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Recombinant pUC57 vector having kanamycin resistance, and constructing method thereof

A kanamycin and construction method technology, applied in the field of genetic engineering, to achieve the effect of wide use

Inactive Publication Date: 2013-12-18
GENSCRIPT NANJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the selectable marker on the pUCk57 vector is the ampicillin resistance gene, and there is no pUC plasmid with the Kanna resistance marker

Method used

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  • Recombinant pUC57 vector having kanamycin resistance, and constructing method thereof
  • Recombinant pUC57 vector having kanamycin resistance, and constructing method thereof
  • Recombinant pUC57 vector having kanamycin resistance, and constructing method thereof

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Embodiment 1

[0021] (1) pUC57 backbone cloning and purification

[0022] Add pUC-57 upstream primer pUC57-F (5'-TCGCGCGTTTCGGTGATGACGGTG-3', SEQ ID NO.2) and pUC-57 downstream primer pUC57-R (5'-TTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTG-3', SEQ ID NO. .3) Each 1ul (concentration is 10uM), pUC-57 (purchased from GenScript company) plasmid template 1ng, 10ul 10×PBO buffer, 1ul 10mM dNTP, 1ul PBO DNA polymerase, make up the reaction system to 100ul with sterile water . The reaction conditions are: 94°C denaturation for 2 minutes; 94°C for 10 seconds, 58°C for 10 seconds, 72°C for 1 minute, a total of 30 cycles; 72°C for 5 minutes.

[0023] The fragment with a length of 1640 bp was purified by 1.5% agarose gel electrophoresis, and the purification kit model AP-96-GX-1 was purchased from Axygen Company.

[0024] (2) Cloning and purification of kanamycin resistance gene

[0025] Add APH upstream primer APH-F (5'-AAATGAAGTTTTAAATCAAGCCCAATCTGAATAATGTTACAACC-3', SEQ ID NO.4) and APH downstream ...

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Abstract

The invention belongs to the genetic engineering field, and discloses a recombinant pUC57 vector having a kanamycin resistance, and a constructing method thereof. The recombinant pUC57 vector having a kanamycin resistance is characterized in that the sequence of the recombinant pUC57 vector having a kanamycin resistance is represented by SEQ ID NO.1. The recombinant pUC57 vector having a kanamycin resistance can be obtained through homologous recombination of a pUC57 vector skeleton obtained through cloning and a fragment containing kanamycin resistance gene.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a recombinant pUC57 vector with kanamycin resistance and a construction method thereof. Background technique [0002] Gene recombination is a commonly used research method in molecular biology, in which plasmid vectors are inevitably used. Plasmid is a small circular DNA molecule. As the most commonly used and simplest carrier in genetic engineering, it must include three parts: genetic marker gene, replication region, and target gene. Plasmids are found in all bacterial taxa and are DNA molecules that self-replicate independently of the bacterial extrachromosomal. In nature, plasmids appear when nutrients are sufficient, and they will change in terms of structure, size, replication mode, copy number of each bacterium, fecundity in different bacteria, and transferability between strains. Perhaps most important are changes in the characteristics carried by the plasmid. Most prok...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/65C12N15/64
Inventor 董亢陈文柱徐士军卢圣东王珠银
Owner GENSCRIPT NANJING
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