Part:BBa_K4035014:Design
Expression of the CUP1-GGGGSEAAAKGGGGS-CUP1 dimer on the extracellular membrane of S. cerevisiae
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1014
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 150
- 1000COMPATIBLE WITH RFC[1000]
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 to avoid loops during syntethis.
Source
The CUP1 sequence is the genomic sequence of the yeast copper metallothionein 1 protein. The linker sequence is the reverse transcription of the GGGGSEAAAKGGGGS amino acid sequence. The full CUP1-linker-CUP1 sequence was synthetized and inserted by Gibson Assembly in the pCTcon2-V5 plasmid (1) 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.