Part:BBa_K1861030

eYFP gene for expression in yeast

This part includes the CMV promoter BBa_K1861303, the Kozak sequence BBa_K792001, the reporter gene for eYFP BBa_K1861302, and the adh1 terminator BBa_J63002. This construct is flanked by sites for homologous recombination (HR) into the his3 locus on chromosome XV of S. cerevisiae, BBa_K1861301 and BBa_K1861304. Additionally, there are two TALE binding sites inside the CMV promoter, one for TALE1 BBa_K1861050 and one for TALE2 BBa_K1861060. The two TALE3 BBa_K1861050 binding sites at the 3' end of the 5' HR and at the 5' end of the 3' HR complete the construct. The following enzymes can be used to cut parts out of this construct:

CMV promoter: SacI and BamHI

Kozak sequence and the start codon ATG: BamHI and XhoI

the CDS without the start codon ATG: XhoI and SalI

the terminator: SalI and BclI.

Due to this part's structure, any protein expressed this way will begin with the amino acids MLE.

It is advised not to attempt a HR into the his3 locus due to screening difficulties. Instead, this part (without HR sites, unless you want to keep the TALE3 binding site) should be cloned into an integration plasmid that can complement a certain deficiency, and then transformed into a yeast strain possessing that deficiency. For example, we used YIplac204 to complement the trp1 mutation of the strain K699.


Figure 1. Laser confocal scanning microscope picture of untransformed K699 strain (left) and the YIYFP (yeast inserted YFP) strain created by transforming K699 with this part in YIplac204 (right).

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 276
  Illegal XhoI site found at 300
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 967