Coding

Part:BBa_K4035002:Design

Designed by: Anissa Hammi   Group: iGEM21_EPFL   (2021-08-31)
Revision as of 19:14, 21 October 2021 by Hammi (Talk | contribs) (→‎Design Notes)


Dimerization of the copper metallothionein 1 : CUP1-(GGGGS)3-CUP1


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 190
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The sequence has been cloned using Gibson Assembly. The start and stop codon of the CUP1 genomic sequences were removed in order to fuse the proteins together with the linker.

Source

The CUP1 sequence is the genomic sequence of the copper metallotionein 1 protein. The linker sequence is the reverse transcription of the GGGGS amino acid sequence. After having linked two copies with a linker in between, the full sequence was codon optimized by the software of the company that synthetized the sequence in order to avoid loops during syntethis. The CUP1-linker-CUP1 sequence was then inserted by Gibson Assembly in the pCTcon2_V5 plasmid containing Aga2, V5 tag and the Gal1 promoter.

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.