Difference between revisions of "Part:BBa K2340012"
 
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When supplied with a guide RNA (gRNA), this enzyme will process it into a CRISPR RNA (crRNA). The crRNA contains a sequence that is complementary to the RNA it is targetting.
 
When supplied with a guide RNA (gRNA), this enzyme will process it into a CRISPR RNA (crRNA). The crRNA contains a sequence that is complementary to the RNA it is targetting.
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<b><font size="+1"> Additional comments </font></b>
 
<b><font size="+1"> Additional comments </font></b>

Latest revision as of 21:17, 15 December 2017

dCAS13a linked to GFP with NLS (improved version)


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1094
    Illegal BglII site found at 2030
    Illegal BglII site found at 2294
    Illegal BglII site found at 2798
    Illegal BglII site found at 3284
    Illegal BglII site found at 3392
    Illegal BglII site found at 3722
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 3403


Usage and Biology

dead CAS13a (or dCas13) with a double HEPN nuclease 1 and 2 inactive mutation, linked to an enhanced green fluorescent protein (wtGFP) via a linker sequence (ggctcctccggc). On the N-terminal and the C-terminal of the construct there are nuclear localization sequences (NLS; DNA sequence: cccaagaaaaaacgcaaggtg. Amino acid sequence: PKKKRKV) that will reduce background noise when expressed and used in mammalian cell lines.

When supplied with a guide RNA (gRNA), this enzyme will process it into a CRISPR RNA (crRNA). The crRNA contains a sequence that is complementary to the RNA it is targetting.


Additional comments

This part is the improved version of BBa_K2340000 by iGEM team Kent 2017. BBa_K2340000 was constructed using a wild type GFP (BBa_K648013) which did not work in mammalian cell cultured at 37 degrees Celsius. This improved version of the dCAS13a-GFP construct has a enhanced GFP (eGFP) instead of the wtGFP which should improve its function at 37o Celsius. This is not tested and validated due to time and resource restrictions.

Apart from the type of GFP used in this construct the design of this biobrick is identical to BBa_K2340000.