Composite

Part:BBa_K4687036

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


MAD7+recE/T+pJYS1+PlacM:MADE/TJlacM-g5

A recombinant plasmid with pJYS1 as the vector backbone, PlacM as the promoter of MAD7, a recE/T recombination system and a gRNA synthesized after a thought-designed design was constructed by utilizing the PCR method as well as a one-step cloning method. Under the action of PlacM promoter, CRISPR-MAD7 gene is expressed, and under the guidance of gRNA, CRISPR-MAD7 can be accurately localized on the target gene for gene editing, and under the action of recE/T recombination system it can help to repair the sheared gene by homologous end recombination. In this study, we combined the recE/T and CRISPR-MAD7 systems. We optimized the MAD7 promoter sequence and selected pJYS1 as the vector backbone for improving the editing efficiency of CRISPR-MAD7 system in Corynebacterium glutamicum.In this study we transferred the constructed recombinant plasmid into E. coli by chemical transformation method to verify the accuracy of the recombinant plasmid. Afterwards, the recombinant plasmid was transferred into Corynebacterium glutamicum by electroporation to observe the colony growth.

Sequence and Features


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
  • 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
  • 1000
    COMPATIBLE WITH RFC[1000]


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