Part:BBa_K2429008

pENTR L. shahii Cas13a-DDX6

This part includes the 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 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. 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 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 5835
  Illegal PstI site found at 1867
  Illegal PstI site found at 2236
  Illegal PstI site found at 2965
  
• 12
  INCOMPATIBLE 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
  
• 21
  INCOMPATIBLE 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
  
• 23
  INCOMPATIBLE 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
  
• 25
  INCOMPATIBLE 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
  
• 1000
  INCOMPATIBLE 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