Plasmid

Part:BBa_K4686062:Design

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


pPB_antiGFP_SNIPR_IRES_mCherry


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 695
    Illegal EcoRI site found at 1096
    Illegal XbaI site found at 3391
    Illegal XbaI site found at 6860
    Illegal SpeI site found at 441
    Illegal SpeI site found at 4089
    Illegal PstI site found at 1646
    Illegal PstI site found at 1836
    Illegal PstI site found at 2975
    Illegal PstI site found at 3336
    Illegal PstI site found at 3403
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 695
    Illegal EcoRI site found at 1096
    Illegal NheI site found at 7188
    Illegal SpeI site found at 441
    Illegal SpeI site found at 4089
    Illegal PstI site found at 1646
    Illegal PstI site found at 1836
    Illegal PstI site found at 2975
    Illegal PstI site found at 3336
    Illegal PstI site found at 3403
    Illegal NotI site found at 3342
    Illegal NotI site found at 4353
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 695
    Illegal EcoRI site found at 1096
    Illegal BglII site found at 7202
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 695
    Illegal EcoRI site found at 1096
    Illegal XbaI site found at 3391
    Illegal XbaI site found at 6860
    Illegal SpeI site found at 441
    Illegal SpeI site found at 4089
    Illegal PstI site found at 1646
    Illegal PstI site found at 1836
    Illegal PstI site found at 2975
    Illegal PstI site found at 3336
    Illegal PstI site found at 3403
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 695
    Illegal EcoRI site found at 1096
    Illegal XbaI site found at 3391
    Illegal XbaI site found at 6860
    Illegal SpeI site found at 441
    Illegal SpeI site found at 4089
    Illegal PstI site found at 1646
    Illegal PstI site found at 1836
    Illegal PstI site found at 2975
    Illegal PstI site found at 3336
    Illegal PstI site found at 3403
    Illegal NgoMIV site found at 1431
    Illegal NgoMIV site found at 1566
    Illegal NgoMIV site found at 3951
    Illegal AgeI site found at 306
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 5529
    Illegal SapI.rc site found at 6611


Design Notes

We designed this plasmid to isolate the Reporter gene from pHR_PGK_SNIPR_Hinge Notch SNIPR (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_PGK_SNIPR_Hinge Notch SNIPR (LaG17) was a gift from Kole Roybal (Addgene plasmid # 188380 ; http://n2t.net/addgene:188380 ; RRID:Addgene_188380)

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