Multi-fragment connected positive clone screening system and construction method thereof
By constructing a positive clone screening system with multiple fragment ligation and utilizing linearized cloning vectors and promoter switches, the false positive clone rate was reduced, the efficiency of multiple fragment recombination was improved, the problem of high false positive clone rate in Gibson homologous recombination technology was solved, and efficient DNA storage was achieved.
Patent Information
- Application Number
- CN202411094559.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-10
- Publication Date
- 2026-02-10
AI Technical Summary
Existing Gibson homologous recombination technology has a high false positive cloning rate when assembling multiple short DNA fragments, which is difficult to reduce effectively, leading to problems with the accuracy and cost of DNA storage data.
A positive clone screening system with multiple fragment ligation was constructed. Using a linearized cloning vector, resistance gene and promoter switch, multiple short DNA fragments were ligated through homologous recombination, and false positive clones were removed by screening with double resistance genes.
It effectively reduced the false positive cloning rate, increased the positive rate of multi-fragment recombinant clones, reduced DNA storage costs, improved the accuracy of DNA storage data, and provided an efficient information storage method for distributed DNA array storage.
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Figure CN121495960A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and in particular to a highly efficient method for screening positive clones by linking two or more fragments. Background Technology
[0002] With the continuous improvement of molecular biology techniques, common techniques such as yeast homologous recombination, TPA ligation, and Golden Gate assembly are mostly used for the assembly of long DNA fragments. Gibson homologous recombination technology can effectively realize the distributed storage of multiple short DNA fragments. Based on the recombination of DNA fragments with 15-25 nt homologous sequences at the ends of linearized vectors, several ligation fragments can be cloned into any site of any linear vector.
[0003] When Gibson homologous recombination technology is used to assemble more than nine short DNA fragments, the false positive clone rate can reach 94.6%. Therefore, reducing the false positive clone rate is an urgent problem to be solved in the assembly of multiple short DNA fragments. Gibson homologous recombination technology assembles fragments by recognizing homologous sequences between them, making it less likely to lose individual fragments during multi-fragment assembly. The assembly results show that most fragments are completely assembled or completely unassembled, while a minority are partially assembled. To remove most of the completely unassembled false positive clones, this invention constructs a rapid and efficient screening system to improve the storage efficiency of distributed DNA arrays. The promoter is used as one of the movable fragments and is ligated into the pUC19K vector along with the assembled fragments, which can quickly remove false positive clones in KanR resistance screening. This invention successfully applies a rapid screening system to a distributed DNA movable-type storage system with nine movable fragments, reducing the false positive clone rate to below 5%, improving the accuracy of DNA storage data, reducing DNA storage costs, laying the foundation for subsequent DNA data reading, and providing a new and efficient screening method for the ligation of two or more short DNA fragments. Summary of the Invention
[0004] In view of this, the present invention provides a positive clone screening system with multiple fragment ligation, comprising: a linearized cloning vector (linearized pUC-19, etc.), a small target DNA fragment, an resistance gene, and a promoter switch for initiating the expression of the resistance gene.
[0005] In a specific embodiment of the present invention, the resistance gene is located at one end of the linearized vector and forms a complete DNA chain with the linearized vector.
[0006] In a specific embodiment of the present invention, the resistance gene is a KanR gene, a GmR gene, or a CmR gene.
[0007] In a specific embodiment of the present invention, the promoter switch for initiating the expression of the resistance gene is the pJ23119 promoter, the Pc promoter, or the cat promoter.
[0008] The present invention also provides a method for constructing a positive clone screening system with multiple fragment ligation, comprising the following steps: inserting a promoterless resistance gene that has different resistance from the cloning vector itself into the multiple cloning site of the cloning vector to construct a dual resistance gene vector, wherein after the vector is linearized, the promoterless resistance gene is located at one end of the linearized vector.
[0009] In a specific embodiment of the present invention, the assembled fragments contain 20-30 bp of overlapping sequences between the previous and next fragments, and the foremost and last fragments have 20-30 bp of overlapping sequences with the linearized dual-resistance cloning vector at the foremost and last ends, respectively.
[0010] In a specific embodiment of the present invention, the promoter switch of the resistance gene is assembled upstream of the promoterless resistance gene located at one end of the linear vector.
[0011] In a specific embodiment of the present invention, the length of the assembled target fragment is 50-100 bp, and the number of target fragments is 2-12.
[0012] In a specific embodiment of the present invention, the linearized vector, the target fragment, and the DNA fragment of the promoter switch are mixed in a certain proportion for homologous recombination ligation.
[0013] This invention provides a method for constructing a dual-resistance gene vector by inserting a promoterless resistance gene, different from the resistance of the cloning vector itself, into the multiple cloning site of the cloning vector, and then linearizing the vector so that the promoterless resistance gene is located at one end of the linearized vector. It also provides a method for assembling a target fragment, a dual-resistance vector, and a resistance gene promoter switch gene using homologous recombination, and using the dual-resistance gene for clone screening, thereby reducing the false positive rate and increasing the positive rate of multi-fragment recombination clones. Furthermore, it provides a method for efficient screening of ligated positive clones suitable for short DNA fragments, enabling efficient screening of ligated positive clones from multiple DNA fragments. Finally, it provides a method for constructing a DNA storage database suitable for distributed DNA mobile type storage, enabling efficient ligation of short DNA fragments and efficient, low-cost storage of DNA information. Attached Figure Description
[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.
[0015] Figure 1This is a schematic diagram of the construction of the pUC19K recombinant plasmid.
[0016] Figure 2 It is pUC19K P Schematic diagram of recombinant plasmid construction.
[0017] Figure 3 This verifies the effect of the pJ23119 promoter on KanR resistance;
[0018] Figure 4 This is a schematic diagram of the nine-fragment positive clone screening process;
[0019] Figure 5 This is a screening and verification of positive clones with double antibodies;
[0020] Figure 6 The positive clone rate of recombination ligation systems with different numbers of fragments; Detailed Implementation
[0021] The present invention will now be described in detail:
[0022] Example 1
[0023] AmpR and KanR dual antibody screening vector was constructed on pUC-19 using the 119 promoter:
[0024] Step 1: Construction of an efficient positive clone screening system:
[0025] 1.1 Preparation of vectors and fragments
[0026] The pUC-19 linear vector was obtained by PCR amplification using pUC-19 as a template.
[0027] The KanR gene was obtained by PCR using pET-28a as a template.
[0028] The pJ23119 promoter was obtained by PCR using pFS-91g as a template.
[0029] 1 μL DNA template, 1 μL upstream primer, 1 μL downstream primer, 2× Hieff Add 25 μL of Plus PCR MasterMix (With Dye) high-fidelity enzyme premix to a total PCR volume of 50 μL with ddH2O; pre-denature at 98℃ for 5 min, then denature at 98℃ for 10 s, anneal at 60℃ (according to primer settings) for 20 s, extend at 72℃ for 30 s / kb, cycle 30-35 times, and finally extend at 72℃ for 5 min.
[0030] The KanR gene, pJ23119 promoter, and pUC-19 linear vector were recovered by gel extraction.
[0031] 1.2 Construction of Recombinant Plasmids
[0032] The KanR gene fragment, the pUC-19 linearized fragment, and the 2x homologous recombinase were added to the recombinant pUC19K system.
[0033] In the recombination of pUC19K P The system was supplemented with the pJ23119 promoter, KanR gene fragment, pUC-19 linearized fragment, and 2x homologous recombinase.
[0034] The recombinant system was incubated in a 50°C metal bath for 1 hour, followed by an ice bath for 2 minutes, before further conversion and use.
[0035] pUC19K and pUC19K P Construction of plasmid recombination system, such as Figure 1 and Figure 2 As shown.
[0036] 1.3 Build and Verify
[0037] The recombined pUC19K and pUC19K have completed their connection. P The plasmids were transformed into E. coli DH5α competent cells, plated on Amp plates with a final concentration of 50 μg / mL, and incubated overnight at 37°C. Single clones were picked and sequenced for verification. The plasmids were also plated on Kan plates with a final concentration of 25 μg / mL and incubated overnight at 37°C to verify the resistance of the recombinant system.
[0038] The KanR gene serves as a selection marker, and the pJ23119 promoter acts as a switch regulating KanR gene function. Colony growth only occurs on double-antibody plates containing Amp and Kan when the pJ23119 promoter is present. The specific results of verifying the pJ23119 promoter's regulation of the KanR gene switch are as follows: Figure 3 As shown.
[0039] Step 2: Design and preparation of short DNA fragments:
[0040] 2.1 Design of DNA Short Fragment Complementation
[0041] The length of the homologous arm region between each fragment and the vector is 25 bp, the similarity between each overlap region should be low, and the GC content should be moderate (approximately 44%-50%). The homologous arms of the main parts of this invention are shown in Table 1.
[0042]
[0043]
[0044] 2.2 Preparation of short DNA fragments
[0045] Mix the following mixture: 2 μL of upstream primer (10 μM) and 2 μL of downstream primer (10 μM) of DNA fragment, and 2× Hieff Add 25 μL of Plus PCR Master Mix (With Dye) high-fidelity enzyme premix to a total PCR volume of 50 μL by adding ddH2O.
[0046] The reaction was performed according to the following procedure: 98℃ for 5 min, 98℃ for 10 s, 60℃ for 20 s, 72℃ for 20 s, and 72℃ for 5 min. 1 / 10 volume of pre-chilled 3M sodium acetate buffer was added to the annealed product and mixed by inversion; twice the volume of pre-chilled anhydrous ethanol was added and mixed by inversion, and the mixture was incubated at -20℃ for 30 min; the mixture was centrifuged at 12000 rpm for 15 min at 4℃, and the supernatant was gently removed using a pipette, then left exposed at room temperature for 5 min; 1 / 2 volume of pre-chilled 70% ethanol solution was added to an EP tube to resuspend the precipitate; the mixture was centrifuged at 12000 rpm for 5 min at 4℃; the supernatant was gently removed using a pipette, and after drying, an appropriate amount of sterile ddH2O was added to dissolve the precipitate; the concentration of the recovered short fragment was measured using an ultra-micro spectrophotometer and diluted to 60 ng / μL.
[0047] The promoter regulates KanR to play a role in antibiotic selection; the majority of positive clones were selected from double antibody plates, and PCR verification showed that approximately 83.3% of the clones were positive. Figure 4 .
[0048] Step 3: Construct a positive clone screening system with multiple fragment ligations
[0049] 3.1 Efficiency of multi-segment connection before filtering
[0050] Set a fragment number gradient of 5-10, and ligate the prepared DNA fragments into the pUC19 linear vector (without selection marker). Add 1 μL of each fragment and 5 μL of the vector (60 ng / μL), along with 2x homologous recombinase, and mix well. Incubate at 50°C in a metal bath for 1 h, briefly centrifuge, and place on ice for 5 min. Transfer 5 μL of the recombinant system to 50 μL of DH5α, incubate on ice for 30 min, heat shock at 42°C for 90 s, and incubate on ice for 2 min. Add 850 μL of LB medium and incubate at 37°C for 45 min. Spread 50 μL onto Kan medium (25 μg / mL) and incubate overnight at 37°C.
[0051] The positive clone rate was statistically analyzed in recombination ligation systems with different numbers of fragments.
[0052] 3.2 Efficiency of multi-segment connection after filtering
[0053] The pJ23119 promoter was used as the last fragment in the assembly process with the remaining DNA fragments. The DNA fragment number gradient was set to "n+1" (n = 4, 5, 6…9; "1" represents the fragment carrying the pJ23119 promoter). The ligation efficiency is shown in Table 2. Figure 6 As shown.
[0054] Table 2: Positive clone rate of recombination ligation systems with different fragment numbers
[0055]
[0056] Example 2
[0057] AmpR and GmR dual antibody screening vector was constructed on pUC-19 using the Pc promoter:
[0058] Step 1: Construction of an efficient positive clone screening system:
[0059] 1.1 Preparation of vectors and fragments
[0060] The pUC-19 linear vector was obtained by PCR amplification using pUC-19 as a template.
[0061] The GmR gene was obtained by PCR using pFS-91g as a template.
[0062] The Pc promoter was obtained by PCR using pFS-91g as a template.
[0063] 1 μL DNA template, 1 μL upstream primer, 1 μL downstream primer, 2× Hieff Add 25 μL of Plus PCR MasterMix (With Dye) high-fidelity enzyme premix to a total PCR volume of 50 μL with ddH2O; pre-denature at 98℃ for 5 min, then denature at 98℃ for 10 s, anneal at 60℃ (according to primer settings) for 20 s, extend at 72℃ for 30 s / kb, cycle 30-35 times, and finally extend at 72℃ for 5 min.
[0064] Gel recovery of GmR gene, Pc promoter and linear vector.
[0065] 1.2 Construction of Recombinant Plasmids
[0066] The GmR gene fragment, the pUC-19 linearized fragment, and the 2x homologous recombinase were added to the recombinant pUC19G system.
[0067] In the recombination of pUC19K Pc The Pc promoter, GmR gene fragment, pUC-19 linearization fragment, and 2x homologous recombinase were added to the system.
[0068] The recombinant system was incubated in a 50°C metal bath for 1 hour, followed by an ice bath for 2 minutes, before further conversion and use.
[0069] 1.3 Build and Verify
[0070] The recombined pUC19G and pUC19K will be connected. Pc The plasmids were transformed into E. coli DH5α competent cells, plated on Amp plates with a final concentration of 50 μg / mL, and incubated overnight at 37°C. Single clones were picked and sequenced for verification. The plasmids were also plated on Gm plates with a final concentration of 25 μg / mL and incubated overnight at 37°C to verify the resistance of the recombinant system.
[0071] The GmR gene serves as a selection marker, and the Pc promoter acts as a switch to regulate the function of the GmR gene. When the Pc promoter is present, colony growth only occurs on double antibody plates containing Amp and Gm, and the Pc promoter's regulation of the GmR gene switch is verified.
[0072] Step 2: Design and preparation of short DNA fragments:
[0073] 2.1 Design of DNA Short Fragment Complementation
[0074] The length of the homologous arm region between each fragment and the vector is 25 bp, the similarity between each overlap region should be low, and the GC content should be moderate (approximately 44%-50%).
[0075] 2.2 Preparation of short DNA fragments
[0076] Mix the following mixture: 2 μL of upstream primer (10 μM) and 2 μL of downstream primer (10 μM) of DNA fragment, and 2× Hieff Add 25 μL of Plus PCR Master Mix (With Dye) high-fidelity enzyme premix to a total PCR volume of 50 μL by adding ddH2O.
[0077] The reaction was performed according to the following procedure: 98℃ for 5 min, 98℃ for 10 s, 60℃ for 20 s, 72℃ for 20 s, and 72℃ for 5 min. 1 / 10 volume of pre-chilled 3M sodium acetate buffer was added to the annealed product and mixed by inversion; twice the volume of pre-chilled anhydrous ethanol was added and mixed by inversion, and the mixture was incubated at -20℃ for 30 min; the mixture was centrifuged at 12000 rpm for 15 min at 4℃, and the supernatant was gently removed using a pipette, then left exposed at room temperature for 5 min; 1 / 2 volume of pre-chilled 70% ethanol solution was added to an EP tube to resuspend the precipitate; the mixture was centrifuged at 12000 rpm for 5 min at 4℃; the supernatant was gently removed using a pipette, and after drying, an appropriate amount of sterile ddH2O was added to dissolve the precipitate; the concentration of the recovered short fragment was measured using an ultra-micro spectrophotometer and diluted to 60 ng / μL.
[0078] Step 3: Construct a positive clone screening system with multiple fragment ligations
[0079] 3.1 Efficiency of multi-segment connection before filtering
[0080] Set up a fragment number gradient of 5-10, and ligate the prepared DNA fragments into the pUC19 linear vector (without selection marker). Add 1 μL of each fragment and 5 μL of the vector (60 ng / μL), along with 2x homologous recombinase, and mix well. Incubate at 50°C for 1 h, briefly centrifuge, and place on ice for 5 min. Transfer 5 μL of the recombination system to 50 μL of DH5α, incubate on ice for 30 min, heat shock at 42°C for 90 s, and incubate on ice for 2 min. Add 850 μL of LB medium and incubate at 37°C for 45 min. Spread 50 μL onto Gm medium (25 μg / mL) and incubate overnight at 37°C. Calculate the positive clone rate under different fragment number recombination ligation systems.
[0081] 3.2 Efficiency of multi-segment connection after filtering
[0082] The Pc promoter is the last fragment assembled with the remaining DNA fragments, and the number of DNA fragments is set to a gradient of "n+1" (n = 4, 5, 6...9; "1" is the fragment carrying the Pc promoter).
[0083] Example 3
[0084] AmpR and CmR dual antibody screening vector was constructed on pUC-19 using the cat promoter:
[0085] Step 1: Construction of an efficient positive clone screening system:
[0086] 1.1 Preparation of vectors and fragments
[0087] The pUC-19 linear vector was obtained by PCR amplification using pUC-19 as a template.
[0088] The CmR gene was obtained by PCR using pSmartBAC-S as a template.
[0089] The cat promoter was obtained by PCR using pSmartBAC-S as a template.
[0090] 1 μL DNA template, 1 μL upstream primer, 1 μL downstream primer, 2× Hieff Add 25 μL of Plus PCR MasterMix (With Dye) high-fidelity enzyme premix to a total PCR volume of 50 μL with ddH2O; pre-denature at 98℃ for 5 min, then denature at 98℃ for 10 s, anneal at 60℃ (according to primer settings) for 20 s, extend at 72℃ for 30 s / kb, cycle 30-35 times, and finally extend at 72℃ for 5 min.
[0091] Gel recovery of CmR gene, cat promoter and linear vector.
[0092] 1.2 Construction of Recombinant Plasmids
[0093] The CmR gene fragment, the pUC-19 linearized fragment, and the 2x homologous recombinase were added to the recombinant pUC19C system.
[0094] In the recombination of pUC19K Pcat The system was supplemented with the cat promoter, CmR gene fragment, pUC-19 linearized fragment, and 2x homologous recombinase.
[0095] The recombinant system was incubated in a 50°C metal bath for 1 hour, followed by an ice bath for 2 minutes, before further conversion and use.
[0096] 1.3 Build and Verify
[0097] The recombined pUC19C and pUC19K were reconnected. Pcat The plasmids were transformed into E. coli DH5α competent cells, plated on Amp plates with a final concentration of 50 μg / mL, and incubated overnight at 37°C. Single clones were picked and sequenced for verification. The plasmids were also plated on Cm plates with a final concentration of 25 μg / mL and incubated overnight at 37°C to verify the resistance of the recombinant system.
[0098] The CmR gene serves as a selection marker, and the cat promoter acts as a switch to regulate the function of the CmR gene. When the cat promoter is present, colony growth only occurs on double antibody plates containing Amp and Cm, and the cat promoter's regulation of the CmR gene switch is verified.
[0099] Step 2: Design and preparation of short DNA fragments:
[0100] 2.1 Design of DNA Short Fragment Complementation
[0101] The length of the homologous arm region between each fragment and the vector is 25 bp, the similarity between each overlap region should be low, and the GC content should be moderate (approximately 44%-50%).
[0102] 2.2 Preparation of short DNA fragments
[0103] Mix the following mixture: 2 μL of upstream primer (10 μM) and 2 μL of downstream primer (10 μM) of DNA fragment, and 2× Hieff Add 25 μL of Plus PCR Master Mix (With Dye) high-fidelity enzyme premix to a total PCR volume of 50 μL with ddH2O; react according to the following program: 98℃ for 5 min, 98℃ for 10 s, 60℃ for 20 s, 72℃ for 20 s, and 72℃ for 5 min. Add 1 / 10 volume of pre-chilled 3M sodium acetate buffer to the annealed product and mix by inversion; add twice the volume of pre-chilled anhydrous ethanol, mix by inversion, and incubate at -20°C for 30 min; centrifuge at 12000 rpm for 15 min at 4°C, gently remove the supernatant with a pipette, and leave exposed at room temperature for 5 min; add 1 / 2 volume of pre-chilled 70% ethanol solution to an EP tube to resuspend the precipitate; centrifuge at 12000 rpm for 5 min at 4°C; gently remove the supernatant with a pipette, dry, and add an appropriate amount of sterile ddH2O to dissolve the precipitate; measure the concentration of the recovered short fragment using an ultra-micro spectrophotometer and dilute to 60 ng / μL.
[0104] Step 3: Construct a positive clone screening system with multiple fragment ligations
[0105] 3.1 Efficiency of multi-segment connection before filtering
[0106] Set up a fragment number gradient of 5-10, and ligate the prepared DNA fragments into the pUC19 linear vector (without selection marker). Add 1 μL of each fragment and 5 μL of the vector (60 ng / μL), along with 2x homologous recombinase, and mix well. Incubate at 50°C for 1 h, briefly centrifuge, and place on ice for 5 min. Transfer 5 μL of the recombination system to 50 μL of DH5α, incubate on ice for 30 min, heat shock at 42°C for 90 s, and incubate on ice for 2 min. Add 850 μL of LB medium and incubate at 37°C for 45 min. Spread 50 μL of the recombinant system onto Cm medium (25 μg / mL) and incubate overnight at 37°C. Calculate the positive clone rate under different fragment number recombination ligation systems.
[0107] 3.2 Efficiency of multi-segment connection after filtering
[0108] The cat promoter is used as the last fragment to be assembled with the remaining DNA fragments, and the number of DNA fragments is set to a gradient of "n+1" (n = 4, 5, 6...9; "1" is the fragment carrying the cat promoter).
[0109] This invention constructs a highly efficient double-antibody positive clone screening system, which can rapidly screen out 5-10 DNA fragment homologous recombination false positive clones. Comparing the positive clone rates before and after using the double-antibody screening system, when the number of ligated fragments exceeds 8, the false positive clone rate after double-antibody screening is less than 5%; with nine fragments, the positive clone rate exceeds 95%; and with ten fragments, the positive clone rate was 4.7% before screening and 94.4% after double-antibody screening, an improvement of 89.7% compared to before screening. The results are as follows... Figure 6 As shown.
[0110] Compared with traditional DNA ligation techniques, the positive clone screening method of this invention effectively solves the problem of excessive false-positive clones in multi-fragment DNA recombination. This invention prepares short double-stranded DNA fragments through single-stranded DNA annealing and extension, reducing DNA fragment preparation costs. It constructs a pUC19K double antibody screening system and establishes a promoter-based method for screening false-positive clones. This invention effectively improves the efficiency of multi-fragment ligation and significantly reduces the false-positive clone rate.
[0111] This invention addresses the problem of low efficiency in ligating short DNA fragments by providing a method for screening positive clones using multiple DNA fragment ligations, thereby improving the storage efficiency of distributed DNA arrays.
[0112] This invention successfully applies a rapid screening system to a distributed DNA type storage system with nine type fragments, improving the accuracy of DNA storage data, reducing DNA storage costs, laying the foundation for subsequent DNA data reading, and providing a new and efficient screening method for the ligation of two or more short DNA fragments.
Claims
1. A positive clone screening system with multi-fragment ligation, characterized in that, include: Linearized cloning vector, small target DNA fragment, resistance gene, and promoter switch that initiates resistance gene expression.
2. The positive clone screening system based on multi-fragment ligation according to claim 1, characterized in that, The resistance gene is located at one end of the linearized vector, forming a complete DNA strand with the linearized vector.
3. The positive clone screening system based on any one of claims 1 to 2, characterized in that, The resistance gene mentioned is the KanR gene, GmR gene, or CmR gene.
4. The positive clone screening system based on multi-fragment ligation according to claim 1, characterized in that, The promoter switch for initiating the expression of the resistance gene is the pJ23119 promoter, the Pc promoter, or the cat promoter.
5. A method for constructing a positive clone screening system with multi-fragment ligation according to any one of claims 1 to 4, characterized in that, Includes the following steps: A promoterless resistance gene, which has a different resistance from that of the cloning vector itself, is inserted into the multiple cloning site of the cloning vector to construct a dual resistance gene vector. After the vector is linearized, the promoterless resistance gene is located at one end of the linearized vector.
6. The method for constructing a positive clone screening system with multi-fragment ligation according to claim 5, characterized in that: Each assembled fragment contains a 20-30 bp overlap sequence between the preceding and following fragments, and the foremost and last fragments have a 20-30 bp overlap sequence with the linearized dual-resistance cloning vector at the foremost and last ends, respectively.
7. The method for constructing a positive clone screening system with multi-fragment ligation according to claim 6, characterized in that: The promoter switch of the resistance gene is assembled upstream of the promoterless resistance gene located at one end of the linear vector.
8. The method for constructing a positive clone screening system with multi-fragment ligation according to claim 7, characterized in that: The assembled target fragment is 50-100 bp in length, and the number of target fragments is 2-12. The linearized vector, target fragment, and promoter switch DNA fragment are mixed in a certain proportion for homologous recombination ligation.
9. The method for constructing a positive clone screening system with multi-fragment ligation according to claim 8, characterized in that: The DNA obtained after homologous recombination was transformed into E. coli competent cells for double antibiotic selection.
10. The application of the multi-fragment ligation positive clone screening system according to claims 1-4 in the multi-fragment assembly of DNA cloning and DNA storage.