Part:BBa_K2429049

pTRE mKate FF4

This is a 2-exon split mKate reporter construct and has an intron that contains 3 microRNA sites for FF4. These lie downstream of the tetracycline response element (TRE) promoter. When in the presence of the rtTA3 protein, this promoter is activated. Our team used this to determine whether our system was controlling the inclusion of the second exon by measuring the amount of fluorescence. In the absence of dCas13a or Ms2, the intron should be spliced out as normal, leading to a complete mKate mRNA transcript and flouresence would be seen. In the presence of dCas13a or Ms2, which targets the 3' splice site of the intron, the second exon would be spliced out along with the intron, and the final mRNA transcript would not include the second exon. Thus, with out system, we expect a knock down of flourescence.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 322
  Illegal EcoRI site found at 878
  Illegal EcoRI site found at 1495
  Illegal XbaI site found at 30
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 322
  Illegal EcoRI site found at 878
  Illegal EcoRI site found at 1495
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 322
  Illegal EcoRI site found at 878
  Illegal EcoRI site found at 1495
  Illegal BamHI site found at 339
  Illegal XhoI site found at 775
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 322
  Illegal EcoRI site found at 878
  Illegal EcoRI site found at 1495
  Illegal XbaI site found at 30
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 322
  Illegal EcoRI site found at 878
  Illegal EcoRI site found at 1495
  Illegal XbaI site found at 30
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1400
  Illegal SapI.rc site found at 393