Part:BBa_K2429035

phEF1a 3 Exon mKate-HBG Reporter

This part splits the red fluorescent mKate reporter into two exons, with human beta globin (HBG) intron 2 in between The first mKate exon and an exon that contains a stop codon. HBG intron 1 is placed between the exon with a stop codon and the second mKate exon. We used this split mKate construct as a reporter construct that we used to determine if our dCas13 or Ms2 systems were successful in controlling the inclusion of an exon. We used HBG Introns because it is a standard intron used in reporter constructs. Upstream of the coding sequences lies the human elongation factor 1a (hEF1a) promoter, which is a low level constitutive promoter.

In the absence of our system, the following mRNA transcript would be made. The presence of the stop codon would lead to a truncated protein, and there would be a knock down of fluorescence.

In the presence of our system, the following mRNA transcript would be made. The dCas13a or Ms2 protein would bind to the 3' splice site, and cause HBG intron 2, REST codon, and HBG intron 1 to be spliced out, and would result in more fluorescence than if our system wasn't present.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 2944
  Illegal XbaI site found at 2349
  Illegal PstI site found at 315
  Illegal PstI site found at 820
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2944
  Illegal PstI site found at 315
  Illegal PstI site found at 820
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2944
  Illegal BglII site found at 569
  Illegal BamHI site found at 1183
  Illegal XhoI site found at 968
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 2944
  Illegal XbaI site found at 2349
  Illegal PstI site found at 315
  Illegal PstI site found at 820
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 2944
  Illegal XbaI site found at 2349
  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 SapI.rc site found at 1237