Part:BBa_K3017066:Design

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
  

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.