Coding

Part:BBa_K2957095:Design

Designed by: Ye Cheng Zheng   Group: iGEM19_MIT   (2019-10-19)


NeonGreen-C5a


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 3730
    Illegal SpeI site found at 2842
    Illegal PstI site found at 223
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 3730
    Illegal SpeI site found at 2842
    Illegal PstI site found at 223
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 3730
    Illegal BglII site found at 1691
    Illegal BamHI site found at 239
    Illegal XhoI site found at 1807
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 3730
    Illegal SpeI site found at 2842
    Illegal PstI site found at 223
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 3730
    Illegal SpeI site found at 2842
    Illegal PstI site found at 223
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 3370
    Illegal SapI.rc site found at 3343


Design Notes

We used Modular Cloning Golden Gate Type II Assembly, so we had to consider how parts came together. We also used the natural secretion tag given in the part so we could use it to test chemokine secretion success by HEKs. We also had to consider which terminal the fluorescence/protein tag was added so as not to render the chemokine dysfunctional. See more on the MIT iGEM 2019 wiki: https://2019.igem.org/Team:MIT.


Source

This part was ordered from IDT/Twist Biosciences as a gBlock and then assembled into a plasmid. The sequence was taken from this site: https://www.uniprot.org/uniprot/P01031, and then codon-optimized using IDT's codon optimization tool. The NeonGreen tag was a part in the Weiss lab database.

References