Composite

Part:BBa_K3017067:Design

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


Construct for testing CRISPRi sgRNA cross-talk with non-target DNA


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 3336
    Illegal PstI site found at 4758
    Illegal PstI site found at 4962
    Illegal PstI site found at 4992
    Illegal PstI site found at 6204
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 919
    Illegal NheI site found at 942
    Illegal NheI site found at 2312
    Illegal NheI site found at 2492
    Illegal NheI site found at 2515
    Illegal PstI site found at 3336
    Illegal PstI site found at 4758
    Illegal PstI site found at 4962
    Illegal PstI site found at 4992
    Illegal PstI site found at 6204
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2797
    Illegal BamHI site found at 2251
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 3336
    Illegal PstI site found at 4758
    Illegal PstI site found at 4962
    Illegal PstI site found at 4992
    Illegal PstI site found at 6204
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 3336
    Illegal PstI site found at 4758
    Illegal PstI site found at 4962
    Illegal PstI site found at 4992
    Illegal PstI site found at 6204
    Illegal NgoMIV site found at 3624
    Illegal NgoMIV site found at 4728
    Illegal NgoMIV site found at 4801
    Illegal NgoMIV site found at 5286
    Illegal NgoMIV site found at 6195
    Illegal AgeI site found at 616
    Illegal AgeI site found at 728
    Illegal AgeI site found at 2086
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 2068


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.