Part:BBa_K3600002

YCF1 promoter part plasmid

YCF1 promoter part plasmid was made in order to create a part plasmid which contains the YCF1 promoter and is compatible with the Yeast Toolkit. To create a promoter part plasmid compatible with the yeast toolkit, at first, we designed a gBlock consisting of the 500bp promoter region, that was retrieved from the yeast genome database, and two linking sequences which were added to the ends of the promoter.These linking sequences contained a BsaI and a BsmBI cutting site each to make Golden Gate assembly possible.Then, the gBlock was inserted into the part plasmid entry vector (pYTK001) through a BsmBI assembly.

Usage and Biology

The YCF1 gene encodes the Yeast cadmium factor 1 ¹ and is required for yeast cells to better resist exposure to cadmium ². The YCF1 promoter is regulated by the transcription factor Yap1p. The binding site for Yap1p, in the promoter YCF1, is TTACTAA (ref). Using the yeast toolkit for modular assembly, the YCF1 promoter plasmid was used as a part of type II ⁴.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 935
  Illegal EcoRI site found at 1665
  Illegal XbaI site found at 944
  Illegal XbaI site found at 1842
  Illegal SpeI site found at 2810
  Illegal PstI site found at 2819
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 935
  Illegal EcoRI site found at 1665
  Illegal SpeI site found at 2810
  Illegal PstI site found at 2819
  Illegal NotI site found at 923
  Illegal NotI site found at 2829
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal suffix found in sequence at 2412
  Illegal EcoRI site found at 935
  Illegal EcoRI site found at 1665
  Illegal BglII site found at 1280
  Illegal BglII site found at 2283
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 935
  Illegal EcoRI site found at 1665
  Illegal XbaI site found at 944
  Illegal XbaI site found at 1842
  Illegal SpeI site found at 2810
  Illegal PstI site found at 2819
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 935
  Illegal EcoRI site found at 1665
  Illegal XbaI site found at 944
  Illegal XbaI site found at 1842
  Illegal SpeI site found at 2810
  Illegal PstI site found at 2819
  Illegal NgoMIV site found at 2767
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

[1] Bhuiyan, M. S. U. et al. Overexpression of a yeast cadmium factor 1 (YCF1) enhances heavy metal tolerance and accumulation in Brassica juncea. Plant Cell Tiss Organ Cult 105, 85–91 (2011).

[2] Li, Z.-S., Szczypka, M., Lu, Y.-P., Thiele, D. J. & Rea, P. A. The Yeast Cadmium Factor Protein (YCF1) Is a Vacuolar Glutathione S-Conjugate Pump. J. Biol. Chem. 271, 6509–6517 (1996).

[3] 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).

[4] Kuge, S. & Jones, N. YAP1 dependent activation of TRX2 is essential for the response of Saccharomyces cerevisiae to oxidative stress by hydroperoxides. The EMBO Journal 13, 655–664 (1994).