Composite

Part:BBa_K3017070:Design

Designed by: Luk Hau Ching   Group: iGEM19_Hong_Kong_HKUST   (2019-10-17)


Combined CRISPRi and antisense RNA Toggle Switch - Model circuit


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 852
    Illegal PstI site found at 2274
    Illegal PstI site found at 2478
    Illegal PstI site found at 2508
    Illegal PstI site found at 3720
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 5543
    Illegal NheI site found at 6921
    Illegal NheI site found at 7093
    Illegal NheI site found at 7116
    Illegal NheI site found at 8733
    Illegal PstI site found at 852
    Illegal PstI site found at 2274
    Illegal PstI site found at 2478
    Illegal PstI site found at 2508
    Illegal PstI site found at 3720
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 313
    Illegal BamHI site found at 5482
    Illegal BamHI site found at 6860
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 852
    Illegal PstI site found at 2274
    Illegal PstI site found at 2478
    Illegal PstI site found at 2508
    Illegal PstI site found at 3720
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 852
    Illegal PstI site found at 2274
    Illegal PstI site found at 2478
    Illegal PstI site found at 2508
    Illegal PstI site found at 3720
    Illegal NgoMIV site found at 1140
    Illegal NgoMIV site found at 2244
    Illegal NgoMIV site found at 2317
    Illegal NgoMIV site found at 2802
    Illegal NgoMIV site found at 3711
    Illegal AgeI site found at 5317
    Illegal AgeI site found at 6695
    Illegal AgeI site found at 7702
    Illegal AgeI site found at 7814
    Illegal AgeI site found at 9342
    Illegal AgeI site found at 9454
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 5299
    Illegal SapI site found at 6677


Design Notes

Source

dCas9 gene comes from Streptococcus pyogenes

References

Y. J. Lee, A. Hoynes-Oconnor, M. C. Leong, and T. S. Moon, “Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system,” Nucleic Acids Research, vol. 44, no. 5, pp. 2462–2473, Feb. 2016.

C. Anders, O. Niewoehner, A. Duerst, and M. Jinek, “Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease,” Nature, vol. 513, no. 7519, pp. 569–573, 2014.

S. H. Sternberg, S. Redding, M. Jinek, E. C. Greene, and J. A. Doudna, “DNA Interrogation by the CRISPR RNA-Guided Endonuclease Cas9,” Biophysical Journal, vol. 106, no. 2, 2014.

T. Karvelis, G. Gasiunas, A. Miksys, R. Barrangou, P. Horvath, and V. Siksnys, “crRNA and tracrRNA guide Cas9-mediated DNA interference inStreptococcus thermophilus,” RNA Biology, vol. 10, no. 5, pp. 841–851, 2013.

T. Møller, T. Franch, P. Højrup, D. R. Keene, H. P. Bächinger, R. G. Brennan, and P. Valentin-Hansen, “Hfq,” Molecular Cell, vol. 9, no. 1, pp. 23–30, 2002.

G. M. Cech, A. Szalewska-Pałasz, K. Kubiak, A. Malabirade, W. Grange, V. Arluison, and G. Węgrzyn, “The Escherichia Coli Hfq Protein: An Unattended DNA-Transactions Regulator,” Frontiers in Molecular Biosciences, vol. 3, 2016.