Part:BBa_K1486030

rLucN + ADH1 terminator + Kanamycin resistance

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 N-terminus without initial methionine (the 109 first amino acids, otherly called rLucN) followed by the ADH1 terminator and a KanMX cassette containing a constitutive TEF promoter, a Kanamycin resistance (works against geneticin) and a constitutive TEF terminator. The rLucN tag needs to come in physical contact with the the rLuc C-terminus to form a functional luciferase, as in a protein complementation assay.

Use this part

To use this part and tag the protein Pbs2, 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 Pbs2 and the 50 first non-coding bases after the gene. To perform a protein complementation assay, we tagged the protein Hog1 with another BioBrick : the BBa_K1486036.

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 PstI site found at 723
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 723
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 723
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 723
  
• 1000
  COMPATIBLE WITH RFC[1000]