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. doi:10.1038/nature13769
Alexandra E. Briner, Paul D. Donohoue, Ahmed A. Gomaa, Kurt Selle, Euan M. Slorach, Christopher H. Nye,Rachel E. Haurwitz, Chase L. Beisel, Andrew P. May, and Rodolphe Barrangou (2014). Guide RNA Functional Modules Direct Cas9 Activity and Orthogonality. Molecular Cell 56, 333–339, October 23, 2014 ª2014 Elsevier Inc. 333
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 4
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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