Part:BBa_K2429009
pENTR L. shahii dCas13a-DDX6
Leptotrichia shahii deactivated Cas13a-DDX6 protein coding region. This part produces a deactivated version of the CRISPR protein Cas13a, 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 DDX6 domain. This added domain prohibits the initiation of RNA translation.
This part includes the deactivated Leptotrichia shahii Cas13a protein coding region. This basic part is a pENTR vector that serves as an intermediate in the production process of a final expression vector. The 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 5835
Illegal PstI site found at 1867
Illegal PstI site found at 2236
Illegal PstI site found at 2965 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 5835
Illegal PstI site found at 1867
Illegal PstI site found at 2236
Illegal PstI site found at 2965 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 5835
Illegal BglII site found at 611
Illegal BglII site found at 1235
Illegal BglII site found at 1631
Illegal BglII site found at 1922
Illegal BglII site found at 2012
Illegal BglII site found at 3173
Illegal BglII site found at 3260
Illegal BamHI site found at 1
Illegal BamHI site found at 4255
Illegal BamHI site found at 4357 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 5835
Illegal PstI site found at 1867
Illegal PstI site found at 2236
Illegal PstI site found at 2965 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 5835
Illegal PstI site found at 1867
Illegal PstI site found at 2236
Illegal PstI site found at 2965
Illegal NgoMIV site found at 4212
Illegal NgoMIV site found at 4231 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 597
Illegal SapI.rc site found at 703
Illegal SapI.rc site found at 1573
Illegal SapI.rc site found at 2407
Illegal SapI.rc site found at 5713
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