Reporter

Part:BBa_K2429034

Designed by: Nia Myrie   Group: iGEM17_MIT   (2017-10-24)


phEF1a 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 human elongation factor 1a (hEF1a) promoter, which is a low level constitutive promoter.

"2ex.png"

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 2764
    Illegal PstI site found at 315
    Illegal PstI site found at 820
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2764
    Illegal PstI site found at 315
    Illegal PstI site found at 820
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2764
    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 2764
    Illegal PstI site found at 315
    Illegal PstI site found at 820
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 2764
    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 2669
    Illegal SapI.rc site found at 1237


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