Composite

Part:BBa_K5159028

Designed by: Tianyue WU   Group: iGEM24_HKUST   (2024-10-01)

PEX10 Knockout Cassette

This part was designed with the 5’ and 3’ homologous arms of Yarrowia lipolytica PEX10 gene flanking a LEU2 selection marker expression cassette, which can be released by the Cre-loxP system after use. The construct enables part of the PEX10 to be replaced by the LEU2 expression cassette through homologous recombination (HR). The two homologous arms can locate the PEX10 gene in the genome of Yarrowia lipolytica and help perform the recombination to disable the PEX10 expression, leading to a disrupted peroxisome biogenesis and improved lipid accumulation in the cells. It is directly transformed as a linear fragment into Yarrowia lipolytica to perform the knockout.

As the unconventional yeast Yarrowia lipolytica shows a higher tendency of utilizing non-homologous end-joining (NHEJ) instead of HR for genomic integration, two 1000 bp homologous arm in length is necessary for the efficient knockout to occur.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1035
    Illegal PstI site found at 196
    Illegal PstI site found at 415
    Illegal PstI site found at 4250
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1035
    Illegal PstI site found at 196
    Illegal PstI site found at 415
    Illegal PstI site found at 4250
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1035
    Illegal BglII site found at 3267
    Illegal BamHI site found at 251
    Illegal BamHI site found at 4136
    Illegal XhoI site found at 1981
    Illegal XhoI site found at 2010
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1035
    Illegal PstI site found at 196
    Illegal PstI site found at 415
    Illegal PstI site found at 4250
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1035
    Illegal PstI site found at 196
    Illegal PstI site found at 415
    Illegal PstI site found at 4250
    Illegal NgoMIV site found at 1123
    Illegal AgeI site found at 2595
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1641
    Illegal BsaI site found at 1924
    Illegal BsaI.rc site found at 1839
    Illegal SapI site found at 2392


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