Plasmid

Part:BBa_K4231017:Design

Designed by: Shi Han   Group: iGEM22_USTC   (2022-09-30)


pUC19 carrying with ERG10, ERG13 and URA3 tag from Y.lipolytica


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 634
    Illegal PstI site found at 3745
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 634
    Illegal PstI site found at 3745
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2741
    Illegal BamHI site found at 255
    Illegal XhoI site found at 1391
    Illegal XhoI site found at 2737
    Illegal XhoI site found at 2779
    Illegal XhoI site found at 4247
    Illegal XhoI site found at 4280
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 634
    Illegal PstI site found at 3745
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 634
    Illegal PstI site found at 3745
    Illegal NgoMIV site found at 8
    Illegal NgoMIV site found at 889
    Illegal NgoMIV site found at 1295
    Illegal NgoMIV site found at 3187
    Illegal NgoMIV site found at 4435
    Illegal AgeI site found at 3673
    Illegal AgeI site found at 4538
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1415
    Illegal BsaI site found at 2140
    Illegal SapI site found at 1248


Design Notes

At the C-terminus of the ERG10 and ERG13 enzyme genes, we added the PTS1 sequence ( GGGSSKL ) to ensure that the enzyme protein can be localized in the peroxisome. When assembling the plasmid, we chose restricted endonuclease digestion and T4 DNA ligase treatment to successfully complete the construction.

Source

The coding sequences of ERG10 and ERG13 were amplified from Y.lipolytica strain po1f. The URA3 tag was amplified from Y.lipolytica strain W29

References