Part:BBa_K4687044:Design
MAD7+recE/T+pJYS1+PlacM:MADE/TJlacM-g5d3
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Illegal PstI site found at 15500 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 9481
Illegal EcoRI site found at 9724
Illegal EcoRI site found at 10600
Illegal EcoRI site found at 15996
Illegal XbaI site found at 8698
Illegal SpeI site found at 6464
Illegal SpeI site found at 14418
Illegal SpeI site found at 14797
Illegal PstI site found at 7654
Illegal PstI site found at 8944
Illegal PstI site found at 9211
Illegal PstI site found at 10405
Illegal PstI site found at 12574
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Design Notes
In the combination of CRISPR-MAD7 gene editing system and recE/T recombinase system, we improved the efficiency of gene editing after optimizing the system. We need to knock out crtEb, a downstream gene of lycopene in the terpenoid biosynthesis pathway. After cleaving the DNA gap by CRISPR-MAD7 nuclease, the donor DNA is repaired by homologous recombination to achieve the knockout of the targeted gene.
Source
CRISPR-MAD7 nuclease was identified in Eubacterium rectale. RecE/T is derived from Bacterial,Archaeal and Plant Plastid Product. Strong promoter PlacM based on the sacB gene of Bacillus subtilis to be synthesized artificially. The vector skeleton pJYS1 was derived from the constitutive expression of FnCpf1 and recT in the C.glutamitum. Donor DNA is derived from a continuous random sequence of 1000bp in length with homology effects at the front and end of the CRISPR-MAD7 nuclease binding to the PAM region.The main components in this recombinant plasmid are derived from the basic components: BBa_K4687000、BBa_K4687001、BBa_K4687002、BBa_K4687009、BBa_K4687014、BBa_K4687022