Coding

Part:BBa_K4035004:Design

Designed by: Anissa Hammi   Group: iGEM21_EPFL   (2021-09-09)
Revision as of 06:10, 13 September 2021 by Hammi (Talk | contribs) (→‎Source)


Dimerization of the copper metallothionein 1 : CUP1-(AP)7-CUP1


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 90
    Illegal PstI site found at 312
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 90
    Illegal PstI site found at 312
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 90
    Illegal PstI site found at 312
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 90
    Illegal PstI site found at 312
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 306


Design Notes

The start and stop codon of the CUP1 genomic sequences were removed in order to fuse the proteins together with the linker, Aga2 and V5. In addition, the full sequence was codon optimized before being ordered in order to avoid loops formation during synthesis. The synthetized CUP1-linker-CUP1 sequence was then inserted in the pCTcon2_V5 plasmid that was already containing Aga2, V5 tag and the Gal1 promoter by Gibson assembly.


Source

The CUP1 sequence is the genomic sequence of the copper metallotionein 1 protein. The sequence of the linker comes from a reverse translation of the amino acid sequence AP and was codon optimized for the yeast S. cerevisiae (1).

References

(1) Chao, G.; Lau, W. L.; Hackel, B. J.; Sazinsky, S. L.; Lippow, S. M.; Wittrup, K. D. Isolating and Engineering Human Antibodies Using Yeast Surface Display. Nat Protoc 2006, 1 (2), 755–768. https://doi.org/10.1038/nprot.2006.94.