Part:BBa_K2429005

pTRE 2-fluroescent protein (2-FP) reporter construct

This is a 3 exon, 2 intron report construct that allows use to test if we can splice together different protein isoforms conditionally. The output is the translation eYFP or eBFP.

This part consists of 3 exons: the first exon is made of sequence for a "conserved" region that most fluorescent proteins share. The second intron is made of the The second exon is made up of the sequences unique to YFP, as well as the remaining parts of the fluorescent protein. The third exon is very similar to the second, except instead of sequences unique to yellow fluorescence, it has sequences unique to blue. In between the conserved region and YFP exon is the second human beta globin (HBG) intron, and in between the YFP exon and BFP exon is the first HBG intron. These sequences lie downstream of the human elongation factor 1a (hEF1a) promoter, which is a low level constitutive promoter

We used this construct to determine whether our RNA binding proteins (dCas13a or Ms2) were successful in controlling the exclusion of an exon.

In the absence of our system, the following mRNA transcript would be made. The presence of the stop codon in between the eYFP and eBFP proteins would lead to a truncaated protein in which only the conserved region and eYFP sequence would remain, and only yellow fluorescent protein would be produced.

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, eYFP exon, and HBG intron 1 to be spliced out, and would result in only blue fluorescent protein to be produced.

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 2620
  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 2620
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 322
  Illegal EcoRI site found at 2620
  Illegal BamHI site found at 339
  Illegal BamHI site found at 1750
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 322
  Illegal EcoRI site found at 2620
  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 2620
  Illegal XbaI site found at 30
  
• 1000
  COMPATIBLE WITH RFC[1000]