Plasmid_Backbone

Part:BBa_K5103005

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


PCG004 plasmid cut with BsaI for cloning Cry8Da

Sequence and Features


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


Profile

Name: PCG004 plasmid cut with BsaI for cloning Cry8Da
Base Pairs: 7947 bp
Origin: Modified version of plasmid derived form pHT01 [1]
Properties: Shuttle plasmid cut with BsaI

Usage and Biology

This part represents the pGC004 plasmid cut with the BsaI restriction enzyme, allowing for the integration of the Cry8Da gene. PCG004 is a commonly used plasmid developed for high efficiency cloning and expression in E. coli and Bacillus subtilis (B. subtilis).[1] It is derived from pHT01, to be an entry vector (“shuttle”) to replicate within both E. coli and B. subtilis and transfers a plasmid transformed within E. coli to the final destination of B. subtilis. Due to its “shuttle abilities”, it has potential applications within bioagriculture and biocontrol, where genes with insecticidal properties can be inserted into Bacillus species in the pests’ environment. Previous research has also shown the use of the PCG004 plasmid in fermented food and beverage applications.[1]

Due to the BsaI cut, this part (BBa_K5103005) is now a specific and complimentary to only part Part:BBa_K5103000. BsaI is a type IIS enzyme that cuts outside of its recognition site and causes an overhang[2]. This specificity limits this part's application to other expression vectors using the same restriction enzyme.

References

[1] Gilbert, C., Howarth, M., Harwood, C. R., & Ellis, T. (2017). Extracellular Self-Assembly of Functional and Tunable Protein Conjugates from Bacillus subtilis. ACS synthetic biology, 6(6), 957–967. https://doi.org/10.1021/acssynbio.6b00292
[2] New England Biolabs. (2020). BsaI. https://www.neb.com/en/products/r0535-bsai

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