Plasmid
Part:BBa_K3697010:Design
Designed by: Lauren Ramlan, Christopher Neimeth Group: iGEM20_Stanford (2020-10-22)
mCherry_BSU Plasmid
Assembly Compatibility:
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 4308
Illegal XbaI site found at 1568
Illegal SpeI site found at 1472
Illegal PstI site found at 117
Illegal PstI site found at 1713
Illegal PstI site found at 3862
Illegal PstI site found at 4314 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 4308
Illegal SpeI site found at 1472
Illegal PstI site found at 117
Illegal PstI site found at 1713
Illegal PstI site found at 3862
Illegal PstI site found at 4314 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 4308
Illegal BglII site found at 1585
Illegal XhoI site found at 1218 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 4308
Illegal XbaI site found at 1568
Illegal SpeI site found at 1472
Illegal PstI site found at 117
Illegal PstI site found at 1713
Illegal PstI site found at 3862
Illegal PstI site found at 4314 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 4308
Illegal XbaI site found at 1568
Illegal SpeI site found at 1472
Illegal PstI site found at 117
Illegal PstI site found at 1713
Illegal PstI site found at 3862
Illegal PstI site found at 4314
Illegal NgoMIV site found at 1254
Illegal AgeI site found at 3532 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 291
Illegal BsaI site found at 5940
Illegal BsaI site found at 5972
Illegal BsaI.rc site found at 5928
Illegal BsaI.rc site found at 5960
Design Notes
This plasmid was already optimized for use in B. subtilis, but we had to consider which expression level and resistances were optimal for our purposes. We selected pVeg as the mCherry_BSU promotor due to its high expression levels in B. subtilis, allowing us to easily visualize the production of the mCherry reporter. We selected Kanamycin resistance in B. subtilis because KanR RNA was the target for the RNA toehold system we were testing. With KanR on the plasmid, the target for our detection system would be produced in the cell, and could be easily verified by plating on selective media.
Source
Pamela Silver Laboratory at Harvard Medical School