Composite

Part:BBa_K4035012:Design

Designed by: Anissa Hammi   Group: iGEM21_EPFL   (2021-09-13)
Revision as of 06:20, 13 September 2021 by Hammi (Talk | contribs) (→‎References)


Expression of the CUP1-(EAAAK)3-CUP1 dimer on the extracellular membrane of S. cerevisiae


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 150
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1194


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. After having designed the dimer sequence and before synthetizing it, the full sequence was codon optimized by the software of the company that synthetized it in order to avoid loops during syntethis.


Source

The CUP1 sequence is the genomic sequence of the copper metallotionein 1 protein. The linker sequence is the reverse transcription of the EAAAK amino acid sequence. The full CUP1-linker-CUP1 sequence was synthetized and inserted by Gibson Assembly in the pCTcon2_V5 plasmid containing Aga2, V5 tag and the Gal1 promoter (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.