Difference between revisions of "Part:BBa K1723000:Design"

 
(→‎Design Notes)
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===Design Notes===
 
===Design Notes===
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The original dCas9-ω given to us by the Bikard lab contains an EcoRI restriction site. We performed a site-directed mutagenesis to remove it.
+
dCas9 is a Cas9 double mutant, with mutations at amino acid positions D10A and H840A. These mutations inactivate Cas9 nuclease and nickase activities.
−
 
+
Cas9-&#969 contains an EcoRI restriction site which was removed via site-directed mutagenesis.
  
 +
Fusion of the &#969 subunit (rpoZ) to dCas9 was achieved by Gibson assembly.
  
 
===Source===
 
===Source===

Revision as of 07:51, 11 August 2015


dCas9-ω


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
    Illegal BamHI site found at 4212
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

dCas9 is a Cas9 double mutant, with mutations at amino acid positions D10A and H840A. These mutations inactivate Cas9 nuclease and nickase activities. Cas9-&#969 contains an EcoRI restriction site which was removed via site-directed mutagenesis. 

Fusion of the &#969 subunit (rpoZ) to dCas9 was achieved by Gibson assembly.

Source

A plasmid containing dCas9_ω (pdCas9) was given to our iGEM team by David Bikard.

References