Difference between revisions of "Part:BBa K3081033"

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R1+ssrA 18bp
 
R1+ssrA 18bp
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Reference:
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[1]Wolański M, Donczew R, Zawilakpawlik A, et al. oriC-encoded instructions for the initiation of bacterial chromosome replication.[J]. Frontiers in Microbiology, 2015, 5(735):735.
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Revision as of 19:17, 21 October 2019


pBAD-dCas9-ssrA-J23119-R1+(19bp)

This composite part is the principal design of the inducible CRISPR-based DNA replication interference system, with the 19 bp sgRNA targeting to the R1+ DnaA box on E.coli genome replication initiation region, OriC. In natural situations, R1+ is a high affinity box for DnaA binding. By blocking the binding of DnaA protein to R1+ box using a 18bp sgRNA, alleviation of severe arrest and inhibition to the genome replication initiation is achieved. This part is an improvement of BBa_K3081009, which we add a degradation signal peptide ssrA to the dCas9. This largely accelerates the degradation rate of dCas9 and weakens its overinhibtion on genome replication initiation targeted to the R1 box.

T--Peking--R1%2B%2820bp%29_ssrA.gif

R1+ssrA 20bp

T--Peking--R1%2B%2819bp%29_ssrA.gif

R1+ssrA 19bp

T--Peking--R1%2B%2818bp%29_ssrA.gif

R1+ssrA 18bp

Reference:

[1]Wolański M, Donczew R, Zawilakpawlik A, et al. oriC-encoded instructions for the initiation of bacterial chromosome replication.[J]. Frontiers in Microbiology, 2015, 5(735):735.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
    Illegal NheI site found at 5459
    Illegal NheI site found at 5482
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1470
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961