Part:BBa_K1400000

PTRE(4)GX Dual input promoter. Activation at tetO binding sites, repression at gal4 sites.

This is an engineered variant of the pGAL1 promoter native to S. cerevisiae. This dual input promoter has four upstream activating sequences (UAS) and two repressing sequences. The four UAS sites are tetO binding sites that can bind to the tetracycline responsive activator protein, rtTA (reverse tetracycline-controlled transactivator), to induce transcription. The third and fourth GAL4 binding sites of the native pGAL1 promoter are replaced with tetO sites in this version and the first two GAL4 sites are replaced with random sequences with identical C-G content . The Mig1 sequences that are native to the pGAL1 promoter are replaced with two tetO sites. The two repressing sequences are binding sites for the GAL4 DNA binding domain proximal to the TATA box, causing transcriptional repression by steric hindrance and prevention of transcription machinery assembly at the promoter. In cells expressing rtTA and GEV (GAL4 binding domain-human estrogen receptor-VP16 activator domain), this promoter can be used to drive transcription of a downstream gene by the addition of aTc (anhydrotetracycline). The level of transcription can be modulated or repressed with the addition of β-estradiol.

Figure 1: Characterization of pTREgx via dual drug induction. pTREgx has 4 activating tetO sites and 2 repressing gal4 sites 10bp away from the TATA box. Thus, activation increases with aTc concentration and repression increases with estradiol concentration. Activation is caused by the protein rtTA (reverse-tetracycline transactivator), and repression is caused by the protein GEV (GAL4 binding domain-human estrogen receptor-VP16 activator domain), which is expressed by the strong consititutive promoter, pADH1. 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 35
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 166