Plasmid

Part:BBa_K4686062

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


pPB_antiGFP_SNIPR_IRES_mCherry

A piggybac vector that constitutively expresses an antiGFP SNIPR. SNIPRs are essentially SynNotch receptors. The specific one that we use releases Gal4 (a transcription factor) upon binding and exertion of a mechanical force on the receptor. Our design also has an IRES site linked to mCherry expression as a selective marker for transfected cells (they will have cytoplasmic mCherry). This receptor plasmid is intended to work with the reporter plasmid (part BBa_K4686061), which has a UAS site driving the expression of tBFP. UAS sites specifically only respond to Gal4.

This system allows us to select for cells that have been properly co-transfected with both plasmids by sorting for cells that have both mCherry and mCitrine. Using properly sorted cells, we can know if the SNIPR is bound with sufficient force by looking for expression of tBFP.

Uses primers BBa_K4686021, BBa_K4686022, BBa_K4686023, BBa_K4686024, BBa_K4686025, and BBa_K4686026 to amplify pHR_PGK_SNIPR_Hinge_Notch_SNIPR and pBR70. pHR_PGK_SNIPR_Hinge_Notch_SNIPR is from Kole Roybal, and pBR70 was given to us by a mentor.

This plasmid confers Carbenicillin resistance to the bacteria for selection while cloning. It was assembled via Gibson Assembly.

Sequence and Features


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


[edit]
Categories
Parameters
None