Difference between revisions of "Part:BBa K3037002:Design"

(→‎Design Notes)
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Primer 3: CATAATAAGGAATaCGAAAAGTCAAG
 
Primer 3: CATAATAAGGAATaCGAAAAGTCAAG
 
Primer 4: gatcTCTGCAGCGGCCGCTACTAGTAttaaccggtGTCACCTCCTAGCTGACTCAAATC
 
Primer 4: gatcTCTGCAGCGGCCGCTACTAGTAttaaccggtGTCACCTCCTAGCTGACTCAAATC
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[[File:DCas9 site directed mutagenesis.jpeg|center|400px|thumb|none|dCas9 site directed mutagenesis]]
  
 
===Source===
 
===Source===

Revision as of 00:31, 19 October 2019


dead CRISPR Associated Protein (dCas9)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1096
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 3375
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

In the middle of the coding sequence there was an EcoRI site. As a forbidden restriction enzyme site, this needed to be mutated. Therefore a site directed mutagenesis PCR was preformed with the following primers:

Primer 1: gatcGAATTCGCGGCCGCTTCTAGATAAGGAGGTCAAAAATGgccggcGATAAGAAATACTCAATAGGC Primer 2: CATAATAAGGAATaCGAAAAGTCAAG Primer 3: CATAATAAGGAATaCGAAAAGTCAAG Primer 4: gatcTCTGCAGCGGCCGCTACTAGTAttaaccggtGTCACCTCCTAGCTGACTCAAATC

dCas9 site directed mutagenesis

Source

Synthesized by Integrated DNA Technologies (IDT)

References