Part:BBa_K3600009

prGSH1-yomScarlet-I-tADH1

This cassette was assembled from 6 part plasmids via Golden Gate assembly using the BsmbBI restriction enzyme. The two connectors (pYTK002 and pYTK072), as well as the terminator (pYTK053) and the Ampicilin resistance/backbone (pYTK095) part plasmids are part of the YTK toolkit developed by Lee et al.¹ and can be ordered online. The promoter part plasmid (BBa_K3600003) and the reporter part plasmid (BBa_K3600010) were synthesized by us.

Usage and Biology

This cassette contains the GSH1 promoter upstream of a reporter gene (mScarlet-i). GSH1 is a stress response gene in s.cerevisiae that is regulated by the YAP1 master regulator². This cassette is then assembled with a part plasmid containing Lys2 homology sequences (BBa_K36000012) in another Golden Gate BsmBI assembly, to give a final integration cassette (BBa_K3600016).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 936
  Illegal EcoRI site found at 1666
  Illegal XbaI site found at 945
  Illegal XbaI site found at 1843
  Illegal SpeI site found at 2811
  Illegal PstI site found at 2820
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 936
  Illegal EcoRI site found at 1666
  Illegal SpeI site found at 2811
  Illegal PstI site found at 2820
  Illegal NotI site found at 924
  Illegal NotI site found at 2830
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal suffix found in sequence at 2413
  Illegal EcoRI site found at 936
  Illegal EcoRI site found at 1666
  Illegal BglII site found at 2284
  Illegal XhoI site found at 1531
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 936
  Illegal EcoRI site found at 1666
  Illegal XbaI site found at 945
  Illegal XbaI site found at 1843
  Illegal SpeI site found at 2811
  Illegal PstI site found at 2820
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 936
  Illegal EcoRI site found at 1666
  Illegal XbaI site found at 945
  Illegal XbaI site found at 1843
  Illegal SpeI site found at 2811
  Illegal PstI site found at 2820
  Illegal NgoMIV site found at 2768
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1528
  

References

[1] Lee, M. E., DeLoache, W. C., Cervantes, B. & Dueber, J. E. A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth. Biol. 4, 975–986 (2015).

[2] Gomes DS, Fragoso LC, Riger CJ, Panek AD, Eleutherio EC. Regulation of cadmium uptake by Saccharomyces cerevisiae. Biochimica et Biophysica Acta (BBA)-General Subjects. 2002 Oct 10;1573(1):21-5.