Part:BBa_K2100004

pENTR pTRE

This construct is an entry vector containing the TRE promoter. The promoter is structured as six tetO sites upstream of a minimal CMV promoter. The induction of doxycycline activates a tetracycline transactivator protein which in turn binds to the tetO sites to initiate transcription of the gene.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 322
  Illegal XbaI site found at 30
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 322
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 322
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 322
  Illegal XbaI site found at 30
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 322
  Illegal XbaI site found at 30
  
• 1000
  COMPATIBLE WITH RFC[1000]

We characterized our TRE promoter induced with a fine sweep of Doxycycline from a concentration of 0 uM to 2000 uM. We transfected HEK293 cells with 250ng of TRE-mKate and 250 ng hEF1a-eYFP. We analyzed fluorescent output in the PE Texas Red channel to measure the activity of the TRE promoter under different DOX concentrations. We used 0 uM of DOX as a control.

The results indicate the optimal DOX concentration was 500 uM for maximum TRE activity. Beyond that point, adding a higher concentration of DOX does not significantly increase fluorescent output from the TRE-mKate construct.