Part:BBa_K4604019:Design
piG_03 (tetR_RiboK12_mazF)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 710
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 883
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1518
Design Notes
A stop codon was added to terminate translation after mazF.
Cloning of piG_03
Plasmid piG_02b (BBa_K4604018) was used as a template. For the PCR we used the general protocol for the Q5 polymerase with an annealing temperature of 62°C and an elongation time of 2 minutes. A Dpn1 digest was done at 37°C for an hour, afterwards the DNA was loaded onto a 1% agarose gel. The correct bands were cut out and extracted. Gibson Assembly was used according to the protocol to assemble the plasmid. A transformation was done and the resulting colonies after an approximate 12-14h incubation time were screened by colony PCR. DNA of potential colonies containing the insert was isolated from overnight cultures (5mL LB-medium, 34 mg/mL chloramphenicol) and sent for sequencing for correct insertion and no mutation.
Source
This BioBrick is BBa_K4604018 without the fluorescent protein mTurquoise, cloned via Gibson Assembly.