Difference between revisions of "Part:BBa K4630201:Design"

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The plasmid is temperature sensitive.
 
The plasmid is temperature sensitive.
  
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As the length of the segments vary, we decided to remove redundant part step by step, especially the functional sgRNA. We designed primers for the backbone to delete the sgRNA sequence using Gibson Assembly and Golden Gate Assembly (fig 1).
  
  

Revision as of 17:11, 11 October 2023


The optimized pCas


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1344
    Illegal EcoRI site found at 7542
    Illegal EcoRI site found at 11785
    Illegal SpeI site found at 5392
    Illegal PstI site found at 11075
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1344
    Illegal EcoRI site found at 7542
    Illegal EcoRI site found at 11785
    Illegal NheI site found at 1103
    Illegal SpeI site found at 5392
    Illegal PstI site found at 11075
    Illegal NotI site found at 5711
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1344
    Illegal EcoRI site found at 7542
    Illegal EcoRI site found at 11785
    Illegal BglII site found at 4205
    Illegal BamHI site found at 3382
    Illegal BamHI site found at 7476
    Illegal BamHI site found at 11664
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1344
    Illegal EcoRI site found at 7542
    Illegal EcoRI site found at 11785
    Illegal SpeI site found at 5392
    Illegal PstI site found at 11075
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1344
    Illegal EcoRI site found at 7542
    Illegal EcoRI site found at 11785
    Illegal SpeI site found at 5392
    Illegal PstI site found at 11075
    Illegal NgoMIV site found at 10624
    Illegal AgeI site found at 7311
    Illegal AgeI site found at 8973
    Illegal AgeI site found at 11499
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 4231
    Illegal BsaI.rc site found at 4219
    Illegal SapI site found at 7293
    Illegal SapI site found at 10564
    Illegal SapI site found at 10774
    Illegal SapI site found at 11481


Design Notes

The plasmid is temperature sensitive.


As the length of the segments vary, we decided to remove redundant part step by step, especially the functional sgRNA. We designed primers for the backbone to delete the sgRNA sequence using Gibson Assembly and Golden Gate Assembly (fig 1).


Source

Zhao, D., Yuan, S., Xiong, B. et al. Development of a fast and easy method for Escherichia coli genome editing with CRISPR/Cas9. Microb Cell Fact 15, 205 (2016).

References

Zhao, D., Yuan, S., Xiong, B. et al. Development of a fast and easy method for Escherichia coli genome editing with CRISPR/Cas9. Microb Cell Fact 15, 205 (2016).