Coding

Part:BBa_K2429040

Designed by: Nia Myrie   Group: iGEM17_MIT   (2017-10-24)
Revision as of 18:59, 24 October 2017 by Registry (Talk | contribs)

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pTRE L. shahii dCas13a

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, 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. 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. 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


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 322
    Illegal EcoRI site found at 632
    Illegal XbaI site found at 30
    Illegal PstI site found at 2232
    Illegal PstI site found at 2601
    Illegal PstI site found at 3330
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 322
    Illegal EcoRI site found at 632
    Illegal PstI site found at 2232
    Illegal PstI site found at 2601
    Illegal PstI site found at 3330
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 322
    Illegal EcoRI site found at 632
    Illegal BglII site found at 976
    Illegal BglII site found at 1600
    Illegal BglII site found at 1996
    Illegal BglII site found at 2287
    Illegal BglII site found at 2377
    Illegal BglII site found at 3538
    Illegal BglII site found at 3625
    Illegal BamHI site found at 339
    Illegal BamHI site found at 4620
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 322
    Illegal EcoRI site found at 632
    Illegal XbaI site found at 30
    Illegal PstI site found at 2232
    Illegal PstI site found at 2601
    Illegal PstI site found at 3330
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 322
    Illegal EcoRI site found at 632
    Illegal XbaI site found at 30
    Illegal PstI site found at 2232
    Illegal PstI site found at 2601
    Illegal PstI site found at 3330
    Illegal NgoMIV site found at 4577
    Illegal NgoMIV site found at 4596
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 962
    Illegal SapI.rc site found at 1068
    Illegal SapI.rc site found at 1938
    Illegal SapI.rc site found at 2772


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