Coding

Part:BBa_K1982008:Design

Designed by: Zexu Li   Group: iGEM16_NEU-China   (2016-10-09)


tCas9-CIBN (Eukaryotic LACE system)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2758
    Illegal NgoMIV site found at 3667
    Illegal NgoMIV site found at 4231
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 3786
    Illegal SapI.rc site found at 1177
    Illegal SapI.rc site found at 1419


Design Notes

The CRY2/CIBN interaction is entirely genetically encoded. The binding reverses within minutes in the dark, allowing rapid shutoff of transcription by placing samples in the dark. This fusion protein is for use in LACE(light-activated CRISPR/Cas9 effector) system, and a tCas9 fused to its N terminus. To regulate DNA transcription by blue light, the system is based on CRY2/CIBN interaction in which a light-mediated protein interaction brings together two protein (tCas9 and an activation domain VP64) . If we remove the stimulation of blue light, dark reversion of CRY2 will dissociate the interaction with CIBN and shut off transcription.


Source

CRY2 and CIB1 are extracted from Arabidopsis thaliana. tCas9 is from GE Share company. VP64 is synthetic construct.

References