Difference between revisions of "Part:BBa K4035010: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 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 in order to avoid loops during syntethis.  
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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.  
  
  
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===Source===
 
===Source===
  
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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. The full CUP1-linker-CUP1 sequence was synthetized and inserted by Gibson Assembly in the pCTcon2_V5 plasmid containing Aga2, V5 tag and the Gal1 promoter.  
+
The CUP1 sequence is the genomic sequence of the yeast copper metallothionein 1 protein (2). The linker sequence is the reverse transcription of the GGGGS 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===
 
===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.
 
(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.
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(2) https://www.uniprot.org/uniprot/P0CX80

Latest revision as of 21:08, 21 October 2021


Expression of the CUP1-(GGGGS)4-CUP1 dimer on the extracellular membrane of S. cerevisiae


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 900
    Illegal PstI site found at 1218
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 900
    Illegal PstI site found at 1218
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1045
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 900
    Illegal PstI site found at 1218
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 900
    Illegal PstI site found at 1218
    Illegal AgeI site found at 150
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 894


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 (2). The linker sequence is the reverse transcription of the GGGGS 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.

(2) https://www.uniprot.org/uniprot/P0CX80