Plasmid_Backbone

Part:BBa_K5103005:Design

Designed by: Nikoo Mansourian   Group: iGEM24_Guelph   (2024-09-22)


PCG004 plasmid cut with BsaI for cloning Cry8Da


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 595
    Illegal EcoRI site found at 6398
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 595
    Illegal EcoRI site found at 6398
    Illegal NheI site found at 6234
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 595
    Illegal EcoRI site found at 6398
    Illegal BglII site found at 66
    Illegal BglII site found at 3147
    Illegal XhoI site found at 3151
  • 23
    INCOMPATIBLE WITH RFC[23]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 595
    Illegal EcoRI site found at 6398
  • 25
    INCOMPATIBLE WITH RFC[25]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 595
    Illegal EcoRI site found at 6398
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal SapI.rc site found at 1219
    Illegal SapI.rc site found at 5124


Design Notes

We specifically engineered Cry8Da part: BBa_K5103000 to be compatible with this part, through using BsaI, a type IIS enzyme. A benefit of type IIS enzymes is that cuts outside its recognition sequence (GGTCTCN | NNNN NNN), allowing the creation of custom overhangs for flexible cloning. It enables seamless, scar-free ligation of DNA fragments and is commonly used in Golden Gate Assembly for efficient multi-part assembly.

Source

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References