Coding

Part:BBa_K4212002:Design

Designed by: Shirin Bamezai   Group: iGEM22_Imperial_College_London   (2022-09-29)


CotG_4Gx3_ChiS


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 894
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 894
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 894
    Illegal BamHI site found at 879
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 894
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 894
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

4Gx3 - flexible linker


Source

The CotG CDS comes from the genome of B. subtilis, and was produced through genome amplification with appropriately designed primers. The reverse primer included the sequence for the synthetic flexible linker. The chitinase protein sequence comes from the B. pumilus, however this was codon-optimised for B. subtilis and synthesized as a gBlock (IDT). The individual CDSs were also either ordered or amplified with Type IIS restriction sites on either end, to enable scarless fusion as a single CDS in a level 0 plasmid within a Golden Gate system.

References