Part:BBa_K2957046

CMV-2xGal4 (Gal4 repressible promoter), CCL5, NeonGreen Tag

Contents:CMV-2xGal4 (Gal4 repressible promoter), CCL5, NeonGreen Tag, SynthpA, with Carb resistance

What is it?: A Composite part; it is a genetic circuit with CMV-2xGal4 promoter and CCL5 gene allowing for inducible expression of CCL5. The CCL5 has a NeonGreen protein attached to it.

What does it do?: This part has a promoter that is only active when induced by the syn-notch receptor and was put in our genetic circuits to show inducible production of the CCL5, which is involved in an inflammatory response, we were testing. It was important for our project in particular because of its involvement in the immune system and is known to create neutrophil swarming. This specific part was important to show a wave mechanic since when neutrophils expressing the syn-notch antigen would bind to the receptor on HEK cells, the promoter can activate more or less CCL5 and NeonGreen production.

How to use it?: This part was created with Type IIS Cloning and transfected into HEKs. It also has a NeonGreen tag attached to the CCL5 so this allows us to visualize it and determine transfection efficiency. See more on the Parts Overview Page or MIT iGEM 2019 wiki: <a href=https://2019.igem.org/Team:MIT/Parts>. "

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 1668
  Illegal SpeI site found at 92
  Illegal PstI site found at 1111
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 92
  Illegal PstI site found at 1111
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1127
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 1668
  Illegal SpeI site found at 92
  Illegal PstI site found at 1111
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 1668
  Illegal SpeI site found at 92
  Illegal PstI site found at 1111
  Illegal AgeI site found at 1674
  
• 1000
  COMPATIBLE WITH RFC[1000]