Difference between revisions of "Part:BBa K4035007:Design"

 
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===Design Notes===
 
===Design Notes===
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The start and stop codon of the CUP1 genomic sequences were removed in order to fuse the proteins together. In addition, the full sequence was codon optimized before being ordered in order to avoid loops formation during synthesis. The synthetized CUP1-linker-CUP1 sequence was then inserted by Gibson Assembly in the pCTcon2_V5 plasmid containing Aga2, V5 tag and the Gal1 promoter.  
+
The start and stop codon of the CUP1 genomic sequences were removed in order to fuse the proteins together. In addition, the full sequence was codon optimized before being ordered in order to avoid loops formation during synthesis. The synthetized CUP1-linker-CUP1 sequence was then inserted by Gibson Assembly in the pCTcon2_V5 plasmid containing Aga2, V5 tag and the Gal1 promoter (1).  
  
  
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===References===
 
===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.

Revision as of 06:35, 13 September 2021


Dimerization of the copper metallothionein 1 : CUP1-GGGGSEAAAKGGGGS-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 start and stop codon of the CUP1 genomic sequences were removed in order to fuse the proteins together. In addition, the full sequence was codon optimized before being ordered in order to avoid loops formation during synthesis. The synthetized CUP1-linker-CUP1 sequence was then inserted by Gibson Assembly in the pCTcon2_V5 plasmid containing Aga2, V5 tag and the Gal1 promoter (1).


Source

The CUP1 sequence is the genomic sequence of the copper metallotionein 1 protein. The sequence of the linker comes from a reverse translation of the amino acid sequence GGGGSEAAAKGGGGS and was codon optimized for the yeast S. cerevisiae. The fusion protein was fully synthetized.

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.