Part:BBa_K4687028:Design
MAD7+recE/T+pEC+Ptuf:MADE/TEtuf
- 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
Design Notes
Traditional gene editing methods are not very efficient in Corynebacterium glutamicum. In order to improve the efficiency of gene editing in Corynebacterium glutamicum, we tried to construct a new system that can be expressed in Corynebacterium glutamicum that can perform gene editing efficiently. Whereas CRISPR-MAD7 nuclease has been described to target a wider range of PAM sequences, i.e., 5'-YTTN-3', and exhibit high gene editing activity in microbial systems with small molecular weights, CRISPR-MAD7 can be used for a variety of gene editing. Therefore we wanted to improve the gene editing efficiency of CRISPR-MAD7 system in Corynebacterium glutamicum by optimizing its processing. Therefore we designed pEC-XK99E as the vector backbone to carry recE/T recombination system as well as MAD7 protein expressed under the action of Ptuf promoter to observe the gene editing efficiency in Corynebacterium glutamicum.
Source
CRISPR-MAD7 nuclease was identified in Eubacterium rectale. RecE/T is derived from Bacterial,Archaeal and Plant Plastid Product.pEC-XK99E is an E. coli-Corynebacterium glutamicum shuttle expression vector based on the medium-sized copyless plasmid pGA1.The Ptuf promoter is the natural promoter of Corynebacterium glutamicum.The major components in this recombinant plasmid are derived from the base components:BBa_K4687000，BBa_K4687001，BBa_K4687005，BBa_K4687006