Part:BBa_K1486036

rLucC + ADH1 terminator + CaUra3 cassette

This part is a protein tag to be used with the genomic integration technique (homologous recombination) on the budding yeast S.cerevisiae. It consists of a 13 a.a long linker, the Renilla reniformis luciferase C-terminus without initial methionine (the 201 last amino acids, otherly called rLucC) followed by the ADH1 terminator and a CaUra3 cassette containing a constitutive TEF promoter, a Ura3 gene from Candida albicans and a constitutive TEF terminator. The rLucC tag needs to come in physical contact with the the rLuc N-terminus to form a functional luciferase, as in a protein complementation assay.

Use this part

To use this part and tag the protein Hog1, we attached 50 bases flanking regions to both 5' and 3' ends via PCR. Flanking regions are DNA sequences homologous to the yeast genome, targeting the genomic site of insertion. In our case, they were the last 50 coding bases of the protein Hog1 and the 50 first non-coding bases after the gene. To perform a protein complementation assay, we tagged the protein Pbs2 with another BioBrick : the BBa_K1486030.

Reference

Studies of Dynamic Protein-Protein Interactions in Bacteria Using Renilla Luciferase Complementation Are Undermined by Nonspecific Enzyme Inhibition: Stavroula K. Hatzios, Simon Ringgaard, Brigid M. Davis, Matthew K. Waldor.

Source

The coding sequences of the Renilla reniformis luciferase and its fragments were generously given by Dr. Matthew Waldor's laboratory.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1310
  Illegal PstI site found at 999
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1310
  Illegal PstI site found at 999
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1310
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1310
  Illegal PstI site found at 999
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1310
  Illegal PstI site found at 999
  
• 1000
  COMPATIBLE WITH RFC[1000]