Part:BBa_K4035003:Design
Dimerization of the copper metallothionein 1 : CUP1-(GGGGS)4-CUP1
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 90
Illegal PstI site found at 408 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 90
Illegal PstI site found at 408 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 235
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 90
Illegal PstI site found at 408 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 90
Illegal PstI site found at 408 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 84
Design Notes
The start and stop codon of the CUP1 genomic sequences were removed in order to fuse the proteins together with the linker. In addition, the full sequence was codon optimized before being ordered in order to avoid loops formation during synthesis.
Source
The CUP1 sequence is the genomic sequence of the yeast copper metallotionein 1 protein (2). The sequence of the linker comes from a reverse translation of the amino acid sequence GGGGS and codon optimized for yeast S. cerevisiae. The CUP1-linker-CUP1 sequence was then 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.