Part:BBa_K2429023

phEF1a L. shahii Cas13a

This part includes the Leptotrichia shahii Cas13a protein coding region downstream of the human elongation factor 1a (hEF1a) promoter, which is a low level constitutive promoter. This final expression vector produces a CRISPR protein known as Cas13a, 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 active in bacteria; however, since we used it in mammalian cells, the protein's endonuclease activity isn't expected to be seen. 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 5541
  Illegal PstI site found at 315
  Illegal PstI site found at 820
  Illegal PstI site found at 3049
  Illegal PstI site found at 3418
  Illegal PstI site found at 4147
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 5541
  Illegal PstI site found at 315
  Illegal PstI site found at 820
  Illegal PstI site found at 3049
  Illegal PstI site found at 3418
  Illegal PstI site found at 4147
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 5541
  Illegal BglII site found at 569
  Illegal BglII site found at 1793
  Illegal BglII site found at 2417
  Illegal BglII site found at 2813
  Illegal BglII site found at 3104
  Illegal BglII site found at 3194
  Illegal BglII site found at 4355
  Illegal BamHI site found at 1183
  Illegal BamHI site found at 5437
  Illegal XhoI site found at 968
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 5541
  Illegal PstI site found at 315
  Illegal PstI site found at 820
  Illegal PstI site found at 3049
  Illegal PstI site found at 3418
  Illegal PstI site found at 4147
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 5541
  Illegal PstI site found at 315
  Illegal PstI site found at 820
  Illegal PstI site found at 3049
  Illegal PstI site found at 3418
  Illegal PstI site found at 4147
  Illegal NgoMIV site found at 703
  Illegal NgoMIV site found at 5394
  Illegal NgoMIV site found at 5413
  Illegal AgeI site found at 81
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1779
  Illegal SapI.rc site found at 1885
  Illegal SapI.rc site found at 2755
  Illegal SapI.rc site found at 3589