Part:BBa_K2715044:Design
Constitutive expression of dCas9, and regulatory region of Toxin A in C. difficile driving GusA
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1515
Illegal XbaI site found at 4831
Illegal XbaI site found at 5047 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1515
Illegal NheI site found at 1274 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1515
Illegal BamHI site found at 3553 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1515
Illegal XbaI site found at 4831
Illegal XbaI site found at 5047 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1515
Illegal XbaI site found at 4831
Illegal XbaI site found at 5047 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
This part contains a dcas9 coding sequence under constitutive expression of a strong clostridial promoter Pcac_thl, itself characterised in the following part BBa_K2715001. It also contains the regulatory region and promoter for Toxin A, driving expression of the reporter gene gusA, and has the alternative sigma factor required for activity of the promoter under control of the clostridial ferredoxin promoter, itself characterised in the following part BBa_K2715002. Each of these 3 modules are separated by a terminator sequence BBa_K2715014 , in order to represent read-through of the strong promoters into the native Toxin A promoter. The composite module is not RFC10 compatible because the dCas protein that we used contains restriction sites, we are not physically submitting this module but we do use it for experiments demonstrated on the wiki.
Source
The dcas9 is from S. pyrogenes, the promoter is from C. acetobutylicum, the toxin A regulatory regions including TcdR are from C. difficile, and the GusA protein is taken from E. coli.