Part:BBa_K4687028
MAD7+recE/T+pEC+Ptuf:MADE/TEtuf
A recombinant plasmid with pEC-XK99E as the vector backbone, Ptuf 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 Ptuf 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 allows the CRISPR-MAD7 system to be expressed in Corynebacterium glutamicum and to perform gene editing.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 3919
Illegal NheI site found at 4677 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 677
Illegal BglII site found at 725
Illegal BglII site found at 1046
Illegal BglII site found at 2261
Illegal BglII site found at 2913
Illegal BglII site found at 3767
Illegal BglII site found at 5209 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 4297
Illegal NgoMIV site found at 4371
Illegal NgoMIV site found at 5873
Illegal AgeI site found at 1828 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 5722
Illegal SapI.rc site found at 5932
Illegal SapI.rc site found at 6905
| None |