阅读说明：本技术 提高目的片段与psiCHECK2载体连接效率的方法 (Method for improving connection efficiency of target fragment and psiCHECK2 vector ) 是由 孙伟 曹修凯 王珊 袁泽湖 王善禾 于 2021-09-30 设计创作，主要内容包括：本发明提出了一种提高目的片段与psiCHECK2载体连接效率的方法,该方法以牛基因组DNA为模板,利用载体同源序列引物替换传统引物中的保护碱基序列,通过PCR扩增目的片段,将PCR扩增产物与psiCHECK2载体连接后导入感受态细胞,判断目的片段与psiCHECK2载体的载体连接效率。本发明的同源片段实验组的连接效率要比常规引物实验组和细菌内源性同源重实验组效率高,但该方法并不是基于同源重组原理,而是通过改变保护碱基的长度(本发明中采用的是载体同源序列)提高双酶切效率达到改善重组效率的目的。本发明可为其他载体构建提供技术依据。(The invention provides a method for improving the connection efficiency of a target fragment and a psiCHECK2 vector, which comprises the steps of using bovine genomic DNA as a template, replacing a protective base sequence in a traditional primer by using a vector homologous sequence primer, amplifying the target fragment through PCR, connecting a PCR amplification product with a psiCHECK2 vector, introducing the vector into a competent cell, and judging the connection efficiency of the target fragment and the psiCHECK2 vector. The connection efficiency of the homologous fragment experimental group is higher than that of a conventional primer experimental group and a bacterial endogenous homologous recombination experimental group, but the method is not based on the homologous recombination principle, and the aim of improving the recombination efficiency is fulfilled by improving the double enzyme digestion efficiency by changing the length of a protective base (the vector homologous sequence is adopted in the invention). The invention can provide technical basis for the construction of other vectors.)

1. A method for improving the efficiency of connecting a target fragment with a psiCHECK2 vector, which is characterized by comprising the following steps: a bovine genome DNA is taken as a template, a vector homologous sequence primer is designed to replace a protective base in a traditional primer, a target segment is amplified by utilizing PCR, a PCR amplification product is connected with a psiCHECK2 vector and then is led into a competent cell, and the vector recombination efficiency of the target segment and a psiCHECK2 vector is judged.

2. The method for improving the efficiency of the ligation of the target fragment to the psiCHECK2 vector according to claim 1, wherein: the target fragment is cattleIGFBP3Partial fragment of gene located in cattleIGFBP3The gene candidate region chr4: 76123453-76124382.

3. The method for improving the efficiency of the ligation of the target fragment to the psiCHECK2 vector according to claim 1, wherein: the connection efficiency of the vector is determined according to the bacterial monoclonal number, the PCR positive rate of the bacterial liquid and the sequencing accuracy, and the connection efficiency of the target fragment and the psiCHECK2 vector is determined.

4. The method for improving the efficiency of the ligation of the target fragment to the psiCHECK2 vector according to claim 2, wherein: the method is used for amplifying cattleIGFBP3The introduction vector homologous fragment primer pair of the gene partial fragment is as follows:

the upstream primer F1:

5′-TAGGCGATCGCTCGAGGCACAAAAGACTGCCAAGGACA -3′，

the downstream primer R1:

5′- TTGCGGCCAGCGGCCGCCACCAAGCAAGGGCGATT -3′。

5. the method of claim 4, wherein the method comprises the steps of: the homologous sequence of the carrier in the primer is as follows:

upstream primer vector homology sequence: TAGGCGATCG

Downstream primer vector homology sequence: TTGCGGCCA are provided.

6. The method for improving the efficiency of the ligation of the target fragment to the psiCHECK2 vector according to claim 1, wherein: the fragment size of the PCR amplification product is 963 bp.

7. The method for improving the efficiency of the ligation of the target fragment to the psiCHECK2 vector according to claim 1, wherein: the PCR amplification product and the psiCHECK2 vector are treated by endonucleases Xho I and Not I, are connected by T4 ligase, and are introduced into DH5 alpha competent cells.

8. The method for improving the efficiency of the ligation of the target fragment to the psiCHECK2 vector according to claim 1, wherein: the amplification system used for PCR comprises 1.0. mu.L of 50 ng/. mu.L of template DNA, 1.0. mu.L of each of the upstream and downstream primers of a 10 mM primer pair, 12.5. mu.L of 2 XMASTERMix (Sangon), and 9.5. mu.L of deionized water; the reaction procedure of the PCR is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30 s, annealing at 60 ℃ for 30 s, and extension at 72 ℃ for 60 s for 35 cycles.

9. Use of the method of any one of claims 1 to 8 in the construction of a recombinant vector.

10. Use according to claim 9, characterized in that: the bacterial monoclonal number, the bacterial liquid PCR positive rate and the sequencing accuracy of the homologous sequence primer recombinant vector are higher than those of a conventional primer recombinant vector and a bacterial endogenous homologous recombinant vector.

Technical Field

The invention relates to a method for improving the connection efficiency of a target fragment and a psiCHECK2 vector, belonging to the technical field of molecular biology.

Background

It is known that the gene engineering research includes four major steps, namely, the acquisition of target genes, the construction of gene expression vectors, the transfer of the gene expression vectors into recipient cells, and the monitoring and identification of the target genes, wherein the most important of the four steps is the construction of the most complicated gene expression vectors. Vector construction is an important experimental technique in molecular biology and genetic engineering, is a necessary means for researching gene function, and generally requires higher recombination efficiency and accuracy. At present, plasmid vectors, phage vectors and cosmids are commonly used as vectors, wherein the plasmid vectors are most widely applied in animal experiments. Plasmid vectors are derived from bacteria, exogenous plasmids can be introduced into DH5 alpha competent bacteria, large-scale extraction of plasmids is realized by culturing the bacteria, and various commercial plasmid extraction kits are available at present. Plasmids can replicate themselves in cells of other species and contain abundant endonuclease sites in their DNA sequences. These properties have led to the widespread use of plasmid vectors in molecular biology and genetic engineering.

The psiCHECK2 vector is a plasmid vector that monitors changes in the expression of a gene of interest fused to a reporter gene. The vector uses Renilla luciferase as a major reporter gene, and the target fragment is cloned into a multiple cloning site downstream of the translation stop codon of Renilla luciferase. RNAi processes against the gene of interest, triggered by synthetic sirnas or shrnas expressed in vivo, result in cleavage and subsequent degradation of the fused mRNA. Whether the siRNA or shRNA expressed in vivo has a targeting relationship with the target fragment can be judged by detecting the change of the renilla luciferase activity. The vector has important significance in researching interaction experiments of microRNA and gene 3' UTR at present. There are two methods for constructing the psiCHECK2 vector, one is a conventional primer method, and the other is a homologous recombination method. At present, a conventional primer method is generally adopted, namely a protective base, an enzyme cutting site and a target fragment sequence are adopted, but the method can greatly reduce the double enzyme cutting efficiency of a PCR product due to the fact that the protective base is short, and therefore the experimental connection efficiency is low. The construction process of the homologous recombination method is relatively simple, much time and energy can be saved, but the false positive rate is slightly higher. Although commercial homologous recombination vector construction kits are available to reduce the false positive rate, they are expensive.

Disclosure of Invention

To overcome the deficiencies of the prior art, it is an object of the present invention to provide, but not limited to, a method for improving the efficiency of ligation of a desired fragment to the psiCHECK2 vector.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a method for improving the connection efficiency of a target fragment and a psiCHECK2 vector, which comprises the following steps: a bovine genome DNA is used as a template, a vector homologous sequence is designed to replace a protective base in a traditional primer, a target fragment is amplified by utilizing PCR, a PCR amplification product is connected with a psiCHECK2 vector and then is led into a competent cell, after 2 hours of air shaking culture at 37 ℃, a plate is coated, then the competent cell is cultured in a constant temperature incubator at 37 ℃ for 16 hours, the single clone is counted and randomly picked, bacteria liquid PCR and Sanger sequencing is carried out, and the vector recombination efficiency of the target fragment and the psiCHECK2 vector is judged.

The method of the invention uses cattle genome DNA as a template, replaces protective basic groups in conventional primers by using vector homologous sequences, and amplifies cattle by PCRIGFBP3And (3) gene partial fragments, treating the PCR product and psiCHECK2 by endonucleases Xho I and Not I, connecting by using T4 ligase, introducing the connecting product into DH5 alpha competent cells, and finally judging the recombination efficiency of the target fragment and psiCHECK2 vector according to the bacterial monoclonal number, agarose gel electrophoresis result and sequencing result.

The further optimized technical scheme of the invention is as follows:

preferably, the fragment of interest is bovineIGFBP3Partial fragment of gene located in cattleIGFBP3The nucleotide sequence of the gene candidate region chr4: 76123453-76124382 is shown in SEQ ID NO.1, and the nucleotide sequence of the psiCHECK2 vector is shown in SEQ ID NO. 2.

Preferably, the recombination efficiency of the vector is the connection efficiency of the target fragment and the psiCHECK2 vector according to the bacterial monoclonal number, the PCR positive rate of a bacterial liquid and the sequencing accuracy.

Preferably, the introduction vector homologous fragment primer pair P1 for amplifying the partial fragment of the bovine IGFBP3 gene is:

the upstream primer F1 (the nucleotide sequence of which is shown in SEQ ID NO. 3):

5′-TAGGCGATCGCTCGAGGCACAAAAGACTGCCAAGGACA -3′，

the downstream primer R1 (the nucleotide sequence of which is shown in SEQ ID NO. 4):

5′- TTGCGGCCAGCGGCCGCCACCAAGCAAGGGCGATT -3′；

the conventional amplification primer pair P2 is:

the upstream primer F2 (the nucleotide sequence of which is shown in SEQ ID NO. 5):

5′- CCGCTCGAGGCACAAAAGACTGCCAAGGA -3′，

the downstream primer R2 (the nucleotide sequence of which is shown in SEQ ID NO. 6):

5′- ATAAGAATGCGGCCGCCACCAAGCAAGGGCGATTTT -3′。

preferably, the vector homologous sequence in the vector homologous sequence primer is:

upstream primer vector homology sequence: TAGGCGATCG

Downstream primer vector homology sequence: TTGCGGCCA, respectively;

the conventional primer comprises the following protective basic groups:

the upstream primer protects the base: CCG

Protecting a base by a downstream primer: ataaagaat.

Preferably, the fragment size of the PCR amplification product based on the primer pair P1 is 963 bp, and the fragment size of the PCR amplification product based on the primer pair P2 is 955 bp.

Preferably, the PCR amplification product based on the primer pair P1 and the psiCHECK2 vector are treated by endonucleases Xho I and Not I, and are then introduced into DH5 alpha competent cells after being connected by T4 ligase, and the cells are marked as a homologous fragment experimental group.

PCR amplification products based on the primer pair P1 and psiCHECK2 vector treated by endonucleases Xho I and Not I are directly introduced into DH5 alpha competent cells and are marked as a bacterial endogenous homologous recombination test group.

PCR amplification products based on a primer pair P2 and a psiCHECK2 vector are treated by endonucleases Xho I and Not I, are connected by T4 ligase, and are introduced into DH5 alpha competent cells, and the cells are marked as a conventional primer test group.

Preferably, the amplification system used in the PCR comprises 1.0. mu.L of 50 ng/. mu.L template DNA, 1.0. mu.L of each of the upstream and downstream primers corresponding to 10 mM primer pair P1 or P2, 12.5. mu.L of 2 XMastermix (Sangon), and 9.5. mu.L of deionized water; the reaction procedure of the PCR is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30 s, annealing at 60 ℃ for 30 s, and extension at 72 ℃ for 60 s for 35 cycles.

The invention also provides application of the method in construction of the recombinant vector.

The application is that the bacterial monoclonal number, the bacterial liquid PCR positive rate and the sequencing accuracy of the homologous sequence primer recombinant vector are higher than those of a conventional primer recombinant vector and a bacterial endogenous homologous recombinant vector, namely the connection efficiency of a homologous fragment experimental group is higher than that of a conventional primer experimental group and a bacterial endogenous homologous recombination experimental group, and a technical basis can be provided for the construction of other vectors.

In the invention, cattle are usedIGFBP3The gene chr4: 76123453-76124382 is used as an amplification region. And respectively carrying out PCR amplification by utilizing two groups of primers, wherein the homologous sequence primer is a sequence of a vector homologous fragment + enzyme cutting site + target fragment, and the conventional primer is a sequence of a protective base + enzyme cutting site + target fragment. And introducing the amplified product into DH5 alpha after different treatments, wherein a homologous fragment experimental group is formed by connecting a double-enzyme digestion P1 PCR + double-enzyme digestion psiCHECK2 vector + T4, a bacterial endogenous homologous recombination experimental group is formed by connecting a P1 PCR + double-enzyme digestion psiCHECK2 vector, and a conventional primer experimental group is formed by connecting a double-enzyme digestion P2 PCR product with a double-enzyme digestion psiCHECK2 vector + T4. And (3) judging the recombination efficiency of the target fragment and the psiCHECK2 vector according to the bacterial monoclonal number, the bacterial liquid PCR positive rate and the sequencing accuracy result.

The invention is not based on the principle of homologous recombination, but improves the double enzyme cutting efficiency by changing the length of a protective basic group (the vector homologous sequence is adopted in the invention) to achieve the aim of improving the recombination efficiency. The cleavage efficiency of the current commercial endonuclease is affected by the number of protected bases. Based on the PCR product amplified by the traditional primer, the 2-hour enzyme digestion efficiency of Xho I is only 10%, and the 2-hour enzyme digestion efficiency of Not I is 25%. And the double enzyme cutting has a barrel effect, even if the efficiency of one enzyme cutting reaches 100%, if the efficiency of the other enzyme cutting is very low, the connection efficiency of the vector is also low. Compared with the connection efficiency of a conventional primer method and a homologous recombination method (non-kit), the method finds that the bacterial monoclonal number, the bacterial liquid PCR positive rate and the sequencing accuracy of the homologous sequence primer method are higher than those of other two methods.

Compared with the prior art, the invention has the following advantages:

(1) the connection efficiency of the homologous fragment experimental group is higher than that of a conventional primer experimental group and a bacterial endogenous homologous recombination experimental group;

(2) the method is accurate and reliable, simple to operate and low in cost;

(3) provides technical basis for the construction of other vectors.

Drawings

FIG. 1 is a graph showing the result of monoclonal image, PCR of bacterial liquid and sequencing of the homologous fragment experimental group of the present invention.

FIG. 2 is a graph showing the result of the single clone picture, the PCR of the bacterial liquid and the sequencing of the endogenous homologous recombination experimental group of bacteria.

FIG. 3 is a graph showing the result of PCR of bacteria liquid and monoclonal image of the conventional primer experimental group of the present invention.

Detailed Description

The technical scheme of the invention is further explained in detail by combining the embodiment as follows: the present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation and a specific operation process are given, but the protection authority of the present invention is not limited to the implementation described below. Experimental procedures, for which specific conditions are not noted in the following examples, were selected according to methods and conditions conventional in the art, or according to commercial instructions. The reagents and materials involved in the following examples are commercially available and are not listed here.

The PCR related to the invention takes bovine genome DNA as a template and cattleIGFBP3Gene candidate region chr76123453-76124382 is an amplification region, and two sets of primers are used for PCR amplification respectively, wherein the homologous sequence primer is designed as a vector homologous fragment + enzyme cutting site + target fragment sequence, and the conventional primer is a protective base + enzyme cutting site + target fragment sequence. And introducing the amplified product into DH5 alpha after different treatments, wherein a homologous fragment experimental group is formed by connecting a double-enzyme digestion P1 PCR + double-enzyme digestion psiCHECK2 vector + T4, a bacterial endogenous homologous recombination experimental group is formed by connecting a P1 PCR + double-enzyme digestion psiCHECK2 vector, and a conventional primer experimental group is formed by connecting a double-enzyme digestion P2 PCR product with a double-enzyme digestion psiCHECK2 vector + T4. And (3) judging the recombination efficiency of the target fragment and the psiCHECK2 vector according to the bacterial monoclonal number, the bacterial liquid PCR positive rate and the sequencing accuracy result.

Example 1

1. Bovine sample collection

A cattle is taken as a detection object, and 2 cattle jugular vein blood samples are collected from Shanxi Qinbao animal husbandry Limited company.

2. Separation, extraction and purification of genome DNA

Refer to Sambrock et al (2002) method.

3. Amplification of target and reference sequences

Cattle published in NCBI database (http:// www.ncbi.nlm.nih.gov /)IGFBP1The gene sequence is a reference sequence, and the Primer 5.0 is used for designing and amplifyingIGFBP3And (3) amplifying primers, and adding a restriction enzyme sequence and a protective base or a restriction enzyme sequence and a vector homologous fragment at the 5' end of the primers according to Xho I and Not I restriction enzyme sites. The primer pair sequence information is shown in Table 1.

TABLE 1 PCR primer information

The amplification system used for PCR was 25. mu.L: the amplification system used for PCR included 1.0. mu.L of 50 ng/. mu.L template DNA, 1.0. mu.L each of the upstream and downstream primers for 10 mM primer pair P1 or P2, 12.5. mu.L of 2 XMastermix (Sangon), and 9.5. mu.L of deionized water. .

The reaction procedure used to perform the PCR was: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30 s, annealing at 60 ℃ for 30 s, and extension at 72 ℃ for 60 s for 35 cycles.

4. Treatment of PCR products

The homologous fragment experimental group is that a PCR amplification product based on a P1 primer and a psiCHECK2 vector are treated by endonuclease Xho I and Not I, are connected by T4 ligase and are introduced into DH5 alpha competent cells.

The bacterial endogenous homologous recombination experimental group is that a PCR amplification product based on a P1 primer and a psiCHECK2 vector treated by endonucleases Xho I and Not I are directly introduced into DH5 alpha competent cells.

The conventional primer experimental group is based on that after the PCR amplification product of the P2 primer and the psiCHECK2 vector are treated by endonucleases Xho I and Not I and are connected by T4 ligase, DH5 alpha competent cells are introduced.

5. Carrier attachment efficiency determination

And respectively coating the DH5 alpha transformed by the homologous fragment experimental group, the bacterial endogenous homologous recombination experimental group and the conventional primer experimental group, counting the number of monoclonals of 3 plates in each group, and obtaining the results shown in the figures 1 to 3. And (4) judging the difference of the number of the monoclones in each group by using SPSS 18 software one-factor variance analysis.

Randomly picking 8 monoclonals from each plate, picking less than 8 monoclonals, carrying out PCR amplification on bacteria liquid by using respective corresponding primers P1 or P2, and counting the PCR positive rate of the bacteria liquid of three experimental groups, wherein the results are shown in figures 1 to 3. And (4) judging the difference of the PCR positive rate of each group of bacteria liquid by using SPSS 18 software chi-square test.

8 monoclonals are randomly picked again from each plate, less than 8 monoclonals are all picked, sequencing is carried out, and the sequence accuracy is determined, and the results are shown in the figures 1 to 3. The SPSS 18 software chi-square test is used to determine the difference in correctness of each set of connection sequences.

The analysis results are shown in table 2, the monoclonal number, the bacterial liquid PCR positive rate and the sequence correctness of the homologous fragment experimental group and the bacterial endogenous homologous recombination experimental group are all obviously higher than those of the conventional primer experimental group, and the sequence correctness of the homologous fragment experimental group is obviously higher than that of the bacterial endogenous homologous recombination experimental group, so that the construction power of the recombinant vector can be obviously improved.

TABLE 2IGFBP3Analysis of ligation efficiency of gene fragment and psiCHECK2 vector

6. Application of the above vector construction method in molecular biology

The method has the advantages of accuracy, reliability, simple operation and low cost, and can provide technical basis for the construction of other vectors.

The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can understand that the modifications or substitutions within the technical scope of the present invention are included in the scope of the present invention, and therefore, the scope of the present invention should be subject to the protection scope of the claims. .

Sequence listing

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acaccggcaa gaccctggga gtgaaccagc gcggcgagct gtgtgtgcgc ggccctatga 3720

ttatgtccgg ctacgtgaat aaccctgagg ccacaaacgc cctgatcgac aaggacggct 3780

ggctgcactc tggcgacatt gcctactggg acgaggacga gcacttcttc atcgtggacc 3840

gcctgaagtc tctgatcaag tacaagggct accaggtggc cccagccgag ctggagtcta 3900

tcctgctgca gcaccctaac attttcgacg ccggagtggc cggcctgccc gacgacgatg 3960

ccggcgagct gcctgccgcc gtcgtcgtgc tggaacacgg caagaccatg accgagaagg 4020

agatcgtgga ctatgtggcc agccaggtga caaccgccaa gaagctgcgc ggcggagtgg 4080

tgttcgtgga cgaggtgccc aagggcctga ccggcaagct ggacgcccgc aagatccgcg 4140

agatcctgat caaggctaag aaaggcggca agatcgccgt gtaataattc tagagtcggg 4200

gcggccggcc gcttcgagca gacatgataa gatacattga tgagtttgga caaaccacaa 4260

ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg 4320

taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc 4380

aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta 4440

aaatcgataa ggatccaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 4500

tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc 4560

ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc 4620

ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa 4680

aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg 4740

gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag 4800

ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc 4860

gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta 4920

cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg 4980

cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca 5040

acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac 5100

caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat 5160

taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg 5220

ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata 5280

aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta 5340

agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa 5400

atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag 5460

tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg 5520

tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact 5580

gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 5640

taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 5700

aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 5760

ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 5820

catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 5880

ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 5940

ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 6000

agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 6060

taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 6120

atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 6180

cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 6240

ccttttgctg gccttttgct cacatggctc gac 6273

<210> 3

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

taggcgatcg ctcgaggcac aaaagactgc caaggaca 38

<210> 4

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ttgcggccag cggccgccac caagcaaggg cgatt 35

<210> 5

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ccgctcgagg cacaaaagac tgccaagga 29

<210> 6

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ataagaatgc ggccgccacc aagcaagggc gatttt 36