Resistance expression cassette for efficiently constructing recombinant mycobacterium without resistance markers
A technology without resistance marker and expression cassette
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Embodiment 1
[0035] Example 1 Construction of plasmid pUCDHmKE
[0036] The schematic diagram of the structure of plasmid pUCDHmKE is shown in figure 1 , see restriction site map figure 2 , which contains the above dif- ΩHYG -dif Resistance expression cassette. Depend on figure 1 It can be seen that the plasmid pUCDHmKE contains in a clockwise direction: Escherichia coli origin of replication (pMB1 ori), dif- ΩHYG -dif Resistance expression cassette, promoter P (BLA) (for promoting the ampicillin resistance gene Amp ( which is bla) , ampicillin resistance gene Amps Can be used for plasmid screening.
[0037] said dif- ΩHYG -dif The resistance expression cassette contains a simplified modified hygromycin resistance gene ( Hyg ), as described in SEQ ID NO:1, the 24th to 73bp on the gene is the promoter sequence (Pr), which is used to start Hyg gene expression. Hyg DNA sequences joined at both ends dif The sequences are shown in SEQ ID NO:7 and SEQ ID NO:8.
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Embodiment 2
[0049] Example 2 Construction and Application of Integrated Plasmid pMH94DHmKE
[0050] The schematic diagram of the structure of the integrated plasmid pMH94DHmKE is shown in Figure 4 , the plasmid pMH94DHmKE contains in a clockwise direction: LacZ promoter (can be used for blue and white screening, and to promote the expression of the entire plasmid backbone behind), phage integration site attP , integrase gene Int (can express integrase, allowing plasmids to pass through attP sites integrated into the mycobacterial genome), dif- ΩHYG -dif Resistance expression cassette, E. coli origin of replication ( oriE ), the ampicillin resistance gene Amps (for plasmid screening). dif- ΩHYG -dif The two ends of the resistance expression cassette were connected sequentially Hin dIII restriction site, xho I restriction site, can be used to cut out the dif- ΩHYG -dif Resistance expression cassette.
[0051] For the construction procedure of the integrated plasm...
Embodiment 3
[0058] Example 3 Construction and application of integrated plasmid pblDHCiGn
[0059] In the integrated plasmid pblDHCiGn, the resistance expression cassette exists in the form of double restriction sites on both sides. The construction of this plasmid is to prove that the resistance expression cassette is still effective in this form. Plasmid structure see Image 6 , in a clockwise direction, which in turn contains: promoter P(BLA) (used to start the following Amps expression), ampicillin resistance gene Amps (can be used for plasmid screening), E. coli origin of replication ( OriE ), phage integration site ( attP ), integrase gene Int (can express integrase, allowing plasmids to pass through attP integrated into the mycobacterial genome), dif- ΩHYG -dif Resistance expression cassette, enhanced green fluorescent protein gene eGFP . dif- ΩHYG -dif The two ends of the resistance expression cassette are connected with different enzyme cutting sites.
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