Part:BBa_K4035004:Design
Dimerization of the copper metallothionein 1 : CUP1-(AP)7-CUP1
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 90
Illegal PstI site found at 312 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 90
Illegal PstI site found at 312 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 90
Illegal PstI site found at 312 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 90
Illegal PstI site found at 312 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 306
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. In addition, the full sequence was codon optimized before being ordered to avoid loops formation during synthesis. The synthetized CUP1-linker-CUP1 sequence was then inserted in the pCTcon2-V5 plasmid that was already containing Aga2, V5 tag and the Gal1 promoter by Gibson assembly.
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 AP and was codon optimized for the yeast S. cerevisiae (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.