Coding

Part:BBa_K4035006:Design

Designed by: Anissa Hammi   Group: iGEM21_EPFL   (2021-09-10)
Revision as of 20:18, 21 October 2021 by Hammi (Talk | contribs)


Dimerization of the copper metallothionein 1 : CUP1-(EAAAK)4-CUP1


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 408
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 408
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 408
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 408
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The start and stop codon of the CUP1 genomic sequences were removed in order to fuse the proteins together. 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 by Gibson Assembly in the pCTcon2-V5 plasmid (1) containing Aga2, V5 tag and the Gal1 promoter.


Source

The CUP1 sequence is the genomic sequence of the copper metallothionein 1 protein. The sequence of the linker comes from a reverse translation of the amino acid sequence EAAAK and was codon optimized for the yeast S. cerevisiae. The fusion protein was fully synthetized.

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.

(2) https://www.uniprot.org/uniprot/P0CX80