Coding

Part:BBa_K3037002:Design

Designed by: Arnau Pérez Roig   Group: iGEM19_TU_Dresden   (2019-10-03)
Revision as of 16:46, 21 October 2019 by ArnauP (Talk | contribs) (→‎Design Notes)

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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

The primers used to adapt this BioBrick to the Freiburg RFC25 standard were the following ones:

Prefix: GAATTCGCGGCCGCTTCTAGATAAGGAGGTCAAAAATGgccggc

Suffix: accggttaaTACTAGTAGCGGCCGCTGCAG

Find more information in here

Source

Synthesized by Integrated DNA Technologies (IDT)

References