Composite

Part:BBa_K2019001

Designed by: John Lazar   Group: iGEM16_Northwestern   (2016-07-15)


mRFP+gRNA Template (for saCas9)

Part BBa_K2019001 is a part consisting of part BBa_J04450 followed by BBa_J23119-Guide Template-gRNA Scaffolding downstream. This part has a customizable guide template, which is flanked on both ends by BsaI sites to allow for a new guide to be inserted in place of the template through Golden Gate Assembly.


Usage and Biology

The primary goal of this part is to test the functionality of saCas9. By using Golden Gate Assembly, one can insert a 20 bp guide sequence, which will be expressed constitutively with the correct sgRNA scaffolding. Using this part and a pair of overlapping primers with correct sticky ends, we created part BBa_K2019002 through Golden Gate Assembly. Sequence and Features


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


[edit]
Categories
Parameters
None