Composite

Part:BBa_K4687031:Design

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


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


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]


Design Notes

For gene editing using CRISPR technology, we need to transfect the plasmid into cells, allow it to express the CRISPR system and edit the target gene. To improve the efficiency of gene editing, we optimized the CRISPR system to determine the optimal CRISPR-Cas system.The CRISPR-MAD7 system initiates the genome editing process by creating targeted DNA breaks, while the recE/T system ensures the accurate and efficient integration of foreign DNA sequences into the bacterial genome at those breaks. And we have experimentally determined the optimal promoter PlacM.On this basis, we changed the vector skeleton to pJYS1 to obtain the new recombinant plasmid, introduced it into Corynebacterium glutamicum, and observed the colony growth to determine the optimal plasmid skeleton.


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. The main components in this recombinant plasmid are derived from the basic components: BBa_K4687000、BBa_K4687002、BBa_K4687009

References