Composite

Part:BBa_K4687043:Design

Designed by: Yiming Jiang   Group: iGEM23_HBUT-China   (2023-10-07)


MAD7+recE/T+pJYS1+PlacM:MADE/TJlacM-g5d2


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 9481
    Illegal EcoRI site found at 9724
    Illegal EcoRI site found at 10600
    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
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 9481
    Illegal EcoRI site found at 9724
    Illegal EcoRI site found at 10600
    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
    Illegal NotI site found at 9811
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 9481
    Illegal EcoRI site found at 9724
    Illegal EcoRI site found at 10600
    Illegal BglII site found at 714
    Illegal BglII site found at 762
    Illegal BglII site found at 1083
    Illegal BglII site found at 2298
    Illegal BglII site found at 2950
    Illegal BglII site found at 3804
    Illegal BglII site found at 8507
    Illegal XhoI site found at 16814
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 9481
    Illegal EcoRI site found at 9724
    Illegal EcoRI site found at 10600
    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
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 9481
    Illegal EcoRI site found at 9724
    Illegal EcoRI site found at 10600
    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
    Illegal NgoMIV site found at 5134
    Illegal NgoMIV site found at 13112
    Illegal AgeI site found at 1865
    Illegal AgeI site found at 16740
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 15327
    Illegal BsaI.rc site found at 15970


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 crtR, 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.glutamicum. 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_K4687002、BBa_K4687009、BBa_K4687014、BBa_K4687021

References