Part:BBa_K2176001

Cassette for the constitutive production of GAL4-KaiCp and LexA-SasA, linked by a self-cleaving P2A

This part is a composite of four parts:

1) a (constitutive) yeast TEF1 promoter

2) a protein coding sequence for a fusion of the GAL4 activator domain and phosphorylated KaiC

3) a protein coding sequence for a fusion of LexA and SasA

4) and a yeast ADH1 terminator

The two protein coding sequences are linked by a P2A linker domain, which is self-cleaving.

This part works in tandem with a GFP reporter construct we designed (BBa_K2176000) that is regulated by a LexA operator. The LexA protein from this part (that is, BBa_K2176001) binds to the LexA operator, carrying with it the SasA protein to which it is fused. SasA binds phosphorylated KaiC (KaiCp), which is made by this cassette in a 1:1 ratio with the LexA-SasA fusion. This KaiCp is fused with a GAL4 activator domain, which recruits RNA polymerase. As a result, an organism expressing both this cassette and the GFP reporter BBa_K2176000 will be constitutively expressing GFP.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 6
  Illegal SpeI site found at 4479
  Illegal PstI site found at 4465
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 6
  Illegal SpeI site found at 4479
  Illegal PstI site found at 4465
  Illegal NotI site found at 12
  Illegal NotI site found at 4470
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 6
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 6
  Illegal SpeI site found at 4479
  Illegal PstI site found at 4465
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 6
  Illegal XbaI site found at 21
  Illegal SpeI site found at 4479
  Illegal PstI site found at 4465
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 206
  Illegal BsaI.rc site found at 3479