Generator

Part:BBa_K1179071:Design

Designed by: Nelson Hall   Group: iGEM13_MIT   (2013-09-18)


CMV_Acyl-TyA-eGFP


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 2062
    Illegal SpeI site found at 2037
    Illegal PstI site found at 1282
    Illegal PstI site found at 1531
    Illegal PstI site found at 1553
    Illegal PstI site found at 2067
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2062
    Illegal SpeI site found at 2037
    Illegal PstI site found at 1282
    Illegal PstI site found at 1531
    Illegal PstI site found at 1553
    Illegal PstI site found at 2067
    Illegal NotI site found at 2088
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2062
    Illegal BglII site found at 1382
    Illegal BamHI site found at 2031
    Illegal XhoI site found at 2095
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 2062
    Illegal SpeI site found at 2037
    Illegal PstI site found at 1282
    Illegal PstI site found at 1531
    Illegal PstI site found at 1553
    Illegal PstI site found at 2067
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 2062
    Illegal SpeI site found at 2037
    Illegal PstI site found at 1282
    Illegal PstI site found at 1531
    Illegal PstI site found at 1553
    Illegal PstI site found at 2067
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1215
    Illegal BsaI.rc site found at 1567
    Illegal BsaI.rc site found at 2020


Design Notes

It is recommended that people interested in using this part for mammalian cell transfection experiments use a transfection fluorescent marker to calculate the efficiency of the Acyl-TyA-eGFP because the green fluorescent proteins get exported from the cell. The transfection marker acts as the efficiency of the Acyl-TyA-eGFP assuming multiple plasmids are either all integrated into the same cell or not integrated at all.

Source

The eGFP is derived from the jellyfish Aequorea victoria. The TyA protein is a yeast viral protein and when added with the Acylation tag it acts as an exosome export protein, allowing the eGFP to be exported when fused to the TyA. We got the pcDNA_CMV_Acyl-TyA-eGFP part from the Addgene repository and created an entry vector by PCRing out the Acyl-TyA-eGFP and adding the L1 and L2 sites onto it. Then we performed a gateway reaction with a CMV promoter and the entry vector to create the CMV_Acyl-TyA-eGFP expression.

References