Plasmid_Backbone

Part:BBa_K2448037:Design

Designed by: jeremy armetta   Group: iGEM17_Evry_Paris-Saclay   (2017-10-27)


pSB3K3 BsaI free


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2729
    Illegal NheI site found at 1384
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 2735
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2729
    Illegal XhoI site found at 177
    Illegal XhoI site found at 1020
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found at 2729
    Illegal suffix found at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found at 2729
    Plasmid lacks a suffix.
    Illegal XbaI site found at 2744
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal AgeI site found at 1470
    Illegal AgeI site found at 1793
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.


Design Notes

To disrupt the BsaI recognition site GGTCTC by site-directed mutagenesis we choose to change its sequence to GGTCAC. This site is present in the non coding region between the p15A origin of replication and the VF2 primer binding site, so non special considerations had to be taken into account.

Source

site directed mutagenesis on the pSB3K3 bearing the BBa_J04450 reporter.

References

[1] Engler C, Kandzia R, Marillonnet S. A one pot, one step, precision cloning method with high throughput capability. PLoS One (2008) 3, e3647.