Part:BBa_K2429024

phEF1a L. shahii dCas13a

This part includes the deactivated 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 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 1476
  Illegal PstI site found at 315
  Illegal PstI site found at 820
  Illegal PstI site found at 3076
  Illegal PstI site found at 3445
  Illegal PstI site found at 4174
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1476
  Illegal PstI site found at 315
  Illegal PstI site found at 820
  Illegal PstI site found at 3076
  Illegal PstI site found at 3445
  Illegal PstI site found at 4174
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1476
  Illegal BglII site found at 569
  Illegal BglII site found at 1820
  Illegal BglII site found at 2444
  Illegal BglII site found at 2840
  Illegal BglII site found at 3131
  Illegal BglII site found at 3221
  Illegal BglII site found at 4382
  Illegal BamHI site found at 1183
  Illegal BamHI site found at 5464
  Illegal XhoI site found at 968
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1476
  Illegal PstI site found at 315
  Illegal PstI site found at 820
  Illegal PstI site found at 3076
  Illegal PstI site found at 3445
  Illegal PstI site found at 4174
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1476
  Illegal PstI site found at 315
  Illegal PstI site found at 820
  Illegal PstI site found at 3076
  Illegal PstI site found at 3445
  Illegal PstI site found at 4174
  Illegal NgoMIV site found at 703
  Illegal NgoMIV site found at 5421
  Illegal NgoMIV site found at 5440
  Illegal AgeI site found at 81
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1806
  Illegal SapI.rc site found at 1912
  Illegal SapI.rc site found at 2782
  Illegal SapI.rc site found at 3616