Part:BBa_K1559002:Design

rearranged CRISPR/Cas9 system without promoter

Assembly Compatibility:

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

  Illegal NheI site found at 1099
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 3378
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 4320
  Illegal BsaI.rc site found at 4297
  

Design Notes

This part is formed by joining Part:BBa_K1559001 upstream of Part:BBa_K1559000.

Source

pCas9

We obtained our pCas9 sample by way of Part:BBa_K1218011 (not annotated)

Original pCas9 plasmid, available from addgene: http://www.addgene.org/42876/

pCas9 sequence in genbank format (Our annotations are sourced from here):

http://www.addgene.org/static/data/42/17/509f1fc8-85d2-11e2-92ba-003048dd6500.gb

Email reply from Wenyan Jiang:

The type II CRISPR-Cas system, i.e. tracr, Cas9 and crRNA array was directly cloned from s.pyogenes, with a modification of the array to facilitate the cloning of spacers as you know. I’m not sure which unannotated region of pCas9 you were referring to, perhaps from nucleotides 2015 to 2224? This region contains a bi-directional promoter that drives the expression of both tracr and cas9. And of course they were cloned directly from s. pyogenes without any engineering.

Email reply from Luciano Marraffini:

We did not engineer any promoter or terminator. The sequences are those present in the crispr system of Streptococcus pyogenes and work constitutively in E. coli

Terminators

Terminator/stem-loop prediction was done using ARNold:

http://rna.igmors.u-psud.fr/toolbox/arnold/index.php

References

Jiang, W., Bikard, D., Cox, D., Zhang, F., & Marraffini, L. A. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology, 31(3), 233 – 239. doi:10.1038/nbt.2508