Part:BBa_J100333

repClone 2 Red

This is the same part as J100205 except the TT has been removed and the Bsa1 site in-between the RBS and promoter has been moved to the end of the GFP sequence. We found J100205 as well as its modified J100324 part to be unstable. We designed repClone Red (J100205) to test the function of repressors. Internal BsaI sites allow the insertion of the Ptet promoter and removal of the backwards P2 promoter pointed towards GFP. Successful cloning of the Ptet promoter will change colonies from green to not green. Variations on the Ptet promoter, or introduction of anhydrotetrocycline (aTc), will affect the activity of the repressor, as indicated by the fluorescence of colonies containing the plasmid. This can be used in synthetic biology laboratories or for undergraduate introductory biology lab modules. From the previous J100324 sequence, excised the Bsa1 site in-between the RBS and P2 promoter. Using the Short Sequence FW primer, will insert this BSa1 sequence in-between the Promoter P2 and the end of the GFP.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 7
  Illegal SpeI site found at 21
  Illegal PstI site found at 2317
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 7
  Illegal SpeI site found at 21
  Illegal PstI site found at 2317
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 7
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 7
  Illegal SpeI site found at 21
  Illegal PstI site found at 2317
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 7
  Illegal SpeI site found at 21
  Illegal PstI site found at 2317
  Illegal AgeI site found at 2190
  Illegal AgeI site found at 2302
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1606
  Illegal BsaI.rc site found at 728