Part:BBa_K4687025
MAD7+recE/T+pEC+Ptrc:MADE/TEtrc
A recombinant plasmid with pEC-XK99E as the vector backbone, Ptrc 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 Ptrc 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 3901
Illegal NheI site found at 4659 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 667
Illegal BglII site found at 715
Illegal BglII site found at 1036
Illegal BglII site found at 2251
Illegal BglII site found at 2903
Illegal BglII site found at 3757
Illegal BglII site found at 5191 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 4279
Illegal NgoMIV site found at 4353
Illegal NgoMIV site found at 5855
Illegal AgeI site found at 1818 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 5704
Illegal SapI.rc site found at 5914
Illegal SapI.rc site found at 6887
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