Plasmid

Part:BBa_K4686061:Design

Designed by: Maia Weatherly   Group: iGEM23_Princeton   (2023-10-10)


pPB_Gal4UAS_tBFP_PGK_mCitrine


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 175
    Illegal EcoRI site found at 1658
    Illegal XbaI site found at 312
    Illegal XbaI site found at 1146
    Illegal XbaI site found at 5881
    Illegal SpeI site found at 307
    Illegal SpeI site found at 336
    Illegal SpeI site found at 1404
    Illegal SpeI site found at 3110
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 175
    Illegal EcoRI site found at 1658
    Illegal NheI site found at 360
    Illegal NheI site found at 6209
    Illegal SpeI site found at 307
    Illegal SpeI site found at 336
    Illegal SpeI site found at 1404
    Illegal SpeI site found at 3110
    Illegal NotI site found at 1097
    Illegal NotI site found at 3374
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 175
    Illegal EcoRI site found at 1658
    Illegal BglII site found at 6223
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 175
    Illegal EcoRI site found at 1658
    Illegal XbaI site found at 312
    Illegal XbaI site found at 1146
    Illegal XbaI site found at 5881
    Illegal SpeI site found at 307
    Illegal SpeI site found at 336
    Illegal SpeI site found at 1404
    Illegal SpeI site found at 3110
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 175
    Illegal EcoRI site found at 1658
    Illegal XbaI site found at 312
    Illegal XbaI site found at 1146
    Illegal XbaI site found at 5881
    Illegal SpeI site found at 307
    Illegal SpeI site found at 336
    Illegal SpeI site found at 1404
    Illegal SpeI site found at 3110
    Illegal NgoMIV site found at 2972
    Illegal AgeI site found at 1269
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 4550
    Illegal BsaI.rc site found at 1004
    Illegal SapI.rc site found at 5632


Design Notes

We designed this plasmid to isolate the Reporter gene from pHR_Gal4UAS_tBFP_PGK_mCitrine (a lentiviral vector) and put it into the piggybac vector of pBR70 in order to make the construct more safe to work with. We also had to consider two step PCR amplification to add overhangs to the PCR insert for Gibson assembly due to repeat regions on the ends where the primers bound.


Source

The primers used were designed by a member of our team.

pHR_Gal4UAS_tBFP_PGK_mCitrine was a gift from Kole Roybal (Addgene plasmid # 188386 ; http://n2t.net/addgene:188386 ; RRID:Addgene_188386)

pBR70 was given to us by a mentor. She assembled it using TfRΔEctoR1-mRFP-GFPnb was a gift from Jeanne Stachowiak (Addgene plasmid # 113555 ; http://n2t.net/addgene:113555 ; RRID:Addgene_113555) and the HeraBiolabs Piggybac vector (https://www.herabiolabs.com/piggybac-products/)

References