Part:BBa_K2306013

Cas13a spacer with flanking double repeats (with constitutive promoter)

This part is a composition of a constitutive promoter (BBa_J23119) and the Cas13a spacer with flanking double repeats (BBa_K2306015). This promoter is also used by [http://science.sciencemag.org/content/356/6336/438 Gootenberg et al. 2017] for the CRISPR array of Cas13a.

Usage and Biology

This biobrick can be used in combination with parts encoding for Cas13a. This part encodes for the transcription of CRISPR guide RNA (crRNA).

By digestion with BsaI the spacer will be removed leaving two sticky ends. A new spacer can then be ligated in the array using sticky end ligation.

When designing a new spacer to ligate into the array, make sure it contains the right sticky ends at both ends. One might consider to use our Cas13a spacer [http://2017.igem.org/Team:TUDelft/Model/spacerdesign design tool] to design crRNAs that meet all the requirements.

A terminator should be added downstream of the array.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 94
  Illegal BsaI.rc site found at 81
  

References

Gootenberg, J. S., Abudayyeh, O. O., Lee, J. W., Essletzbichler, P., Dy, A. J., Joung, J., … Koonin, E. V. (2017). Nucleic acid detection with CRISPR-Cas13a/C2c2, 9321(April). https://doi.org/10.1126/science.aam9321