Composite

Part:BBa_K2019001:Design

Designed by: John Lazar   Group: iGEM16_Northwestern   (2016-07-15)
Revision as of 00:11, 13 October 2016 by T lazar (Talk | contribs) (→‎References)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)


mRFP+gRNA Template (for saCas9)


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 1099
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1076
    Illegal SpeI site found at 1099
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 1099
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 1099
    Illegal AgeI site found at 781
    Illegal AgeI site found at 893
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1154
    Illegal BsaI.rc site found at 1106


Design Notes

First, we ordered a gblock that included J23119-Guide Template-sgRNA scaffolding (saCas9) with homology to the BBa_J04450-PSB1T3 backbone. We then linearized a BBa_J04450 in PSB1T3 backbone and performed a 2 piece Gibson. Results were then sequenced to confirm that the assembly was successful.

Source

gRNA scaffolding for sgRNA and the J23119 constitutive promoter were both suggestions from the supplementary information of "In vivo genome editing using Staphylococcus aureus Cas9".

References

F. Ann Ran, Le Cong, Winston X. Yan, David A. Scott, Jonathan S. Gootenberg, Andrea J. Kriz, Bernd Zetsche, Ophir Shalem, Xuebing Wu, Kira S. Makarova, Eugene V. Koonin, Phillip A. Sharp, Feng Zhang. In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015;520.