Part:BBa_K4687026
MAD7+recE/T+pEC+PlacM:MADE/TElacM
A recombinant plasmid with pEC-XK99E as the vector backbone, PlacM as the promoter of CRISPR-MAD7 and recE/T recombination system was constructed by using PCR and one-step cloning method. Under the action of PlacM promoter, CRISPR-MAD7 gene is expressed, and under the action of recE/T recombination system it can help the clipped gene to repair by homologous end recombination. Under the action of recE/T system can improve the editing efficiency of CRISPR-MAD7 system in Corynebacterium glutamicum.In the experimental process, we firstly transferred the constructed recombinant plasmid into E. coli by chemical transformation method to initially verify whether the recombinant plasmid is correct or not and amplify the recombinant plasmid. After extracting the recombinant plasmid from E. coli, the recombinant plasmid was transferred into Corynebacterium glutamicum by electroporation. This allowed the CRISPR-MAD7 system to be expressed in Corynebacterium glutamicum and gene editing to be performed.The results were observed and analyzed to determine whether the recombinant plasmid could be efficiently used in Corynebacterium glutamicum.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 3956
Illegal NheI site found at 4714 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 714
Illegal BglII site found at 762
Illegal BglII site found at 1083
Illegal BglII site found at 2298
Illegal BglII site found at 2950
Illegal BglII site found at 3804
Illegal BglII site found at 5246 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 4334
Illegal NgoMIV site found at 4408
Illegal NgoMIV site found at 5910
Illegal AgeI site found at 1865 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 5759
Illegal SapI.rc site found at 5969
Illegal SapI.rc site found at 6942
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