Part:BBa_K1463002:Design
Recombinase Switch With GFP (weaker RBS)
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 82
Illegal NheI site found at 105 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 62
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 938
Design Notes
The promoter had to face leftwards, the attP and attB sites had to be far enough apart to recombine efficiently and be in head-to-head (inverted repeat) orientation. GFP had to be to the right of the switch.
Restriction sites in switch - HindIII
HindIII was designed into the switch downstream of the promoter. This allowed for confirmation of the "flip" of the switch by restriction digest.
Using both HindIII and PstI produces different sized fragments dependent on the orientation of the switch promoter.
Transcriptional terminators were designed into the switch construct in order to prevent transcriptional leakage of GFP when the switch is in the "OFF" position.
A spacer was included in the switch design to ensure the att sites were a suitable distance apart for the recombinase enzyme to work without steric hindrance.
Source
Composite biobrick consisting of attP, reversed BBa_J23100 promoter, forward facing BBa_B0010 terminator, PhiC31 attB site and the BBa_E0040 GFP gene with a medium strength BBa_B0032 ribosome binding site.