Part:BBa_K2429042
pTRE L. shahii dCas13a-DDX6
This part includes the deactivated Leptotrichia shahii Cas13a protein coding region downstream of the tetracycline response element (TRE) promoter. When in the presence of the rtTA3 protein, this promoter is activated. This final expression vector produces a deactivated version of the CRISPR protein Cas13a. Without the deactivation sequence, a CRISPR protein known as Cas13a would be produced, which binds and cuts mRNA. Upon recognition of the mRNA, this protein exhibits "promiscuous" ribonuclease activity, not only cutting at base pairs along the targeted RNA molecule, but other nearby RNA molecules as well.which normally binds and cuts mRNA. With the deactivation sequence, the protein becomes catalytically inactive, and unable to cut the mRNA; however, it can still bind to the mRNA molecule. Linked to the sequence coding for the protein is a sequence coding for the DEAD-box helicase DDX6. This added domain prohibits the initiation of RNA translation and can allow the mRNA strand to exist for a longer period of time.
Typically, during the process of alternative splicing in eukaryotic cells, specific RNA binding proteins will bind to sequences or motifs on a pre-mRNA strand and eventually come together to form a spliceosome protein. This resulting protein will cleave the mRNA strand to exclude portions of the pre-mRNA (called introns) and the remaining sequences in the mature mRNA are known as exons. Our team used this protein in an attempt to control what exons would be included in an mRNA transcript by targeting the motifs in an intron, thus blocking splicing factors from binding and retaining an exon.
Furthermore, our team tested variations of this protein (e.g. catalytically deactivated, additional domains) to see whether such variations would affect the splicing capabilities. This specific variation of the protein is catalytically inactive in bacteria, and this behavior is expected to carry over into mammalian cells. Additionally, the focus is on the protein's binding and blocking ability rather than the catalytic activity.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 6
Illegal EcoRI site found at 322
Illegal EcoRI site found at 6173
Illegal XbaI site found at 30
Illegal PstI site found at 2205
Illegal PstI site found at 2574
Illegal PstI site found at 3303 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 6
Illegal EcoRI site found at 322
Illegal EcoRI site found at 6173
Illegal PstI site found at 2205
Illegal PstI site found at 2574
Illegal PstI site found at 3303 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 6
Illegal EcoRI site found at 322
Illegal EcoRI site found at 6173
Illegal BglII site found at 949
Illegal BglII site found at 1573
Illegal BglII site found at 1969
Illegal BglII site found at 2260
Illegal BglII site found at 2350
Illegal BglII site found at 3511
Illegal BglII site found at 3598
Illegal BamHI site found at 339
Illegal BamHI site found at 4593
Illegal BamHI site found at 4695 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 6
Illegal EcoRI site found at 322
Illegal EcoRI site found at 6173
Illegal XbaI site found at 30
Illegal PstI site found at 2205
Illegal PstI site found at 2574
Illegal PstI site found at 3303 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 6
Illegal EcoRI site found at 322
Illegal EcoRI site found at 6173
Illegal XbaI site found at 30
Illegal PstI site found at 2205
Illegal PstI site found at 2574
Illegal PstI site found at 3303
Illegal NgoMIV site found at 4550
Illegal NgoMIV site found at 4569 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 935
Illegal SapI.rc site found at 1041
Illegal SapI.rc site found at 1911
Illegal SapI.rc site found at 2745
Illegal SapI.rc site found at 6051
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