Part:BBa_M50439:Design

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 150
  Illegal AgeI site found at 759
  Illegal AgeI site found at 1648
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1500
  Illegal BsaI.rc site found at 1194
  Illegal SapI.rc site found at 1513
  

Design Notes

As this part was taken from previously described research, our design decisions were mostly left to address the induction of the protein and termination of translation. We chose GAL1 as our promoter because we were unsure of the toxicity the construct might have in the cell. This way, we were able to grow the cells up in glucose media, which inhibits GAL1, until we had robust colonies and were comfortable expressing the novel construct. The specific terminator was chosen because the codon was optimized for S. cerevisiae. The entire sequence was optimized for S. cerevisiae using the IDT Codon Optimization tool.

Source

GAL1 promoter sequence: BBa_J63006

pHlash sequence: pHlash: A New Genetically Encoded and Ratiometric Luminescence Sensor of Intracellular pH, PLOS

Terminator sequence: BBa_K1462070

References

https://parts.igem.org/Part:BBa_J63006

https://parts.igem.org/Part:BBa_K1462070

Zhang, Yunfei et al. “pHlash: A New Genetically Encoded and Ratiometric Luminescence Sensor of Intracellular pH.” Ed. Valentin Ceña. PLoS ONE 7.8 (2012): e43072. PMC. Web. 9 May 2018.