Part:BBa_K2429034
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.
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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
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 2764
Illegal PstI site found at 315
Illegal PstI site found at 820 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2764
Illegal PstI site found at 315
Illegal PstI site found at 820 - 21INCOMPATIBLE 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 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2764
Illegal PstI site found at 315
Illegal PstI site found at 820 - 25INCOMPATIBLE 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 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2669
Illegal SapI.rc site found at 1237
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