Plasmid

Part:BBa_K4231019:Design

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


pUC19 carrying with ERG12, ERG20, BbTps3 and LEU tag from Y.lipolytica


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 6096
    Illegal EcoRI site found at 6146
    Illegal EcoRI site found at 7108
    Illegal XbaI site found at 5583
    Illegal SpeI site found at 1468
    Illegal PstI site found at 1285
    Illegal PstI site found at 2071
    Illegal PstI site found at 2826
    Illegal PstI site found at 3406
    Illegal PstI site found at 3796
    Illegal PstI site found at 5209
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 6096
    Illegal EcoRI site found at 6146
    Illegal EcoRI site found at 7108
    Illegal SpeI site found at 1468
    Illegal PstI site found at 1285
    Illegal PstI site found at 2071
    Illegal PstI site found at 2826
    Illegal PstI site found at 3406
    Illegal PstI site found at 3796
    Illegal PstI site found at 5209
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 6096
    Illegal EcoRI site found at 6146
    Illegal EcoRI site found at 7108
    Illegal BglII site found at 812
    Illegal BglII site found at 944
    Illegal BglII site found at 8377
    Illegal BamHI site found at 2447
    Illegal BamHI site found at 5528
    Illegal XhoI site found at 1073
    Illegal XhoI site found at 1229
    Illegal XhoI site found at 3979
    Illegal XhoI site found at 7091
    Illegal XhoI site found at 7120
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 6096
    Illegal EcoRI site found at 6146
    Illegal EcoRI site found at 7108
    Illegal XbaI site found at 5583
    Illegal SpeI site found at 1468
    Illegal PstI site found at 1285
    Illegal PstI site found at 2071
    Illegal PstI site found at 2826
    Illegal PstI site found at 3406
    Illegal PstI site found at 3796
    Illegal PstI site found at 5209
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 6096
    Illegal EcoRI site found at 6146
    Illegal EcoRI site found at 7108
    Illegal XbaI site found at 5583
    Illegal SpeI site found at 1468
    Illegal PstI site found at 1285
    Illegal PstI site found at 2071
    Illegal PstI site found at 2826
    Illegal PstI site found at 3406
    Illegal PstI site found at 3796
    Illegal PstI site found at 5209
    Illegal NgoMIV site found at 2200
    Illegal NgoMIV site found at 3081
    Illegal NgoMIV site found at 3772
    Illegal NgoMIV site found at 6234
    Illegal AgeI site found at 3680
    Illegal AgeI site found at 7705
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1966
    Illegal BsaI.rc site found at 3872
    Illegal BsaI.rc site found at 5826
    Illegal SapI site found at 5179


Design Notes

At the C-terminus of the BbTps3, ERG12 and ERG20 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 In-Fusion® Snap Assembly Master Mix to successfully complete the construction.


Source

The coding sequences of ERG12, ERG20, Leu tag were amplified from Y.lipolytica.The ORF of BbTps3 was found in NCBI and was synthesized according to the sequence.

References