Part:BBa_K2429033

phEF1a 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 human elongation factor 1a (hEF1a) promoter, which is a low level constitutive promoter 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 1722
  Illegal EcoRI site found at 2339
  Illegal PstI site found at 315
  Illegal PstI site found at 820
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1722
  Illegal EcoRI site found at 2339
  Illegal PstI site found at 315
  Illegal PstI site found at 820
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1722
  Illegal EcoRI site found at 2339
  Illegal BglII site found at 569
  Illegal BamHI site found at 1183
  Illegal XhoI site found at 968
  Illegal XhoI site found at 1619
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1722
  Illegal EcoRI site found at 2339
  Illegal PstI site found at 315
  Illegal PstI site found at 820
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1722
  Illegal EcoRI site found at 2339
  Illegal PstI site found at 315
  Illegal PstI site found at 820
  Illegal NgoMIV site found at 703
  Illegal AgeI site found at 81
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2244
  Illegal SapI.rc site found at 1237