CN110129356B - Method for assembling multiple gene segments into py-2u vector by using yeast and application - Google Patents

Abstract

The invention discloses a method for assembling multiple gene segments to a py-2u vector by using yeast and application thereof, relating to the technical field of biological engineering. The method adds homologous sequences homologous with a py-2u vector to two segments of target genes to be cloned; dividing the obtained gene into a plurality of small fragments, and adding separate enzyme cutting sites in two sections; the enzyme cutting sites at the two ends of each small fragment are the same enzyme cutting site or different independent enzyme cutting sites; each small fragment is obtained by gene synthesis or PCR amplification, and the blunt end of the product is connected to a cloning vector or TA is cloned to a small fragment vector; and (3) carrying out enzyme digestion on each fragment by using a separate enzyme digestion site, and recovering a product for the next step of yeast transformation. The constructed Py-2uL can be induced by arabinose to improve the copy number of the Py-2uL in escherichia coli, thereby being beneficial to subsequent gene operations such as plasmid extraction and the like; can stably and quickly complete the gene synthesis and assembly of the growth gene, and is simpler, more convenient, economic and efficient compared with the conventional cloning process.

Description

Method for assembling multiple gene segments into py-2u vector by using yeast and application

Technical Field

The invention relates to the technical field of bioengineering, in particular to a method for assembling a plurality of gene segments to a py-2u vector by using yeast and application thereof.

Background

Synthetic biology has great potential of use in the fields of biomedicine, biofuel, fine chemicals, agriculture and the like, and with the rapid development of synthetic biology, the synthetic biology gradually enters the era of whole gene combination, and the technical significance of synthetic genomics is great. The DNA genome cloning assembly technology is an important molecular biological tool and plays a key role in the rapid and effective assembly of large-fragment DNA elements. DNA synthesis is the core technology supporting the development of synthetic biology, and it is not dependent on DNA templates, can be synthesized directly from known DNA sequences, and plays a significant role in the synthesis of genes and biological elements, the re-design and assembly of gene circuits and biosynthetic pathways, and the artificial synthesis of whole genomes.

The patent application No. 201310389392.0 discloses a method for rapid yeast assembly of multi-fragment DNA, comprising the steps of: (1) co-transforming yeast with a plurality of DNA molecules comprising homology arms and a linearized yeast shuttle vector; (2) washing off all transformed yeast colonies on the whole screening culture plate, centrifuging, and discarding supernatant to obtain transformed yeast cells; (3) extracting plasmid DNA of yeast cells, and transforming escherichia coli competent cells; (4) screening colibacillus clone to obtain large fragment DNA. The method has the advantages of high speed, simplicity, feasibility, high success rate, low cost, high efficiency and easy operation of assembling the large-fragment DNA molecules, and can assemble a plurality of small-fragment DNA molecules into one large-fragment DNA molecule.

Because of the limited length of chemically synthesized DNA fragments, short oligonucleotides are synthesized and then spliced to synthesize long DNA fragments. Therefore, the basic idea of gene synthesis is: firstly, designing and synthesizing oligonucleotides according to an original genome sequence; splicing oligonucleotides into longer DNA sequences by various methods; thirdly, further splicing to obtain a longer DNA sequence on the basis of the longer sequence to splice into a complete genome; and fourthly, transplanting the synthesized genome into cells and verifying the function of the synthesized genome. The traditional technology for obtaining the target large fragment by the large fragment cloning technology has various defects, for example, random library construction cloning needs high-throughput screening; polymerase Chain Reaction (PCR) is difficult to amplify fragments of more than 10kb, and assembly from small fragments is time-consuming and labor-consuming and has high mutation rate; it is difficult to find suitable restriction enzyme cutting sites at both ends of the fragment based on restriction enzyme ligation.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a method for assembling a plurality of gene fragments into a Py-2u vector by using yeast and application thereof, wherein the constructed Py-2uL can be induced by arabinose to improve the copy number of the gene fragments in escherichia coli, and is beneficial to subsequent gene operations such as plasmid extraction and the like; can stably and quickly complete the gene synthesis and assembly of the growth gene, including the assembly of the genome, and is simpler, more convenient, economic and efficient compared with the conventional cloning process.

The purpose of the invention can be realized by the following technical scheme:

the invention provides a method for assembling a plurality of gene segments into a py-2u vector by using yeast, which comprises the following steps:

s1, adding two segments of homologous sequences which are homologous with the py-2u vector and have the length of 40-60bp in the target gene to be cloned;

s2, dividing the gene obtained in the step S1 into a plurality of small fragments, and adding separate enzyme cutting sites in two sections; the enzyme cutting sites at the two ends of each small fragment are the same enzyme cutting site or different independent enzyme cutting sites;

s3, each small fragment is obtained through gene synthesis or PCR amplification, and the blunt end of the product is connected to a cloning vector or TA is cloned to a small fragment vector;

and S4, carrying out enzyme digestion on each fragment by using the single enzyme digestion site, and recovering a product for the next yeast transformation.

As a further embodiment of the present invention, the yeast transformation step S4 comprises the following steps:

1) selecting yeast monoclonal shake on the plate, culturing in 4mL liquid YPD culture medium overnight, transferring 2mL culture medium to 50mL liquid YPD culture medium the next day, and continuously culturing for 4-6h until OD reaches 0.5-0.8; collecting the bacterial liquid, carrying out centrifugal precipitation, adding 0.1M lithium acetate solution for resuspension for transformation;

2) adding the DNA fragment and the linearized vector into the yeast obtained in the previous step;

3) adding DMSO-lithium acetate-PEG into the liquid in the previous step, uniformly mixing, placing the mixture into a constant-temperature incubation device for incubation treatment, closing a sealed door, starting a servo motor, and further driving a rotating shaft and a multi-test-tube mounting plate to rotate by the servo motor, so that the samples are stirred at a high speed, and centrifugal mixing of the samples in the test tubes is promoted;

4) centrifuging, discarding the supernatant, and treating with ddH₂O resuspension of cells, plating on SC-TRP plates, and culturing at 30 ℃ for 2-3 days.

As a further embodiment of the invention, the amount of each DNA fragment added in step 2) is 50-100ng each in a molar ratio of 1:1, and 200-500ng of linearized vector is added.

As a further embodiment of the present invention, the incubation conditions in step 3) are incubation at 37 ℃ for 30min and reaction at 42 ℃ for 30 min.

As a further embodiment of the invention, the Py-2u vector comprises a high copy type Py-2uH and a low copy type Py-2 uL.

As a further scheme of the invention, the homologous sequences of the PY-2u vector are respectively as follows:

(1) homologous sequence 1:

TGAGCCcgccagggttttcccagtcacgacgttgtaaaacgacggccagt；

(2) homologous sequence 2:

atcggatgccgggaccgacgagtgcagaggcgtgcaagcgagcgtcgacGC。

the invention also provides the use of a method for assembling multiple gene fragments into a py-2u vector using yeast for gene synthesis and gene assembly of long genes, including assembly of genomes.

As a further embodiment of the present invention, the plasmid construction for gene assembly comprises the following steps: (1) cloning the gene with the target vector homologous sequence to Py-2u in full length; (2) the full-length gene is cut by enzyme and recombined to a target vector through Gibson.

The invention has the beneficial effects that:

1. the method for assembling a plurality of gene fragments into a Py-2u vector by using yeast is different from the conventional yeast large intestine shuttle vector, and the constructed Py-2uL can be induced by arabinose to improve the copy number of the gene fragments in escherichia coli, thereby being beneficial to subsequent gene operations such as plasmid extraction and the like. In the uninduced state, it is single copy in the large intestine, a feature that makes it possible to clone 10-200Kb of genes, including unstable viral genomes.

2. The method for assembling a plurality of gene segments to the py-2u vector by using the yeast can stably and quickly complete the gene synthesis and assembly of long genes, including the assembly of genomes; and can also quickly complete the plasmid construction work of assembling a plurality of gene segments. The first step is to clone the gene with the target vector homologous sequence to Py-2u in full length, and the second step is to enzyme-cut the full-length gene and recombine to the target vector through Gibson recombination. The method of the invention only needs 13-16 days to complete the gene synthesis and assembly of a gene of nearly 10k, while the conventional method at least needs 19-22 days, and has obvious advantages in terms of time and process simplicity. Compared with the conventional cloning process, the method is simpler, more convenient, economic and efficient.

3. Hatch the in-process through the constant temperature device of hatching, use different external diameter's test tube to hold the sample, and insert little test tube holding jar or big test tube holding jar with the test tube according to test tube size in, the external diameter of the test tube that big test tube holding jar was suitable for is 20-30mm, the external diameter of the test tube that little test tube holding jar was suitable for is 10-20mm, made things convenient for the high-speed stirring and the centrifugal separation of the sample of a plurality of different capacities, saved the time that the constant temperature was hatched greatly, improved and hatched efficiency.

Drawings

The invention will be further described with reference to the accompanying drawings.

FIG. 1 is an electrophoretogram of plasmids extracted after arabinose induction in example 2 of the present invention.

FIG. 2 is the plasmid cleavage map finally obtained in example 2 of the present invention.

FIG. 3 is a three-dimensional view of the isothermal incubation apparatus of the present invention.

FIG. 4 is a three-dimensional view of the housing centrifugal mechanism of the present invention.

FIG. 5 is a vertical center sectional view of the invention housing a centrifugal mechanism.

Figure 6 is a three-dimensional view of the damper disc of the present invention.

In the figure: 100. a heat-insulating shell; 200. a sealing door; 300. a cylinder telescopic rod; 400. a damper disc; 410. a rotating shaft mounting hole; 420. a triangular shock absorbing plate; 430. a shock-absorbing post; 431. a damping spring; 432. a post mount; 440. a guard plate; 450. a servo motor; 451. a motor mounting seat; 452. a screw; 460. a rotating shaft; 510. a multi-test tube mounting plate; 511. clamping a large test tube hole; 512. clamping the small test tube; 513. a nut; 520. l-shaped flap plates; 530. a large test tube accommodating tank; 540. a slide rail; 550. a slide plate; 560. a small test tube accommodating tank; 570. a connecting plate.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The idea of the invention is to add homologous sequences at both ends of a plurality of gene segments during design, and add independent enzyme cutting sites besides the homologous sequences. After the 500-3000bp small fragment is completed, obtaining a gene fragment by enzyme digestion, transforming the gene fragment and the py-2u vector into yeast together, and obtaining a successfully assembled full-length gene by utilizing a repair system in the yeast body. Py-2u vectors include high copy type Py-2uH and low copy type Py-2uL, respectively, suitable for conventional long gene synthesis, genomic or viral gene assembly.

Example 1

Referring to FIGS. 1-6, a method for assembling multiple gene segments into a py-2u vector using yeast includes the following steps:

s1, adding two homologous sequences which are homologous with the py-2u vector in two sections of the target gene to be cloned, wherein the length of the homologous sequences is 40-60 bp;

s2, dividing the gene obtained in the step S1 into a plurality of small fragments, wherein the small fragments can be divided into two segments, and the two segments are usually divided into two segments according to the length, wherein the two segments are respectively added with a single enzyme cutting site (the enzyme cutting site does not exist in the target gene, otherwise, the target gene containing the enzyme cutting site is cut off when the small fragment plasmid is cut by the enzyme cutting site in the following step). The enzyme cutting sites at both ends of each small fragment are the same enzyme cutting site, and can also be different independent enzyme cutting sites.

S3, each small fragment can be obtained by gene synthesis or PCR amplification, and the blunt end of the product is connected to a cloning vector or TA is cloned to a small fragment vector. The gene of each small fragment is verified to be correct by sequencing, and then the next step is carried out.

S4, each fragment is cut by the single enzyme cutting site, and the product is recovered and used for the next step of yeast transformation.

The specific transformation method comprises the following steps:

1) selecting yeast monoclonal shake on the plate, culturing overnight in 4mL liquid YPD medium, transferring 2mL medium to 50mL liquid YPD medium the next day, and culturing for 4-6h until OD reaches 0.5-0.8. The bacterial solution was collected and pelleted by centrifugation and resuspended in 0.1M lithium acetate solution for transformation.

2) Adding DNA and linearized vector to the yeast obtained in the previous step. The addition amount of each fragment was 50-100ng each, and 200-500ng of vector was added in a molar ratio of 1: 1.

3) Adding DMSO-lithium acetate-PEG into the liquid in the previous step, uniformly mixing, putting the mixture into a constant-temperature incubation device for incubation treatment, closing the sealed door 200, starting the servo motor 450, and further driving the rotating shaft 460 and the multi-test-tube mounting plate 510 to rotate by the servo motor 450, so that the sample is stirred at a high speed, and centrifugal mixing of the sample in the test tube is promoted;

4) centrifuging, discarding the supernatant, and treating with ddH₂O resuspension of cells, plating on SC-TRP plates, and culturing at 30 ℃ for 2-3 days.

Different from the conventional yeast large intestine shuttle vector, the constructed Py-2uL can be induced by arabinose to improve the copy number of the Py-2uL in escherichia coli, and is beneficial to subsequent gene operations such as plasmid extraction and the like. In the uninduced state, it is single copy in the large intestine, a feature that makes it possible to clone 10-200Kb of genes, including unstable viral genomes. The method can be used for stably and rapidly completing the gene synthesis and assembly of the long gene, including the assembly of the genome. The method can quickly complete the plasmid construction work of assembling a plurality of gene segments. The first step is to clone the gene with the target vector homologous sequence to Py-2u in full length, and the second step is to enzyme-cut the full-length gene and recombine to the target vector through Gibson recombination. Compared with the conventional cloning process, the method is simpler, more convenient, economic and efficient.

Example 2

Referring to FIG. 1, 3 vector plasmids were randomly picked, plasmids were extracted after arabinose induction, and the plasmids were electrophoresed, as shown in lanes 1, 2, and 3, and after plasmids were extracted from the uninduced control group, as shown in lanes 4, 5, and 6, it was found that the concentration of uninduced plasmids in lanes was low, and the supercoiled plasmid content was lower than that of the induced plasmids. The values were measured with an onedrop spectrophotometer as follows:

referring to FIG. 2, the final plasmid map obtained was digested with AscI and PmlI to a correct size of 9k/5.2 k. The results of the time-recording of the relevant operations are given in the following table:

in this embodiment, 8 gene fragments can be assembled at a time by using yeast, which greatly exceeds the upper limit of fragments that can be assembled by a conventional assembly method (2-3 gene fragments, even if Gibson, the success rate of assembly of more than 4 fragments is reduced very quickly), the time consumption of an actual experiment is as shown in the above table, the success rate is 100%, 3 clones are randomly picked from a yeast plate, and escherichia coli is transformed respectively, and it is verified that the clones on all plates are successfully assembled clones.

The method of the invention only needs 13-16 days to complete the gene synthesis and assembly of the gene with the approximate 10k, while the conventional method at least needs 19-22 days, thereby having obvious advantages in terms of time and process simplicity. The associated operations and time consumption of the conventional process are shown in the following table:

example 3

Referring to fig. 3-6, the present embodiment provides a constant temperature incubation device, which can accommodate, centrifugally stir, and heat test tubes and slides of different sizes. Specifically, the constant-temperature incubation device comprises a heat-insulating shell 100, a sealed door 200 and a centrifugal mechanism, wherein a cylindrical cavity is formed in the heat-insulating shell 100, the sealed door 200 is used for containing the centrifugal mechanism to be opened and closed, the side edge of the sealed door 200 is hinged to the top of the heat-insulating shell 100, and the bottom of the sealed door 200 and the bottom of the heat-insulating shell 100 are connected with an air cylinder telescopic rod 300.

The setting centrifugation mechanism sets up in the inside cylindric cavity of heat preservation shell 100, includes: damping disk 400, shock attenuation installation mechanism, many test tubes receiving mechanism. The damper disc 400 is a hollow cylinder with an open top, and a rotation shaft mounting hole 410 is formed at the bottom of the damper disc 400. The damping installation mechanism comprises a triangular damping plate 420, damping columns 430, a guard plate 440, a servo motor 450 and a rotating shaft 460, wherein the cross section of the triangular damping plate 420 is triangular, and the three damping columns 430 are distributed at three corners of the triangular damping plate 420. The shock-absorbing column 430 comprises a shock-absorbing spring 431 and a column mounting seat 432, wherein one end of the shock-absorbing spring 431 is fixedly connected with the bottom of the triangular shock-absorbing plate 420, and the other end of the shock-absorbing spring is fixed in a hollow cavity of the column mounting seat 432 in an extending manner. The guard plate 440 has a rectangular parallelepiped shape and is disposed inside the damper cylinder 430. The servo motor 450 is disposed at the bottom center position of the triangular shock absorbing plate 420, and is fixedly connected with the triangular shock absorbing plate 420 through the motor mounting seat 451 and the screw 452, and the rotation shaft 460 extends upward through the center position of the motor mounting seat 451 and the rotation shaft mounting hole 410, so that the multi-tube accommodating mechanism is disposed in the shock absorbing plate 400.

Many test tubes holding mechanism includes many test tubes mounting panel 510, big test tube holding mechanism, little test tube holding mechanism, and many test tubes mounting panel 510's main part is the type ring form, and central point puts and is equipped with the fixed orifices, and the periphery side is equipped with the big test tube card hole 511 that the cross-section is waist shape, and inside is equipped with little test tube card hole 512, and big test tube card hole 511 and the equal ring array distribution in little test tube card hole 512 are on many test tubes mounting panel 510. The top of the rotation shaft 460 passes through the fixing hole and is fastened by the nut 513, so that the multi-cuvette mounting plate 510 is fixedly coupled to the rotation shaft 460. Big test tube holding mechanism includes L shape and turns over folded plate 520, big test tube holding jar 530, and L shape turns over the vertical limit of folded plate 520 and inserts big test tube card hole 511, with big test tube card hole 511 clearance fit, is equipped with on the horizontal limit and holds big test tube holding jar 530 male mounting hole. The cuvette accommodating mechanism comprises a slide rail 540, a slide plate 550 and a cuvette accommodating tank 560, the slide rail 540 is arranged on the inner side edge of the cuvette clamping hole 512, the slide plate 550 is inserted on the slide rail 540, and the cuvette accommodating tank 560 is arranged on the back surface of the slide plate 550 and is connected with the slide plate 550 through a connecting plate 570.

The working method of the constant temperature incubation device in the embodiment is as follows:

s1, the sealed door 200 is opened, the cylinder telescopic rod 300 is upwards pushed and extended, the sliding plate 550 is inserted into the sliding rail 540, the small test tube accommodating tank 560 is inserted into the small test tube clamping hole 512, and the small test tube accommodating mechanism is fixed; inserting the L-shaped turnover plate 520 into the large test tube clamping hole 511, and inserting the large test tube accommodating tank 530 into the mounting hole of the L-shaped turnover plate 520 to complete the fixation of the large test tube accommodating mechanism;

s2, inserting test tubes with different sizes and containing samples into the small test tube containing tank 560 and the large test tube containing tank 530, wherein the outer diameter of the test tube suitable for the large test tube containing tank 530 is 20-30mm, and the outer diameter of the test tube suitable for the small test tube containing tank 560 is 10-20 mm;

s3, close airtight door 200, open servo motor 450, servo motor 450 further drives the rotation of pivot 460, many test tube mounting panel 510 for the test tube on little test tube holding tank 560 and the big test tube holding tank 530 takes place high-speed stirring, promotes the centrifugation of sample in the test tube and mixes.

In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The foregoing is illustrative and explanatory only and is not intended to be exhaustive or to limit the invention to the precise embodiments described, and various modifications, additions, and substitutions may be made by those skilled in the art without departing from the scope of the invention or exceeding the scope of the claims.

Sequence listing

<110> general biosystems (Anhui) Ltd

<120> method for assembling multiple gene fragments into py-2u vector using yeast and use thereof

<130> do not

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 9521

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ccgcggcagg ccctccgagc gtggtggagc cgttctgtga gacagccggg tacgagtcgt 60

gacgctggaa ggggcaagcg ggtggtgggc aggaatgcgg tccgccctgc agcaaccgga 120

gggggaggga gaagggagcg gaaaagtctc caccggacgc ggccatggct cggggggggg 180

ggggcagcgg aggagcgctt ccggccgacg tctcgtcgct gattggcttc ttttcctccc 240

gccgtgtgtg aaaacacaaa tggcgtgttt tggttggcgt aaggcgcctg tcagttaacg 300

gcagccggag tgcgcagccg ccggcagcct cgctctgccc actgggtggg gcgggaggta 360

ggtggggtga ggcgagctgg acgtgcgggc gcggtcggcc tctggcgggg cgggggaggg 420

gagggagggt cagcgaaagt agctcgcgcg cgagcggccg cccaccctcc ccttcctctg 480

ggggagtcgt tttacccgcc gccggccggg cctcgtcgtc tgattggctc tcggggccca 540

gaaaactggc ccttgccatt ggctcgtgtt cgtgcaagtt gagtccatcc gccggccagc 600

gggggcggcg aggaggcgct cccaggttcc ggccctcccc tcggccccgc gccgcagagt 660

ctggccgcgc gcccctgcgc aacgtggcag gaagcgcgcg ctgggggcgg ggacgggcag 720

tagggctgag cggctgcggg gcgggtgcaa gcacgtttcc gacttgagtt gcctcaagag 780

gggcgtgctg agccagacct ccatcgcgca ctccggggag tggagggaag gagcgagggc 840

tcagttgggc tgttttggag gcaggaagca cttgctctcc caaagtcgct ctgagttgtt 900

atcagtaagg gagctgcagt ggagtaggcg gggagaaggc cgcacccttc tccggagggg 960

ggaggggagt gttgcaatac ctttctggga gttctctgct gcctcctggc ttctgaggac 1020

cgccctgggc ctgggagaat cccttccccc tcttccctcg tgatctgcaa ctccagtcac 1080

tagtctgcag cccaagctag cgtttaaacg acgtccgtac gcctaggacg cgtgccacca 1140

tgagccccaa gaagaagaga aaggtggagg ccagcatcga aaaaaaaaag tccttcgcca 1200

agggcatggg cgtgaagtcc acactcgtgt ccggctccaa agtgtacatg acaaccttcg 1260

ccgaaggcag cgacgccagg ctggaaaaga tcgtggaggg cgacagcatc aggagcgtga 1320

atgagggcga ggccttcagc gctgaaatgg ccgataaaaa cgccggctat aagatcggca 1380

acgccaaatt cagccatcct aagggctacg ccgtggtggc taacaaccct ctgtatacag 1440

gacccgtcca gcaggatatg ctcggcctga aggaaactct ggaaaagagg tacttcggcg 1500

agagcgctga tggcaatgac aatatttgta tccaggtgat ccataacatc ctggacattg 1560

aaaaaatcct cgccgaatac attaccaacg ccgcctacgc cgtcaacaat atctccggcc 1620

tggataagga cattattgga ttcggcaagt tctccacagt gtatacctac gacgaattca 1680

aagaccccga gcaccatagg gccgctttca acaataacga taagctcatc aacgccatca 1740

aggcccagta tgacgagttc gacaacttcc tcgataaccc cagactcggc tatttcggcc 1800

aggccttttt cagcaaggag ggcagaaatt acatcatcaa ttacggcaac gaatgctatg 1860

acattctggc cctcctgagc ggactgaggc actgggtggt ccataacaac gaagaagagt 1920

ccaggatctc caggacctgg ctctacaacc tcgataagaa cctcgacaac gaatacatct 1980

ccaccctcaa ctacctctac gacaggatca ccaatgagct gaccaactcc ttctccaaga 2040

actccgccgc caacgtgaac tatattgccg aaactctggg aatcaaccct gccgaattcg 2100

ccgaacaata tttcagattc agcattatga aagagcagaa aaacctcgga ttcaatatca 2160

ccaagctcag ggaagtgatg ctggacagga aggatatgtc cgagatcagg aaaaatcata 2220

aggtgttcga ctccatcagg accaaggtct acaccatgat ggactttgtg atttataggt 2280

attacatcga agaggatgcc aaggtggctg ccgccaataa gtccctcccc gataatgaga 2340

agtccctgag cgagaaggat atctttgtga ttaacctgag gggctccttc aacgacgacc 2400

agaaggatgc cctctactac gatgaagcta atagaatttg gagaaagctc gaaaatatca 2460

tgcacaacat caaggaattt aggggaaaca agacaagaga gtataagaag aaggacgccc 2520

ctagactgcc cagaatcctg cccgctggcc gtgatgtttc cgccttcagc aaactcatgt 2580

atgccctgac catgttcctg gatggcaagg agatcaacga cctcctgacc accctgatta 2640

ataaattcga taacatccag agcttcctga aggtgatgcc tctcatcgga gtcaacgcta 2700

agttcgtgga ggaatacgcc tttttcaaag actccgccaa gatcgccgat gagctgaggc 2760

tgatcaagtc cttcgctaga atgggagaac ctattgccga tgccaggagg gccatgtata 2820

tcgacgccat ccgtatttta ggaaccaacc tgtcctatga tgagctcaag gccctcgccg 2880

acaccttttc cctggacgag aacggaaaca agctcaagaa aggcaagcac ggcatgagaa 2940

atttcattat taataacgtg atcagcaata aaaggttcca ctacctgatc agatacggtg 3000

atcctgccca cctccatgag atcgccaaaa acgaggccgt ggtgaagttc gtgctcggca 3060

ggatcgctga catccagaaa aaacagggcc agaacggcaa gaaccagatc gacaggtact 3120

acgaaacttg tatcggaaag gataagggca agagcgtgag cgaaaaggtg gacgctctca 3180

caaagatcat caccggaatg aactacgacc aattcgacaa gaaaaggagc gtcattgagg 3240

acaccggcag ggaaaacgcc gagagggaga agtttaaaaa gatcatcagc ctgtacctca 3300

ccgtgatcta ccacatcctc aagaatattg tcaatatcaa cgccaggtac gtcatcggat 3360

tccattgcgt cgagcgtgat gctcaactgt acaaggagaa aggctacgac atcaatctca 3420

agaaactgga agagaaggga ttcagctccg tcaccaagct ctgcgctggc attgatgaaa 3480

ctgcccccga taagagaaag gacgtggaaa aggagatggc tgaaagagcc aaggagagca 3540

ttgacagcct cgagagcgcc aaccccaagc tgtatgccaa ttacatcaaa tacagcgacg 3600

agaagaaagc cgaggagttc accaggcaga ttaacaggga gaaggccaaa accgccctga 3660

acgcctacct gaggaacacc aagtggaatg tgatcatcag ggaggacctc ctgagaattg 3720

acaacaagac atgtaccctg ttcagaaaca aggccgtcca cctggaagtg gccaggtatg 3780

tccacgccta tatcaacgac attgccgagg tcaattccta cttccaactg taccattaca 3840

tcatgcagag aattatcatg aatgagaggt acgagaaaag cagcggaaag gtgtccgagt 3900

acttcgacgc tgtgaatgac gagaagaagt acaacgatag gctcctgaaa ctgctgtgtg 3960

tgcctttcgg ctactgtatc cccaggttta agaacctgag catcgaggcc ctgttcgata 4020

ggaacgaggc cgccaagttc gacaaggaga aaaagaaggt gtccggcaat tccggatccg 4080

gacctaagaa aaagaggaag gtggcggccg cttacccata cgatgttcca gattacgctg 4140

ctagcggcag tggagccact aacttcagcc tgctgaagca ggctggagac gtggaggaaa 4200

accctggacc tggcgcgcct gtggcgcgta aggtcgatct cacctcctgc gatcgcgagc 4260

cgatccacat ccccggcagc attcagccgt gcggctgcct gctagcctgc gacgcgcagg 4320

cggtgcggat cacgcgcatt acggaaaatg ccggcgcgtt ctttggacgc gaaactccgc 4380

gggtcggtga gctactcgcc gattacttcg gcgagaccga agcccatgcg ctgcgcaacg 4440

cactggcgca gtcctccgat ccaaagcgac cggcgctgat cttcggttgg cgcgacggcc 4500

tgaccggccg caccttcgac atctcactgc atcgccatga cggtacatcg atcatcgagt 4560

tcgagcctgc ggcggccgaa caggccgaca atccgctgcg gctgacgcgg cagatcatcg 4620

cgcgcaccaa agaactgaag tcgctcgaag agatggccgc acgggtgccg cgctatctgc 4680

aggcgatgct cggctatcac cgcgtgatgt tgtaccgctt cgcggacgac ggctccggga 4740

tggtgatcgg cgaggcgaag cgcagcgacc tcgagagctt tctcggtcag cactttccgg 4800

cgtcgctggt cccgcagcag gcgcggctac tgtacttgaa gaacgcgatc cgcgtggtct 4860

cggattcgcg cggcatcagc agccggatcg tgcccgagca cgacgcctcc ggcgccgcgc 4920

tcgatctgtc gttcgcgcac ctgcgcagca tctcgccctg ccatctcgaa tttctgcgga 4980

acatgggcgt cagcgcctcg atgtcgctgt cgatcatcat tgacggcacg ctatggggat 5040

tgatcatctg tcatcattac gagccgcgtg ccgtgccgat ggcgcagcgc gtcgcggccg 5100

aaatgttcgc cgacttctta tcgctgcact tcaccgccgc ccaccaccaa cgctaattaa 5160

ttaagatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc 5220

cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta 5280

ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat 5340

ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct taggtaccca 5400

gttaaccggt gttttcggga gtagtgcccc aactggggta acctttgagt tctctcagtt 5460

gggggcgtag ggtcgccgac gttttcggga gtagtgcccc aactggggta acctttgagt 5520

tctctcagtt gggggcgtag ggtcgccgac gttttcggga gtagtgcccc aactggggta 5580

acctttgagt tctctcagtt gggggcgtag ggtcgccgac ggaagttcct attctctaga 5640

aagtatagga acttcgccac catgggatcg gccattgaac aagatggatt gcacgcaggt 5700

tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc 5760

tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag 5820

accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg 5880

gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac 5940

tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc 6000

gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc 6060

tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc 6120

ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg 6180

ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat 6240

gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc 6300

cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa 6360

gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat 6420

tcgcagcgca tcgccttcta tcgccttctt gacgagttct tcggcagtgg agccactaac 6480

ttcagcctgc tgaagcaggc tggagacgtg gaggaaaacc ctggacctgt gagcaagggc 6540

gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc 6600

cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg 6660

aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg 6720

acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc 6780

aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc 6840

aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag 6900

ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac 6960

tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac 7020

ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag 7080

aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag 7140

tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg 7200

accgccgccg ggatcactct cggcatggac gagctgtaca agtgagatca taatcagcca 7260

taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct 7320

gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta 7380

caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag 7440

ttgtggtttg tccaaactca tcaatgtatc ttagaagttc ctattctcta gaaagtatag 7500

gaacttccta gaagatgggc gggagtcttc tgggcaggct taaaggctaa cctggtgtgt 7560

gggcgttgtc ctgcagggga attgaacagg tgtaaaattg gagggacaag acttcccaca 7620

gattttcggt tttgtcggga agttttttaa taggggcaaa taaggaaaat gggaggatag 7680

gtagtcatct ggggttttat gcagcaaaac tacaggttat tattgcttgt gatccgcctc 7740

ggagtatttt ccatcgaggt agattaaaga catgctcacc cgagttttat actctcctgc 7800

ttgagatcct tactacagta tgaaattaca gtgtcgcgag ttagactatg taagcagaat 7860

tttaatcatt tttaaagagc ccagtacttc atatccattt ctcccgctcc ttctgcagcc 7920

ttatcaaaag gtattttaga acactcattt tagccccatt ttcatttatt atactggctt 7980

atccaacccc tagacagagc attggcattt tccctttcct gatcttagaa gtctgatgac 8040

tcatgaaacc agacagatta gttacataca ccacaaatcg aggctgtagc tggggcctca 8100

acactgcagt tcttttataa ctccttagta cactttttgt tgatcctttg ccttgatcct 8160

taattttcag tgtctatcac ctctcccgtc aggtggtgtt ccacatttgg gcctattctc 8220

agtccaggga gttttacaac aatagatgta ttgagaatcc aacctaaagc ttaactttcc 8280

actcccatga atgcctctct cctttttctc catttataaa ctgagctatt aaccattaat 8340

ggtttccagg tggatgtctc ctcccccaat attacctgat gtatcttaca tattgccagg 8400

ctgatatttt aagacattaa aaggtatatt tcattattga gccacatggt attgattact 8460

gcttactaaa attttgtcat tgtacacatc tgtaaaaggt ggttcctttt ggaatgcaaa 8520

gttcaggtgt ttgttgtctt tcctgaccta aggtcttgtg agcttgtatt ttttctattt 8580

aagcagtgct ttctcttgga ctggcttgac tcatggcatt ctacacgtta ttgctggtct 8640

aaatgtgatt ttgccaagct tcttcaggac ctataatttt gcttgacttg tagccaaaca 8700

caagtaaaat gattaagcaa caaatgtatt tgtgaagctt ggtttttagg ttgttgtgtt 8760

gtgtgtgctt gtgctctata ataatactat ccaggggctg gagaggtggc tcggagttca 8820

agagcacaga ctgctcttcc agaagtcctg agttcaattc ccagcaacca catggtggct 8880

cacaaccatc tgtaatggga tctgatgccc tcttctggtg tgtctgaaga ccacaagtgt 8940

attcacatta aataaataaa tcctccttct tcttcttttt ttttttttta aagagaatac 9000

tgtctccagt agaatttact gaagtaatga aatactttgt gtttgttcca atatggtagc 9060

caataatcaa attactcttt aagcactgga aatgttacca aggaactaat ttttatttga 9120

agtgtaactg tggacagagg agccataact gcagacttgt gggatacaga agaccaatgc 9180

agactttaat gtcttttctc ttacactaag caataaagaa ataaaaattg aacttctagt 9240

atcctatttg tttaaactgc tagctttact taacttttgt gcttcatcta tacaaagctg 9300

aaagctaagt ctgcagccat tactaaacat gaaagcaagt aatgataatt ttggatttca 9360

aaaatgtagg gccagagttt agccagccag tggtggtgct tgcctttatg cctttaatcc 9420

cagcactctg gaggcagaga caggcagatc tctgagtttg agcccagcct ggtctacaca 9480

tcaagttcta tctaggatag ccaggaatac acacagaaac c 9521

<210> 2

<211> 1183

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cccgggtgag cccgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtccgc 60

ggcaggccct ccgagcgtgg tggagccgtt ctgtgagaca gccgggtacg agtcgtgacg 120

ctggaagggg caagcgggtg gtgggcagga atgcggtccg ccctgcagca accggagggg 180

gagggagaag ggagcggaaa agtctccacc ggacgcggcc atggctcggg gggggggggg 240

cagcggagga gcgcttccgg ccgacgtctc gtcgctgatt ggcttctttt cctcccgccg 300

tgtgtgaaaa cacaaatggc gtgttttggt tggcgtaagg cgcctgtcag ttaacggcag 360

ccggagtgcg cagccgccgg cagcctcgct ctgcccactg ggtggggcgg gaggtaggtg 420

gggtgaggcg agctggacgt gcgggcgcgg tcggcctctg gcggggcggg ggaggggagg 480

gagggtcagc gaaagtagct cgcgcgcgag cggccgccca ccctcccctt cctctggggg 540

agtcgtttta cccgccgccg gccgggcctc gtcgtctgat tggctctcgg ggcccagaaa 600

actggccctt gccattggct cgtgttcgtg caagttgagt ccatccgccg gccagcgggg 660

gcggcgagga ggcgctccca ggttccggcc ctcccctcgg ccccgcgccg cagagtctgg 720

ccgcgcgccc ctgcgcaacg tggcaggaag cgcgcgctgg gggcggggac gggcagtagg 780

gctgagcggc tgcggggcgg gtgcaagcac gtttccgact tgagttgcct caagaggggc 840

gtgctgagcc agacctccat cgcgcactcc ggggagtgga gggaaggagc gagggctcag 900

ttgggctgtt ttggaggcag gaagcacttg ctctcccaaa gtcgctctga gttgttatca 960

gtaagggagc tgcagtggag taggcgggga gaaggccgca cccttctccg gaggggggag 1020

gggagtgttg caataccttt ctgggagttc tctgctgcct cctggcttct gaggaccgcc 1080

ctgggcctgg gagaatccct tccccctctt ccctcgtgat ctgcaactcc agtcactagt 1140

ctgcagccca agctagcgtt taaacgacgt ccgtacgccc ggg 1183

<210> 3

<211> 1244

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cccgggccag tcactagtct gcagcccaag ctagcgttta aacgacgtcc gtacgcctag 60

gacgcgtgcc accatgagcc ccaagaagaa gagaaaggtg gaggccagca tcgaaaaaaa 120

aaagtccttc gccaagggca tgggcgtgaa gtccacactc gtgtccggct ccaaagtgta 180

catgacaacc ttcgccgaag gcagcgacgc caggctggaa aagatcgtgg agggcgacag 240

catcaggagc gtgaatgagg gcgaggcctt cagcgctgaa atggccgata aaaacgccgg 300

ctataagatc ggcaacgcca aattcagcca tcctaagggc tacgccgtgg tggctaacaa 360

ccctctgtat acaggacccg tccagcagga tatgctcggc ctgaaggaaa ctctggaaaa 420

gaggtacttc ggcgagagcg ctgatggcaa tgacaatatt tgtatccagg tgatccataa 480

catcctggac attgaaaaaa tcctcgccga atacattacc aacgccgcct acgccgtcaa 540

caatatctcc ggcctggata aggacattat tggattcggc aagttctcca cagtgtatac 600

ctacgacgaa ttcaaagacc ccgagcacca tagggccgct ttcaacaata acgataagct 660

catcaacgcc atcaaggccc agtatgacga gttcgacaac ttcctcgata accccagact 720

cggctatttc ggccaggcct ttttcagcaa ggagggcaga aattacatca tcaattacgg 780

caacgaatgc tatgacattc tggccctcct gagcggactg aggcactggg tggtccataa 840

caacgaagaa gagtccagga tctccaggac ctggctctac aacctcgata agaacctcga 900

caacgaatac atctccaccc tcaactacct ctacgacagg atcaccaatg agctgaccaa 960

ctccttctcc aagaactccg ccgccaacgt gaactatatt gccgaaactc tgggaatcaa 1020

ccctgccgaa ttcgccgaac aatatttcag attcagcatt atgaaagagc agaaaaacct 1080

cggattcaat atcaccaagc tcagggaagt gatgctggac aggaaggata tgtccgagat 1140

caggaaaaat cataaggtgt tcgactccat caggaccaag gtctacacca tgatggactt 1200

tgtgatttat aggtattaca tcgaagagga tgccaaggcc cggg 1244

<210> 4

<211> 1232

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

cccgggggtc tacaccatga tggactttgt gatttatagg tattacatcg aagaggatgc 60

caaggtggct gccgccaata agtccctccc cgataatgag aagtccctga gcgagaagga 120

tatctttgtg attaacctga ggggctcctt caacgacgac cagaaggatg ccctctacta 180

cgatgaagct aatagaattt ggagaaagct cgaaaatatc atgcacaaca tcaaggaatt 240

taggggaaac aagacaagag agtataagaa gaaggacgcc cctagactgc ccagaatcct 300

gcccgctggc cgtgatgttt ccgccttcag caaactcatg tatgccctga ccatgttcct 360

ggatggcaag gagatcaacg acctcctgac caccctgatt aataaattcg ataacatcca 420

gagcttcctg aaggtgatgc ctctcatcgg agtcaacgct aagttcgtgg aggaatacgc 480

ctttttcaaa gactccgcca agatcgccga tgagctgagg ctgatcaagt ccttcgctag 540

aatgggagaa cctattgccg atgccaggag ggccatgtat atcgacgcca tccgtatttt 600

aggaaccaac ctgtcctatg atgagctcaa ggccctcgcc gacacctttt ccctggacga 660

gaacggaaac aagctcaaga aaggcaagca cggcatgaga aatttcatta ttaataacgt 720

gatcagcaat aaaaggttcc actacctgat cagatacggt gatcctgccc acctccatga 780

gatcgccaaa aacgaggccg tggtgaagtt cgtgctcggc aggatcgctg acatccagaa 840

aaaacagggc cagaacggca agaaccagat cgacaggtac tacgaaactt gtatcggaaa 900

ggataagggc aagagcgtga gcgaaaaggt ggacgctctc acaaagatca tcaccggaat 960

gaactacgac caattcgaca agaaaaggag cgtcattgag gacaccggca gggaaaacgc 1020

cgagagggag aagtttaaaa agatcatcag cctgtacctc accgtgatct accacatcct 1080

caagaatatt gtcaatatca acgccaggta cgtcatcgga ttccattgcg tcgagcgtga 1140

tgctcaactg tacaaggaga aaggctacga catcaatctc aagaaactgg aagagaaggg 1200

attcagctcc gtcaccaagc tctgcgcccg gg 1232

<210> 5

<211> 1235

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cccgggctac gacatcaatc tcaagaaact ggaagagaag ggattcagct ccgtcaccaa 60

gctctgcgct ggcattgatg aaactgcccc cgataagaga aaggacgtgg aaaaggagat 120

ggctgaaaga gccaaggaga gcattgacag cctcgagagc gccaacccca agctgtatgc 180

caattacatc aaatacagcg acgagaagaa agccgaggag ttcaccaggc agattaacag 240

ggagaaggcc aaaaccgccc tgaacgccta cctgaggaac accaagtgga atgtgatcat 300

cagggaggac ctcctgagaa ttgacaacaa gacatgtacc ctgttcagaa acaaggccgt 360

ccacctggaa gtggccaggt atgtccacgc ctatatcaac gacattgccg aggtcaattc 420

ctacttccaa ctgtaccatt acatcatgca gagaattatc atgaatgaga ggtacgagaa 480

aagcagcgga aaggtgtccg agtacttcga cgctgtgaat gacgagaaga agtacaacga 540

taggctcctg aaactgctgt gtgtgccttt cggctactgt atccccaggt ttaagaacct 600

gagcatcgag gccctgttcg ataggaacga ggccgccaag ttcgacaagg agaaaaagaa 660

ggtgtccggc aattccggat ccggacctaa gaaaaagagg aaggtggcgg ccgcttaccc 720

atacgatgtt ccagattacg ctgctagcgg cagtggagcc actaacttca gcctgctgaa 780

gcaggctgga gacgtggagg aaaaccctgg acctggcgcg cctgtggcgc gtaaggtcga 840

tctcacctcc tgcgatcgcg agccgatcca catccccggc agcattcagc cgtgcggctg 900

cctgctagcc tgcgacgcgc aggcggtgcg gatcacgcgc attacggaaa atgccggcgc 960

gttctttgga cgcgaaactc cgcgggtcgg tgagctactc gccgattact tcggcgagac 1020

cgaagcccat gcgctgcgca acgcactggc gcagtcctcc gatccaaagc gaccggcgct 1080

gatcttcggt tggcgcgacg gcctgaccgg ccgcaccttc gacatctcac tgcatcgcca 1140

tgacggtaca tcgatcatcg agttcgagcc tgcggcggcc gaacaggccg acaatccgct 1200

gcggctgacg cggcagatca tcgcgcgcac ccggg 1235

<210> 6

<211> 1238

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cccgggccga acaggccgac aatccgctgc ggctgacgcg gcagatcatc gcgcgcacca 60

aagaactgaa gtcgctcgaa gagatggccg cacgggtgcc gcgctatctg caggcgatgc 120

tcggctatca ccgcgtgatg ttgtaccgct tcgcggacga cggctccggg atggtgatcg 180

gcgaggcgaa gcgcagcgac ctcgagagct ttctcggtca gcactttccg gcgtcgctgg 240

tcccgcagca ggcgcggcta ctgtacttga agaacgcgat ccgcgtggtc tcggattcgc 300

gcggcatcag cagccggatc gtgcccgagc acgacgcctc cggcgccgcg ctcgatctgt 360

cgttcgcgca cctgcgcagc atctcgccct gccatctcga atttctgcgg aacatgggcg 420

tcagcgcctc gatgtcgctg tcgatcatca ttgacggcac gctatgggga ttgatcatct 480

gtcatcatta cgagccgcgt gccgtgccga tggcgcagcg cgtcgcggcc gaaatgttcg 540

ccgacttctt atcgctgcac ttcaccgccg cccaccacca acgctaatta attaagatca 600

taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc 660

ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt 720

ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac 780

tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaggtaccc agttaaccgg 840

tgttttcggg agtagtgccc caactggggt aacctttgag ttctctcagt tgggggcgta 900

gggtcgccga cgttttcggg agtagtgccc caactggggt aacctttgag ttctctcagt 960

tgggggcgta gggtcgccga cgttttcggg agtagtgccc caactggggt aacctttgag 1020

ttctctcagt tgggggcgta gggtcgccga cggaagttcc tattctctag aaagtatagg 1080

aacttcgcca ccatgggatc ggccattgaa caagatggat tgcacgcagg ttctccggcc 1140

gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat 1200

gccgccgtgt tccggctgtc agcgcagggg cgcccggg 1238

<210> 7

<211> 1237

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cccgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg 60

ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg 120

aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg 180

ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc 240

tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc 300

tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc 360

gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc 420

aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg 480

atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct 540

tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt 600

tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc 660

tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt 720

tcttcggcag tggagccact aacttcagcc tgctgaagca ggctggagac gtggaggaaa 780

accctggacc tgtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg 840

agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg 900

ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct 960

ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc 1020

acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca 1080

ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg 1140

acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc 1200

tggggcacaa gctggagtac aactacaaca gcccggg 1237

<210> 8

<211> 1366

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cccgggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta 60

caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt 120

caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa 180

cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc 240

cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac 300

cgccgccggg atcactctcg gcatggacga gctgtacaag tgagatcata atcagccata 360

ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga 420

aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca 480

aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt 540

gtggtttgtc caaactcatc aatgtatctt agaagttcct attctctaga aagtatagga 600

acttcctaga agatgggcgg gagtcttctg ggcaggctta aaggctaacc tggtgtgtgg 660

gcgttgtcct gcaggggaat tgaacaggtg taaaattgga gggacaagac ttcccacaga 720

ttttcggttt tgtcgggaag ttttttaata ggggcaaata aggaaaatgg gaggataggt 780

agtcatctgg ggttttatgc agcaaaacta caggttatta ttgcttgtga tccgcctcgg 840

agtattttcc atcgaggtag attaaagaca tgctcacccg agttttatac tctcctgctt 900

gagatcctta ctacagtatg aaattacagt gtcgcgagtt agactatgta agcagaattt 960

taatcatttt taaagagccc agtacttcat atccatttct cccgctcctt ctgcagcctt 1020

atcaaaaggt attttagaac actcatttta gccccatttt catttattat actggcttat 1080

ccaaccccta gacagagcat tggcattttc cctttcctga tcttagaagt ctgatgactc 1140

atgaaaccag acagattagt tacatacacc acaaatcgag gctgtagctg gggcctcaac 1200

actgcagttc ttttataact ccttagtaca ctttttgttg atcctttgcc ttgatcctta 1260

attttcagtg tctatcacct ctcccgtcag gtggtgttcc acatttgggc ctattctcag 1320

tccagggagt tttacaacaa tagatgtatt gagaatccaa cccggg 1366

<210> 9

<211> 1373

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

cccgggccta ttctcagtcc agggagtttt acaacaatag atgtattgag aatccaacct 60

aaagcttaac tttccactcc catgaatgcc tctctccttt ttctccattt ataaactgag 120

ctattaacca ttaatggttt ccaggtggat gtctcctccc ccaatattac ctgatgtatc 180

ttacatattg ccaggctgat attttaagac attaaaaggt atatttcatt attgagccac 240

atggtattga ttactgctta ctaaaatttt gtcattgtac acatctgtaa aaggtggttc 300

cttttggaat gcaaagttca ggtgtttgtt gtctttcctg acctaaggtc ttgtgagctt 360

gtattttttc tatttaagca gtgctttctc ttggactggc ttgactcatg gcattctaca 420

cgttattgct ggtctaaatg tgattttgcc aagcttcttc aggacctata attttgcttg 480

acttgtagcc aaacacaagt aaaatgatta agcaacaaat gtatttgtga agcttggttt 540

ttaggttgtt gtgttgtgtg tgcttgtgct ctataataat actatccagg ggctggagag 600

gtggctcgga gttcaagagc acagactgct cttccagaag tcctgagttc aattcccagc 660

aaccacatgg tggctcacaa ccatctgtaa tgggatctga tgccctcttc tggtgtgtct 720

gaagaccaca agtgtattca cattaaataa ataaatcctc cttcttcttc tttttttttt 780

ttttaaagag aatactgtct ccagtagaat ttactgaagt aatgaaatac tttgtgtttg 840

ttccaatatg gtagccaata atcaaattac tctttaagca ctggaaatgt taccaaggaa 900

ctaattttta tttgaagtgt aactgtggac agaggagcca taactgcaga cttgtgggat 960

acagaagacc aatgcagact ttaatgtctt ttctcttaca ctaagcaata aagaaataaa 1020

aattgaactt ctagtatcct atttgtttaa actgctagct ttacttaact tttgtgcttc 1080

atctatacaa agctgaaagc taagtctgca gccattacta aacatgaaag caagtaatga 1140

taattttgga tttcaaaaat gtagggccag agtttagcca gccagtggtg gtgcttgcct 1200

ttatgccttt aatcccagca ctctggaggc agagacaggc agatctctga gtttgagccc 1260

agcctggtct acacatcaag ttctatctag gatagccagg aatacacaca gaaaccatcg 1320

gatgccggga ccgacgagtg cagaggcgtg caagcgagcg tcgacgcccc ggg 1373

Claims (1)

1. A method for assembling a plurality of gene fragments into a py-2u vector using yeast, comprising the steps of:

s1, adding two segments of homologous sequences which are homologous with the py-2u vector and have the length of 40-60bp in the target gene to be cloned;

s2, dividing the gene obtained in the step S1 into a plurality of small fragments, and adding separate enzyme cutting sites in two sections; the enzyme cutting sites at the two ends of each small fragment are the same enzyme cutting site or different independent enzyme cutting sites;

s3, each small fragment is obtained through gene synthesis or PCR amplification, and the blunt end of the product is connected to a cloning vector or TA is cloned to a small fragment vector;

s4, performing enzyme digestion on each fragment by using the single enzyme digestion site, and recovering a product for the next yeast transformation;

the specific steps of step S4 yeast transformation are as follows:

1) selecting yeast monoclonal shake on the plate, culturing in 4mL liquid YPD culture medium overnight, transferring 2mL culture medium to 50mL liquid YPD culture medium the next day, and continuously culturing for 4-6h until OD reaches 0.5-0.8; collecting the bacterial liquid, carrying out centrifugal precipitation, adding 0.1M lithium acetate solution for resuspension for transformation;

2) adding the DNA fragment and the linearized vector into the yeast obtained in the previous step;

3) adding DMSO-lithium acetate-PEG into the liquid in the previous step, uniformly mixing, putting the mixture into a constant-temperature incubation device for incubation treatment, closing a sealed door (200), starting a servo motor (450), and further driving a rotating shaft (460) and a multi-test-tube mounting plate (510) to rotate by the servo motor (450), so that a sample is stirred at a high speed, and centrifugal mixing of the sample in the test tube is promoted;

4) centrifuging, discarding the supernatant, and treating with ddH₂O resuspending the cells, plating, coating on SC-TRP plate, and culturing at 30 deg.C for 2-3 days;

adding the addition amount of each DNA fragment in the step 2) according to the molar ratio of 1:1, wherein 50-100ng of each DNA fragment is added, and 200-500ng of linearized vector is added;

the incubation condition of the step 3) is incubation at 37 ℃ for 30min and reaction at 42 ℃ for 30 min;

the Py-2u vector comprises a high copy type Py-2uH and a low copy type Py-2 uL;

the homologous sequences of the PY-2u vector are respectively as follows:

(1) homologous sequence 1:

TGAGCCcgccagggttttcccagtcacgacgttgtaaaacgacggccagt；

(2) homologous sequence 2:

atcggatgccgggaccgacgagtgcagaggcgtgcaagcgagcgtcgacGC；

the method is used for gene synthesis and gene assembly of long genes, wherein the gene assembly comprises assembly of genomes;

the plasmid construction procedure for gene assembly was as follows: (1) cloning the gene with the target vector homologous sequence to Py-2u in full length; (2) the full-length gene is cut by enzyme and recombined to a target vector through Gibson.

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