
tctgaaagac acaaggttaa cttgttgatc gcttctaagc aaccaagaca acaatggttc 3660
actgttgaag aagctgaagt tttggctaac gaaggtttga agatcatcga ctgggcttct 3720
actgctccat cttctgacgt tgacatcact ttcgcttctg ctggtactga accaactatc 3780
gaaactttgg ctgctttgtg gttgatcaac caagctttcc cagacgttaa gttcagatac 3840
gttaacgttg ttgaattgtt gagattgcaa aagaagtctg aaccaaacat gaacgacgaa 3900
agagaattgt ctgctgaaga attcaacaag tacttccaag ctgacactcc agttatcttc 3960
ggtttccacg cttacgaaaa cttgatcgaa tctttcttct tcgaaagaaa gttcactggt 4020
gacgtttacg ttcacggtta cagagaagac ggtgacatca ctactactta cgacatgaga 4080
gtttactctc acttggacag attccaccaa gctaaggaag ctgctgaaat cttgtctgct 4140
aacggtaaga tcgaccaagc tgctgctgac actttcatcg ctaagatgga cgacactttg 4200
gctaagcact tccaagttac tagaaacgaa ggtagagaca tcgaagaatt cactgactgg 4260
acttggtctc cattgaagta atcatgtaat tagttatgtc acgcttacat tcacgccctc 4320
cctccacatc cgctctaacc gaaaaggaag gagttagaca acctgaagtc taggtcccta 4380
tttatttttt tatagttatg ttagtattaa gaacgttatt tatatttcaa atttttcttt 4440
tttttctgta cagacgcgtg tacgcatgta acattatact gaaaaccttg cttgagaagg 4500
ttttgggacg ctcgaaggct ttaatttgca tcgcggagtt tggcgcccgt tttttcgagc 4560
cccacacgtt tcggtgagta tgagcggcgg cagattcgag cgtttccggt ttccgcggct 4620
ggacgagagc ccatgatggg ggctcccacc accagcaatc agggccctga ttacacaccc 4680
acctgtaatg tcatgctgtt catcgtggtt aatgctgctg tgtgctgtgt gtgtgtgttg 4740
tttggcgctc attgttgcgt tatgcagcgt acaccacaat attggaagct tattagcctt 4800
tctatttttt cgtttgcaag gcttaacaac attgctgtgg agagggatgg ggatatggag 4860
gccgctggag ggagtcggag aggcgttttg gagcggcttg gcctggcgcc cagctcgcga 4920
aacgcaccta ggaccctttg gcacgccgaa atgtgccact tttcagtcta gtaacgcctt 4980
acctacgtca ttccatgcat gcatgtttgc gccttttttc ccttgccctt gatcgccaca 5040
cagtacagtg cactgtacag tggaggtttt gggggggtct tagatgggag ctaaaagcgg 5100
cctagcggta cactagtggg attgtatgga gtggcatgga gcctaggtgg agcctgacag 5160
gacgcacgac cggctagccc gtgacagacg atgggtggct cctgttgtcc accgcgtaca 5220
aatgtttggg ccaaagtctt gtcagccttg cttgcgaacc taattcccaa ttttgtcact 5280
tcgcaccccc attgatcgag ccctaacccc tgcccatcag gcaatccaat taagctcgca 5340
ttgtctgcct tgtttagttt ggctcctgcc cgtttcggcg tccacttgca caaacacaaa 5400
caagcattat atataaggct cgtctctccc tcccaaccac actcactttt ttgcccgtct 5460
tcccttgcta acacaaaagt caagaacaca aacaaccacc ccaaccccct tacacacaag 5520
acatatctac agcaatgaag ttgatggaaa acatcttcga cttggctaag gctaacaaga 5580
agaagatcgt tttggctgaa ggtgaagaag aaagaaacat cagagcttct gaagaaatca 5640
tcaaggacgg tatcgctgac atcatcttgg ttggttctga atctgttatc aaggaatctg 5700
ctgctaagtt cggtgttaac ttggctggtg ttgaaatcgt tgacccagaa acttcttcta 5760
agactgctgg ttacgctaac gctttctacg aaatcagaaa gaacaagggt gttactttgg 5820
aaaaggctga caagatcgtt agagacccaa tctacttcgc tactatgatg gttaagttgg 5880
gtgacgctga aggtttggtt tctggtgcta tccacactac tggtgacttg ttgagaccag 5940
gtttgcaaat cgttaagact gttccaggtg cttctgttgt ttcttctgtt ttcttgatgt 6000
ctgttccaga ctgtgaatac ggtgaagacg gtttcttgtt gttcgctgac tgtgctgtta 6060
acgtttgtcc aactgctgaa gaattgtctt ctatcgctat cactactgct gaaactgcta 6120
agaacttgtg taagatcgaa ccaagagttg ctatgttgtc tttctctact atgggttctg 6180
cttctcacga attggttgac aaggttacta aggctactaa gttggctaag gaagctagac 6240
cagacttgga catcgacggt gaattgcaat tggacgcttc tatcgttaag aaggttgctg 6300
acttgaaggc tccaggttct aaggttgctg gtaaggctaa cgttttgatc ttcccagaca 6360
tccaagctgg taacatcggt tacaagttgg ttcaaagatt cgctaaggct gaagctatcg 6420
gtccaatctg tcaaggtttc gctaagccaa tcaacgactt gtctagaggt tgttctgttg 6480
acgacatcgt taaggttgtt gctgttactg ctgttcaagc tcaagctcaa ggttaagtac 6540
ctccatggcc tgtccccacg ttgccggtct tgcctcctac tacctgtcca tcaatgacga 6600
ggttctcacc cctgcccagg tcgaggctct tattactgag tccaacaccg gtgttcttcc 6660
caccaccaac ctcaagggct ctcccaacgc tgttgcctac aacggtgttg gcatttaggc 6720
aattaacaga tagtttgccg gtgataattc tcttaacctc ccacactcct ttgacataac 6780
gatttatgta acgaaactga aatttgacca gatattgttg taaatagaaa atctggcttg 6840
taggtggcaa aatcccgtct ttgttcatca attccctctg tgactactcg tcatcccttt 6900
atgttcgact gtcgtatttt tattttccat acatacgcaa gtgagatggc 6950
<210> 59
<211> 8883
<212> DNA
<213> Artificial sequence
<400> 59
ggccgcatcg cttcggataa ctcctgctat acgaagttat acgaattcgc gcccagagag 60
ccattgacgt tctttctaat ttggaccgat agccgtatag tccagtctat ctataagttc 120
aactaactcg taactattac cataacatat acttcactgc cccagataag gttccgataa 180
aaagttctgc agactaaatt tatttcagtc tcctcttcac caccaaaatg ccctcctacg 240
aagctcgagc taacgtccac aagtccgcct ttgccgctcg agtgctcaag ctcgtggcag 300
ccaagaaaac caacctgtgt gcttctctgg atgttaccac caccaaggag ctcattgagc 360
ttgccgataa ggtcggacct tatgtgtgca tgatcaagac ccatatcgac atcattgacg 420
acttcaccta cgccagcact gtgctccccc tcaaggaact tgctcttaag cacggtttct 480
tcctgttcga ggacagaaag ttcgcagata ttggcaacac tgtcaagcac cagtacaaga 540
acggtgtcta ccgaatcgcc gagtggtccg atatcaccaa cgcccacggt gtacccggaa 600
ccggaatcat tgctggcctg cgagctggtg ccgaggaaac tgtctctgaa cagaagaagg 660
aggacgtctc tgactacgag aactcccagt acaaggagtt cctggtcccc tctcccaacg 720
agaagctggc cagaggtctg ctcatgctgg ccgagctgtc ttgcaagggc tctctggcca 780
ctggcgagta ctccaagcag accattgagc ttgcccgatc cgaccccgag tttgtggttg 840
gcttcgttgc ccagaaccga cctaagggcg actctgagga ctggcttatt ctgacccccg 900
gggtgggtct tgacgacaag ggagacgctc tcggacagca gtaccgaact gttgaggatg 960
tcatgtctac cggaacggat atcataattg tcggccgagg tctgtacggc cagaaccgag 1020
atcctatcga ggaggccaag cgataccaga aggctggctg ggaggcttac cagaagatta 1080
actgttagag gttagactat ggatatgtaa tttaactgtg tatatagaga gcgtgcaagt 1140
atggagcgct tgttcagctt gtatgatggt cagacgacct gtctgatcga gtatgtatga 1200
tactgcacaa cctgtgtatc cgcatgatct gtccaatggg gcatgttgtt gtgtttctcg 1260
gaattcagaa taacttcgta tgatgtatgc tatacgaagt tatccgggct gcacccaaca 1320
ataaatgggt agggttgcac caacaaaggg atgggatggg gggtagaaga tacgaggata 1380
acggggctca atggcacaaa taagaacgaa tactgccatt aagactcgtg atccagcgac 1440
tgacaccatt gcatcatcta agggcctcaa aactacctcg gaactgctgc gctgatctgg 1500
acaccacaga ggttccgagc actttaggtt gcaccaaatg tcccaccagg tgcaggcaga 1560
aaacgctgga acagcgtgta cagtttgtct tagcaaaaag tgaaggcgct gaggtcgagc 1620
agggtggtgt gacttgttat agcctttaga gctgcgaaag cgcgtatgga tttggctcat 1680
caggccagat tgagggtctg tggacacatg tcatgttagt gtacttcaat cgccccctgg 1740
atatagcccc gacaataggc cgtggcctca tttttttgcc ttccgcacat ttccattgct 1800
cggtacccac accttgcttc tcctgcactt gccaacctta atactggttt acattgacca 1860
acatcttaca agcggggggc ttgtctaggg tatatataaa cagtggctct cccaatcggt 1920
tgccagtctc ttttttcctt tctttcccca cagattcgaa atctaaacta cacatcacac 1980
aatgcctgtt actgacgtcc ttaagcgaaa gtccggtgtc atcgtcggcg acgatgtccg 2040
agccgtgagt atccacgaca agatcagtgt cgagacgacg cgttttgtgt aatgacacaa 2100
tccgaaagtc gctagcaaca cacactctct acacaaacta acccagatgg ccacccccga 2160
agatctcgct gccaacgccg actacattag aatggccgat cagtacgtcg aggtgcccgg 2220
aggaaccaac aacaacaact acgccaacgt cgagctgatt gtcgacgtgg ctgagcgatt 2280
cggcgtcgat gccgtgtggg ccggatgggg ccatgccagt gaaaatcccc tgctccccga 2340
gtcgctagcg gcctctcccc gcaagattgt cttcatcggc cctcccggag ctgccatgag 2400
atctctggga gacaaaattt cttctaccat tgtggcccag cacgcaaagg tcccgtgtat 2460
cccgtggtct ggaaccggag tggacgaggt tgtggttgac aagagcacca acctcgtgtc 2520
cgtgtccgag gaggtgtaca ccaagggctg caccaccggt cccaagcagg gtctggagaa 2580
ggctaagcag attggattcc ccgtgatgat caaggcttcc gagggaggag gaggaaaggg 2640
tattcgaaag gttgagcgag aggaggactt cgaggctgct taccaccagg tcgagggaga 2700
gatccccggc tcgcccatct tcattatgca gcttgcaggc aatgcccggc atttggaggt 2760
gcagcttctg gctgatcagt acggcaacaa tatttcactg tttggtcgag attgttcggt 2820
tcagcgacgg catcaaaaga ttattgagga ggctcctgtg actgtggctg gccagcagac 2880
cttcactgcc atggagaagg ctgccgtgcg actcggtaag cttgtcggat atgtctctgc 2940
aggtaccgtt gaatatctgt attcccatga ggacgacaag ttctacttct tggagctgaa 3000
tcctcgtctt caggtcgaac atcctaccac cgagatggtc accggtgtca acctgcccgc 3060
tgcccagctt cagatcgcca tgggtatccc cctcgatcga atcaaggaca ttcgtctctt 3120
ttacggtgtt aaccctcaca ccaccactcc aattgatttc gacttctcgg gcgaggatgc 3180
tgataagaca cagcgacgtc ccgtcccccg aggtcacacc actgcttgcc gaatcacatc 3240
cgaggaccct ggagagggtt tcaagccctc cggaggtact atgcacgagc tcaacttccg 3300
atcctcgtcc aacgtgtggg gttacttctc cgttggtaac cagggaggta tccattcgtt 3360
ctcggattcg cagtttggtc acatcttcgc cttcggtgag aaccgaagtg cgtctcgaaa 3420
gcacatggtt gttgctttga aggaactatc tattcgaggt gacttccgaa ccaccgtcga 3480
gtacctcatc aagctgctgg agacaccgga cttcgaggac aacaccatca ccaccggctg 3540
gctggatgag cttatctcca acaagctgac tgccgagcga cccgactcgt tcctcgctgt 3600
tgtttgtggt gctgctacca aggcccatcg agcttccgag gactctattg ccacctacat 3660
ggcttcgcta gagaagggcc aggtccctgc tcgagacatt ctcaagaccc ttttccccgt 3720
tgacttcatc tacgagggcc agcggtacaa gttcaccgcc acccggtcgt ctgaggactc 3780
ttacacgctg ttcatcaacg gttctcgatg cgacattgga gttagacctc tttctgacgg 3840
tggtattctg tgtcttgtag gtgggagatc ccacaatgtc tactggaagg aggaggttgg 3900
agccacgcga ctgtctgttg actccaagac ctgccttctc gaggtggaga acgaccccac 3960
tcagcttcga tctccctctc ccggtaagct ggttaagttc ctggtcgaga acggcgacca 4020
cgtgcgagcc aaccagccct atgccgagat tgaggtcatg aagatgtaca tgactctcac 4080
tgctcaggag gacggtattg tccagctgat gaagcagccc ggttccacca tcgaggctgg 4140
cgacatcctc ggtatcttgg cccttgatga tccttccaag gtcaagcatg ccaagccctt 4200
tgagggccag cttcccgagc ttggaccccc cactctcagc ggtaacaagc ctcatcagcg 4260
atacgagcac tgccagaacg tgctccataa cattctgctt ggtttcgata accaggtggt 4320
gatgaagtcc actcttcagg agatggttgg tctgctccga aaccctgagc ttccttatct 4380
ccagtgggct catcaggtgt cttctctgca cacccgaatg agcgccaagc tggatgctac 4440
tcttgctggt ctcattgaca aggccaagca gcgaggtggc gagtttcctg ccaagcagct 4500
tctgcgagcc cttgagaagg aggcgagctc tggcgaggtc gatgcgctct tccagcaaac 4560
tcttgctcct ctgtttgacc ttgctcgaga gtaccaggac ggtcttgcta tccacgagct 4620
tcaggttgct gcaggccttc tgcaggccta ctacgactct gaggcccggt tctgcggacc 4680
caacgtacgt gacgaggatg tcattctcaa gcttcgagag gagaaccgag attctcttcg 4740
aaaggttgtg atggcccagc tgtctcattc tcgagtcgga gccaagaaca accttgtgct 4800
ggcccttctc gatgaataca aggtggccga ccaggctggc accgactctc ctgcctccaa 4860
cgtgcacgtt gcaaagtact tgcgacctgt gctgcgaaag attgtggagc tggaatctcg 4920
agcttctgcc aaggtatctc tgaaagcccg agagattctc atccagtgcg ctctgccctc 4980
tctaaaggag cgaactgacc agcttgagca cattctgcga tcttctgtcg tcgagtctcg 5040
atacggagag gttggtctgg agcaccgaac tccccgagcc gatattctca aggaggttgt 5100
cgactccaag tacattgtct ttgatgtgct tgcccagttc tttgcccacg atgatccctg 5160
gatcgtcctt gctgccctgg agctgtacat ccgacgagct tgcaaggcct actccatcct 5220
ggacatcaac taccaccagg actcggacct gcctcccgtc atctcgtggc gatttagact 5280
gcctaccatg tcgtctgctt tgtacaactc agtagtgtct tctggctcca aaacccccac 5340
ttccccctcg gtgtctcgag ctgattccgt ctccgacttt tcgtacaccg ttgagcgaga 5400
ctctgctccc gctcgaaccg gagcgattgt tgccgtgcct catctggatg atctggagga 5460
tgctctgact cgtgttctgg agaacctgcc caaacggggc gctggtcttg ccatctctgt 5520
tggtgctagc aacaagagtg ccgctgcttc tgctcgtgac gctgctgctg ctgccgcttc 5580
atccgttgac actggcctgt ccaacatttg caacgttatg attggtcggg ttgatgagtc 5640
tgatgacgac gacactctga ttgcccgaat ctcccaggtc attgaggact ttaaggagga 5700
ctttgaggcc tgttctctgc gacgaatcac cttctccttc ggcaactccc gaggtactta 5760
tcccaagtat ttcacgttcc gaggccccgc atacgaggag gaccccacta tccgacacat 5820
tgagcctgct ctggccttcc agctggagct cgcccgtctg tccaacttcg acatcaagcc 5880
tgtccacacc gacaaccgaa acatccacgt gtacgaggct actggcaaga acgctgcttc 5940
cgacaagcgg ttcttcaccc gaggtatcgt acgacctggt cgtcttcgag agaacatccc 6000
cacctcggag tatctcattt ccgaggctga ccggctcatg agcgatattt tggacgctct 6060
agaggtgatt ggaaccacca actcggatct caaccacatt ttcatcaact tctcagccgt 6120
ctttgctctg aagcccgagg aggttgaagc tgcctttggc ggtttcctgg agcgatttgg 6180
ccgacgtctg tggcgacttc gagtcaccgg tgccgagatc cgaatgatgg tatccgaccc 6240
cgaaactggc tctgctttcc ctctgcgagc aatgatcaac aacgtctctg gttacgttgt 6300
gcagtctgag ctgtacgctg aggccaagaa cgacaagggc cagtggattt tcaagtctct 6360
gggcaagccc ggctccatgc acatgcggtc tatcaacact ccctacccca ccaaggagtg 6420
gctgcagccc aagcggtaca aggcccatct gatgggtacc acctactgct atgacttccc 6480
cgagctgttc cgacagtcca ttgagtcgga ctggaagaag tatgacggca aggctcccga 6540
cgatctcatg acttgcaacg agctgattct cgatgaggac tctggcgagc tgcaggaggt 6600
gaaccgagag cccggcgcca acaacgtcgg tatggttgcg tggaagtttg aggccaagac 6660
ccccgagtac cctcgaggcc gatctttcat cgtggtggcc aacgatatca ccttccagat 6720
tggttcgttt ggccctgctg aggaccagtt cttcttcaag gtgacggagc tggctcgaaa 6780
gctcggtatt cctcgaatct atctgtctgc caactctggt gctcgaatcg gcattgctga 6840
cgagctcgtt ggcaagtaca aggttgcgtg gaacgacgag actgacccct ccaagggctt 6900
caagtacctt tacttcaccc ctgagtctct tgccaccctc aagcccgaca ctgttgtcac 6960
cactgagatt gaggaggagg gtcccaacgg cgtggagaag cgtcatgtga tcgactacat 7020
tgtcggagag aaggacggtc tcggagtcga gtgtctgcgg ggctctggtc tcattgcagg 7080
cgccacttct cgagcctaca aggatatctt cactctcact cttgtcacct gtcgatccgt 7140
tggtatcggt gcttaccttg ttcgtcttgg tcaacgagcc atccagattg agggccagcc 7200
catcattctc actggtgccc ccgccatcaa caagctgctt ggtcgagagg tctactcttc 7260
caacttgcag cttggtggta ctcagatcat gtacaacaac ggtgtgtctc atctgactgc 7320
ccgagatgat ctcaacggtg tccacaagat catgcagtgg ctgtcataca tccctgcttc 7380
tcgaggtctt ccagtgcctg ttctccctca caagaccgat gtgtgggatc gagacgtgac 7440
gttccagcct gtccgaggcg agcagtacga tgttagatgg cttatttctg gccgaactct 7500
cgaggatggt gctttcgagt ctggtctctt tgacaaggac tctttccagg agactctgtc 7560
tggctgggcc aagggtgttg ttgttggtcg agctcgtctt ggcggcattc ccttcggtgt 7620
cattggtgtc gagactgcga ccgtcgacaa tactacccct gccgatcccg ccaacccgga 7680
ctctattgag atgagcacct ctgaagccgg ccaggtttgg taccccaact cggccttcaa 7740
gacctctcag gccatcaacg acttcaacca tggtgaggcg cttcctctca tgattcttgc 7800
taactggcga ggcttttctg gtggtcagcg agacatgtac aatgaggttc tcaagtacgg 7860
atctttcatt gttgatgctc tggttgacta caagcagccc atcatggtgt acatccctcc 7920
caccggtgag ctgcgaggtg gttcttgggt tgtggttgac cccaccatca actcggacat 7980
gatggagatg tacgctgacg tcgagtctcg aggtggtgtg ctggagcccg agggaatggt 8040
cggtatcaag taccgacgag acaagctact ggacaccatg gctcgtctgg atcccgagta 8100
ctcctctctc aagaagcagc ttgaggagtc tcccgattct gaggagctca aggtcaagct 8160
cagcgtgcga gagaagtctc tcatgcccat ctaccagcag atctccgtgc agtttgccga 8220
cttgcatgac cgagctggcc gaatggaggc caagggtgtc attcgtgagg ctcttgtgtg 8280
gaaggatgct cgtcgattct tcttctggcg aatccgacga cgattagtcg aggagtacct 8340
cattaccaag atcaatagca ttctgccctc ttgcactcgg cttgagtgtc tggctcgaat 8400
caagtcgtgg aagcctgcca ctcttgatca gggctctgac cggggtgttg ccgagtggtt 8460
tgacgagaac tctgatgccg tctctgctcg actcagcgag ctcaagaagg acgcttctgc 8520
ccagtcgttt gcttctcaac tgagaaagga ccgacagggt actctccagg gcatgaagca 8580
ggctctcgct tctctttctg aggctgagcg ggctgagctg ctcaaggggt tgtgatcatg 8640
taattagtta tgtcacgctt acattcacgc cctccctcca catccgctct aaccgaaaag 8700
gaaggagtta gacaacctga agtctaggtc cctatttatt tttttatagt tatgttagta 8760
ttaagaacgt tatttatatt tcaaattttt cttttttttc tgtacagacg cgtgtacgca 8820
tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa ggctttaatt 8880
tgc 8883
<210> 60
<211> 9095
<212> DNA
<213> Artificial sequence
<400> 60
ggccgcatcg cttcggataa ctcctgctat acgaagttat acgcgcccag agagccattg 60
acgttctttc taatttggac cgatagccgt atagtccagt ctatctataa gttcaactaa 120
ctcgtaacta ttaccataac atatacttca ctgccccaga taaggttccg ataaaaagtt 180
ctgcagacta aatttatttc agtctcctct tcaccaccaa aatgccctcc tacgaagctc 240
gagctaacgt ccacaagtcc gcctttgccg ctcgagtgct caagctcgtg gcagccaaga 300
aaaccaacct gtgtgcttct ctggatgtta ccaccaccaa ggagctcatt gagcttgccg 360
ataaggtcgg accttatgtg tgcatgatca agacccatat cgacatcatt gacgacttca 420
cctacgccag cactgtgctc cccctcaagg aacttgctct taagcacggt ttcttcctgt 480
tcgaggacag aaagttcgca gatattggca acactgtcaa gcaccagtac aagaacggtg 540
tctaccgaat cgccgagtgg tccgatatca ccaacgccca cggtgtaccc ggaaccggaa 600
tcattgctgg cctgcgagct ggtgccgagg aaactgtctc tgaacagaag aaggaggacg 660
tctctgacta cgagaactcc cagtacaagg agttcctggt cccctctccc aacgagaagc 720
tggccagagg tctgctcatg ctggccgagc tgtcttgcaa gggctctctg gccactggcg 780
agtactccaa gcagaccatt gagcttgccc gatccgaccc cgagtttgtg gttggcttcg 840
ttgcccagaa ccgacctaag ggcgactctg aggactggct tattctgacc cccggggtgg 900
gtcttgacga caagggagac gctctcggac agcagtaccg aactgttgag gatgtcatgt 960
ctaccggaac ggatatcata attgtcggcc gaggtctgta cggccagaac cgagatccta 1020
tcgaggaggc caagcgatac cagaaggctg gctgggaggc ttaccagaag attaactgtt 1080
agaggttaga ctatggatat gtaatttaac tgtgtatata gagagcgtgc aagtatggag 1140
cgcttgttca gcttgtatga tggtcagacg acctgtctga tcgagtatgt atgatactgc 1200
acaacctgtg tatccgcatg atctgtccaa tggggcatgt tgttgtgttt ctcgagaata 1260
acttcgtatg atgtatgcta tacgaagtta tgcgatcgca gagaccgggt tggcggcgta 1320
tttgtgtccc aaaaaacagc cccaattgcc ccaattgacc ccaaattgac ccagtagcgg 1380
gcccaacccc ggcgagagcc cccttcaccc cacatatcaa acctcccccg gttcccacac 1440
ttgccgttaa gggcgtaggg tactgcagtc tggaatctac gcttgttcag actttgtact 1500
agtttctttg tctggccatc cgggtaaccc atgccggacg caaaatagac tactgaaaat 1560
ttttttgctt tgtggttggg actttagcca agggtataaa agaccaccgt ccccgaatta 1620
cctttcctct tcttttctct ctctccttgt caactcacac ccgaaatcgt taagcatttc 1680
cttctgagta taagaatcat tcaaaatggt gagtttcaga ggcagcagca attgccacgg 1740
gctttgagca cacggccggg tgtggtccca ttcccatcga cacaagacgc cacgtcatcc 1800
gaccagcact ttttgcagta ctaaccgcag atttatgact atcgactcac aatactacaa 1860
gtcgcgagac aaaaacgaca cggcacccaa aatcgcggga atccgatatg ccccgctatc 1920
gacaccatta ctcaaccgat gtgagacctt ctctctggtc tggcacattt tcagcattcc 1980
cactttcctc acaattttca tgctatgctg cgcaattcca ctgctctggc catttgtgat 2040
tgcgtatgta gtgtacgctg ttaaagacga ctccccgtcc aacggaggag tggtcaagcg 2100
atactcgcct atttcaagaa acttcttcat ctggaagctc tttggccgct acttccccat 2160
aactctgcac aagacggtgg atctggagcc cacgcacaca tactaccctc tggacgtcca 2220
ggagtatcac ctgattgctg agagatactg gccgcagaac aagtacctcc gagcaatcat 2280
ctccaccatc gagtactttc tgcccgcctt catgaaacgg tctctttcta tcaacgagca 2340
ggagcagcct gccgagcgag atcctctcct gtctcccgtt tctcccagct ctccgggttc 2400
tcaacctgac aagtggatta accacgacag cagatatagc cgtggagaat catctggctc 2460
caacggccac gcctcgggct ccgaacttaa cggcaacggc aacaatggca ccactaaccg 2520
acgacctttg tcgtccgcct ctgctggctc cactgcatct gattccacgc ttcttaacgg 2580
gtccctcaac tcctacgcca accagatcat tggcgaaaac gacccacagc tgtcgcccac 2640
aaaactcaag cccactggca gaaaatacat cttcggctac cacccccacg gcattatcgg 2700
catgggagcc tttggtggaa ttgccaccga gggagctgga tggtccaagc tctttccggg 2760
catccctgtt tctcttatga ctctcaccaa caacttccga gtgcctctct acagagagta 2820
cctcatgagt ctgggagtcg cttctgtctc caagaagtcc tgcaaggccc tcctcaagcg 2880
aaaccagtct atctgcattg tcgttggtgg agcacaggaa agtcttctgg ccagacccgg 2940
tgtcatggac ctggtgctac tcaagcgaaa gggttttgtt cgacttggta tggaggtcgg 3000
aaatgtcgcc cttgttccca tcatggcctt tggtgagaac gacctctatg accaggttag 3060
caacgacaag tcgtccaagc tgtaccgatt ccagcagttt gtcaagaact tccttggatt 3120
cacccttcct ttgatgcatg cccgaggcgt cttcaactac gatgtcggtc ttgtccccta 3180
caggcgaccc gtcaacattg tggttggttc ccccattgac ttgccttatc tcccacaccc 3240
caccgacgaa gaagtgtccg aataccacga ccgatacatc gccgagctgc agcgaatcta 3300
caacgagcac aaggatgaat atttcatcga ttggaccgag gagggcaaag gagccccaga 3360
gttccgaatg attgagtaaa aattcatgta attagttatg tcacgcttac attcacgccc 3420
tccctccaca tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc 3480
tatttatttt tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct 3540
tttttttctg tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa 3600
ggttttggga cgctcgaagg ctttaatttg catcgcggag tttggcgccc gttttttcga 3660
gccccacacg tttcggtgag tatgagcggc ggcagattcg agcgtttccg gtttccgcgg 3720
ctggacgaga gcccatgatg ggggctccca ccaccagcaa tcagggccct gattacacac 3780
ccacctgtaa tgtcatgctg ttcatcgtgg ttaatgctgc tgtgtgctgt gtgtgtgtgt 3840
tgtttggcgc tcattgttgc gttatgcagc gtacaccaca atattggaag cttattagcc 3900
tttctatttt ttcgtttgca aggcttaaca acattgctgt ggagagggat ggggatatgg 3960
aggccgctgg agggagtcgg agaggcgttt tggagcggct tggcctggcg cccagctcgc 4020
gaaacgcacc taggaccctt tggcacgccg aaatgtgcca cttttcagtc tagtaacgcc 4080
ttacctacgt cattccatgc atgcatgttt gcgccttttt tcccttgccc ttgatcgcca 4140
cacagtacag tgcactgtac agtggaggtt ttgggggggt cttagatggg agctaaaagc 4200
ggcctagcgg tacactagtg ggattgtatg gagtggcatg gagcctaggt ggagcctgac 4260
aggacgcacg accggctagc ccgtgacaga cgatgggtgg ctcctgttgt ccaccgcgta 4320
caaatgtttg ggccaaagtc ttgtcagcct tgcttgcgaa cctaattccc aattttgtca 4380
cttcgcaccc ccattgatcg agccctaacc cctgcccatc aggcaatcca attaagctcg 4440
cattgtctgc cttgtttagt ttggctcctg cccgtttcgg cgtccacttg cacaaacaca 4500
aacaagcatt atatataagg ctcgtctctc cctcccaacc acactcactt ttttgcccgt 4560
cttcccttgc taacacaaaa gtcaagaaca caaacaacca ccccaacccc cttacacaca 4620
agacatatct acagcattta tggtgaaaaa cgtggaccaa gtggatctct cgcaggtcga 4680
caccattgcc tccggccgag atgtcaacta caaggtcaag tacacctccg gcgttaagat 4740
gagccagggc gcctacgacg acaagggccg ccacatttcc gagcagccct tcacctgggc 4800
caactggcac cagcacatca actggctcaa cttcattctg gtgattgcgc tgcctctgtc 4860
gtcctttgct gccgctccct tcgtctcctt caactggaag accgccgcgt ttgctgtcgg 4920
ctattacatg tgcaccggtc tcggtatcac cgccggctac caccgaatgt gggcccatcg 4980
agcctacaag gccgctctgc ccgttcgaat catccttgct ctgtttggag gaggagctgt 5040
cgagggctcc atccgatggt gggcctcgtc tcaccgagtc caccaccgat ggaccgactc 5100
caacaaggac ccttacgacg cccgaaaggg attctggttc tcccactttg gctggatgct 5160
gcttgtgccc aaccccaaga acaagggccg aactgacatt tctgacctca acaacgactg 5220
ggttgtccga ctccagcaca agtactacgt ttacgttctc gtcttcatgg ccattgttct 5280
gcccaccctc gtctgtggct ttggctgggg cgactggaag ggaggtcttg tctacgccgg 5340
tatcatgcga tacacctttg tgcagcaggt gactttctgt gtcaactccc ttgcccactg 5400
gattggagag cagcccttcg acgaccgacg aactccccga gaccacgctc ttaccgccct 5460
ggtcaccttt ggagagggct accacaactt ccaccacgag ttcccctcgg actaccgaaa 5520
cgccctcatc tggtaccagt acgaccccac caagtggctc atctggaccc tcaagcaggt 5580
tggtctcgcc tgggacctcc agaccttctc ccagaacgcc atcgagcagg gtctcgtgca 5640
gcagcgacag aagaagctgg acaagtggcg aaacaacctc aactggggta tccccattga 5700
gcagctgcct gtcattgagt ttgaggagtt ccaagagcag gccaagaccc gagatctggt 5760
tctcatttct ggcattgtcc acgacgtgtc tgcctttgtc gagcaccacc ctggtggaaa 5820
ggccctcatt atgagcgccg tcggcaagga cggtaccgct gtcttcaacg gaggtgtcta 5880
ccgacactcc aacgctggcc acaacctgct tgccaccatg cgagtttcgg tcattcgagg 5940
cggcatggag gttgaggtgt ggaagactgc ccagaacgaa aagaaggacc agaacattgt 6000
ctccgatgag agtggaaacc gaatccaccg agctggtctc caggccaccc gggtcgagaa 6060
ccccggtatg tctggcatgg ctgcttagaa atgcagtttg tttagcaaaa tatatttaac 6120
gagtttgata gaggcgctgg actacataat tactgaatca cgcgtacatg tttcagctca 6180
aattgtatca cggtttcttt gtagcaatgg agggggagag ttgacaaggc attagagaag 6240
agagcgagag gagaagacaa gtggatagac gactgcaatc atatgatctg cacaaactgc 6300
gatgttttcc tgtcagatca tgttcttttg ctcatagtta agctatcgtg actttacgga 6360
tccgccgagc atcttagtag cgaggtttgc ggtctgggct gatcggcttt tgttgatcgg 6420
gttcggaacg aattagaagt gggtcgatag caatgatcaa ttcggggtga gtgagtgcgg 6480
ttagtgggaa accggggaga tactgtgggg atttagggga cagtgttaag aagaggaagg 6540
ggtgagggag gtgaggtgag ggagagggaa gtatagaggt ttatgtagta acatcaatca 6600
tacagtttaa actgcaccca acaataaatg ggtagggttg caccaacaaa gggatgggat 6660
ggggggtaga agatacgagg ataacggggc tcaatggcac aaataagaac gaatactgcc 6720
attaagactc gtgatccagc gactgacacc attgcatcat ctaagggcct caaaactacc 6780
tcggaactgc tgcgctgatc tggacaccac agaggttccg agcactttag gttgcaccaa 6840
atgtcccacc aggtgcaggc agaaaacgct ggaacagcgt gtacagtttg tcttagcaaa 6900
aagtgaaggc gctgaggtcg agcagggtgg tgtgacttgt tatagccttt agagctgcga 6960
aagcgcgtat ggatttggct catcaggcca gattgagggt ctgtggacac atgtcatgtt 7020
agtgtacttc aatcgccccc tggatatagc cccgacaata ggccgtggcc tcattttttt 7080
gccttccgca catttccatt gctcggtacc cacaccttgc ttctcctgca cttgccaacc 7140
ttaatactgg tttacattga ccaacatctt acaagcgggg ggcttgtcta gggtatatat 7200
aaacagtggc tctcccaatc ggttgccagt ctcttttttc ctttctttcc ccacagattc 7260
gaaatctaaa ctacacatca cacaatgcct gttactgacg tccttaagcg aaagtccggt 7320
gtcatcgtcg gcgacgatgt ccgagccgtg agtatccacg acaagatcag tgtcgagacg 7380
acgcgttttg tgtaatgaca caatccgaaa gtcgctagca acacacactc tctacacaaa 7440
ctaacccaga tgtctagcaa atggtttaat gctatacacc tactggtgtg cccgttgacg 7500
gtactggtag gatatctcat gaacacgtat ggctacggtg cggcgctgca agcaaccctg 7560
aataaggatg gtctggtaaa tgctatgttg gtaaagaaag ggtggttttg gacttccttg 7620
gtcggatggt ggtgtattat acgctaccgt gcggtgccag gggcaaccga cagagaccgg 7680
agacacattg tccagtcatt caaaaggtat gccatactga cagtatggtg gtacgtattc 7740
acgcaaggta tatggtttgg cgtaggcccc atcatggact tggtattcgt atatacgggt 7800
ggccattgtc actatgacgt cttcgatgat gcaggtcacg tgaacgaaga cttccagggt 7860
tctgtcaccc ggaccaatcg cgcgttggcg ctcattcaca atgtcctcac tttgcacgga 7920
caccaccaag aacaccgtca gcaacaactc tgggaccgct ccatagggtc gatccagggc 7980
gccctgcagg cgacgcaacc gaaaacccca aaaaacgtaa cggccagcgc tgccgctgcc 8040
atcaatactt ttattcatga ccagatgcac aggtggcaag gtccgttaac cacctcggca 8100
cagtgcagac gttttggagg ccattgggct ggaggacacg atccatccgg gcacgtgttc 8160
ttggccacct tgatgtgcat gtttcttcta ggtgagctac gtgtgtttgg gcgtcgtgca 8220
ctggttcact tgtacgcgca gaaatggcag cttgtacgat tggtgacccg ccttttcgac 8280
actggaccgc tatggacgtg gcggcggtgt ggcggcggct caatgacctg tggcgcccgt 8340
ttgtggcgtg cgatagtcga gccgcctgtc acgtgcgctg ccgccctgct ccgtttgacg 8400
cgatgcatag catgcgacca cccagtaatc atactgctga cgctattggt cacgtggtta 8460
tggcagctgc tgttgactgc ggtggcgtcc cgtttccaca ccgtacgtga gcacatgtct 8520
ggattgctag ccgcgtacat agtgacaggc cttgtctacg ctcgcgacgc agccgcgcta 8580
cgtccagtat gacgctattt atcactcttt acaacttcta cctcaactat ctactttaat 8640
aaatgaatat cgtttattct ctatgattac tgtatatgcg ttcctctaag acaaatcgaa 8700
accagcatgc gatcgaatgg catacaaaag tttcttccga agttgatcaa tgtcctgata 8760
gtcaggcagc ttgagaagat tgacacaggt ggaggccgta gggaaccgat caacctgtct 8820
accagcgtta cgaatggcaa atgacgggtt caaagccttg aatccttgca atggtgcctt 8880
ggatactgat gtcacaaact taagaagcag ccgcttgtcc tcttcctcga aactctcaaa 8940
cacagtccag aggtccttta tagcttgatc tgtatccaga tagcctccgt aattggtgtg 9000
tgtcttcaaa tcccagacgt ccacattggc atgtcctcca ctgataagca tttgaagttc 9060
atctgcgttg aacattgaga cccacgaagg gtcgc 9095
<210> 61
<211> 861
<212> DNA
<213> Artificial sequence
<400> 61
atgccctcct acgaagctcg agctaacgtc cacaagtccg cctttgccgc tcgagtgctc 60
aagctcgtgg cagccaagaa aaccaacctg tgtgcttctc tggatgttac caccaccaag 120
gagctcattg agcttgccga taaggtcgga ccttatgtgt gcatgatcaa gacccatatc 180
gacatcattg acgacttcac ctacgccagc actgtgctcc ccctcaagga acttgctctt 240
aagcacggtt tcttcctgtt cgaggacaga aagttcgcag atattggcaa cactgtcaag 300
caccagtaca agaacggtgt ctaccgaatc gccgagtggt ccgatatcac caacgcccac 360
ggtgtacccg gaaccggaat cattgctggc ctgcgagctg gtgccgagga aactgtctct 420
gaacagaaga aggaggacgt ctctgactac gagaactccc agtacaagga gttcctggtc 480
ccctctccca acgagaagct ggccagaggt ctgctcatgc tggccgagct gtcttgcaag 540
ggctctctgg ccactggcga gtactccaag cagaccattg agcttgcccg atccgacccc 600
gagtttgtgg ttggcttcgt tgcccagaac cgacctaagg gcgactctga ggactggctt 660
attctgaccc ccggggtggg tcttgacgac aagggagacg ctctcggaca gcagtaccga 720
actgttgagg atgtcatgtc taccggaacg gatatcataa ttgtcggccg aggtctgtac 780
ggccagaacc gagatcctat cgaggaggcc aagcgatacc agaaggctgg ctgggaggct 840
taccagaaga ttaactgtta g 861
<210> 62
<211> 34
<212> DNA
<213> Artificial sequence
<400> 62
atcgcttcgg ataactcctg ctatacgaag ttat 34
<210> 63
<211> 34
<212> DNA
<213> Artificial sequence
<400> 63
ataacttcgt atgatgtatg ctatacgaag ttat 34

Claims (2)

1. A recombinant engineering bacterium with a preservation number of CCTCC NO: m2020606, which is classified and named Yarrowia lipolytica (Yarrowia lipolytica) ZTQ5320.
2. A method for producing beta-carotene, characterized in that beta-carotene is extracted by fermenting the recombinant engineered bacterium of claim 1.
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CN114277040B (en) * 2021-12-24 2023-08-25 江南大学 Construction method and application of efficient synthesis beta-carotene strain
CN114317307B (en) * 2021-12-30 2024-05-10 广州智特奇生物科技股份有限公司 Genetically engineered bacterium capable of improving astaxanthin biosynthesis yield as well as construction method and application thereof
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Denomination of invention: A genetically engineered bacterium for increasing b - carotene production and its application

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