Composite

Part:BBa_K4825059:Design

Designed by: Ye Siyi   Group: iGEM23_GreatBay-SCIE   (2023-10-12)


pTEF2-CDC9-LEU1-tSSA1-pPGK1-COX6-LEU2-tPGK1-pTDH3-COX4-LEU4^S547∆-tTDH1


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1572
    Illegal EcoRI site found at 2036
    Illegal EcoRI site found at 3326
    Illegal EcoRI site found at 4115
    Illegal EcoRI site found at 6579
    Illegal EcoRI site found at 7518
    Illegal XbaI site found at 1813
    Illegal XbaI site found at 8965
    Illegal XbaI site found at 9775
    Illegal XbaI site found at 9889
    Illegal SpeI site found at 9930
    Illegal PstI site found at 2502
    Illegal PstI site found at 3300
    Illegal PstI site found at 3795
    Illegal PstI site found at 4080
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1572
    Illegal EcoRI site found at 2036
    Illegal EcoRI site found at 3326
    Illegal EcoRI site found at 4115
    Illegal EcoRI site found at 6579
    Illegal EcoRI site found at 7518
    Illegal NheI site found at 9453
    Illegal SpeI site found at 9930
    Illegal PstI site found at 2502
    Illegal PstI site found at 3300
    Illegal PstI site found at 3795
    Illegal PstI site found at 4080
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1572
    Illegal EcoRI site found at 2036
    Illegal EcoRI site found at 3326
    Illegal EcoRI site found at 4115
    Illegal EcoRI site found at 6579
    Illegal EcoRI site found at 7518
    Illegal BglII site found at 6405
    Illegal BglII site found at 10660
    Illegal BamHI site found at 9606
    Illegal BamHI site found at 10572
    Illegal XhoI site found at 5048
    Illegal XhoI site found at 11251
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1572
    Illegal EcoRI site found at 2036
    Illegal EcoRI site found at 3326
    Illegal EcoRI site found at 4115
    Illegal EcoRI site found at 6579
    Illegal EcoRI site found at 7518
    Illegal XbaI site found at 1813
    Illegal XbaI site found at 8965
    Illegal XbaI site found at 9775
    Illegal XbaI site found at 9889
    Illegal SpeI site found at 9930
    Illegal PstI site found at 2502
    Illegal PstI site found at 3300
    Illegal PstI site found at 3795
    Illegal PstI site found at 4080
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1572
    Illegal EcoRI site found at 2036
    Illegal EcoRI site found at 3326
    Illegal EcoRI site found at 4115
    Illegal EcoRI site found at 6579
    Illegal EcoRI site found at 7518
    Illegal XbaI site found at 1813
    Illegal XbaI site found at 8965
    Illegal XbaI site found at 9775
    Illegal XbaI site found at 9889
    Illegal SpeI site found at 9930
    Illegal PstI site found at 2502
    Illegal PstI site found at 3300
    Illegal PstI site found at 3795
    Illegal PstI site found at 4080
    Illegal NgoMIV site found at 2843
    Illegal AgeI site found at 425
    Illegal AgeI site found at 7131
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 5034
    Illegal BsaI site found at 5303
    Illegal BsaI.rc site found at 5284
    Illegal BsaI.rc site found at 6415
    Illegal SapI site found at 4305
    Illegal SapI site found at 6865
    Illegal SapI.rc site found at 6849


Design Notes

we engineered LEU1, LEU2, and LEU4 with three different signal peptide, which all lead them into mitochondria. Therefore, the concentration of enzymes that involve in 2-ketoacid elongation would become much higher in mitochondria, so that KIV will be convert to KIC before encountering KDC. In the end, KIC would be secreted out into cytosol and convert to Isopentanol.


Source

Saccharomyces cerevisiae

References