Difference between revisions of "Part:BBa K524005:Design"
(→‎Design Notes)
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===Design Notes===
 
===Design Notes===
  
−
The heat sensitive replication origin oriR101 & repA101-ts from the pKD46 backbone was replaced by the ori-gamma of the R6K origin of replication. Since the function of the nascent orf60a was unknown, it was retained in pToolkit.
+
The heat sensitive replication origin oriR101 & repA101-ts from the pKD46 backbone was replaced by the ori-gamma of the R6K origin of replication. Since the function of the adjacent orf60a to oriR101 & repA101ts was unknown, it was retained in pToolkit.
  
 
===Source===
 
===Source===

Revision as of 14:01, 5 October 2011

pToolkit


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1215
    Illegal EcoRI site found at 2724
    Illegal PstI site found at 2219
    Illegal PstI site found at 2466
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1215
    Illegal EcoRI site found at 2724
    Illegal PstI site found at 2219
    Illegal PstI site found at 2466
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1215
    Illegal EcoRI site found at 2724
    Illegal BamHI site found at 1154
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1215
    Illegal EcoRI site found at 2724
    Illegal PstI site found at 2219
    Illegal PstI site found at 2466
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1215
    Illegal EcoRI site found at 2724
    Illegal PstI site found at 2219
    Illegal PstI site found at 2466
    Illegal AgeI site found at 989
    Illegal AgeI site found at 2646
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 4422
    Illegal SapI site found at 971


Design Notes

The heat sensitive replication origin oriR101 & repA101-ts from the pKD46 backbone was replaced by the ori-gamma of the R6K origin of replication. Since the function of the adjacent orf60a to oriR101 & repA101ts was unknown, it was retained in pToolkit.

Source

The backbone of the pToolkit, including the arabinose inducible lambda RED recombination system and the ampicillin resistance gene (bla) comes from the RED recombinase plasmid pKD46, courtesy of E.coli Genetic Resources at Yale CGSC, The Coli Genetic Stock Center.

References

Datsenko KA, Wanner BL.(2000).One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5.

F Wu, I Goldberg, and M Filutowicz.(1992).Roles of a 106-bp origin enhancer and Escherichia coli DnaA protein in replication of plasmid R6K, Nucleic Acids Res. 1992 February 25; 20(4): 811–817.

F Wu, I Goldberg, and M Filutowicz.(1994).Binding of DnaA protein to a replication enhancer counteracts the inhibition of plasmid R6K gamma origin replication mediated by elevated levels of R6K pi protein, J Bacteriol. 1994 November; 176(22): 6795–6801.