Part:BBa_K1486023

Yeast optimized superfolder GFP

Purpose of the Biobrick

This part corresponds to the sequence of the superfolded GFP optimized for yeast cells. We used it as a tag to control the expression of our genes of interest Hog1 and Pbs2. It was extracted by PCR from plasmids we ordered to addgene:Plasmid 44901 pFA6a-link-yoSuperfolderGFP-Kan and Plasmid 44873 pFA6a-link-yoSuperfolderGFP-CaURA3.

Experiment 1: CpxR dimerization & Dimerization Orientation

Associated Biobricks

: We assembled this part with the yeast resistance genes and the terminator ADH1. We also made a split sfGFP to evaluate the dimerization of hog1 and pbs2 in response to osmotic stress.

• sfGFP-ADH1-Kan ( https://parts.igem.org/Part:BBa_K1486026)
• sfGFP-N ( https://parts.igem.org/Part:BBa_K1486028)
• sfGFP_N-ADH1-Kan ( https://parts.igem.org/Part:BBa_K1486029)
• sfGFP-ADH1-Ura ( https://parts.igem.org/Part:BBa_K1486032)
• sfGFP-C( https://parts.igem.org/Part:BBa_K1486034)
• sfGFP_C-ADH1-Ura( https://parts.igem.org/Part:BBa_K1486035)

References

Improved Blue, Green, and Red Fluorescent Protein Tagging Vectors for S. cerevisiae. Lee et al (PLoS One. 2013 Jul 2;8(7):e67902. doi: 10.1371/journal.pone.0067902. Print 2013. PubMed)

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 2213
  Illegal PstI site found at 3446
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  Illegal PstI site found at 2213
  Illegal PstI site found at 3446
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 3657
  Illegal BamHI site found at 1144
  Illegal XhoI site found at 1283
  Illegal XhoI site found at 2470
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 2213
  Illegal PstI site found at 3446
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 2213
  Illegal PstI site found at 3446
  Illegal AgeI site found at 979
  Illegal AgeI site found at 1694
  Illegal AgeI site found at 2881
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961