Part:BBa_K4687025:Design
MAD7+recE/T+pEC+Ptrc:MADE/TEtrc
- 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
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 would like 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 CRISPR-MAD7 protein expressed under the action of Ptrc 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.Ptrc is derived from the strong promoter of E. coli; Trp hybridizes to the lac UV 5 promoter.The major components in this recombinant plasmid are derived from the base components:BBa_K4687000，BBa_K4687003，BBa_K4687006