Part:BBa_K2381012

3'-OriC gRNA

It’s a gRNA plasmid which helps our CRISPR-System target to the MG1655 OriC sequence in order to achieve our expectation this year. Its backbone is taken from iGEM Kit Plate named BBa_K165005, but the BsaI restriction site which was in Amp. coding sequence has been deleted and a synonymous mutation has occurred by overlapping PCR. We used J23119 promoter, a high-intensity promoter, to obtain a higher expression of gRNA than dCas9. The 21bp-sequence comes after is a kind of sequence types taken from the article that is complementary to the Oric sequence and has a nice effect on our project. The full length gRNA scaffold is also designed after consulting literatures.

Reference

Mitchell R. O’Connell, Benjamin L. Oakes, Samuel H. Sternberg, Alexandra East-Seletsky, Matias Kaplan & Jennifer A. Doudna (2014). Programmable RNA recognition and cleavage by CRISPR/Cas9. Nat Commun, 6, 6256. doi:10.1038/nature13769

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 4
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]