Part:BBa_K4687027:Design
MAD7+recE/T+pEC+Ptac:MADE/TEtac
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 3914
Illegal NheI site found at 4672 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 672
Illegal BglII site found at 720
Illegal BglII site found at 1041
Illegal BglII site found at 2256
Illegal BglII site found at 2908
Illegal BglII site found at 3762
Illegal BglII site found at 5204 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 4292
Illegal NgoMIV site found at 4366
Illegal NgoMIV site found at 5868
Illegal AgeI site found at 1823 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 5717
Illegal SapI.rc site found at 5927
Illegal SapI.rc site found at 6900
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 Ptac 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.Ptac is formed by hybridizing a promoter of tryptophan manipulator origin with a promoter of Lac manipulator origin.The major components in this recombinant plasmid are derived from the base components:BBa_K4687000，BBa_K4687001，BBa_K4687004，BBa_K4687006