Difference between revisions of "Part:BBa K2429006"

 
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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.
 
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.
+
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.
  
 
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Latest revision as of 18:04, 23 October 2017


pENTR L. shahii Cas13a

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.

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 4359
    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 4359
    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 4359
    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
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 4359
    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 4359
    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