CN114875098B - Kit for carrying out seamless assembly on multiple DNA fragments and assembly vector and application method thereof - Google Patents

Abstract

The present invention relates to the fields of biochemistry and molecular biology. The invention provides a kit for seamlessly assembling a plurality of DNA fragments and an assembly carrier, which comprises T5 exonuclease, DNA polymerase, single-stranded DNA binding protein and assembly buffer solution. The invention also provides an application method of the kit, wherein the application method not only has the advantages of the traditional seamless recombinant cloning technology, but also has very high recombination efficiency and low background rate when being applied to seamless cloning of a plurality of DNA fragments, and has the advantages of simple principle, convenient operation, high positive rate, low background, wide application range, low price and the like when realizing the in-vitro assembly of a plurality of DNA fragments.

Description

A kit for seamlessly assembling multiple DNA fragments and assembly vectors and its application method

technical field

The invention relates to the technical fields of biochemistry and molecular biology, in particular to a kit for seamlessly assembling DNA fragments.

Background technique

Molecular cloning technology, as an essential technology in modern molecular biology laboratories, has always been the basic method for gene identification, gene expression research and gene biological function analysis. With the in-depth study of non-coding sequences in the genome, More and more attention has been paid to the cloning and functional analysis of regulatory element sequences. Traditional molecular cloning is often based on the use of the same restriction endonuclease to digest the vector and the target fragment, so that the vector and the target fragment have the same sticky end or blunt end, and then use T4 DNA ligase to connect them in vitro to achieve different DNA fragments assembly. However, the enzymatic ligation method is not only inefficient and time-consuming, but also has disadvantages such as sequence dependence, sequence residue, and difficulty in achieving high-throughput operations. In addition, modern molecular laboratories occupy more and more mainstream methods based on single-strand annealing and splicing, which are not limited by restriction enzyme sites, including SLIC, Gibson Assembly, In-Fusion, Hot Fusion and TEDA, etc. .

Some of the above methods require patented recombinant enzymes, etc., resulting in high prices, and some methods require many types of proteins, which increases the cost of the experiment. Moreover, the assembly efficiency and correct rate of the above methods will decrease with the increase in the number of assembled fragments, and still have Optimized space.

Contents of the invention

The purpose of the present invention is to provide a kit and application method for seamlessly assembling multiple DNA fragments and assembly vectors. The technical scheme described in the present invention can optimize the seamless assembly of multiple DNA fragments, and can also achieve Very high recombination efficiency and low background rate.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The invention provides a kit for seamlessly assembling multiple DNA fragments and assembly vectors, the kit includes T5 exonuclease, DNA polymerase, single-stranded DNA binding protein and assembly buffer.

As preferably, the use concentration of the T5 exonuclease is 0.001-0.005U/μl; the use concentration of the DNA polymerase is 0.010-0.030U/μl; the use concentration of the single-stranded DNA binding protein is 0.001-0.001- 0.005 μg/μl.

Preferably, the assembly buffer is Pre-assembly buffer. In the present invention, T5 exonuclease, DNA polymerase and single-stranded DNA binding protein can be seamlessly assembled in vitro to obtain a circular target plasmid with a metastable structure; the 3'-5' of T5 exonuclease The exonuclease activity cuts the end of the DNA fragment to expose the single-stranded complementary homology arm region, and the short homologous sequence on the end anneals to make the vector and the inserted DNA fragment closely connected together to form a circular plasmid with a gap, which is transformed into E. coli Then rely on the cell's own repair mechanism to complete the gap repair of the plasmid; DNA polymerase repairs the missing DNA near the homology arm region along the 5'-3' to form a stable subplasmid structure containing the gap in vitro; single-stranded DNA-binding proteins can participate in maintaining the integrity and stability of ssDNA ends formed by exonuclease cleavage, greatly improving the assembly efficiency of multiple DNA fragments or DNA fragments with hairpin ends.

Preferably, both ends of the DNA fragment have homologous arm sequences that are homologous to adjacent DNA fragments, and the length of the homologous arm sequences is 9bp-50bp.

The present invention also provides an application method of the kit, the application method is: adding 30-50ng/μl linearized vector and multiple 20-100ng/μl DNA fragments into the seamless assembly system for assembly reaction.

Preferably, the seamless assembly system includes T5 exonuclease, DNA polymerase, single-stranded DNA binding protein, assembly buffer and water in the kit.

Preferably, when the linearized carrier is used in an amount of 1ng-300ng, the molar ratio between the linearized carrier and the DNA fragment is 1:1-1:10.

As a preference, the total reaction system of the assembly reaction includes the following components in 20 μl:

As a preference, the 2×seamless assembly system includes the following components in 400 μl:

Preferably, the procedure of the assembly reaction is: after incubating at 50±10°C for 0.1-1.0h, the temperature is lowered to 4°C.

By adopting the above technical scheme, the present invention has the following beneficial effects:

1. The kit provided by the technical solution of the present invention can realize the seamless cloning, assembly and connection of multiple DNA fragments. Compared with other DNA connection techniques, it is not limited by restriction enzyme sites, and does not need to add specific recombination sites to realize In the true sense of seamless cloning, it still has high assembly efficiency and correct rate in the case of multiple DNA fragment assembly.

2. The application of the single-stranded DNA binding protein in the present invention greatly improves the assembly efficiency of multiple DNA fragments or DNA fragments with hairpin ends, realizes efficient and accurate seamless cloning, and increases the success rate and application of DNA assembly prospect. Especially the assembly improvement effect involving more than 4 DNA fragments is more obvious.

3. The technical solution of the present invention is not only efficient and high-quality, but also cost-effective, simple in principle, convenient in operation and wide in application.

Description of drawings

Fig. 1 is the schematic diagram of the principle of TSP assembly technology of the present invention;

Fig. 2 is the plasmid map that embodiment 1 makes;

Figure 3 is the results related to the cloning process of TP and TSP methods (wherein A is the electrophoretic gel image of the 6 fragments selected by the plasmid vector; B is the number of corresponding clones formed by two-fragment, three-fragment and four-fragment assembly under TP and TSP methods respectively Schematic diagram; C is the electrophoretic gel map of the fragments formed by the corresponding clones assembled by two-fragment, three-fragment, and four-fragment respectively under TP and TSP methods);

Fig. 4 is the plasmid map that embodiment 2 makes;

Figure 5 is the results related to the cloning process of TEDA, TP, GA and TSP methods (where A is the electrophoretic gel image of the six fragments selected by the plasmid vector; B is the corresponding clone of the six fragments assembled under the TEDA, TP, GA and TSP methods respectively Schematic diagram of the formation number; C is a schematic diagram of the corresponding colony formation number under different treatment times such as 10 minutes, 20 minutes, 40 minutes and 60 minutes under the TSP method; D is the six-fragment assembly under the TEDA, TP, GA and TSP methods respectively Electrophoresis gel images of fragments formed by the corresponding clones).

Detailed ways

The invention provides a kit for seamlessly assembling multiple DNA fragments and assembly vectors, the kit includes T5 exonuclease, DNA polymerase, single-stranded DNA binding protein and assembly buffer.

In the present invention, the DNA polymerase is a common thermostable DNA polymerase with exonuclease activity, preferably Taq DNA polymerase with 5'-3' exonuclease activity or a 3'-5' Pfu DNA polymerase with exonuclease activity.

In the present invention, the single-stranded DNA binding protein preferably includes any of the extreme heat-resistant single-stranded DNA binding protein (ET-SSB protein), RecA, T4 gene 32 single-stranded DNA binding protein and E. coli single-stranded DNA binding protein. A sort of.

In the present invention, the use concentration of the T5 exonuclease is preferably 0.001-0.005U/μl, further preferably 0.004U/μl; the use concentration of the DNA polymerase is preferably 0.010-0.030U/μl, It is further preferably 0.025 U/μl; the concentration of the single-stranded DNA binding protein used is preferably 0.001-0.005 U/μl, further preferably 0.003 μg/μl.

In the present invention, the assembly buffer is Pre-assembly buffer, which specifically includes Tris-HCl with pH=7.5, 10mM MgCl ₂ , 10mM DTT, 0.2mM dNTP Mix and PEG8000 with a mass ratio of 5%. Any of 1-10mg/l NaCl, 0.1-10mg/l EDTA, 10-200g/l glycerin, 10-50mg/l bovine serum albumin, 0.1-10mg/l adenosine-5'-triphosphate.

The present invention also provides an application method of the kit, the application method is: adding 30-50ng/μl linearized vector and multiple 20-100ng/μl DNA fragments into the seamless assembly system for assembly Reaction; when there are preferably 2 DNA fragments, the concentration of the DNA fragments is preferably 30-100ng/μl; when the DNA fragments are preferably 3-4, the concentration of the DNA fragments is preferably 20-50ng/μl; when the DNA fragments are preferably When there are 6 DNA fragments, the concentration of DNA fragments is preferably 20-50 ng/μl.

In the present invention, both ends of the DNA fragment have homology arm sequences homologous to adjacent DNA fragments, and the length of the homology arm sequence is preferably 9bp-50bp, more preferably 20bp-30bp.

In the present invention, the seamless assembly system includes T5 exonuclease, DNA polymerase, single-stranded DNA binding protein, assembly buffer and water in the kit.

In the present invention, when the amount of the linearized carrier is 1ng-300ng, the molar ratio between the linearized carrier and the DNA fragment is 1:1-1:10, and a further preferred molar ratio is 1:2-1:4. Wherein, the amount of the linearized carrier is 1ng-300ng, more preferably 20ng-200ng, and still more preferably 40ng-120ng.

In the present invention, the total reaction system of the assembly reaction comprises the following components in terms of 20 μl:

In the present invention, the 2×seamless assembly system includes the following components in 400 μl:

In the present invention, the procedure of the assembly reaction is: after incubation at 50±10° C. for 0.1-1.0 h, the temperature is lowered to 4° C.

In the present invention, after the completion of the assembly reaction, it also includes the process of transforming the obtained assembly system into competent cells of Escherichia coli for verification; the competent cells preferably include DH5a, JM109, Top10, GB05, S17-1 , CC118, SM10 and any one of XL-1Blue MRF.

The technical solutions provided by the present invention will be described in detail below in conjunction with the examples, but they should not be interpreted as limiting the protection scope of the present invention.

Example 1: Application of TSP kit in two-fragment, three-fragment or four-fragment assembly

Taking the construction of pcDNA3.1-TSHR-EGFP expression vector as an example (Figure 2), the recombination efficiency test of different numbers of DNA fragments was carried out.

(1) Material preparation

TSP kit consists of assembly buffer, T5 exonuclease, DNA polymerase and single-strand binding protein;

Concentrate T5 exonuclease, DNA polymerase and single-strand binding protein at 2×, 3×, 4× or 5× concentrations, which are reserved for the subsequent addition of varying amounts of DNA;

The dosage of T5 exonuclease is 0.004U/total reaction system, the dosage of DNA polymerase is 0.025U/total reaction system, and the dosage of single-chain binding protein is 0.003U/total reaction system;

TSP buffer components are 100mM Tris-HCl with pH=7.5, 10mM MgCl ₂ , 10mM DTT, 0.2mM dNTPMix and PEG8000 with a mass ratio of 5%;

TSP assembly system includes TSP kit and water;

The above concentration conditions do not affect the assembly effect; the three enzymes and the reaction buffer in the kit can be pre-mixed or added later; the kit has been tested and stored in an environment between -20°C and -80°C. It does not affect the assembly efficiency, and it can be stored in this temperature range for more than 6 months without affecting the assembly effect.

(2) Design primers

Firstly, different primers are designed according to the vector sequence to amplify the plasmid into DNA fragments of different lengths, including two fragments, three fragments or four fragments. The length of the homologous arm sequences used for recombination between different DNA fragments ranges from 20bp to 30bp. The primers used in this step are as follows:

Frag-A-F1:TGGATAGCGGTTTGACTCAC

Frag-A-R1:GCTTCCTTTGCGGCGGCTTCTAGCTCAGTCTGCTTGCATT

Frag-B-F1:GAAGCCGCCGCAAAGGAAGCTG

Frag-B-R1: GTGAGTCAAAACCGCTATCCA

Frag-C-F1:GATCAGCCTCGACTGTGCCT

Frag-C-R1:GTGAGTCAAAACCGCTATCCA

Frag-D-F1:GAAGCCGCCGCAAAGGAAGCTG

Frag-D-R1:TAGAAGGCACAGTCGAGG

Frag-E-F1:GATCAGCCTCGACTGTGCCT

Frag-E-R1:AGGTAACTGGCTTCAGCAGA

Frag-F-F1:GTATCTGCGCTCTGCTGAAG

Frag-F-R1:GTGAGTCAAAACCGCTATCCA

In the connection of two fragments (A+B), use primers Frag-A-F1 and Frag-A-R1 to amplify fragment A, and use primers Frag-B-F1 and Frag-B-R1 to amplify fragment B;

In a three-fragment (A+C+D) ligation, use primers Frag-A-F1 and Frag-A-R1 to amplify Fragment A, and use primers Frag-C-F1 and Frag-C-R1 to amplify Fragment C , Fragment D was amplified using primers Frag-D-F1 and Frag-D-R1;

In a four-fragment (A+D+E+F) ligation, fragment A was amplified using primers Frag-A-F1 and Frag-A-R1, and fragment A was amplified using primers Frag-D-F1 and Frag-D-R1 Fragment D, Fragment E was amplified using primers Frag-E-F1 and Frag-E-R1, and Fragment F was amplified using primers Frag-F-F1 and Frag-F-R1.

(3) PCR amplification of assembled fragments

Reactions in a total volume of 50 μl contained 25 μl KOD One ^™ PCR MasterMix, 1.5 μl primer F1, 1.5 μl primer R1, 5 μl DNA template (plasmid DNA diluted to <1 μg/ml) and sterile water 17 μl.

The PCR cycle program is as follows: Step 1: 10 seconds at 98°C; Step 2: 10 seconds at 60°C; Step 3: 25 seconds at 68°C (5sec./kb); Step 4: Return to Step 1 29 times; the fifth step: 72°C for 5 minutes, take out the PCR product and store it at 4°C or use it for subsequent experiments. The PCR product is usually detected by electrophoresis on an agarose gel (1-2%), and then recovered by cutting the gel. The PCR product is recovered according to the method recommended in the instructions of the gel recovery kit.

(4) Assembly

For 2-fragment assembly, add 1 μl 40ng/μl linearized vector and 2 μl 60ng/μl PCR insert into a PCR microtube containing 10 μl 2X TSP assembly system, add sterile ultrapure water to the tube, Make up to a final volume of 20 μl. The reaction solution was incubated at 50° C. for 1 hour. After the reaction, it could be ice-bathed for 10 minutes to obtain the TSP assembly product (A in FIG. 3 ).

For 3-fragment assembly, use 1 μl (40ng/μl, try to adjust the molar ratio of different fragments to 1:1) for each insert, and the other operations are the same as above.

For 4-fragment assembly, use 1 μl (40ng/μl, try to adjust the molar ratio of different fragments to 1:1) for each insert, and the other operations are the same as above.

(5) Transformation Competent Cells

Take 5 μl of the obtained TSP assembly product and add it to DH5α competent cells. After adding LB, resume culture at 37°C and 250 rpm for 1 hour, then spread the transformation product on the LB plate containing the corresponding antibiotic, and culture at constant temperature (37°C) After 16 hours, the number of clones was counted (as shown in B in Figure 3).

(6) Screening of positive colonies

In addition, gene-specific primers (Frag-A-F1 and Frag-D-R1) were used to conduct bacteria screening PCR experiments to obtain the positive rate (C in Figure 3).

Positive rate = positive colonies / total colonies

In this example, in order to prove the importance and advantages of adding single-chain binding protein to the kit formula, a control group TP kit (ie Hot Fusion method, refer to Fu, Changlin, et al., PloS one, 2014) was set up , compared with the TSP method, the only difference is that the single-chain binding protein is missing in the kit, and other experimental conditions are exactly the same.

The results of this example are shown in Figure 3, and A in Figure 3 shows that DNA fragments of different sizes for assembly were successfully prepared in this example by PCR.

B in Figure 3 reveals that both the TP kit and the TSP kit can achieve the assembly of different numbers of fragments, and in the two-fragment assembly method, the number of clones obtained by TSP kit assembly is significantly higher than that of the TP group, and the number of clones increases by nearly 40%; in the three-fragment assembly method, the number of clones obtained by the TSP kit increased significantly by nearly 410%, which greatly improved the cloning efficiency; and in the four-fragment assembly, the number of clones obtained by the TSP kit also significantly increased by 420 %. The above results show that the optimized TSP kit has extremely significant advantages in multi-fragment assembly and can form more clones.

C in Figure 3 reveals part of the results of colony PCR using gene-specific primers. First, it reveals that there are a large number of positive clones in the clones assembled by the TP kit and the TSP kit, which can be used as a reliable DNA fragment assembly kit ; At the same time, in two-fragment and three-fragment assembly, the positive rate of the two kits is greater than 95%; in four-fragment recombination, the TSP kit still has a positive rate of more than 90%, while the positive rate of the TP kit is Nearly 70%, highlighting the high efficiency and accuracy of the TSP kit in multi-fragment assembly.

Example 2: Application of TSP kit in six-fragment assembly

In this embodiment, the construction of pcDNA3.1-TSHR-EGFP expression vector is still taken as an example ( FIG. 4 ), and the recombination application test of six-fragment assembly is carried out.

(1) Material preparation

TSP kit consists of assembly buffer, T5 exonuclease, DNA polymerase and single-strand binding protein;

Concentrate T5 exonuclease, DNA polymerase and single-strand binding protein at 2×, 3×, 4× or 5× concentrations, which are reserved for the subsequent addition of varying amounts of DNA;

The dosage of T5 exonuclease is 0.004U/total reaction system, the dosage of DNA polymerase is 0.025U/total reaction system, and the dosage of single-chain binding protein is 0.003U/total reaction system;

TSP buffer components are 100mM Tris-HCl with pH=7.5, 10mM MgCl ₂ , 10mM DTT, 0.2mM dNTPMix and PEG8000 with a mass ratio of 5%;

TSP assembly system includes TSP kit and water;

The above concentration conditions do not affect the assembly effect; the three enzymes and the reaction buffer in the kit can be pre-mixed or added later; the kit has been tested and stored in an environment between -20°C and -80°C. It does not affect the assembly efficiency, and it can be stored in this temperature range for more than 6 months without affecting the assembly effect.

(2) Design primers

First, different primers were designed according to the vector sequence to amplify the plasmid into six DNA fragments (Frag-I to Frag-VI) of different lengths. The lengths of the homology arm sequences used for recombination between different DNA fragments ranged from 20bp to 30bp. The primers used in this step are as follows:

Frag-I-F1:TGGATAGCGGTTTGACTCAC

Frag-Ⅰ-R1:GCTTCCTTTGCGGCGGCTTCTAGCTCAGTCTGCTTGCATT

Frag-Ⅱ-F1:GAAGCCGCCGCAAAGGAAGCTG

Frag-Ⅱ-R1:TAGAAGGCACAGTCGAGG

Frag-Ⅲ-F1:GATCAGCCTCGACTGTGCCT

Frag-Ⅲ-R1:CGATCCTCATCCTGTCTCTT

Frag-Ⅳ-F1: GGATCTGATCAAGAGACAGG

Frag-Ⅳ-R1: TTACCGCCTTTGAGTGAGCT

Frag-Ⅴ-F1:GAGCGGTATCAGCTCACTC

Frag-Ⅴ-R1: AGGTAACTGGCTTCAGCAGA

Frag-Ⅵ-F1: GTATCTGCGCTCTGCTGAAG

Frag-Ⅵ-R1:GTGAGTCAAACCGCTATCCA

(3) PCR amplification of assembled fragments

Reactions in a total volume of 50 μl contained 25 μl KOD One ^™ PCR MasterMix, 1.5 μl primer F1, 1.5 μl primer R1, 5 μl DNA template (plasmid DNA diluted to <1 μg/ml) and sterile water 17 μl.

The PCR cycle program is as follows: Step 1: 10 seconds at 98°C; Step 2: 10 seconds at 60°C; Step 3: 25 seconds at 68°C (5sec./kb); Step 4: Return to Step 1 29 times; the fifth step: 72°C for 5 minutes, take out the PCR product and store it at 4°C or use it for subsequent experiments. The PCR product is usually detected by electrophoresis on an agarose gel (1-2%), and then recovered by cutting the gel. The PCR product is recovered according to the method recommended in the instructions of the gel recovery kit.

(4) Assembly

Add 1 μl of 40ng/μl linearized vector and 2 μl of 40ng/μl PCR insert into a PCR microtube containing 10 μl of 2XTSP assembly system, and add sterile ultrapure water to the tube to make up the final volume to 20 μl. The reaction solution was incubated at 50° C. for 1 hour. After the reaction, it could be ice-bathed for 10 minutes to obtain the TSP assembly product (A in FIG. 5 ).

(5) Transformation Competent Cells

Take 5 μl of the obtained TSP assembly product and add it to DH5α competent cells. After adding LB, restore the culture at 37°C and 250rpm for 1 hour, then spread the transformation product on the LB plate containing the corresponding antibiotic, and culture at constant temperature (37°C) After 16 hours, the number of clones was counted (as shown in B in Figure 5).

(6) Screening of positive colonies

In addition, gene-specific primers (Frag-I-F1 and Frag-II-R1) were used to perform bacteria screening PCR experiments to obtain the positive rate (D in Figure 5).

Positive rate = positive colonies / total colonies

In this example, in order to prove the importance and advantages of adding single-chain binding proteins to the kit formula, multiple control groups were set up. First, the TEDA method was selected, referring to Xia, Yongzhen, et al. Nucleic acids research 47.3 (2019 ) method to prepare the reaction solution, the core of which is that the kit only uses 0.04U/total reaction system of T5 exonuclease, the reaction condition is 30°C for 30 minutes, and the other conditions are similar to the TSP method; secondly, the TP kit was selected , compared with the TSP method, the only difference is that the single-chain binding protein is missing in the kit, and other experimental conditions are exactly the same; in addition, the core of the purchased commercial GibsonAssembly cloning kit is that compared with the TSP kit, it lacks For single-strand binding proteins, a DNA ligase with a higher cost is added, and the remaining conditions are close to those of the TSP kit.

The results of this example are shown in Figure 5. The above four assembly methods can all realize the assembly of six fragments. According to the comparison of the average number of colony forming units, the TSP kit formed the largest number of clones, with an average number of about 1.65×10 ⁴ colonyforming units (CFU)/μg plasmaid, followed by the commercial Gibson Assembly kit, the average number is about 1.04×10 ⁴ CFU/μg plasmamid, and the third is the TP kit, the average number is about 0.32×10 ⁴ CFU/μg plasmamid, and the number of clones is the least The TEDA method is used, the average number is about 0.12×10 ⁴ CFU/μgplasmid, and the difference in the number of clones obtained by the four methods is significant. This result shows that this TSP kit has the best ability to form clones, and it is significantly better than other assembly methods at least in six-fragment recombination.

In order to further explore the optimal conditions of the TSP kit, its assembly ability was tested under different incubation times at 50°C. The results are shown in C in Figure 5, and a large number of recombinant clones can be obtained after 10 minutes of incubation , the average number is about 0.88×10 ⁴ CFU/μg plasmamid, and the highest number of clones is obtained after 20 minutes of incubation, the average number is about 2.33×10 ⁴ CFU/μg plasmamid, and a large number of clones can also be obtained at 40 minutes and 60 minutes , the average number is 1.51×10 ⁴ and 1.21×10 ⁴ CFU/μgplasmid, revealing that the incubation time of TSP can range from 10 minutes to 60 minutes, and 20 minutes of incubation has the optimal effect in this example.

The result (D in Fig. 5) of determining the positive rate of clones by colony PCR shows that in the formation of clones, the TSP kits incubated at different times all have the best positive clone rates, and their efficiencies are all greater than 95%; followed by the GibsonAssembly method , the positive rate is greater than 80%; while the positive rate of the clones obtained by the TP method and TEDA method is only about 50%, which is significantly lower than that of the TSP kit. The above results reveal that this TSP kit has the best six-fragment assembly efficiency and the highest clone positive rate; in addition, the TSP kit abandons the addition of high-cost DNA ligase on the basis of the innovative addition of single-strand binding proteins , significantly reducing the cost of using the kit.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

sequence listing

<110> Sichuan University

<120> A kit for seamlessly assembling multiple DNA fragments and assembly vectors and its application method

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<170> SIPOSequenceListing 1.0

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cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

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tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

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atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

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tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900

gtttaaactt aagcttggta ccgagctcgg ccaccatgct gtggctgcct gtcgccttcc 960

agttggtgct ggtgctgtgc agccagggca cggaaaggtg cccgtcagcc ttctgcgagt 1020

gctccgactg ggatgactac aaaattactt gcagggatat ccacttcatt cccagccttc 1080

cagaggacac tcagaccctg aggtttatgg agacccatct aagaacaatt cccagtgaag 1140

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ttggtatttt taatactgga ctcaaggtat ttcctgacct cactaaaatc tattcatctg 1380

atgtgaactt tttacttgaa attgcagata atcctttcat gacttcagtg cctgcaaatg 1440

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aatacctgaa agttatcaat gaagatgcat ttctgggagt gcatagtgga ccaactctac 1620

tggatatctc cagaacagtc attgttaatc ttcctgtcaa aggattggaa agccttaaag 1680

aactaatggc ccggaacaca tggactttaa agaaattacc agctgtaaag atatttctcc 1740

agctaattcg agctgatcta tcctatccaa gtcactgctg tgctttcaag aactggaaaa 1800

aaaacagtgg aattctggag tacttgatgt gtaaccagtc tggcagtcac agttctcgaa 1860

atagaagatc gttcagagct atcagaggac cattttacaa agattatgca gaagaataca 1920

cagagcgcac tgatactgtt tatgacaaaa acaccaagtt cacaaatttc catgaaaatt 1980

cccaatatta tatatttttg gaagagcatg gggaaggaaa tgttgggttt ggccaagagc 2040

tcaaaaaccc tcaaactgaa gacatgcagg cctttgacag tcatttatgac tatcttgtct 2100

gtggaggcag tgaggatgta atatgcactc cagagcctga tgagtttaat ccctgtgagg 2160

acataatggg ataccagttt ctgaggattg tggtatggtt tgtgaacctg ctggccatca 2220

taggtaatgt ttttgtcctt tttatccttc taaccagcca ttacaaacta actgtaccac 2280

gctttcttat gtgcaacctg gcctttgctg atttttgtat ggggttatac cttctcctga 2340

ttgcttcagt ggacctctac accagtcag agtactataa tcatgctata gagtggcaga 2400

cagggcctgg ttgcaacaca gcaggtttct tcacagtctt tgctagtgag ctttctgtgt 2460

acacacttac tgtgatcacc ctggagcgct ggtatgccat cacctttgcc atgcgtccca 2520

atcgaaagat tcgcctccgc catgctttgg ttatcatgct gggaggttgg ctttcatgct 2580

ttcttcttgc ccttttgcca ctggttagag tcagcagcta cagcaaagtc agcatctgct 2640

tacctatgga cactgaaaca ccagtggccg aagcctatgt tgtctttgtt ttaatatgta 2700

acattattgc ttttgtcatc atttgtgcct gctacataaa aatctatatc actgtacgaa 2760

accctcagta taagtcagga gacaaagaca ccaaaattgc caagaggatg gctgtgctga 2820

ttttcactga ctttctgtgc atggctccta tctctttcca tgctctatct gctatcatga 2880

acaaaccact gataacagtc actaattcca agattttgct ggtacttttc tacccactca 2940

actcttgcgc caacccattt ctatatgcta ttttcaccaa agctttccga agagatgtgt 3000

ttatcctgct aagcaagttt ggcatatgtg agcatcaggc tcaagtctac agaggtcaaa 3060

caatctcagt caaaaacagc agcggctctt acgggcagag aatcagccaa gggataggtc 3120

agattcttac aagcatccaa gacccagtca atgattacct tcctgctatg acaatgcaaa 3180

accaaattct tgcagaagaa tgcaagcaga ctgagctaga agccgccgca aaggaagctg 3240

ctgccaaaga agcagccgct aagatggtga gcaagggcga ggagctgttc accggggtgg 3300

tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg 3360

agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca 3420

agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca 3480

gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct 3540

acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg 3600

tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg 3660

aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata 3720

tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg 3780

aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc 3840

ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca 3900

acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg 3960

gcatggacga gctgtacaag taagaattct gcagatatcc agcacagtgg cggccgctcg 4020

agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc 4080

catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg 4140

tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc 4200

tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg 4260

ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg 4320

ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca 4380

gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct 4440

ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt 4500

tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattaggggt gatggttcac 4560

gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct 4620

ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt 4680

ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac 4740

aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc 4800

aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg 4860

tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc 4920

agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc 4980

ccattctccg ccccatggct gactaatttt ttttattatt gcagaggccg aggccgcctc 5040

tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa 5100

aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc 5160

gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag 5220

gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg 5280

gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa 5340

tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc 5400

agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc 5460

ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga 5520

tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa 5580

acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct 5640

ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat 5700

gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt 5760

ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta 5820

tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga 5880

ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg 5940

ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg 6000

cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc 6060

ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag 6120

ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc 6180

atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa 6240

ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa 6300

tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 6360

cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 6420

attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 6480

tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 6540

ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 6600

gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 6660

ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 6720

cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 6780

ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 6840

accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 6900

catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 6960

gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 7020

tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 7080

agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 7140

actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 7200

gttggtagct cttgatccgg caaacaaacc accgctggta gcggtttttt tgtttgcaag 7260

cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 7320

tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 7380

aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 7440

tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 7500

atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 7560

cgggaggggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 7620

gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 7680

gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 7740

tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 7800

tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 7860

tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 7920

aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 7980

atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 8040

tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 8100

catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 8160

aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 8220

tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 8280

gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 8340

tattattgaa gcatttatca gggttatgt ctcatgagcg gatacatatt tgaatgtatt 8400

tagaaaaata aacaaatagg ggttccgcgc aatttcccc gaaaagtgcc acctgacgtc 8460

Claims (8)

1. A kit for seamlessly assembling a plurality of DNA fragments and an assembly carrier, characterized in that, the kit includes T5 exonuclease, DNA polymerase, single-stranded DNA binding protein and assembly buffer; The use concentration of the T5 exonuclease is 0.001-0.005U/μl; the use concentration of the DNA polymerase is 0.010-0.030U/μl; the use concentration of the single-stranded DNA binding protein is 0.001-0.005U/μl µl; Both ends of the DNA fragment have homology arm sequences homologous to adjacent DNA fragments, and the length of the homology arm sequence is 9bp-50bp; The DNA polymerase includes Taq DNA polymerase with 5'-3' exonuclease activity or Pfu DNA polymerase with 3'-5' exonuclease activity. 2. The kit according to claim 1, wherein the assembly buffer is Pre-assemblybuffer. 3. An application method of the kit according to claim 1, characterized in that, the application method is: adding 30-50ng/μl linearized carrier and multiple 20-100ng/μl DNA fragments to the seamless assembly The assembly reaction is carried out in the system. 4. The application method according to claim 3, wherein the seamless assembly system comprises T5 exonuclease, DNA polymerase, single-stranded DNA binding protein, assembly buffer in the kit according to claim 1 liquid and water. 5. The application method according to claim 3, characterized in that, when the amount of the linearized carrier is 1ng-300ng, the molar ratio between the linearized carrier and the DNA fragment is 1:1-1:10. 6. The application method according to claim 3, wherein the total reaction system of the assembly reaction comprises the following components in 20 μl:

7. The application method according to claim 6, wherein the 2×seamless assembly system comprises the following components in 400 μl:

8 . The application method according to claim 3 , wherein the procedure of the assembly reaction is: after incubation at 50±10° C. for 0.1-1.0 h, the temperature is lowered to 4° C. 8 .

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