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Expression vector for Gateway cloning system

A technology of expression vector and vector plasmid, applied in biological field

Inactive Publication Date: 2014-12-31
ANHUI AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0005] Vector systems compatible with Gateway technology have begun to be applied to the study of plant functional genomes, but a bottleneck problem in the application of these vector systems is how to simply, economically and efficiently construct PCR products or target DNA fragments from other sources onto entry vectors to obtain entry clone

Method used

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  • Expression vector for Gateway cloning system
  • Expression vector for Gateway cloning system
  • Expression vector for Gateway cloning system

Examples

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Embodiment Construction

[0024] In the following examples, the plasmids and enzymes used can be purchased from Biologics, and GatewayKit can be purchased from Invitrogen.

[0025] refer to Figure 2a-2f , the present invention obtains the Gateway clone expression vector through this process:

[0026] 1. Mix 50-100 ng of pART7 vector plasmid DNA with 0.5-1 μL of restriction endonuclease Not I, incubate at 37°C for 2 hours for digestion, and obtain an expression fragment.

[0027] 2. Digest 50-100ng of binary vector pART27 plasmid DNA with 0.5-1 μL restriction endonuclease Not I, and insert the fragment obtained in the above step 1 into the digested pART27 to construct pART27-35Sa. , the Cauliflower Mosaic Virus 35S promoter and the 3′ UTR of the octopine synthase gene were in the same direction as the reporter gene NPT II.

[0028] 3. Mix 50-100 ng of pART27-35Sa plasmid DNA with 0.5-1 μL of restriction endonuclease Sma I, and incubate at 37° C. for 2 hours for enzyme digestion.

[0029] 4. Insert t...

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Abstract

The invention discloses an expression vector for a Gateway cloning system. By connecting double-HA tag sequence fragment with a vector PART27-35Sa-GWB having a Gateway reading frame B fragment, and conducting conversion expression in Escherichia coli, the expression vector can be obtained. The expression vector obtained by the construction method can be used for functional analysis and protein expression of cloned target genes or target DNA fragments of other sources. And the method has the advantages of rapidity, flexibility, high efficiency, time saving, labor saving and the like, thus having important significance in gene function study.

Description

Technical field [0001] The invention discloses an expression vector used in Gateway cloning system and belongs to the field of biotechnology. Background technique [0002] Gateway technology is a new technology for gene cloning developed by Invitrogen. It is a fast and efficient large-scale cloning system. Compared with traditional methods, it has no dependence on vectors and hosts. [0003] The principle of Gateway technology is based on site-specific integration of phage DNA into the bacterial host genome. Under the action of phage and bacterial integration factors (INF, Int), site-specific recombination can occur between the attP site of the lambda phage and the attB site of the E. coli genome. The lambda phage DNA is integrated into the genomic DNA of E. coli, producing on both sides Two new sites: attL and attR. Gateway technology can conveniently and quickly clone one or more genes into any protein expression system, overcoming the shortcomings of the traditional vec...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/66
Inventor 高俊山吴楠巫飞飞孟艳蔡永萍林毅
Owner ANHUI AGRICULTURAL UNIVERSITY
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