Difference between revisions of "Part:BBa K4321005:Design"
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===Source=== | ===Source=== | ||
| − | + | pCG004 originates from the pHT01 plasmid, however this plasmid contains two alterations. The first is the removal of the backbone BsaI site within the AmpR cassette. Secondly, the multiple cloning site was replaced with a BsaI dropout consisting of a constitutive GFP mut3b cassette. This GFP cassette is expressed by the Pveg promoter, spoRBS and the rmB terminator. pCG004 was obtained directly from addgene. | |
===References=== | ===References=== | ||
Tran, D. T., Phan, T. T., Doan, T. T., Tran, T. L., Schumann, W., & Nguyen, H. D. (2020). Integrative expression vectors with Pgrac promoters for inducer-free overproduction of recombinant proteins in bacillus subtilis. Biotechnology Reports, 28. https://doi.org/10.1016/j.btre.2020.e00540 | Tran, D. T., Phan, T. T., Doan, T. T., Tran, T. L., Schumann, W., & Nguyen, H. D. (2020). Integrative expression vectors with Pgrac promoters for inducer-free overproduction of recombinant proteins in bacillus subtilis. Biotechnology Reports, 28. https://doi.org/10.1016/j.btre.2020.e00540 | ||
Revision as of 20:54, 8 October 2022
E.coli to Bacillus subtilis shuttle plasmid (pCG004)
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 9048
Illegal suffix found in sequence at 1
Illegal EcoRI site found at 3615
Illegal EcoRI site found at 6835 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 3615
Illegal EcoRI site found at 6835
Illegal EcoRI site found at 9048
Illegal NheI site found at 3451
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NotI site found at 9
Illegal NotI site found at 9054 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 3615
Illegal EcoRI site found at 6835
Illegal EcoRI site found at 9048
Illegal BglII site found at 361
Illegal BglII site found at 6306
Illegal XhoI site found at 365 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 9048
Illegal suffix found in sequence at 2
Illegal EcoRI site found at 3615
Illegal EcoRI site found at 6835 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 9048
Illegal EcoRI site found at 3615
Illegal EcoRI site found at 6835
Illegal XbaI site found at 9063
Illegal SpeI site found at 2
Illegal PstI site found at 16 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 6234
Illegal BsaI.rc site found at 5170
Illegal SapI.rc site found at 2338
Illegal SapI.rc site found at 7459
Design Notes
pCG004 contains a dropout green fluorescence protein (GFP) which is flanked by two BsaI sites. Replacement of this dropout GFP with our Cyt cassettes (K4321007 and K4321009) was achieved by BsaI digestion and ligation. Upstream of the dropout GFP is a strong IPTG inducible promoter, Pgrac. Insertion of desired genes into this dropout region will enable users to inducible control the expression of downstream genes.
Source
pCG004 originates from the pHT01 plasmid, however this plasmid contains two alterations. The first is the removal of the backbone BsaI site within the AmpR cassette. Secondly, the multiple cloning site was replaced with a BsaI dropout consisting of a constitutive GFP mut3b cassette. This GFP cassette is expressed by the Pveg promoter, spoRBS and the rmB terminator. pCG004 was obtained directly from addgene.
References
Tran, D. T., Phan, T. T., Doan, T. T., Tran, T. L., Schumann, W., & Nguyen, H. D. (2020). Integrative expression vectors with Pgrac promoters for inducer-free overproduction of recombinant proteins in bacillus subtilis. Biotechnology Reports, 28. https://doi.org/10.1016/j.btre.2020.e00540