Composite

Part:BBa_K3017066:Design

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


Construct for testing CRISPRi asRNA de-suppression effect of CRISPRi sgRNA


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 3350
    Illegal PstI site found at 4772
    Illegal PstI site found at 4976
    Illegal PstI site found at 5006
    Illegal PstI site found at 6218
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 933
    Illegal NheI site found at 956
    Illegal NheI site found at 2326
    Illegal NheI site found at 2506
    Illegal NheI site found at 2529
    Illegal PstI site found at 3350
    Illegal PstI site found at 4772
    Illegal PstI site found at 4976
    Illegal PstI site found at 5006
    Illegal PstI site found at 6218
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2811
    Illegal BamHI site found at 2265
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 3350
    Illegal PstI site found at 4772
    Illegal PstI site found at 4976
    Illegal PstI site found at 5006
    Illegal PstI site found at 6218
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 3350
    Illegal PstI site found at 4772
    Illegal PstI site found at 4976
    Illegal PstI site found at 5006
    Illegal PstI site found at 6218
    Illegal NgoMIV site found at 3638
    Illegal NgoMIV site found at 4742
    Illegal NgoMIV site found at 4815
    Illegal NgoMIV site found at 5300
    Illegal NgoMIV site found at 6209
    Illegal AgeI site found at 2100
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 705
    Illegal SapI site found at 2082


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.