Part:BBa_K2429050

pTRE 2 Exon mKate-HBG Reporter

This part splits the red fluorescent mKate reporter into two exons, with human beta globin (HBG) intron 2 in between these two exons. We used this split mKate 2-exon construct as a reporter construct that we used to determine if our dCas13 or Ms2 systems were successful in splicing out the HBG Intron 2. We used HBG Intron 2 because it is a standard intron used in reporter constructs. Upstream of these sequences lies the tetracycline response element (TRE) promoter. When in the presence of the rtTA3 protein, this promoter is activated.

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The figure above represents our construct. The sequence in between the exons represents HBG intron 2. The 3' splice site will be targeted by the guide-dCas13a complex (or guide-Ms2 complex). In the absence of our system, the intron will be spliced out and result in mRNA transcript with both exons included, and will lead to the creation of a red fluorescent protein. In the presence of our system, the RNA binding protein should block the splice site, and the second exon will be spliced out along with the intron. The fluorescent protein wouldn't be produced, and we should see less red fluorescence.

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 1920
  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 1920
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 322
  Illegal EcoRI site found at 1920
  Illegal BamHI site found at 339
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 322
  Illegal EcoRI site found at 1920
  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 1920
  Illegal XbaI site found at 30
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1825
  Illegal SapI.rc site found at 393