CN113481233A - Method for constructing ectoin producing strain - Google Patents

Method for constructing ectoin producing strain Download PDF

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CN113481233A
CN113481233A CN202110750324.7A CN202110750324A CN113481233A CN 113481233 A CN113481233 A CN 113481233A CN 202110750324 A CN202110750324 A CN 202110750324A CN 113481233 A CN113481233 A CN 113481233A
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ectoin
lysc
plasmid
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CN113481233B (en
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范文超
刘映淼
高书良
杨海锋
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Zhejiang Huarui Biotechnology Co ltd
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Abstract

The invention discloses a method for constructing an ectoin producing strain, the constructed genetically engineered strain CGMCC No.22733 can directly produce ectoin by fermenting with glucose as a carbon source for 48 hours in a 5L fermentation tank, and can produce the ectoin 42.7g/L, thereby having development and application potentials.

Description

Method for constructing ectoin producing strain
Technical Field
The invention belongs to the fields of metabolic engineering and genetic engineering, and particularly relates to a method for constructing an ectoine producing strain, a constructed genetic engineering strain and application thereof.
Background
Ectoin (Ectoin) with the chemical name of 2-methyl-1,4,5,6, -tetrahydropyrimidine-4-carboxylic acid, also known as tetrahydromethylpyrimidine carboxylic acid, is readily soluble in water, glycerol, propylene glycol, ethanol, and the like. The molecule is C6H10O2N2The structural formula is as follows:
Figure BDA0003145930490000011
in 1985, Galinski professor finds that halophilic bacteria can generate a natural protective component-ectoin in the cell outer layer under the environment of high temperature, drying, strong UV irradiation and high salinity in Egyptian desert, so that the halophilic bacteria can be prevented from being damaged, and self-repair is carried out; research shows that the ectoin also has good repairing and protecting effects on skin. It is one of the biological engineering preparations adopted by high-grade cosmetics such as Eurya, Yashilan indigo and the like, and can also be used for medical care and health care, such as stress dermatitis, pneumonia, allergic rhinitis, senile dementia and the like. The literature reports that the market price of ectoin reaches $ 1000/kg (Genes 2018,9(4),177.), and the ectoin has a large market potential in the future.
At present, Ectoin is mainly prepared by halophilic bacteria fermentation, and high-salt fermentation not only influences the service life of a fermentation container, but also is a great challenge to subsequent fermentation waste liquid treatment. In 1999, Ono et al reported the synthetic pathway of ectoin in highly halophilic bacteria (Halomonas elongata) and identified the related enzymes (J Bacteriol.1999,181:91-9.) and early fermentation production mainly used this strain while also producing hydrogenated ectoin. With the development of genetic engineering technology, researchers try to use conventional fermentation hosts such as escherichia coli and corynebacterium glutamicum to produce Ectoin through genetic engineering modification (see Microb Cell fact.2021,20:76.), the escherichia coli genetic engineering strain can produce 25g/L Ectoin, and the conversion rate reaches 0.11g/g glucose (Metabolic engineering.2016,36: 10-18.); becker and the like construct a genetic engineering strain for high-yield lysine by taking corynebacterium glutamicum as a host strain, and the fermentation concentration can reach 120g/L (Metabolic engineering.2011,13: 159-; subsequently, they expressed the ectABC gene derived from Pseudomonas stutzeri in LYS-1 of Corynebacterium glutamicum and knocked outThe lysE gene so as to prevent the discharge of lysine can produce 4.5g/L of ectoin at low salt concentration (Microbial Cell factors.2013, 12: 110.); giesselmann et al, by Corynebacterium glutamicum lysCfbrAs a starting strain, the expression of the ectA, the ectB and the ectC is optimized through different promoter combinations, the related metabolic flows are balanced, the strain obtained finally can produce 65g/L ectoin after being fermented for 56 hours (Biotechnol.J.2019,14,1800417), and the conversion rate reaches 0.19g/g glucose.
Disclosure of Invention
The inventor researches the reported biosynthesis route of ectoin in Corynebacterium glutamicum, develops a new metabolic pathway, takes the Corynebacterium glutamicum producing lysine as a starting strain, weakens hom gene, knocks out pck gene, enhances expression of lysC and tkt-tal-zwf by sod promoter, and then integrates ABC ectogene at ddh gene site and phage transposase IS30(IS30-like element, ISCG2 family transposase) gene site to construct ectoin. Therefore, the present invention includes the following technical solutions.
A method for constructing an ectoine-producing strain, comprising the steps of:
A. using corynebacterium glutamicum for producing lysine as an initial strain, knocking down or weakening hom gene of homoserine kinase in genome to obtain a strain A for down-regulating homoserine kinase expression; wherein, the knock-down of the hom gene includes, but is not limited to, knocking out the hom gene, down-regulating the expression of the hom gene, or weakening the enzymatic activity of hom homoserine kinase, for example, the hom gene can be mutated so as to weaken the function of homoserine kinase;
B. knocking out pck gene of coding phosphoenolpyruvate carboxykinase in genome of the strain A to obtain a strain B lacking phosphoenolpyruvate carboxykinase;
C. enhancing the expression of lysC encoding aspartate kinase in the genome of strain B to obtain strain C over-expressing aspartate kinase;
D. enhancing the expression of a coding transketolase gene tkt, a coding tyrosine ammonia lyase gene tal and a coding 6-phosphogluconate dehydrogenase gene zwf in the genome of the strain C to obtain a strain D with an enhanced oxidative phosphorylation pathway;
E. integrating ectoABC genes (namely, ectoA, ectoB and ectoC, which are abbreviated as ECT) derived from Pseudomonas stutzeri at a ddh site of a gene encoding diaminopropionate dehydrogenase in a genome of the strain D to obtain a strain E with enhanced ectoin synthesis pathway;
F. integrating the ectABC gene from Pseudomonas stutzeri at the gene site of bacteriophage transposase IS30 in the genome of the strain E to obtain a strain F with further enhanced ectoin synthesis pathway, and screening positive clones to obtain an ectoin producing strain.
Preferably, the method further comprises the following steps of integrating the ectABC gene in the genome in a superposition manner:
G. further integrating the ectABC gene derived from Pseudomonas stutzeri into the genome of the strain F obtained in step F, and screening for a positive clone with enhanced ectoin synthesis pathway.
In one embodiment, the starting strain in step A is Corynebacterium glutamicum ATCC13032 LysCfbr. The strain can be constructed according to the method reported in Biotechnol.J.2019,14,1800417.
The reduction of the hom gene in the step A can be realized by subjecting the hom gene to T176C mutation, and the homoserine kinase is subjected to T176C mutation, so that the enzyme activity is reduced.
For example, the step of mutating the hom gene by T176C is: introducing a plasmid pK18mobSacB-homT176C with a nucleotide sequence of SEQ ID NO. 4 into a host cell; and carrying out SacB sucrose reverse screening to screen positive clones.
The knock-out of the pck gene in step B above may comprise the steps of: introducing a plasmid pK18mobSacB-KOpck with a nucleotide sequence of SEQ ID NO.2 into a host cell; and carrying out SacB sucrose reverse screening to screen positive clones.
The enhancement of expression of the gene lysC in the above step C can be achieved by placing the lysC gene downstream of the sod promoter. For example, the following steps may be included: introducing a plasmid pK18mobSacB-Psod-lysC with a nucleotide sequence of SEQ ID NO. 1 into a host cell; and carrying out SacB sucrose reverse screening to screen positive clones.
The enhancement of the expression of the genes tkt, tal and zwf in step D above can be achieved by replacing their native promoters with the sod promoter. For example, the following steps may be included: introducing a plasmid pK18mobSacB-Psod-tkt-tal-zwf with a nucleotide sequence of SEQ ID NO. 3 into a host cell; and carrying out SacB sucrose reverse screening to screen positive clones.
The integration of the ectABC gene at the ddh gene site in step E above can include the following steps: introducing a plasmid pK18mobSacB-ddh-ECT with a nucleotide sequence of SEQ ID NO. 5 into a host cell; and carrying out SacB sucrose reverse screening to screen positive clones.
The integration of the ectABC gene at the IS30 gene site in step F above may comprise the following steps: introducing a plasmid pK18mobSacB-IS30-ECT with a nucleotide sequence of SEQ ID NO. 6 into a host cell; and carrying out SacB sucrose reverse screening to screen positive clones.
The introduction of the plasmid in the above step may be calcium chloride transformation or electric transformation, preferably electric transformation.
The second object of the present invention is to provide an ectoine-producing strain which is constructed and selected by the above-mentioned method.
Preferably, the ectoin-producing strain can be Corynebacterium glutamicum (Corynebacterium glutamicum), which is deposited in the common microorganism center of China Committee for culture Collection of microorganisms with the deposit number of CGMCC No. 22733.
The third object of the present invention is to provide the use of the above-mentioned ectoine-producing bacterium such as CGMCC No.22733 for producing ectoine.
For example, ectoin can be produced by fermentation of the above-mentioned ectoin-producing bacterium such as CGMCC No. 22733.
In fermentation, glucose is required as a carbon source.
In one embodiment, the composition of the shake flask fermentation seed medium may be: 10g/L of peptone, 5g/L of beef extract, 5g/L of yeast powder, 2.5g/L of sodium chloride, 2g/L of urea and 10g/L of glucose. It is used by sterilizing at 115 deg.C for 15 min.
The shake flask fermentation medium may be:
Figure BDA0003145930490000041
the fermenter medium can be:
Figure BDA0003145930490000042
Figure BDA0003145930490000051
the fermentation temperature is preferably about 30 ℃.
The invention develops a new ectoin metabolic route, so that corynebacterium glutamicum can directly produce ectoin through fermentation, and the method is worthy of further development and utilization.
The Latin of the constructed high-yield gene engineering bacteria of the ectoin is named Corynebacterium glutamicum, the Chinese name is Corynebacterium glutamicum or Corynebacterium glutamicum, the high-yield gene engineering bacteria are stored in the common microorganism center of China Committee for culture Collection of microorganisms, the storage date is 2021 year 6 month 18 day, the storage address is the microbial research institute of China academy of sciences No. 3 of the morning West Lu No. 1 of the Yangyang area in Beijing, and the storage number is CGMCC No. 22733.
Drawings
FIG. 1 is a diagram showing the metabolic pathway of ectoin in Corynebacterium glutamicum constructed according to the present invention.
FIG. 2 is a schematic structural diagram of plasmid pK18mobsacB constructed according to the present invention.
FIG. 3 is a schematic structural diagram of plasmid pK18mobSacB-Psod-lysC constructed according to the present invention. The nucleotide sequence is SEQ ID NO. 1.
FIG. 4 is a schematic structural view of plasmid pK18mobSacB-KOpck constructed according to the present invention. The nucleotide sequence is SEQ ID NO. 2.
FIG. 5 is a schematic structural diagram of plasmid pK18mobSacB-Psod-tkt-tal-zwf constructed in the present invention. The nucleotide sequence is SEQ ID NO. 3.
FIG. 6 is a schematic structural diagram of plasmid pK18mobSacB-homT176C constructed according to the present invention. The nucleotide sequence is SEQ ID NO. 4.
FIG. 7 is a schematic structural diagram of plasmid pK18mobSacB-ddh-ECT constructed according to the present invention. The nucleotide sequence is SEQ ID NO. 5.
FIG. 8 IS a schematic structural diagram of plasmid pK18mobSacB-IS30-ECT constructed according to the present invention. The nucleotide sequence is SEQ ID NO. 6.
FIG. 9 is a fermentation graph of genetically engineered bacterium ATCC13032-ECT-01 constructed by the present invention. Wherein the abscissa is the fermentation sampling time, and the ordinate is the concentration of ectoin in the fermentation liquid.
Detailed Description
The invention changes the metabolic pathway of the synthesis of the ectoin in the corynebacterium glutamicum producing lysine, and can realize the biosynthesis of the ectoin from glucose through enzyme catalysis as shown in figure 1.
In order to create a new metabolic pathway for ectoin, it is necessary to clone each gene to be knocked out, attenuated, integrated, overexpressed, and enhanced expressed separately on a plasmid vector. Then respectively, sequentially or simultaneously transferring the bacillus glutamicum (C.glutamicum) competent cells into corynebacterium glutamicum competent cells; it is also possible to clone more than two genes on a single plasmid and then to transfer them separately, successively or simultaneously into C.glutamicum competent cells.
Among them, usable plasmid vectors include pK18mobsacB and the like, but are not limited thereto.
It should be understood that in the specific operation of constructing the genetically engineered bacteria of the present invention, the sequence of step A, step B, step C, step D, step E, step F and step G is not fixed from the front to the back according to the English letter sequence, and they can be operated alternately or reversely, as long as each step can achieve its own function and accomplish the oriented change of host cell genotype.
Herein, for the sake of convenience of description, a certain enzyme such as homoserine kinase (hom) is sometimes used in combination with the name of its encoding gene (DNA), and those skilled in the art will understand that they represent different substances in different description occasions. Their meaning will be readily understood by those skilled in the art based on the context and context. For example, for hom, when used to describe the function or class of homoserine kinase, refer to proteins; when described as a gene, refers to the gene encoding the enzyme.
The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.
The addition amount, content and concentration of various substances are referred to herein, wherein the percentage refers to the mass percentage unless otherwise specified.
Examples
Materials and methods
The primers and gene synthesis and sequencing in the examples were performed by Jiangsu Jinzhi Biotechnology GmbH and Anhui Tong Biotechnology GmbH.
The molecular biological experiments in the examples include plasmid construction, digestion, ligation, competent cell preparation, transformation, culture medium preparation, and the like, and are mainly performed with reference to "molecular cloning experimental manual" (third edition), sambrook, d.w. rasel (american), translation of huang peitang et al, scientific press, beijing, 2002). The specific experimental conditions can be determined by simple experiments if necessary.
PCR amplification experiments were performed according to the reaction conditions or kit instructions provided by the supplier of the plasmid or DNA template. If necessary, it can be adjusted by simple experiments.
LB liquid medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride.
LB solid medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride and 20g/L agar powder.
BHIS medium: 37g/L BHI, 91g/L sorbitol.
BHIS-suc Medium: 37g/L BHI, 91g/L sorbitol, 200g/L sucrose, 10g/L glucose.
BHI medium: 37g BHI powder was added to 1L purified water and sterilized at 115 ℃ for 15 min.
LB-SUC 100: 100g of sucrose was added to 1L of LB solid medium.
LB-SUC 100-K25: 100g of sucrose and 25mg of kanamycin were added to 1L of LB solid medium.
20X electrotransfer mother liquor: 80g/L glycine, 2% Tween 80.
In the following examples, when a kanamycin-containing medium was used, the final concentration of kanamycin in the medium was 50. mu.g/ml.
HPLC detection method of ectoin: agilent 1260 high performance liquid chromatograph; the chromatographic column is Agilent AQ-C18, the detector VWD detector, the detection wavelength is 210nm, and the mobile phase is 10Mm potassium dihydrogen phosphate (pH3.5): acetonitrile 99:1, flow rate 0.7ml/min, column oven 20 ℃. The RT of ectoin was 5.4 min.
The starting strain used in the examples was Corynebacterium glutamicum ATCC13032 LysCfbrConstructed by Zhejiang Huari Biotechnology Limited according to the method reported in Biotechnol.J.2019,14,1800417.
The plasmids pK18mobSacB, pK18mobSacB-Psod-lysC, pK18mobSacB-KOpck, pK18mobSacB-Psod-tkt-tal-zwf, pK18mobSacB-homT176C, pK18mobSacB-ddh-ECT and pK18mobSacB-IS30-ECT used in the examples were constructed by Wai, Zhejiang Biotechnology Ltd, and any unit and person could obtain these plasmids for verifying the present invention, but were not allowed to be used for other purposes including development and utilization, scientific research and teaching.
The sequence information of the primers used in the examples is shown in Table 1.
TABLE 1 primer sequences
Figure BDA0003145930490000071
Figure BDA0003145930490000081
Figure BDA0003145930490000091
Note: in the table, the primer name suffix F represents the forward primer and R represents the reverse primer.
Example 1: construction of genetically modified target plasmids
1.1 construction of plasmid pK18-Psod-lysC
Corynebacterium glutamicum ATCC13032 LysCfbrThe genome is taken as a template, and PCR amplification is respectively carried out by using Psod-lysC-up-F/Psod-lysC-up-R and Psod-lysC-down-F/Psod-lysC-down-R primer pairs. PCR amplification conditions: 5min at 95 ℃; 30 cycles of 92 ℃ for 30s, 58 ℃ for 30s, 65 ℃ for 35 s; 10min at 65 ℃. And amplifying to obtain Psod-lysC-up and Psod-lysC-down fragments, wherein the lengths of the Psod-lysC-up and Psod-lysC-down fragments are 1k, and cutting and recovering the gel.
ATCC1303 LysCfbrAnd (3) taking the genome as a template, and taking Psod-lysC-F and Psod-lysC-R as primers for amplification to obtain a Psod-lysC fragment with the length of 200bp, and cutting and recovering the gel. Performing overlap PCR on three fragments of Psod-lysC-up, Psod-lysC-down and Psod-lysC by taking Psod-lysC-up-F and Psod-lysC-down-R as primers, cutting and recycling a product to obtain a Psod-lysC fragment. And (3) taking pK18mobsacB plasmid as a template, taking Psod-lysC-Z-F and Psod-lysC-Z-R as primers for amplification to obtain a Psod-lysC-Z fragment with the length of 5.7kb, and cutting and recovering the gel. The Psod-lysC-Z was assembled with the Psod-lysC fragment (Transgen,
Figure BDA0003145930490000092
seamless Cloning and Assembly Kit), transforming DH5a competent cells, recovering, coating kan plates, performing colony PCR with M13F and M13R as identification primers, selecting positive clones with a 2.2kb band, inoculating the test tubes, and extracting plasmids for use.
The structure of the obtained plasmid pK18-Psod-lysC is shown in FIG. 3, and its nucleotide sequence is SEQ ID NO: 1.
1.2 construction of plasmid pK18-KOpck
ATCC13032 LysCfbrThe genome is taken as a template, and KOpck-up-F/KOpck-up-R and KOpck-down-F/KOpck-down-R primer pairs are respectively used for amplification. PCR amplification conditions: 5min at 95 ℃; 92 deg.C30 cycles of 30s at 58 ℃ for 30s and 65 ℃ for 35 s; 10min at 65 ℃. KOpck-up and KOpck-down fragments are obtained by amplification, the length of each fragment is 1kb, and the fragments are cut and recovered. The KOpck-Z fragment was obtained by amplification using pK18mobsacB plasmid as a template, and the fragment was 5.7kb in length and recovered by cutting the gel. And carrying out Gibson assembly on KOpck-up, KOpck-down and KOpck-Z, transforming a DH5a host, recovering, coating a kan plate, identifying by using M13F and M13R as identification primers, inoculating a colony PCR positive clone, and extracting a plasmid for later use.
The structure of the obtained plasmid pK18-KOpck is shown in FIG. 4, and its nucleotide sequence is SEQ ID NO. 2.
1.3 construction of plasmid pK18-Psod-tkt-tal-zwf
ATCC13032 LysCfbrThe genome is taken as a template, and Psod-ttz-uP-F/Psod-ttz-uP-R and Psod-ttz-down-F/Psod-ttz-down-R are respectively used for amplifying. PCR amplification conditions: 5min at 95 ℃; 30 cycles of 92 ℃ for 30s, 58 ℃ for 30s, 65 ℃ for 35 s; 10min at 65 ℃. The three fragments obtained by amplification are respectively 1kb, 1kb and 200bp in size, ttz-up, ttz-down and Psod-ttz fragments are obtained, and the fragments are cut and recovered. Performing overlap PCR by using Psod-ttz-uP-F, Psod-ttz-down-R as primers and three fragments of ttz-uP, ttz-down and Psod-ttz as templates, wherein the length of the overlap PCR is 2.2kb, and the overlap PCR is cut and recovered to be named as a Psod-ttz-OE fragment. The Psod-ttz-Z-F/Psod-ttz-Z-R primer pair takes pK18mobsacB plasmid as a template for amplification to obtain a Psod-ttz-Z fragment, the length of the Psod-ttz-Z fragment is 5.7kb, and the Psod-ttz-Z fragment is cut and recovered. Gibson assembly is carried out on two fragments of Psod-ttz-OE and Psod-ttz-Z, a DH5a host is transformed, the recovery is carried out, a kan plate is coated, identification is carried out by taking M13F and M13R as identification primers, a positive clone has a 2.2kb band, and a colony PCR positive clone is inoculated for extraction plasmid for standby.
The structure of the obtained plasmid pK18-Psod-tkt-tal-zwf is shown in FIG. 5, and the nucleotide sequence is SEQ ID NO. 3.
1.4 construction of plasmid pK18-homT176C
ATCC13032 LysCfbrThe genome was used as a template, and amplification was performed using homT176C-up-F/homT176C-up-R and homT176C-down-F/homT176C-down-R as primer pairs, respectively. PCR amplification conditions: 5min at 95 ℃; 30 cycles of 92 ℃ for 30s, 58 ℃ for 30s, 65 ℃ for 35 s;10min at 65 ℃. Amplified to obtain homT176C-up and homT176C-down fragments, the length of which are all 1kb, and cut and recovered. The plasmid pK18mobsacB is used as a template, and homT176C-Z-F and homT176C-Z-R are used as primers for amplification to obtain a homT176C-Z fragment with the length of 5.7kb, and the fragment is cut and recovered. Gibson assembly of homT176C-up, homT176C-down and homT176C-Z, transformation of DH5a, recovery, coating of kan plate, colony PCR with M13F and M13R as identifying primers, positive clone band length 2kb, inoculating positive clone to test tube to extract plasmid for use.
The structure of the obtained plasmid pK18-homT176C is shown in FIG. 6, and the nucleotide sequence is SEQ ID NO. 4.
1.5 construction of plasmid pK18mobsacB-ddh-ECT
ATCC13032 LysCfbrThe genome is taken as a template, and Ddh-up-F/Ddh1-up-R and Ddh1-dn-F/Ddh1-dn-R are respectively taken as primer pairs for PCR amplification. PCR amplification conditions: 5min at 95 ℃; 30 cycles of 92 ℃ for 30s, 58 ℃ for 30s, 65 ℃ for 35 s; 10min at 65 ℃. The size of the two amplified fragments is 1kb, and the two fragments are purified and recovered by agarose gel. Performing overlap PCR by using the two obtained purified and recovered fragments as templates and Ddh-up-F/Ddh1-dn-R as a primer pair to obtain a 2kb fragment, and purifying and recovering the fragment by using agarose gel.
The pK18mobsacB plasmid was digested with HindIII/EcoRI, purified and recovered, Gibson ligated to the overlap PCR-recovered fragment, transformed to DH 5. alpha. competent, and coated with kanamycin-resistant plates.
Colony PCR is carried out by taking M13F and M13R as identifying primers, a positive clone strip is 2kb in length, and the positive clone is inoculated to a test tube to extract plasmids. The plasmid was digested with PstI/EcoRI, a single band appeared, and the linearized vector A was recovered by agarose gel purification.
pEKEX2-ECT plasmid (synthesized by Kinzymenia, Suzhou) was digested with PstI/EcoRI, two bands of 3.7kb/8.1kb appeared, and 3.7kb fragment was recovered; the recovered linearized vector fragment was ligated and transformed with the recovered linearized vector A from the above step using T4 ligase and coated with a kanamycin-resistant plate. The transformants were subjected to colony PCR using M13F and M13R as primers for identification, and the positive transformants showed a 5.7kb fragment. Positive transformants were inoculated into resistant tubes, cultured overnight, and plasmids were extracted for use.
The structure of the obtained plasmid pK18mobsacB-ddh-ECT is shown in FIG. 7, and the nucleotide sequence is SEQ ID NO: 5.
1.6 construction of plasmid pK18mobsacB-IS30-ECT
ATCC13032 LysCfbrThe genome IS used as a template, and PCR amplification IS carried out by using primer pairs IS30-up-F/IS30-up-R and IS30-dn-F/IS30-dn-R respectively. PCR amplification conditions: 5min at 95 ℃; 30 cycles of 92 ℃ for 30s, 58 ℃ for 30s, 65 ℃ for 35 s; 10min at 65 ℃. The size of the two amplified fragments is 0.6kb, and the two fragments are purified and recovered by agarose gel.
The two purified fragments obtained above were used as templates, and the primer pair IS30-up-F/IS30-dn-R was used to perform overlap PCR to obtain a 1.2kb fragment, which was then purified and recovered by agarose gel.
The pK18mobsacB plasmid was digested with HindIII/EcoRI, purified and recovered, Gibson ligated to the overlap PCR-recovered fragment, transformed to DH 5. alpha. competent, and coated with kanamycin-resistant plates.
Carrying out colony PCR by taking M13F and M13R as identifying primers, wherein the length of a positive clone strip is 1.2kb, and inoculating the positive clone into a test tube to extract plasmids; the plasmid was digested with PstI/EcoRI, a single band appeared, and the linearized vector B was recovered by agarose gel purification.
pEKEX2-ECT plasmid (synthesized by Kinzymenia, Suzhou) was digested with PstI/EcoRI, two bands of 3.7kb/8.1kb appeared, and 3.7kb fragment was recovered; the recovered linearized vector fragment and the recovered linearized vector B fragment obtained in the above step were ligated and transformed with T4 ligase, and the kanamycin-resistant plate was coated. The transformant carries out colony PCR by taking M13F and M13R as identifying primers, and a positive transformant generates a 4.9kb fragment; positive transformants were inoculated into resistant tubes, cultured overnight, and plasmids were extracted for use.
The structure of the obtained plasmid pK18mobsacB-IS30-ECT IS shown in FIG. 8, and the nucleotide sequence IS SEQ ID NO. 6.
Example 2: construction of genetically engineered strains
2.1 pipetting 3. mu.l (500ng or more) of pK18-Psod-lysC plasmid into Corynebacterium glutamicum ATCC13032 LysCfbrMixing the competent cells, transferring to a 2 μm electric cuvette, and performing electric shock under 2.5kVThe time is 5.3ms, the suspension is immediately transferred into 800 mul of BHIS liquid culture medium preheated at 46 ℃ after electric shock, and is bathed in a water bath kettle at 46 ℃ for 6min, and then is placed in a constant temperature shaking table for culturing at 30 ℃ and 220rpm for 1h, so that the thalli are recovered. After recovery, 100. mu.l of the cells were spread on a BHIS plate containing kanamycin, and the plate was inverted and incubated in a 30 ℃ incubator for 48 hours. Transformants on the kanamycin-containing BHIS plates were picked, inoculated into non-resistant BHIS tube medium, and cultured in a constant temperature shaker at 30 ℃ and 220rpm for 24 hours to allow double crossover. The cells were diluted 1000 times and spread on BHIS-suc plates containing 20% sucrose, and the plates were inverted and cultured in a 30 ℃ incubator for 48 hours. BHIS-suc plate transformants were picked, spotted on a plate BHIS plate and a BHIS plate containing kanamycin, respectively, and inverted and cultured in a 30 ℃ incubator for 24 hours. And carrying out PCR amplification on transformants which can grow on the BHIS plate but cannot grow on the BHIS plate containing kanamycin by using a primer pair Psod-lysC-F/Psod-lysC-down-R, wherein the PCR amplification band of a positive transformant is about 1.2Kb, the PCR amplification band of a negative transformant is not band, and sequencing is carried out to identify a promoter integration positive strain. Positive transformants were picked into 4ml of BHIS tube medium, cultured at 30 ℃ and 220rpm for 16 hours on a constant temperature shaker, and then sterilized with 20% glycerol to obtain ATCC13032PsodlysCfbrAnd (3) strain.
2.2 with reference to the above procedure, the plasmid pK18-KOpck was transformed with the ATCC13032PsodlysC obtained in step 2.1 by the electrotransformation methodfbrThe strain was then subjected to reverse screening with sacB to obtain ATCC13032PsodlysCfbrΔ pck strain.
2.3 similarly, the plasmid pK18-Psod-tkt-tal-zwf, pK18-homT176C, pK18mobsacB-ddh-ECT and pK18mobsacB-IS30-ECT constructed in example 1 were each stacked at once by the above-mentioned method to obtain ATCC13032-ECT-01 strain.
2.4 the procedure of integrating pK18mobsacB-IS30-ECT plasmid into ATCC13032-ECT-01 strain was repeated once to obtain ATCC13032-ECT-02 strain.
Example 3: fermentation of engineering bacteria
3.1 Shake flask fermentation: marking the gene engineering strain on a BHIS flat plate, culturing in an incubator at 30 ℃ for about 2 days, and selecting 2-3 monoclonals to a BHIS test tube; carrying out shake culture at 30 ℃ and 220rpm for about 14h, inoculating seeds according to the proportion of 5 v/v% to shake flasks, and carrying out shake culture at 30 ℃ and 220rpm for about 10 h; then 10 v/v% inoculation amount is inoculated into a fermentation shake flask, the shake culture is carried out at 30 ℃ and 220rpm for about 20h, the sample is taken to detect the content of the ectoin, and the fermentation result is shown in table 2.
TABLE 2 Experimental results of the strains in shake flask fermentation for the production of ectoin
Strain name Shake flask fermentation yield g/L Conversion rate g/g glucose
ATCC13032lysC
fbr 0 0
ATCC13032-ECT-01 1.89 0.189
ATCC13032-ECT-02 2.23 0.223
As can be seen from Table 2, the genetically engineered strains ATCC13032-ECT-01 and ATCC13032-ECT-02 constructed by the present invention changed the original strain ATCC13032 lysCfbrInternal metabolic pathway, thereby enabling the conversion of glucose into ectoin. Overexpression of the ectABC gene is advantageous for increasing the biosynthesis of ectoin.
3.2 fermentation in a fermentation tank: picking ATCC13032-ECT-02 monoclonal from a BHIS plate to a BHI shake flask, and carrying out shake culture at 30 ℃ and 220rpm for about 14 h; inoculating 5 v/v% inoculum size to 5L fermentation tank, fermenting at 30 deg.C, maintaining dissolved oxygen at 30%, maintaining 25% ammonia water at pH of 7.0, fermenting for 64h, and detecting the content of ectoin in the fermentation liquid by HPLC, with the result shown in FIG. 9.
The above examples show that the strain ATCC13032-ECT-02 constructed by the method can realize the accumulation of ectoin in fermentation liquor, and the strain can produce 42.7g/L of ectoin after being fermented in a 5L fermentation tank for 64 hours, has the potential of being further developed and applied to industrial production, and has been subjected to strain preservation with the preservation number of CGMCC No. 22733.
It is to be understood that these examples are for illustrative purposes only and are not limiting upon the present invention. Various changes and modifications of the invention which may occur to those skilled in the art after reading the teachings herein are deemed to be within the scope and equivalents thereof which fall within the scope and spirit of the invention as defined by the appended claims.
Sequence listing
<110> Zhejiang HuaRui Biotechnology Ltd
<120> method for constructing ectoine-producing bacterium
<130> SHPI2110193
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 7884
<212> DNA
<213> Artificial sequence ()
<400> 1
tgccgcaagc actcagggcg caagggctgc taaaggaagc ggaacacgta gaaagccagt 60
ccgcagaaac ggtgctgacc ccggatgaat gtcagctact gggctatctg gacaagggaa 120
aacgcaagcg caaagagaaa gcaggtagct tgcagtgggc ttacatggcg atagctagac 180
tgggcggttt tatggacagc aagcgaaccg gaattgccag ctggggcgcc ctctggtaag 240
gttgggaagc cctgcaaagt aaactggatg gctttcttgc cgccaaggat ctgatggcgc 300
aggggatcaa gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat 360
ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca 420
caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg 480
gttctttttg tcaagaccga cctgtccggt gccctgaatg aactccaaga cgaggcagcg 540
cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact 600
gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct 660
caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg 720
cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt 780
actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc 840
gcgccagccg aactgttcgc caggctcaag gcgcggatgc ccgacggcga ggatctcgtc 900
gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga 960
ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc 1020
cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt 1080
atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga 1140
gcgggactct ggggttcgct agaggatcga tcctttttaa cccatcacat atacctgccg 1200
ttcactatta tttagtgaaa tgagatatta tgatattttc tgaattgtga ttaaaaaggc 1260
aactttatgc ccatgcaaca gaaactataa aaaatacaga gaatgaaaag aaacagatag 1320
attttttagt tctttaggcc cgtagtctgc aaatcctttt atgattttct atcaaacaaa 1380
agaggaaaat agaccagttg caatccaaac gagagtctaa tagaatgagg tcgaaaagta 1440
aatcgcgcgg gtttgttact gataaagcag gcaagaccta aaatgtgtaa agggcaaagt 1500
gtatactttg gcgtcacccc ttacatattt taggtctttt tttattgtgc gtaactaact 1560
tgccatcttc aaacaggagg gctggaagaa gcagaccgct aacacagtac ataaaaaagg 1620
agacatgaac gatgaacatc aaaaagtttg caaaacaagc aacagtatta acctttacta 1680
ccgcactgct ggcaggaggc gcaactcaag cgtttgcgaa agaaacgaac caaaagccat 1740
ataaggaaac atacggcatt tcccatatta cacgccatga tatgctgcaa atccctgaac 1800
agcaaaaaaa tgaaaaatat caagtttctg aatttgattc gtccacaatt aaaaatatct 1860
cttctgcaaa aggcctggac gtttgggaca gctggccatt acaaaacgct gacggcactg 1920
tcgcaaacta tcacggctac cacatcgtct ttgcattagc cggagatcct aaaaatgcgg 1980
atgacacatc gatttacatg ttctatcaaa aagtcggcga aacttctatt gacagctgga 2040
aaaacgctgg ccgcgtcttt aaagacagcg acaaattcga tgcaaatgat tctatcctaa 2100
aagaccaaac acaagaatgg tcaggttcag ccacatttac atctgacgga aaaatccgtt 2160
tattctacac tgatttctcc ggtaaacatt acggcaaaca aacactgaca actgcacaag 2220
ttaacgtatc agcatcagac agctctttga acatcaacgg tgtagaggat tataaatcaa 2280
tctttgacgg tgacggaaaa acgtatcaaa atgtacagca gttcatcgat gaaggcaact 2340
acagctcagg cgacaaccat acgctgagag atcctcacta cgtagaagat aaaggccaca 2400
aatacttagt atttgaagca aacactggaa ctgaagatgg ctaccaaggc gaagaatctt 2460
tatttaacaa agcatactat ggcaaaagca catcattctt ccgtcaagaa agtcaaaaac 2520
ttctgcaaag cgataaaaaa cgcacggctg agttagcaaa cggcgctctc ggtatgattg 2580
agctaaacga tgattacaca ctgaaaaaag tgatgaaacc gctgattgca tctaacacag 2640
taacagatga aattgaacgc gcgaacgtct ttaaaatgaa cggcaaatgg tacctgttca 2700
ctgactcccg cggatcaaaa atgacgattg acggcattac gtctaacgat atttacatgc 2760
ttggttatgt ttctaattct ttaactggcc catacaagcc gctgaacaaa actggccttg 2820
tgttaaaaat ggatcttgat cctaacgatg taacctttac ttactcacac ttcgctgtac 2880
ctcaagcgaa aggaaacaat gtcgtgatta caagctatat gacaaacaga ggattctacg 2940
cagacaaaca atcaacgttt gcgccgagct tcctgctgaa catcaaaggc aagaaaacat 3000
ctgttgtcaa agacagcatc cttgaacaag gacaattaac agttaacaaa taaaaacgca 3060
aaagaaaatg ccgatgggta ccgagcgaaa tgaccgacca agcgacgccc aacctgccat 3120
cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc 3180
gggacgccct cgcggacgtg ctcatagtcc acgacgcccg tgattttgta gccctggccg 3240
acggccagca ggtaggccga caggctcatg ccggccgccg ccgccttttc ctcaatcgct 3300
cttcgttcgt ctggaaggca gtacaccttg ataggtgggc tgcccttcct ggttggcttg 3360
gtttcatcag ccatccgctt gccctcatct gttacgccgg cggtagccgg ccagcctcgc 3420
agagcaggat tcccgttgag caccgccagg tgcgaataag ggacagtgaa gaaggaacac 3480
ccgctcgcgg gtgggcctac ttcacctatc ctgcccggct gacgccgttg gatacaccaa 3540
ggaaagtcta cacgaaccct ttggcaaaat cctgtatatc gtgcgaaaaa ggatggatat 3600
accgaaaaaa tcgctataat gaccccgaag cagggttatg cagcggaaaa gcgctgcttc 3660
cctgctgttt tgtggaatat ctaccgactg gaaacaggca aatgcaggaa attactgaac 3720
tgaggggaca ggcgagagac gatgccaaag agctcctgaa aatctcgata actcaaaaaa 3780
tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccgatc 3840
aacgtctcat tttcgccaaa agttggccca gggcttcccg gtatcaacag ggacaccagg 3900
atttatttat tctgcgaagt gatcttccgt cacaggtatt tattcggcgc aaagtgcgtc 3960
gggtgatgct gccaacttac tgatttagtg tatgatggtg tttttgaggt gctccagtgg 4020
cttctgtttc tatcagctcc tgaaaatctc gataactcaa aaaatacgcc cggtagtgat 4080
cttatttcat tatggtgaaa gttggaacct cttacgtgcc gatcaacgtc tcattttcgc 4140
caaaagttgg cccagggctt cccggtatca acagggacac caggatttat ttattctgcg 4200
aagtgatctt ccgtcacagg tatttattcg gcgcaaagtg cgtcgggtga tgctgccaac 4260
ttactgattt agtgtatgat ggtgtttttg aggtgctcca gtggcttctg tttctatcag 4320
ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaaaagg 4380
atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 4440
ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 4500
ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 4560
ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 4620
ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 4680
ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 4740
tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 4800
tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 4860
tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 4920
tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 4980
gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 5040
tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 5100
ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct 5160
gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc 5220
gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc 5280
cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg 5340
ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta 5400
cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca 5460
ggaaacagct atgacatgat tacgaattct ttcgcccaga accaagtagc ccaaactgaa 5520
ggcaacaatt ggttcggcaa tggtcatggc gggtagcgat ttttgtagtt cgccagcgtt 5580
aaaggaatac tgctgcacga ttgttccaag taatgcggtg aggattaggc cgtagccttc 5640
ccagttcaag atgagtcccg ttatgccttg atggacaaaa agatccaccg cggctttgga 5700
catcactgct acgtagccaa acaatgcacc cgtcacaaga ccaaggatga gggctttgtc 5760
cttctttaat acgtattccg caagcagcca cattccaccc attactgcaa cgccgactaa 5820
aagtactgga atccatcgat cgagtggggg gtgggggttt ccgggaaggg ggcgtcccaa 5880
aacgatcatg atgcccacgg ctacggtgag gagggtagcc cagaagattt cagttcggcg 5940
tagtcggtag ccattgaatc gtgctgagag cggcagcgtg aacatcagcg acaggacaag 6000
cactggttgc actaccaaga gggtgccgaa accaagtgct actgtttgta agaaatatgc 6060
cagcatcgcg gtactcatgc ctgcccacca catcggtgtc atcagagcat tgagtaaagg 6120
tgagctcctt agggagccat cttttggggt gcggagcgcg atccggtgtc tgaccacggt 6180
gccccatgcg attgttaatg ccgatgctag ggcgaaaagc acggcgagca gattgctttg 6240
cacttgattc agggtagttg actaaagagt tgctcgcgaa gtagcacctg tcacttttgt 6300
ctcaaatatt aaatcgaata tcaatatatg gtctgtttat tggaacgcgt cccagtggct 6360
gagacgcatc cgctaaagcc ccaggaaccc tgtgcagaaa gaaaacactc ctctggctag 6420
gtagacacag tttataaagg tagagttgag cgggtaactg tcagcacgta gatcgaaagg 6480
tgcacaaaga ttccgggctt gtgacccgct acccgataaa taggtcggct gaaaaatttc 6540
gttgcaatat caacaaaaag gcctatcatt gggaggtgtc gcaccaagta cttttgcgaa 6600
gcgccatctg acggattttc aaaagatgta tatgctcggt gcggaaacct acgaaaggat 6660
tttttacccg tggccctggt cgtacagaaa tatggcggtt cctcgcttga gagtgcggaa 6720
cgcattagaa acgtcgctga acggatcgtt gccaccaaga aggctggaaa tgatgtcgtg 6780
gttgtctgct ccgcaatggg agacaccacg gatgaacttc tagaacttgc agcggcagtg 6840
aatcccgttc cgccagctcg tgaaatggat atgctcctga ctgctggtga gcgtatttct 6900
aacgctctcg tcgccatggc tattgagtcc cttggcgcag aagcccaatc tttcacgggc 6960
tctcaggctg gtgtgctcac caccgagcgc cacggaaacg cacgcattgt tgatgtcact 7020
ccaggtcgtg tgcgtgaagc actcgatgag ggcaagatct gcattgttgc tggtttccag 7080
ggtgttaata aagaaacccg cgatgtcacc acgttgggtc gtggtggttc tgacaccact 7140
gcagttgcgt tggcagctgc tttgaacgct gatgtgtgtg agatttactc ggacgttgac 7200
ggtgtgtata ccgctgaccc gcgcatcgtt cctaatgcac agaagctgga aaagctcagc 7260
ttcgaagaaa tgctggaact tgctgctgtt ggctccaaga ttttggtgct gcgcagtgtt 7320
gaatacgctc gtgcattcaa tgtgccactt cgcgtacgct cgtcttatag taatgatccc 7380
ggcactttga ttgccggctc tatggaggat attcctgtgg aagaagcagt ccttaccggt 7440
gtcgcaaccg acaagtccga agccaaagta accgttctgg gtatttccga taagccaggc 7500
gaggctgcga aggttttccg tgcgttggct gatgcagaaa tcaacattga catggttctg 7560
cagaacgtct cttctgtaga agacggcacc accgacatca ccttcacctg ccctcgttcc 7620
gacggccgcc gcgcgatgga gatcttgaag aagcttcagg ttcagggcag gatcctctag 7680
agtcgacctg caggcatgca agcttggcac tggccgtcgt tttacaacgt cgtgactggg 7740
aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc 7800
gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg 7860
aatggcgata agctagcttc acgc 7884
<210> 2
<211> 7713
<212> DNA
<213> Artificial sequence ()
<400> 2
tgccgcaagc actcagggcg caagggctgc taaaggaagc ggaacacgta gaaagccagt 60
ccgcagaaac ggtgctgacc ccggatgaat gtcagctact gggctatctg gacaagggaa 120
aacgcaagcg caaagagaaa gcaggtagct tgcagtgggc ttacatggcg atagctagac 180
tgggcggttt tatggacagc aagcgaaccg gaattgccag ctggggcgcc ctctggtaag 240
gttgggaagc cctgcaaagt aaactggatg gctttcttgc cgccaaggat ctgatggcgc 300
aggggatcaa gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat 360
ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca 420
caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg 480
gttctttttg tcaagaccga cctgtccggt gccctgaatg aactccaaga cgaggcagcg 540
cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact 600
gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct 660
caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg 720
cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt 780
actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc 840
gcgccagccg aactgttcgc caggctcaag gcgcggatgc ccgacggcga ggatctcgtc 900
gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga 960
ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc 1020
cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt 1080
atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga 1140
gcgggactct ggggttcgct agaggatcga tcctttttaa cccatcacat atacctgccg 1200
ttcactatta tttagtgaaa tgagatatta tgatattttc tgaattgtga ttaaaaaggc 1260
aactttatgc ccatgcaaca gaaactataa aaaatacaga gaatgaaaag aaacagatag 1320
attttttagt tctttaggcc cgtagtctgc aaatcctttt atgattttct atcaaacaaa 1380
agaggaaaat agaccagttg caatccaaac gagagtctaa tagaatgagg tcgaaaagta 1440
aatcgcgcgg gtttgttact gataaagcag gcaagaccta aaatgtgtaa agggcaaagt 1500
gtatactttg gcgtcacccc ttacatattt taggtctttt tttattgtgc gtaactaact 1560
tgccatcttc aaacaggagg gctggaagaa gcagaccgct aacacagtac ataaaaaagg 1620
agacatgaac gatgaacatc aaaaagtttg caaaacaagc aacagtatta acctttacta 1680
ccgcactgct ggcaggaggc gcaactcaag cgtttgcgaa agaaacgaac caaaagccat 1740
ataaggaaac atacggcatt tcccatatta cacgccatga tatgctgcaa atccctgaac 1800
agcaaaaaaa tgaaaaatat caagtttctg aatttgattc gtccacaatt aaaaatatct 1860
cttctgcaaa aggcctggac gtttgggaca gctggccatt acaaaacgct gacggcactg 1920
tcgcaaacta tcacggctac cacatcgtct ttgcattagc cggagatcct aaaaatgcgg 1980
atgacacatc gatttacatg ttctatcaaa aagtcggcga aacttctatt gacagctgga 2040
aaaacgctgg ccgcgtcttt aaagacagcg acaaattcga tgcaaatgat tctatcctaa 2100
aagaccaaac acaagaatgg tcaggttcag ccacatttac atctgacgga aaaatccgtt 2160
tattctacac tgatttctcc ggtaaacatt acggcaaaca aacactgaca actgcacaag 2220
ttaacgtatc agcatcagac agctctttga acatcaacgg tgtagaggat tataaatcaa 2280
tctttgacgg tgacggaaaa acgtatcaaa atgtacagca gttcatcgat gaaggcaact 2340
acagctcagg cgacaaccat acgctgagag atcctcacta cgtagaagat aaaggccaca 2400
aatacttagt atttgaagca aacactggaa ctgaagatgg ctaccaaggc gaagaatctt 2460
tatttaacaa agcatactat ggcaaaagca catcattctt ccgtcaagaa agtcaaaaac 2520
ttctgcaaag cgataaaaaa cgcacggctg agttagcaaa cggcgctctc ggtatgattg 2580
agctaaacga tgattacaca ctgaaaaaag tgatgaaacc gctgattgca tctaacacag 2640
taacagatga aattgaacgc gcgaacgtct ttaaaatgaa cggcaaatgg tacctgttca 2700
ctgactcccg cggatcaaaa atgacgattg acggcattac gtctaacgat atttacatgc 2760
ttggttatgt ttctaattct ttaactggcc catacaagcc gctgaacaaa actggccttg 2820
tgttaaaaat ggatcttgat cctaacgatg taacctttac ttactcacac ttcgctgtac 2880
ctcaagcgaa aggaaacaat gtcgtgatta caagctatat gacaaacaga ggattctacg 2940
cagacaaaca atcaacgttt gcgccgagct tcctgctgaa catcaaaggc aagaaaacat 3000
ctgttgtcaa agacagcatc cttgaacaag gacaattaac agttaacaaa taaaaacgca 3060
aaagaaaatg ccgatgggta ccgagcgaaa tgaccgacca agcgacgccc aacctgccat 3120
cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc 3180
gggacgccct cgcggacgtg ctcatagtcc acgacgcccg tgattttgta gccctggccg 3240
acggccagca ggtaggccga caggctcatg ccggccgccg ccgccttttc ctcaatcgct 3300
cttcgttcgt ctggaaggca gtacaccttg ataggtgggc tgcccttcct ggttggcttg 3360
gtttcatcag ccatccgctt gccctcatct gttacgccgg cggtagccgg ccagcctcgc 3420
agagcaggat tcccgttgag caccgccagg tgcgaataag ggacagtgaa gaaggaacac 3480
ccgctcgcgg gtgggcctac ttcacctatc ctgcccggct gacgccgttg gatacaccaa 3540
ggaaagtcta cacgaaccct ttggcaaaat cctgtatatc gtgcgaaaaa ggatggatat 3600
accgaaaaaa tcgctataat gaccccgaag cagggttatg cagcggaaaa gcgctgcttc 3660
cctgctgttt tgtggaatat ctaccgactg gaaacaggca aatgcaggaa attactgaac 3720
tgaggggaca ggcgagagac gatgccaaag agctcctgaa aatctcgata actcaaaaaa 3780
tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccgatc 3840
aacgtctcat tttcgccaaa agttggccca gggcttcccg gtatcaacag ggacaccagg 3900
atttatttat tctgcgaagt gatcttccgt cacaggtatt tattcggcgc aaagtgcgtc 3960
gggtgatgct gccaacttac tgatttagtg tatgatggtg tttttgaggt gctccagtgg 4020
cttctgtttc tatcagctcc tgaaaatctc gataactcaa aaaatacgcc cggtagtgat 4080
cttatttcat tatggtgaaa gttggaacct cttacgtgcc gatcaacgtc tcattttcgc 4140
caaaagttgg cccagggctt cccggtatca acagggacac caggatttat ttattctgcg 4200
aagtgatctt ccgtcacagg tatttattcg gcgcaaagtg cgtcgggtga tgctgccaac 4260
ttactgattt agtgtatgat ggtgtttttg aggtgctcca gtggcttctg tttctatcag 4320
ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaaaagg 4380
atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 4440
ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 4500
ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 4560
ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 4620
ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 4680
ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 4740
tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 4800
tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 4860
tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 4920
tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 4980
gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 5040
tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 5100
ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct 5160
gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc 5220
gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc 5280
cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg 5340
ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta 5400
cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca 5460
ggaaacagct atgacatgat tacgaattcg agctcggtac ccggggatcc tctagagtcg 5520
acctgcaggg ctggacccta gaattcggcg tcgcaggagt aaaaaacacc accattgttt 5580
cctccatcga cgatgccatc accaccattt tgcgatggat gaacggcgaa gacatccgcg 5640
acctcaactg gacccgcgca taaatggcct catttccgga gcttccggct cttcgtcgct 5700
tggctacctt gggcaggtcg tggggtttac tgtctgattt caaatacgaa caaacccgac 5760
ctgacatctt ttacggaaac ctggccctcg atacctcgag tctggtggcg gctttgtctg 5820
aagatatttc tggcgccgga ttaaatgacc tgaaagttct cgacgtcggc ggcggacccg 5880
gatacttcgc cgaagccttt gagacactgg gcgccaccta cttctccgtc gaacccgacg 5940
ttggcgaaat gtccgcagct ggcatcgacg tccacggatc agtccgcgga tccggcctcg 6000
acctgccgtt tcttcccgat tcctttgacg tggtgtactc ctccaacgtt gcagaacatg 6060
tctccgcacc gtgggaattg ggagaagaaa tgctccgcgt cacccgcagc ggcggcctgg 6120
caatcctgag ctacaccatt tggttagggc ccttcggcgg ccatgaaacc ggactgtggg 6180
aacactacgt tggcggagaa tttgcccgcg atcgctacac gaagaaacac gggcacccgc 6240
ctaagaacgt tttcggggag tcactgttta atgtgtcctg ccgggagggg ctggaatggg 6300
gagcctccgt gggcaatgcg gaattggttg ccgcttttcc ccgctaccac ccgtattggg 6360
tctggtggat ggttaaagtc ccagtgctcc gagaattcgc ggtaagtaac ttggtgttgg 6420
tgtttaaaaa gcactgaggt tttgaggaat tcatcgctta acgacaagaa aggctcccac 6480
tttcggtggg agcctttctt gttatttagc agttcttaag cgtgaactta cttctccaga 6540
ttttgtgtca ttcgacagag ttctcgcccc ctagcgtagc tttcagatac agaactagtt 6600
aaaactttag gtgagacaac ggacacattt gtcattacca gtggacctac cccctgccca 6660
cacgcatcta cacactttct ttaatatgag agcacccgtt taaatagcct attttggggg 6720
tggtttcaag aattaacctc aaccgttctc cgacagttca ttccccgtcc atggccattg 6780
ggttcagatt tgggcaattc tcacacattc caggggacaa ctttcccagt tttcccacca 6840
ttaacactta acattcggac aataggcaac aaaacgccaa gaacagcggt aatggtaatc 6900
cctttccctt accctgccat cacaatccaa gcactccgct agtggccgac cagcacaaac 6960
cggcccactg tcagtacaca cctttttaaa acaacattta cactcacatg catgcccgca 7020
ctgtcaccac ccgccctcaa ctaccgaact aaagatatgt acttgaagcc aaatttttac 7080
cctagatccc ccttttaaat actttgaaaa ttactcacac acatccccac gttaccccaa 7140
aggttatatc cagttagtcg tatcaaaaag tgctctgatc ttaactttgc cctacctaaa 7200
tacatgaccc caccacgacg gccagtacta acgacagaat ccactagcga acccatttat 7260
taacaaacat tgcaaacaag tgttgaagta ttcgcgaaat tgcaactttt ggagcccaaa 7320
ggtctagacc agtgatcgag cgcacaacgc tcccatttag cggtttcact gccccaaagg 7380
cttaaagtaa tgggcatgcc cactccttct tcgaccaaaa gctacgctgc ggtcttacct 7440
ccacctggcc cctcgtgggc tggttccctc atgggcatct cattgttgtc atcactgttg 7500
aaaatccatg gttttccagt cgtcgcagaa gcttggcact ggccgtcgtt ttacaacgtc 7560
gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg 7620
ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc 7680
tgaatggcga atggcgataa gctagcttca cgc 7713
<210> 3
<211> 7899
<212> DNA
<213> Artificial sequence ()
<400> 3
tgccgcaagc actcagggcg caagggctgc taaaggaagc ggaacacgta gaaagccagt 60
ccgcagaaac ggtgctgacc ccggatgaat gtcagctact gggctatctg gacaagggaa 120
aacgcaagcg caaagagaaa gcaggtagct tgcagtgggc ttacatggcg atagctagac 180
tgggcggttt tatggacagc aagcgaaccg gaattgccag ctggggcgcc ctctggtaag 240
gttgggaagc cctgcaaagt aaactggatg gctttcttgc cgccaaggat ctgatggcgc 300
aggggatcaa gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat 360
ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca 420
caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg 480
gttctttttg tcaagaccga cctgtccggt gccctgaatg aactccaaga cgaggcagcg 540
cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact 600
gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct 660
caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg 720
cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt 780
actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc 840
gcgccagccg aactgttcgc caggctcaag gcgcggatgc ccgacggcga ggatctcgtc 900
gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga 960
ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc 1020
cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt 1080
atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga 1140
gcgggactct ggggttcgct agaggatcga tcctttttaa cccatcacat atacctgccg 1200
ttcactatta tttagtgaaa tgagatatta tgatattttc tgaattgtga ttaaaaaggc 1260
aactttatgc ccatgcaaca gaaactataa aaaatacaga gaatgaaaag aaacagatag 1320
attttttagt tctttaggcc cgtagtctgc aaatcctttt atgattttct atcaaacaaa 1380
agaggaaaat agaccagttg caatccaaac gagagtctaa tagaatgagg tcgaaaagta 1440
aatcgcgcgg gtttgttact gataaagcag gcaagaccta aaatgtgtaa agggcaaagt 1500
gtatactttg gcgtcacccc ttacatattt taggtctttt tttattgtgc gtaactaact 1560
tgccatcttc aaacaggagg gctggaagaa gcagaccgct aacacagtac ataaaaaagg 1620
agacatgaac gatgaacatc aaaaagtttg caaaacaagc aacagtatta acctttacta 1680
ccgcactgct ggcaggaggc gcaactcaag cgtttgcgaa agaaacgaac caaaagccat 1740
ataaggaaac atacggcatt tcccatatta cacgccatga tatgctgcaa atccctgaac 1800
agcaaaaaaa tgaaaaatat caagtttctg aatttgattc gtccacaatt aaaaatatct 1860
cttctgcaaa aggcctggac gtttgggaca gctggccatt acaaaacgct gacggcactg 1920
tcgcaaacta tcacggctac cacatcgtct ttgcattagc cggagatcct aaaaatgcgg 1980
atgacacatc gatttacatg ttctatcaaa aagtcggcga aacttctatt gacagctgga 2040
aaaacgctgg ccgcgtcttt aaagacagcg acaaattcga tgcaaatgat tctatcctaa 2100
aagaccaaac acaagaatgg tcaggttcag ccacatttac atctgacgga aaaatccgtt 2160
tattctacac tgatttctcc ggtaaacatt acggcaaaca aacactgaca actgcacaag 2220
ttaacgtatc agcatcagac agctctttga acatcaacgg tgtagaggat tataaatcaa 2280
tctttgacgg tgacggaaaa acgtatcaaa atgtacagca gttcatcgat gaaggcaact 2340
acagctcagg cgacaaccat acgctgagag atcctcacta cgtagaagat aaaggccaca 2400
aatacttagt atttgaagca aacactggaa ctgaagatgg ctaccaaggc gaagaatctt 2460
tatttaacaa agcatactat ggcaaaagca catcattctt ccgtcaagaa agtcaaaaac 2520
ttctgcaaag cgataaaaaa cgcacggctg agttagcaaa cggcgctctc ggtatgattg 2580
agctaaacga tgattacaca ctgaaaaaag tgatgaaacc gctgattgca tctaacacag 2640
taacagatga aattgaacgc gcgaacgtct ttaaaatgaa cggcaaatgg tacctgttca 2700
ctgactcccg cggatcaaaa atgacgattg acggcattac gtctaacgat atttacatgc 2760
ttggttatgt ttctaattct ttaactggcc catacaagcc gctgaacaaa actggccttg 2820
tgttaaaaat ggatcttgat cctaacgatg taacctttac ttactcacac ttcgctgtac 2880
ctcaagcgaa aggaaacaat gtcgtgatta caagctatat gacaaacaga ggattctacg 2940
cagacaaaca atcaacgttt gcgccgagct tcctgctgaa catcaaaggc aagaaaacat 3000
ctgttgtcaa agacagcatc cttgaacaag gacaattaac agttaacaaa taaaaacgca 3060
aaagaaaatg ccgatgggta ccgagcgaaa tgaccgacca agcgacgccc aacctgccat 3120
cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc 3180
gggacgccct cgcggacgtg ctcatagtcc acgacgcccg tgattttgta gccctggccg 3240
acggccagca ggtaggccga caggctcatg ccggccgccg ccgccttttc ctcaatcgct 3300
cttcgttcgt ctggaaggca gtacaccttg ataggtgggc tgcccttcct ggttggcttg 3360
gtttcatcag ccatccgctt gccctcatct gttacgccgg cggtagccgg ccagcctcgc 3420
agagcaggat tcccgttgag caccgccagg tgcgaataag ggacagtgaa gaaggaacac 3480
ccgctcgcgg gtgggcctac ttcacctatc ctgcccggct gacgccgttg gatacaccaa 3540
ggaaagtcta cacgaaccct ttggcaaaat cctgtatatc gtgcgaaaaa ggatggatat 3600
accgaaaaaa tcgctataat gaccccgaag cagggttatg cagcggaaaa gcgctgcttc 3660
cctgctgttt tgtggaatat ctaccgactg gaaacaggca aatgcaggaa attactgaac 3720
tgaggggaca ggcgagagac gatgccaaag agctcctgaa aatctcgata actcaaaaaa 3780
tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccgatc 3840
aacgtctcat tttcgccaaa agttggccca gggcttcccg gtatcaacag ggacaccagg 3900
atttatttat tctgcgaagt gatcttccgt cacaggtatt tattcggcgc aaagtgcgtc 3960
gggtgatgct gccaacttac tgatttagtg tatgatggtg tttttgaggt gctccagtgg 4020
cttctgtttc tatcagctcc tgaaaatctc gataactcaa aaaatacgcc cggtagtgat 4080
cttatttcat tatggtgaaa gttggaacct cttacgtgcc gatcaacgtc tcattttcgc 4140
caaaagttgg cccagggctt cccggtatca acagggacac caggatttat ttattctgcg 4200
aagtgatctt ccgtcacagg tatttattcg gcgcaaagtg cgtcgggtga tgctgccaac 4260
ttactgattt agtgtatgat ggtgtttttg aggtgctcca gtggcttctg tttctatcag 4320
ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaaaagg 4380
atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 4440
ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 4500
ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 4560
ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 4620
ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 4680
ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 4740
tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 4800
tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 4860
tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 4920
tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 4980
gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 5040
tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 5100
ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct 5160
gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc 5220
gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc 5280
cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg 5340
ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta 5400
cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca 5460
ggaaacagct atgacatgat tacgaattcg agctcggtac ccggggatcc cggccgcacc 5520
catccagcca ccgaacacag tcagcaagat gagcacaatg ttgttctcac cgcgttcagc 5580
ctgaagcatt gtggggattg tggcgacaag gaggagttca ataaccctgg gcttcgttag 5640
cgcaatatag gccttgatcg tgtccaaggg ttctcctcca gaacgttgca ttttcaaatc 5700
actcatatat ttaagttgtg agtccttatt atttaaatat ccctgcggtg agtgtgcacc 5760
ttgcgttgaa ggcccagact ctgacagaag cgtcagagtg tttactcaag acattttcta 5820
agacacacgg caaattagtc ggatgaagtt aattaaaagt tcccgaatca atctttttaa 5880
tgttttcaaa ccatttgaag gtgtgctgac ccaggtggac gccaaccttt aaaaagcttc 5940
agacttttat ttccacttca taaaaactgc ctgtgacgat tccgttaaag attgtgccaa 6000
atcactgcgc aaaactcgcg cggaaccaga ccttgccatg ctatcgccta ttcacactat 6060
ttgagtaatc ggaaatagat gggtgtagac gcttgattgg cggacggttc acagcggacg 6120
atttcaggcc ctcgtagctc gagagtttga aggggtccga ttcgttccgt tcgtgacgct 6180
ttgtgaggtt ttttgacgtt gcaccgtatt gcttgccgaa catttttctt ttcctttcgg 6240
tttttcgaga attttcacct acaaaagccc acgtcacagc tcccagactt aagattgatc 6300
acacctttga cacatttgaa ccacagttgg ttataaaatg ggttcaacat cactatggtt 6360
agaggtgttg acgggtcaga ttaagcaaag actactttcg gggtagatca cctttgccaa 6420
atttgaacca attaacctaa gtcgtagatc tgatcatcgg atctaacgaa aacgaaccaa 6480
aactttggtc ccggtttaac ccaggaagga attccgggct tgtgacccgc tacccgataa 6540
ataggtcggc tgaaaaattt cgttgcaata tcaacaaaaa ggcctatcat tgggaggtgt 6600
cgcaccaagt acttttgcga agcgccatct gacggatttt caaaagatgt atatgctcgg 6660
tgcggaaacc tacgaaagga ttttttaccc ttgaccacct tgacgctgtc acctgaactt 6720
caggcgctca ctgtacgcaa ttacccctct gattggtccg atgtggacac caaggctgta 6780
gacactgttc gtgtcctcgc tgcagacgct gtagaaaact gtggctccgg ccacccaggc 6840
accgcaatga gcctggctcc ccttgcatac accttgtacc agcgggttat gaacgtagat 6900
ccacaggaca ccaactgggc aggccgtgac cgcttcgttc tttcttgtgg ccactcctct 6960
ttgacccagt acatccagct ttacttgggt ggattcggcc ttgagatgga tgacctgaag 7020
gctctgcgca cctgggattc cttgacccca ggacaccctg agtaccgcca caccaagggc 7080
gttgagatca ccactggccc tcttggccag ggtcttgcat ctgcagttgg tatggccatg 7140
gctgctcgtc gtgagcgtgg cctattcgac ccaaccgctg ctgagggcga atccccattc 7200
gaccaccaca tctacgtcat tgcttctgat ggtgacctgc aggaaggtgt cacctctgag 7260
gcatcctcca tcgctggcac ccagcagctg ggcaacctca tcgtgttctg ggatgacaac 7320
cgcatctcca tcgaagacaa cactgagatc gctttcaacg aggacgttgt tgctcgttac 7380
aaggcttacg gctggcagac cattgaggtt gaggctggcg aggacgttgc agcaatcgaa 7440
gctgcagtgg ctgaggctaa gaaggacacc aagcgaccta ccttcatccg cgttcgcacc 7500
atcatcggct tcccagctcc aactatgatg aacaccggtg ctgtgcacgg tgctgctctt 7560
ggcgcagctg aggttgcagc aaccaagact gagcttggat tcgatcctga ggctcacttc 7620
gcgatcgacg atgaggttat cgctcacacc cgctccctcg cagagcgcgc tgcacagaag 7680
aaggctgcat tctagagtcg acctgcaggc atgcaagctt ggcactggcc gtcgttttac 7740
aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc 7800
ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc 7860
gcagcctgaa tggcgaatgg cgataagcta gcttcacgc 7899
<210> 4
<211> 7707
<212> DNA
<213> Artificial sequence ()
<400> 4
tgccgcaagc actcagggcg caagggctgc taaaggaagc ggaacacgta gaaagccagt 60
ccgcagaaac ggtgctgacc ccggatgaat gtcagctact gggctatctg gacaagggaa 120
aacgcaagcg caaagagaaa gcaggtagct tgcagtgggc ttacatggcg atagctagac 180
tgggcggttt tatggacagc aagcgaaccg gaattgccag ctggggcgcc ctctggtaag 240
gttgggaagc cctgcaaagt aaactggatg gctttcttgc cgccaaggat ctgatggcgc 300
aggggatcaa gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat 360
ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca 420
caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg 480
gttctttttg tcaagaccga cctgtccggt gccctgaatg aactccaaga cgaggcagcg 540
cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact 600
gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct 660
caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg 720
cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt 780
actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc 840
gcgccagccg aactgttcgc caggctcaag gcgcggatgc ccgacggcga ggatctcgtc 900
gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga 960
ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc 1020
cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt 1080
atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga 1140
gcgggactct ggggttcgct agaggatcga tcctttttaa cccatcacat atacctgccg 1200
ttcactatta tttagtgaaa tgagatatta tgatattttc tgaattgtga ttaaaaaggc 1260
aactttatgc ccatgcaaca gaaactataa aaaatacaga gaatgaaaag aaacagatag 1320
attttttagt tctttaggcc cgtagtctgc aaatcctttt atgattttct atcaaacaaa 1380
agaggaaaat agaccagttg caatccaaac gagagtctaa tagaatgagg tcgaaaagta 1440
aatcgcgcgg gtttgttact gataaagcag gcaagaccta aaatgtgtaa agggcaaagt 1500
gtatactttg gcgtcacccc ttacatattt taggtctttt tttattgtgc gtaactaact 1560
tgccatcttc aaacaggagg gctggaagaa gcagaccgct aacacagtac ataaaaaagg 1620
agacatgaac gatgaacatc aaaaagtttg caaaacaagc aacagtatta acctttacta 1680
ccgcactgct ggcaggaggc gcaactcaag cgtttgcgaa agaaacgaac caaaagccat 1740
ataaggaaac atacggcatt tcccatatta cacgccatga tatgctgcaa atccctgaac 1800
agcaaaaaaa tgaaaaatat caagtttctg aatttgattc gtccacaatt aaaaatatct 1860
cttctgcaaa aggcctggac gtttgggaca gctggccatt acaaaacgct gacggcactg 1920
tcgcaaacta tcacggctac cacatcgtct ttgcattagc cggagatcct aaaaatgcgg 1980
atgacacatc gatttacatg ttctatcaaa aagtcggcga aacttctatt gacagctgga 2040
aaaacgctgg ccgcgtcttt aaagacagcg acaaattcga tgcaaatgat tctatcctaa 2100
aagaccaaac acaagaatgg tcaggttcag ccacatttac atctgacgga aaaatccgtt 2160
tattctacac tgatttctcc ggtaaacatt acggcaaaca aacactgaca actgcacaag 2220
ttaacgtatc agcatcagac agctctttga acatcaacgg tgtagaggat tataaatcaa 2280
tctttgacgg tgacggaaaa acgtatcaaa atgtacagca gttcatcgat gaaggcaact 2340
acagctcagg cgacaaccat acgctgagag atcctcacta cgtagaagat aaaggccaca 2400
aatacttagt atttgaagca aacactggaa ctgaagatgg ctaccaaggc gaagaatctt 2460
tatttaacaa agcatactat ggcaaaagca catcattctt ccgtcaagaa agtcaaaaac 2520
ttctgcaaag cgataaaaaa cgcacggctg agttagcaaa cggcgctctc ggtatgattg 2580
agctaaacga tgattacaca ctgaaaaaag tgatgaaacc gctgattgca tctaacacag 2640
taacagatga aattgaacgc gcgaacgtct ttaaaatgaa cggcaaatgg tacctgttca 2700
ctgactcccg cggatcaaaa atgacgattg acggcattac gtctaacgat atttacatgc 2760
ttggttatgt ttctaattct ttaactggcc catacaagcc gctgaacaaa actggccttg 2820
tgttaaaaat ggatcttgat cctaacgatg taacctttac ttactcacac ttcgctgtac 2880
ctcaagcgaa aggaaacaat gtcgtgatta caagctatat gacaaacaga ggattctacg 2940
cagacaaaca atcaacgttt gcgccgagct tcctgctgaa catcaaaggc aagaaaacat 3000
ctgttgtcaa agacagcatc cttgaacaag gacaattaac agttaacaaa taaaaacgca 3060
aaagaaaatg ccgatgggta ccgagcgaaa tgaccgacca agcgacgccc aacctgccat 3120
cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc 3180
gggacgccct cgcggacgtg ctcatagtcc acgacgcccg tgattttgta gccctggccg 3240
acggccagca ggtaggccga caggctcatg ccggccgccg ccgccttttc ctcaatcgct 3300
cttcgttcgt ctggaaggca gtacaccttg ataggtgggc tgcccttcct ggttggcttg 3360
gtttcatcag ccatccgctt gccctcatct gttacgccgg cggtagccgg ccagcctcgc 3420
agagcaggat tcccgttgag caccgccagg tgcgaataag ggacagtgaa gaaggaacac 3480
ccgctcgcgg gtgggcctac ttcacctatc ctgcccggct gacgccgttg gatacaccaa 3540
ggaaagtcta cacgaaccct ttggcaaaat cctgtatatc gtgcgaaaaa ggatggatat 3600
accgaaaaaa tcgctataat gaccccgaag cagggttatg cagcggaaaa gcgctgcttc 3660
cctgctgttt tgtggaatat ctaccgactg gaaacaggca aatgcaggaa attactgaac 3720
tgaggggaca ggcgagagac gatgccaaag agctcctgaa aatctcgata actcaaaaaa 3780
tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccgatc 3840
aacgtctcat tttcgccaaa agttggccca gggcttcccg gtatcaacag ggacaccagg 3900
atttatttat tctgcgaagt gatcttccgt cacaggtatt tattcggcgc aaagtgcgtc 3960
gggtgatgct gccaacttac tgatttagtg tatgatggtg tttttgaggt gctccagtgg 4020
cttctgtttc tatcagctcc tgaaaatctc gataactcaa aaaatacgcc cggtagtgat 4080
cttatttcat tatggtgaaa gttggaacct cttacgtgcc gatcaacgtc tcattttcgc 4140
caaaagttgg cccagggctt cccggtatca acagggacac caggatttat ttattctgcg 4200
aagtgatctt ccgtcacagg tatttattcg gcgcaaagtg cgtcgggtga tgctgccaac 4260
ttactgattt agtgtatgat ggtgtttttg aggtgctcca gtggcttctg tttctatcag 4320
ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaaaagg 4380
atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 4440
ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 4500
ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 4560
ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 4620
ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 4680
ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 4740
tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 4800
tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 4860
tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 4920
tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 4980
gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 5040
tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 5100
ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct 5160
gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc 5220
gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc 5280
cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg 5340
ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta 5400
cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca 5460
ggaaacagct atgacatgat tacgaattca ccatggagcc gatgtcagag gcgactgcgg 5520
gcagatcctt ttgaagctgt ttcacaattt ctttgcccag ttcgcggcgg atctggaacc 5580
acttttgcat gcgatcgtcg tcagagtggt tcatgtgaaa aatacactca ccatctcaat 5640
ggtcatggtg aaggcctgta ctggctgcga cagcatggaa ctcagtgcaa tggctgtaag 5700
gcctgcacca acaatgattg agcgaagctc caaaatgtcc tccccgggtt gatattagat 5760
ttcataaata tactaaaaat cttgagagtt tttccgttga aaactaaaaa gctgggaagg 5820
tgaatcgaat ttcggggctt taaagcaaaa atgaacagct tggtctatag tggctaggta 5880
ccctttttgt tttggacaca tgtagggtgg ccgaaacaaa gtaataggac aacaacgctc 5940
gaccgcgatt atttttggag aatcatgacc tcagcatctg ccccaagctt taaccccggc 6000
aagggtcccg gctcagcagt cggaattgcc cttttaggat tcggaacagt cggcactgag 6060
gtgatgcgtc tgatgaccga gtacggtgat gaacttgcgc accgcattgg tggcccactg 6120
gaggttcgtg gcattgctgt ttctgatatc tcaaagccac gtgaaggcgt tgcacctgag 6180
ctgctcactg aggacgcttt tgcactcatc gagcgcgagg atgttgacat cgtcgttgag 6240
gttatcggcg gcattgagta cccacgtgag gtagttctcg cagctctgaa ggccggcaag 6300
tctgttgtta ccgccaataa ggctcttgtt gcagctcact ctgctgagct tgctgatgca 6360
gcggaagccg caaacgttga cctgtacttc gaggctgctg ttgcaggcgc aattccagtg 6420
gttggcccac tgcgtcgctc cctggctggc gatcagatcc agtctgtgat gggcatcgtt 6480
aacggcacct gcaacttcat cttggacgcc atggattcca ccggcgctga ctatgcagat 6540
tctttggctg aggcaactcg tttgggttac gccgaagctg atccaactgc agacgtcgaa 6600
ggccatgacg ccgcatccaa ggctgcaatt ttggcatcca tcgctttcca cacccgtgtt 6660
accgcggatg atgtgtactg cgaaggtatc agcaacatca gcgctgccga cattgaggca 6720
gcacagcagg caggccacac catcaagttg ttggccatct gtgagaagtt caccaacaag 6780
gaaggaaagt cggctatttc tgctcgcgtg cacccgactc tattacctgt gtcccaccca 6840
ctggcgtcgg taaacaagtc ctttaatgca atctttgttg aagcagaagc agctggtcgc 6900
ctgatgttct acggaaacgg tgcaggtggc gcgccaaccg cgtctgctgt gcttggcgac 6960
gtcgttggtg ccgcacgaaa caaggtgcac ggtggccgtg ctccaggtga gtccacctac 7020
gctaacctgc cgatcgctga tttcggtgag accaccactc gttaccacct cgacatggat 7080
gtggaagatc gcgtgggggt tttggctgaa ttggctagcc tgttctctga gcaaggaatc 7140
tccctgcgta caatccgaca ggaagagcgc gatgatgatg cacgtctgat cgtggtcacc 7200
cactctgcgc tggaatctga tctttcccgc accgttgaac tgctgaaggc taagcctgtt 7260
gttaaggcaa tcaacagtgt gatccgcctc gaaagggact aattttactg acatggcaat 7320
tgaactgaac gtcggtcgta aggttaccgt cacggtacct ggatcttctg caaacctcgg 7380
acctggcttt gacactttag gtttggcact gtcggtatac gacactgtcg aagtggaaat 7440
tattccatct ggcttggaag tggaagtttt tggcgaaggc caaggcgaag tcggatcctc 7500
tagagtcgac ctgcaggcat gcaagcttgg cactggccgt cgttttacaa cgtcgtgact 7560
gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct 7620
ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg 7680
gcgaatggcg ataagctagc ttcacgc 7707
<210> 5
<211> 11381
<212> DNA
<213> Artificial sequence ()
<400> 5
gcaattccgg ttcgcttgct gtccataaaa ccgcccagtc tagctatcgc catgtaagcc 60
cactgcaagc tacctgcttt ctctttgcgc ttgcgttttc ccttgtccag atagcccagt 120
agctgacatt catccggggt cagcaccgtt tctgcggact ggctttctac gtgttccgct 180
tcctttagca gcccttgcgc cctgagtgct tgcggcagcg tgaagctagc ttatcgcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gcttcatctt tagccagatc 420
caccaaatcc accactcctt cccccgcaag acgatgatcc ttcggcaaca ccacgcaaaa 480
ctcttccaaa ctaatcagcc gagtctcaat ctctggatcc tcaatgggca aacccatcaa 540
cgtaatatcc aacgccccca agcgcagcaa acttaccgct tccctcgtca acttctgacc 600
aacgagctcc aactccacat taggaagccg tttatgcacc tcactggtca aaagcggcag 660
cgtggaatag ttcagcacac cagaaaaacc aatgcgaaca acaccaacga tctccccctc 720
ggcagcattc acagcttccc tcgccaccgc agcgctcgcc acaatccccc tggcatgtgg 780
caaaaacgcc cgacccgcgg cagttaactc caccgaccgg gtactgcgat caaaaagctt 840
ctgccccaac tccgactcca accgccggat caactgactc aacggcgatt gcggcatccc 900
caaacgaatc gcagcattac taaagtgcaa ttcttccgcg accgcgataa agccttccaa 960
ccaacgaatc tccacgagac ccaatcctat cgtggaaaat cccagctgtt cgtggtttta 1020
gctccaatcg tccatttaag acgtcgagat ttccacatgg gtatcggcct aggttggtcc 1080
gctaaaggcc cttagaagcg atcctgcgag gtcgaacttt tagaagtctt ggccatgaaa 1140
tcacctcatc cgaggcaaac cagacaggcg tgacaagatt ggcgaaaaag ccgaagtttt 1200
ggcacgtgtg tccggtttct catcccctaa accagacagg cgtgccaaaa cctggcggaa 1260
atccagattt ttgtcacgcc tgtctggttt cctcttggtc cagcgaagac accctctgaa 1320
aaggctaaaa gaggcaagga aaccacactg tttccttgcc cctcgagcta aattagacgt 1380
cgcgtgcgat cagatcgtcc aagttctcct gcagggtacc ggtggtcgtg ctgactctac 1440
gggggaggaa gttcagctgg tactgctcgc gttggctgat aaaggagtag agttggatcc 1500
gggcccaagt tcacttaaaa aggagatcaa caatgaaagc aattttcgta ctgaaacatc 1560
ttaatcatgc gagggatggt ttctaatgct gcgccgccca accgatggcg atggctacaa 1620
cctgcatcag ctggtggcac gctgtcagcc actggatacc aactccgtgt actgcaacct 1680
gctgcaatgc tccgatttcg cagataccgc aatcgcagca gaaaacgcac aaggcgaact 1740
ggtgggcttc atctccggct accgcccacc atcccgccct gataccctgt tcgtgtggca 1800
agtggcagtg gattcctcca tgcgcggcca aggcctggca ctgcgcatgc tgctggcact 1860
gaccgcacgc gtggcacgcg aatacggcgt gcgctacatg gaaaccacca tctcccctga 1920
taacggcgca tcccaagcac tgttcaagcg cgcattcgat cgcctggatg caaactgcac 1980
cacccgcacc ctgttcgcac gcgataccca cttcgccggt cagcacgaag atgaagtgct 2040
gtaccgcgcc ggcccattca ccgtgtccca cctggaagag gaactgaagg aacacgcata 2100
atgcctggcg gcagtagcgc ggtggtccca cctgacccca tgccgaactc agaagtgaaa 2160
cgccgtagcg ccgatggtag tgtggggtct ccccatgcga gagtagggaa ctgccaggca 2220
tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc 2280
ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca 2340
acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca 2400
gaaggccatc ctgacggatg gccttttggt accggtggtc gtgctgactc tacgggggag 2460
gaagttcagc tggtactgct cgcgttggct gataaaggag tagagttgga tccgggccca 2520
agttcactta aaaaggagat caacaatgaa agcaattttc gtactgaaac atcttaatca 2580
tgctaaggag gttttctaat gaagaccttc gaactgaacg aatcccgcgt gcgctcctac 2640
tgccgctcct tccctgtggt gttcaagcaa gcacaaggcg cagaactggt gacccaagat 2700
ggcaagcgct acatcgattt cctggccggc gccggcaccc tgaactacgg ccacaaccac 2760
cctgtgctga agcaagcact gctggaatac atcgaatccg atggcatcac ccacggcctg 2820
gatatgtaca ccgaagcaaa ggaacgcttc ctggaaacct tcaaccgcct gatcctggaa 2880
ccacgcggca tgggcgatta ccgcatgcag ttcaccggcc caaccggcac caacgcagtg 2940
gaagcagcaa tgaagctggc acgcaaggtg accggccgca acaacatcat ctccttcacc 3000
aacggcttcc acggctgctc tatcggcgca ctggcagcaa ccggcaatca gcaccaccgc 3060
ggcggctccg gcattggtct gaccgatgtg tcccgcatgc catacgcaaa ctacttcggc 3120
gataagacca acaccatcgg catgatggat aagctgctgt ccgatccatc ctccggcatc 3180
gataagccag cagcagtgat cgtggaagtg gtgcaaggcg aaggcggcct gaacaccgca 3240
tccgcagaat ggatgcgcaa gctggaaaag ctgtgccgca agcacgaaat gctgctgatc 3300
gtggatgata tccaagccgg ctgcggccgc accggcacct tcttctcctt cgaagaaatg 3360
ggcattcagc ctgatatcgt gaccctgtcc aagtccctgt ccggctacgg tctgccattc 3420
gcaatggtgc tgctgcgcca agaactggat cagtggaagc ctggcgaaca caacggcacc 3480
ttccgcggca acaaccacgc attcgtgacc gcagccgcag cagtggaaca cttctggcag 3540
aacgatgcat tcgcaaactc cgtgaaggca aagggcaagc gcatcgcaga tggcatgcag 3600
cgcatcatcc gccgccacgg ccctgattcc ctgttcctga agggccgcgg catgatgatc 3660
ggcatctcct gccctgatgg cgaaatcgca gccgcagtct gtcgccacgc attcgaaaac 3720
ggcctggtga tcgaaacctc cggcgcacac tccgaagtgg tgaagtgcct gtgcccactg 3780
atcatctccg atgaacagat cgatcaagca ctgtccatcc tggataaggc attcgcagca 3840
gtgatgtccg aacagaccga aaaccaagca tcctaatgcc tggcggcagt agcgcggtgg 3900
tcccacctga ccccatgccg aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg 3960
ggtctcccca tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg 4020
aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca 4080
aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga 4140
cgcccgccat aaactgccag gcatcaaatt aagcagaagg ccatcctgac ggatggcctt 4200
ttggtaccgg tggtcgtgct gactctacgg gggaggaagt tcagctggta ctgctcgcgt 4260
tggctgataa aggagtagag ttggatccgg gcccaagttc acttaaaaag gagatcaaca 4320
atgaaagcaa ttttcgtact gaaacatctt aatcatgcta aggaggtttt ctaatgatcg 4380
tgcgcaccct ggcagaatgc gaaaagaccg atcgcaaggt gcactctcag accggcacct 4440
gggattccac ccgcatgctg ctgaaggatg ataaggtggg cttctccttc cacatcacca 4500
ccatctacgc cggctccgaa acccacatcc actatcagaa ccacttcgaa tccgtgtact 4560
gcatctccgg caacggcgaa atcgaaacca tcgcagatgg caagatctac aagatcgaac 4620
ctggcaccct gtacgtgctg gaaaagcacg atgaacacct gctgcgcggc ggctccgaag 4680
atatgaagct ggcatgcgtg ttcaacccac cactgaacgg ccgcgaagtg cacgatgaat 4740
ccggcgtgta cccactggaa gcagaaaccg tgtaatgcct ggcggcagta gcgcggtggt 4800
cccacctgac cccatgccga actcagaagt gaaacgccgt agcgccgatg gtagtgtggg 4860
gtctccccat gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga 4920
aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa 4980
atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac 5040
gcccgccata aactgccagg catcaaatta agcagaaggc catcctgacg gatggccttt 5100
taaagaattc ggtttccgta gcccacgata gctacgcgga tgttggtcat gttcttgtaa 5160
tcctccaaaa ttgtggtggc actgtcctgg tcgagcttac cgagatgcat acttagatga 5220
tgattcaggg acatctcttt catcaggacc gaaagcgaac gtttcgtatt gttgagcctt 5280
ttggttccac cacggatgcg ctgatctatt ttcatggctc ccagcagtca ggatctgtgg 5340
ggcgcagctt caccaacagg acttttgatc cgttgccgtt catggtggtt tatccggatg 5400
gggtggatca gcattggaat gatgcgcggt tgggtttgga tgaaaatacc cgccatttag 5460
gcattgatga tgtggggttc tttgtaaaac tcgccacgca cttgggcaac acgtatggca 5520
tcaagaggat ctttattgtt ggctattcca acggtgggca gatggtgttg cggctcatgc 5580
atgaggttcc caagatgctc agtggcgctg caaccattgc atccaacatg ccagttgcag 5640
agaatacgct gccgcaggtg aaaaccttca agacacatcc ggtgccttat ttggcgatgg 5700
ctggaactgc cgatactttt tcaccgtatg agggtggcga tgccggtatt ggtcgcgaac 5760
accgccgtgg cgtgggcatg tccgcctttg attcagctgc ctatattgcc gcccgaaacg 5820
gactgaccga acaccgccac gacgtgattg atgatgtggt gtcgatcgat acctgggatg 5880
gagaaaatcc cgttgagttt tggacactca acgggatcgg ccacttggta ccaagtggga 5940
aaacttatcc agaatttcta ggcccctcaa ccacatcagt gatagcggct gaggagattg 6000
ggaagttctt tgatggggtc aggcgtcgat aagctcaagc tttaaaaacg cattgcgcaa 6060
gccgtcctgc ccaacagcat ccgtgaccag gtcagcagca gccttgagcg attcggtaat 6120
catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac 6180
gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa 6240
ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 6300
gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc 6360
tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 6420
cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 6480
gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 6540
gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 6600
gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 6660
ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 6720
aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 6780
tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 6840
ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 6900
gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 6960
ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 7020
ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 7080
agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 7140
ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 7200
aaaggatctt cacctagatc cttttggggt gggcgaagaa ctccagcatg agatccccgc 7260
gctggaggat catccagccc tgatagaaac agaagccact ggagcacctc aaaaacacca 7320
tcatacacta aatcagtaag ttggcagcat cacccgacgc actttgcgcc gaataaatac 7380
ctgtgacgga agatcacttc gcagaataaa taaatcctgg tgtccctgtt gataccggga 7440
agccctgggc caacttttgg cgaaaatgag acgttgatcg gcacgtaaga ggttccaact 7500
ttcaccataa tgaaataaga tcactaccgg gcgtattttt tgagttatcg agattttcag 7560
gagctgatag aaacagaagc cactggagca cctcaaaaac accatcatac actaaatcag 7620
taagttggca gcatcacccg acgcactttg cgccgaataa atacctgtga cggaagatca 7680
cttcgcagaa taaataaatc ctggtgtccc tgttgatacc gggaagccct gggccaactt 7740
ttggcgaaaa tgagacgttg atcggcacgt aagaggttcc aactttcacc ataatgaaat 7800
aagatcacta ccgggcgtat tttttgagtt atcgagattt tcaggagctc tttggcatcg 7860
tctctcgcct gtcccctcag ttcagtaatt tcctgcattt gcctgtttcc agtcggtaga 7920
tattccacaa aacagcaggg aagcagcgct tttccgctgc ataaccctgc ttcggggtca 7980
ttatagcgat tttttcggta tatccatcct ttttcgcacg atatacagga ttttgccaaa 8040
gggttcgtgt agactttcct tggtgtatcc aacggcgtca gcggggcagg ataggtgaag 8100
taggcccacc cgcgagcggg tgttccttct tcactgtccc ttattcgcac ctggcggtgc 8160
tcaacgggaa tcctgctctg cgaggctggc cggctaccgc cggcgtaaca gatgagggca 8220
agcggatggc tgatgaaacc aagccaacca ggaagggcag cccacctatc aaggtgtact 8280
gccttccaga cgaacgaaga gcgattgagg aaaaggcggc ggcggccggc atgagcctgt 8340
cggcctacct gctggccgtc ggccagggct acaaaatcac gggcgtcgtg gactatgagc 8400
acgtccgcga gggcgtcccg gaaaacgatt ccgaagccca acctttcata gaaggcggcg 8460
gtggaatcga aatctcgtga tggcaggttg ggcgtcgctt ggtcggtcat ttcgctcggt 8520
accatcggca ttttcttttg cgtttttatt tgttaactgt taattgtcct tgttcaagga 8580
tgctgtcttt gacaacagat gttttcttgc ctttgatgtt cagcaggaag ctcggcgcaa 8640
acgttgattg tttgtctgcg tagaatcctc tgtttgtcat atagcttgta atcacgacat 8700
tgtttccttt cgcttgaggt acagcgaagt gtgagtaagt aaaggttaca tcgttaggat 8760
caagatccat ttttaacaca aggccagttt tgttcagcgg cttgtatggg ccagttaaag 8820
aattagaaac ataaccaagc atgtaaatat cgttagacgt aatgccgtca atcgtcattt 8880
ttgatccgcg ggagtcagtg aacaggtacc atttgccgtt cattttaaag acgttcgcgc 8940
gttcaatttc atctgttact gtgttagatg caatcagcgg tttcatcact tttttcagtg 9000
tgtaatcatc gtttagctca atcataccga gagcgccgtt tgctaactca gccgtgcgtt 9060
ttttatcgct ttgcagaagt ttttgacttt cttgacggaa gaatgatgtg cttttgccat 9120
agtatgcttt gttaaataaa gattcttcgc cttggtagcc atcttcagtt ccagtgtttg 9180
cttcaaatac taagtatttg tggcctttat cttctacgta gtgaggatct ctcagcgtat 9240
ggttgtcgcc tgagctgtag ttgccttcat cgatgaactg ctgtacattt tgatacgttt 9300
ttccgtcacc gtcaaagatt gatttataat cctctacacc gttgatgttc aaagagctgt 9360
ctgatgctga tacgttaact tgtgcagttg tcagtgtttg tttgccgtaa tgtttaccgg 9420
agaaatcagt gtagaataaa cggatttttc cgtcagatgt aaatgtggct gaacctgacc 9480
attcttgtgt ttggtctttt aggatagaat catttgcatc gaatttgtcg ctgtctttaa 9540
agacgcggcc agcgtttttc cagctgtcaa tagaagtttc gccgactttt tgatagaaca 9600
tgtaaatcga tgtgtcatcc gcatttttag gatctccggc taatgcaaag acgatgtggt 9660
agccgtgata gtttgcgaca gtgccgtcag cgttttgtaa tggccagctg tcccaaacgt 9720
ccaggccttt tgcagaagag atatttttaa ttgtggacga atcaaattca gaaacttgat 9780
atttttcatt tttttgctgt tcagggattt gcagcatatc atggcgtgta atatgggaaa 9840
tgccgtatgt ttccttatat ggcttttggt tcgtttcttt cgcaaacgct tgagttgcgc 9900
ctcctgccag cagtgcggta gtaaaggtta atactgttgc ttgttttgca aactttttga 9960
tgttcatcgt tcatgtctcc ttttttatgt actgtgttag cggtctgctt cttccagccc 10020
tcctgtttga agatggcaag ttagttacgc acaataaaaa aagacctaaa atatgtaagg 10080
ggtgacgcca aagtatacac tttgcccttt acacatttta ggtcttgcct gctttatcag 10140
taacaaaccc gcgcgattta cttttcgacc tcattctatt agactctcgt ttggattgca 10200
actggtctat tttcctcttt tgtttgatag aaaatcataa aaggatttgc agactacggg 10260
cctaaagaac taaaaaatct atctgtttct tttcattctc tgtatttttt atagtttctg 10320
ttgcatgggc ataaagttgc ctttttaatc acaattcaga aaatatcata atatctcatt 10380
tcactaaata atagtgaacg gcaggtatat gtgatgggtt aaaaaggatc gatcctctag 10440
cgaaccccag agtcccgctc agaagaactc gtcaagaagg cgatagaagg cgatgcgctg 10500
cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag 10560
ctcttcagca atatcacggg tagccaacgc tatgtcctga tagcggtccg ccacacccag 10620
ccggccacag tcgatgaatc cagaaaagcg gccattttcc accatgatat tcggcaagca 10680
ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc atccgcgcct tgagcctggc 10740
gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacaag 10800
accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg 10860
gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt 10920
ctcggcagga gcaaggtgag atgacaggag atcctgcccc ggcacttcgc ccaatagcag 10980
ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt 11040
ggccagccac gatagccgcg ctgcctcgtc ttggagttca ttcagggcac cggacaggtc 11100
ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga 11160
gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg 11220
agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtctcttg 11280
atcagatctt gatcccctgc gccatcagat ccttggcggc aagaaagcca tccagtttac 11340
tttgcagggc ttcccaacct taccagaggg cgccccagct g 11381
<210> 6
<211> 10575
<212> DNA
<213> Artificial sequence ()
<400> 6
gcaattccgg ttcgcttgct gtccataaaa ccgcccagtc tagctatcgc catgtaagcc 60
cactgcaagc tacctgcttt ctctttgcgc ttgcgttttc ccttgtccag atagcccagt 120
agctgacatt catccggggt cagcaccgtt tctgcggact ggctttctac gtgttccgct 180
tcctttagca gcccttgcgc cctgagtgct tgcggcagcg tgaagctagc ttatcgcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gcttggccct tattatgggc 420
cacgcacact ccatcaagtg ttgcgtgagg actacacaac actgtttgac gagttatctg 480
cgttggggtt gccagcacag gtgtgtgggg ccttacttca tcttgctcca ccaccatcat 540
tacgcttttc ttatatgtcg tgtgtagtgc cgttatttgc tgatgaaatc aaagtcgtag 600
gacaaggcac acgattatcg ttagaagaga aaatgatgat ccaacgtttc catgacaccg 660
gggtcagtgc agcagaaatc ggtcgacgcc tgggtcggtg tcggcaaaca atttccaggg 720
aacttcgacg tggtcaagat gatgatggac gttatcgtgc acgcgactcc tatgaaggtg 780
cgatcaggaa actagcgcgt ccgaaaacac cgaaacttga tgccaatcgt aggcttcggg 840
ctgtggtggt cgaggcgttg aataataaat tatctccgga gcagatttct ggtcttttag 900
ccaccgagca tgctaacgat agctctatgc agattagtca tgaaactatt taccaggcgt 960
tatatgttca aggtaaaggg gcgttgcgtg atgaattgaa gctgcagggt accggtggtc 1020
gtgctgactc tacgggggag gaagttcagc tggtactgct cgcgttggct gataaaggag 1080
tagagttgga tccgggccca agttcactta aaaaggagat caacaatgaa agcaattttc 1140
gtactgaaac atcttaatca tgcgagggat ggtttctaat gctgcgccgc ccaaccgatg 1200
gcgatggcta caacctgcat cagctggtgg cacgctgtca gccactggat accaactccg 1260
tgtactgcaa cctgctgcaa tgctccgatt tcgcagatac cgcaatcgca gcagaaaacg 1320
cacaaggcga actggtgggc ttcatctccg gctaccgccc accatcccgc cctgataccc 1380
tgttcgtgtg gcaagtggca gtggattcct ccatgcgcgg ccaaggcctg gcactgcgca 1440
tgctgctggc actgaccgca cgcgtggcac gcgaatacgg cgtgcgctac atggaaacca 1500
ccatctcccc tgataacggc gcatcccaag cactgttcaa gcgcgcattc gatcgcctgg 1560
atgcaaactg caccacccgc accctgttcg cacgcgatac ccacttcgcc ggtcagcacg 1620
aagatgaagt gctgtaccgc gccggcccat tcaccgtgtc ccacctggaa gaggaactga 1680
aggaacacgc ataatgcctg gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa 1740
ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg tctccccatg cgagagtagg 1800
gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta 1860
tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga gcggatttga 1920
acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa actgccaggc 1980
atcaaattaa gcagaaggcc atcctgacgg atggcctttt ggtaccggtg gtcgtgctga 2040
ctctacgggg gaggaagttc agctggtact gctcgcgttg gctgataaag gagtagagtt 2100
ggatccgggc ccaagttcac ttaaaaagga gatcaacaat gaaagcaatt ttcgtactga 2160
aacatcttaa tcatgctaag gaggttttct aatgaagacc ttcgaactga acgaatcccg 2220
cgtgcgctcc tactgccgct ccttccctgt ggtgttcaag caagcacaag gcgcagaact 2280
ggtgacccaa gatggcaagc gctacatcga tttcctggcc ggcgccggca ccctgaacta 2340
cggccacaac caccctgtgc tgaagcaagc actgctggaa tacatcgaat ccgatggcat 2400
cacccacggc ctggatatgt acaccgaagc aaaggaacgc ttcctggaaa ccttcaaccg 2460
cctgatcctg gaaccacgcg gcatgggcga ttaccgcatg cagttcaccg gcccaaccgg 2520
caccaacgca gtggaagcag caatgaagct ggcacgcaag gtgaccggcc gcaacaacat 2580
catctccttc accaacggct tccacggctg ctctatcggc gcactggcag caaccggcaa 2640
tcagcaccac cgcggcggct ccggcattgg tctgaccgat gtgtcccgca tgccatacgc 2700
aaactacttc ggcgataaga ccaacaccat cggcatgatg gataagctgc tgtccgatcc 2760
atcctccggc atcgataagc cagcagcagt gatcgtggaa gtggtgcaag gcgaaggcgg 2820
cctgaacacc gcatccgcag aatggatgcg caagctggaa aagctgtgcc gcaagcacga 2880
aatgctgctg atcgtggatg atatccaagc cggctgcggc cgcaccggca ccttcttctc 2940
cttcgaagaa atgggcattc agcctgatat cgtgaccctg tccaagtccc tgtccggcta 3000
cggtctgcca ttcgcaatgg tgctgctgcg ccaagaactg gatcagtgga agcctggcga 3060
acacaacggc accttccgcg gcaacaacca cgcattcgtg accgcagccg cagcagtgga 3120
acacttctgg cagaacgatg cattcgcaaa ctccgtgaag gcaaagggca agcgcatcgc 3180
agatggcatg cagcgcatca tccgccgcca cggccctgat tccctgttcc tgaagggccg 3240
cggcatgatg atcggcatct cctgccctga tggcgaaatc gcagccgcag tctgtcgcca 3300
cgcattcgaa aacggcctgg tgatcgaaac ctccggcgca cactccgaag tggtgaagtg 3360
cctgtgccca ctgatcatct ccgatgaaca gatcgatcaa gcactgtcca tcctggataa 3420
ggcattcgca gcagtgatgt ccgaacagac cgaaaaccaa gcatcctaat gcctggcggc 3480
agtagcgcgg tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc 3540
gatggtagtg tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg 3600
aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct 3660
cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg 3720
gtggcgggca ggacgcccgc cataaactgc caggcatcaa attaagcaga aggccatcct 3780
gacggatggc cttttggtac cggtggtcgt gctgactcta cgggggagga agttcagctg 3840
gtactgctcg cgttggctga taaaggagta gagttggatc cgggcccaag ttcacttaaa 3900
aaggagatca acaatgaaag caattttcgt actgaaacat cttaatcatg ctaaggaggt 3960
tttctaatga tcgtgcgcac cctggcagaa tgcgaaaaga ccgatcgcaa ggtgcactct 4020
cagaccggca cctgggattc cacccgcatg ctgctgaagg atgataaggt gggcttctcc 4080
ttccacatca ccaccatcta cgccggctcc gaaacccaca tccactatca gaaccacttc 4140
gaatccgtgt actgcatctc cggcaacggc gaaatcgaaa ccatcgcaga tggcaagatc 4200
tacaagatcg aacctggcac cctgtacgtg ctggaaaagc acgatgaaca cctgctgcgc 4260
ggcggctccg aagatatgaa gctggcatgc gtgttcaacc caccactgaa cggccgcgaa 4320
gtgcacgatg aatccggcgt gtacccactg gaagcagaaa ccgtgtaatg cctggcggca 4380
gtagcgcggt ggtcccacct gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg 4440
atggtagtgt ggggtctccc catgcgagag tagggaactg ccaggcatca aataaaacga 4500
aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc 4560
ctgagtagga caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg 4620
tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg 4680
acggatggcc ttttaaagaa ttcgtggaga aatttcttcg taccggtcgg aagggacgta 4740
aaccgcagtc gaagttgcca tcgagaggta agccgtgggt ggagggtgcg ttgattagtc 4800
aacgcccagc agaagttgct gatcgtgctg tgcctgggca ctgggagggc gatttagtaa 4860
ttggtggtga aaaccaagcg acagcgttgg tgacgttggt ggagcgcacg agccggttga 4920
cgttgattaa gcggttgggg gttaatcatg aggcgtcgac tgtgacggat gcgttggtgg 4980
agatgatggg tgatttgccg caggcgttgc gtcggagttt gacgtgggat cagggtgtgg 5040
agatggcaga gcatgcgcgg tttagcgtgg tgaccaagtg tccggtgttt ttctgtgatc 5100
ctcattcgcc gtggcagcgt gggtcgaatg agaatacgaa tggattggtc agggattttt 5160
tcccgaaggg cactaatttt gctaaagtaa gtgacgaaga agttcagcgg gcacaggatc 5220
tgctgaatta ccggccgcgg aaaatgcatg gttttaaaag cgcgacgcag gtatatgaaa 5280
aaatcgtagt tggtgcatcc accgattgag taatcatggt catagctgtt tcctgtgtga 5340
aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 5400
tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 5460
cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 5520
ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 5580
cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 5640
ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 5700
aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 5760
cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 5820
cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 5880
gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt 5940
tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 6000
cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 6060
ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 6120
gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc 6180
gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 6240
accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 6300
ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 6360
tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatccttttg 6420
gggtgggcga agaactccag catgagatcc ccgcgctgga ggatcatcca gccctgatag 6480
aaacagaagc cactggagca cctcaaaaac accatcatac actaaatcag taagttggca 6540
gcatcacccg acgcactttg cgccgaataa atacctgtga cggaagatca cttcgcagaa 6600
taaataaatc ctggtgtccc tgttgatacc gggaagccct gggccaactt ttggcgaaaa 6660
tgagacgttg atcggcacgt aagaggttcc aactttcacc ataatgaaat aagatcacta 6720
ccgggcgtat tttttgagtt atcgagattt tcaggagctg atagaaacag aagccactgg 6780
agcacctcaa aaacaccatc atacactaaa tcagtaagtt ggcagcatca cccgacgcac 6840
tttgcgccga ataaatacct gtgacggaag atcacttcgc agaataaata aatcctggtg 6900
tccctgttga taccgggaag ccctgggcca acttttggcg aaaatgagac gttgatcggc 6960
acgtaagagg ttccaacttt caccataatg aaataagatc actaccgggc gtattttttg 7020
agttatcgag attttcagga gctctttggc atcgtctctc gcctgtcccc tcagttcagt 7080
aatttcctgc atttgcctgt ttccagtcgg tagatattcc acaaaacagc agggaagcag 7140
cgcttttccg ctgcataacc ctgcttcggg gtcattatag cgattttttc ggtatatcca 7200
tcctttttcg cacgatatac aggattttgc caaagggttc gtgtagactt tccttggtgt 7260
atccaacggc gtcagcgggg caggataggt gaagtaggcc cacccgcgag cgggtgttcc 7320
ttcttcactg tcccttattc gcacctggcg gtgctcaacg ggaatcctgc tctgcgaggc 7380
tggccggcta ccgccggcgt aacagatgag ggcaagcgga tggctgatga aaccaagcca 7440
accaggaagg gcagcccacc tatcaaggtg tactgccttc cagacgaacg aagagcgatt 7500
gaggaaaagg cggcggcggc cggcatgagc ctgtcggcct acctgctggc cgtcggccag 7560
ggctacaaaa tcacgggcgt cgtggactat gagcacgtcc gcgagggcgt cccggaaaac 7620
gattccgaag cccaaccttt catagaaggc ggcggtggaa tcgaaatctc gtgatggcag 7680
gttgggcgtc gcttggtcgg tcatttcgct cggtaccatc ggcattttct tttgcgtttt 7740
tatttgttaa ctgttaattg tccttgttca aggatgctgt ctttgacaac agatgttttc 7800
ttgcctttga tgttcagcag gaagctcggc gcaaacgttg attgtttgtc tgcgtagaat 7860
cctctgtttg tcatatagct tgtaatcacg acattgtttc ctttcgcttg aggtacagcg 7920
aagtgtgagt aagtaaaggt tacatcgtta ggatcaagat ccatttttaa cacaaggcca 7980
gttttgttca gcggcttgta tgggccagtt aaagaattag aaacataacc aagcatgtaa 8040
atatcgttag acgtaatgcc gtcaatcgtc atttttgatc cgcgggagtc agtgaacagg 8100
taccatttgc cgttcatttt aaagacgttc gcgcgttcaa tttcatctgt tactgtgtta 8160
gatgcaatca gcggtttcat cacttttttc agtgtgtaat catcgtttag ctcaatcata 8220
ccgagagcgc cgtttgctaa ctcagccgtg cgttttttat cgctttgcag aagtttttga 8280
ctttcttgac ggaagaatga tgtgcttttg ccatagtatg ctttgttaaa taaagattct 8340
tcgccttggt agccatcttc agttccagtg tttgcttcaa atactaagta tttgtggcct 8400
ttatcttcta cgtagtgagg atctctcagc gtatggttgt cgcctgagct gtagttgcct 8460
tcatcgatga actgctgtac attttgatac gtttttccgt caccgtcaaa gattgattta 8520
taatcctcta caccgttgat gttcaaagag ctgtctgatg ctgatacgtt aacttgtgca 8580
gttgtcagtg tttgtttgcc gtaatgttta ccggagaaat cagtgtagaa taaacggatt 8640
tttccgtcag atgtaaatgt ggctgaacct gaccattctt gtgtttggtc ttttaggata 8700
gaatcatttg catcgaattt gtcgctgtct ttaaagacgc ggccagcgtt tttccagctg 8760
tcaatagaag tttcgccgac tttttgatag aacatgtaaa tcgatgtgtc atccgcattt 8820
ttaggatctc cggctaatgc aaagacgatg tggtagccgt gatagtttgc gacagtgccg 8880
tcagcgtttt gtaatggcca gctgtcccaa acgtccaggc cttttgcaga agagatattt 8940
ttaattgtgg acgaatcaaa ttcagaaact tgatattttt catttttttg ctgttcaggg 9000
atttgcagca tatcatggcg tgtaatatgg gaaatgccgt atgtttcctt atatggcttt 9060
tggttcgttt ctttcgcaaa cgcttgagtt gcgcctcctg ccagcagtgc ggtagtaaag 9120
gttaatactg ttgcttgttt tgcaaacttt ttgatgttca tcgttcatgt ctcctttttt 9180
atgtactgtg ttagcggtct gcttcttcca gccctcctgt ttgaagatgg caagttagtt 9240
acgcacaata aaaaaagacc taaaatatgt aaggggtgac gccaaagtat acactttgcc 9300
ctttacacat tttaggtctt gcctgcttta tcagtaacaa acccgcgcga tttacttttc 9360
gacctcattc tattagactc tcgtttggat tgcaactggt ctattttcct cttttgtttg 9420
atagaaaatc ataaaaggat ttgcagacta cgggcctaaa gaactaaaaa atctatctgt 9480
ttcttttcat tctctgtatt ttttatagtt tctgttgcat gggcataaag ttgccttttt 9540
aatcacaatt cagaaaatat cataatatct catttcacta aataatagtg aacggcaggt 9600
atatgtgatg ggttaaaaag gatcgatcct ctagcgaacc ccagagtccc gctcagaaga 9660
actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa 9720
gcacgaggaa gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca 9780
acgctatgtc ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa 9840
agcggccatt ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat 9900
cctcgccgtc gggcatccgc gccttgagcc tggcgaacag ttcggctggc gcgagcccct 9960
gatgctcttc gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc 10020
gctcgatgcg atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca 10080
gccgccgcat tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca 10140
ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa 10200
cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct 10260
cgtcttggag ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc 10320
cctgcgctga cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt 10380
catagccgaa tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt 10440
caatcatgcg aaacgatcct catcctgtct cttgatcaga tcttgatccc ctgcgccatc 10500
agatccttgg cggcaagaaa gccatccagt ttactttgca gggcttccca accttaccag 10560
agggcgcccc agctg 10575

Claims (10)

1. A method for constructing an ectoine-producing strain, comprising the steps of:
A. taking Corynebacterium glutamicum producing lysine as an initial strain, knocking down hom gene encoding homoserine kinase in genome, and obtaining a strain A which can down-regulate homoserine kinase expression;
B. knocking out pck gene of coding phosphoenolpyruvate carboxykinase in genome of the strain A to obtain a strain B lacking phosphoenolpyruvate carboxykinase;
C. enhancing the expression of lysC encoding aspartate kinase in the genome of strain B to obtain strain C over-expressing aspartate kinase;
D. enhancing the expression of a gene tkt for coding transketolase tkt, a gene tal for coding tyrosine ammonia lyase tal and a gene zwf for coding 6-phosphogluconate dehydrogenase zwf in the genome of the strain C to obtain a strain D with enhanced oxidative phosphorylation pathway;
E. integrating the ectABC gene derived from Pseudomonas stutzeri at the ddh site of a coding gene ddh of diaminopropionic acid dehydrogenase in the genome of the strain D to obtain a strain E with enhanced ectoin synthesis pathway;
F. integrating the ectABC gene from Pseudomonas stutzeri at the gene site of bacteriophage transposase IS30 in the genome of the strain E to obtain a strain F with further enhanced ectoin synthesis pathway, and screening positive clones to obtain an ectoin producing strain.
2. The method of claim 1, further comprising the steps of:
G. further integrating the ectABC gene derived from Pseudomonas stutzeri into the genome of the strain F obtained in step F, and screening for a positive clone with enhanced ectoin synthesis pathway.
3. The method according to claim 1, wherein the starting strain in step a is Corynebacterium glutamicum ATCC13032 LysCfbr。
4. The method of claim 1, wherein the knock-down of the hom gene in step a is achieved by subjecting the hom gene to a T176C mutation.
5. The method of claim 1, wherein the knock-out of the pck gene in step B comprises the steps of: introducing a plasmid pK18mobSacB-KOpck with a nucleotide sequence of SEQ ID NO.2 into a host cell; carrying out SacB sucrose reverse screening, and screening positive clones;
enhancement of expression of the gene lysC in step C is achieved by placing the lysC gene downstream of the sod promoter;
the enhancement of the expression of the genes tkt, tal and zwf in step D is achieved by replacing their native promoter with the sod promoter.
6. The method of claim 1, wherein the integration of ectABC gene at the site of ddh gene in step E comprises the steps of: introducing a plasmid pK18mobSacB-ddh-ECT with a nucleotide sequence of SEQ ID NO. 5 into a host cell; and carrying out SacB sucrose reverse screening to screen positive clones.
7. The method of claim 1, wherein the integration of ectABC gene at the IS30 gene site in step F comprises the steps of: introducing a plasmid pK18mobSacB-IS30-ECT with a nucleotide sequence of SEQ ID NO. 6 into a host cell; and carrying out SacB sucrose reverse screening to screen positive clones.
8. An ectoine-producing bacterium produced by the method according to any one of claims 1 to 7.
9. The bacterium of claim 8, which is Corynebacterium glutamicum (Corynebacterium glutamicum) deposited in the China general microbiological culture Collection center (CGMCC) with the accession number of CGMCC No. 22733.
10. Use of the ectoine-producing bacterium according to claim 8 or 9 for producing ectoine.
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CN111394288A (en) * 2019-01-03 2020-07-10 北京百奥茵诺生物科技有限公司 Recombinant corynebacterium glutamicum, construction method thereof and method for producing tetrahydropyrimidine by using recombinant corynebacterium glutamicum
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