Part:BBa_K2176001:Design
Cassette for the constitutive production of GAL4-KaiCp and LexA-SasA, linked by a self-cleaving P2A
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 6
Illegal SpeI site found at 4479
Illegal PstI site found at 4465 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 6
Illegal SpeI site found at 4479
Illegal PstI site found at 4465
Illegal NotI site found at 12
Illegal NotI site found at 4470 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 6
- 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 6
Illegal SpeI site found at 4479
Illegal PstI site found at 4465 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 6
Illegal XbaI site found at 21
Illegal SpeI site found at 4479
Illegal PstI site found at 4465 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 206
Illegal BsaI.rc site found at 3479
Design Notes
We chose to express both fusion proteins in one cassette, joined by a self-cleaving linker, in order to obtain equal stoichiometric amounts in the cell.
In order to join the promoter and terminator to the protein coding sequence, we used two restriction enzymes, BamHI and BglII, which produce the same sticky ends when cut but have different recognition sites due to differences in the base pairs flanking the sticky ends. The promoter was cut with BamHI, while the side of the protein coding sequence it was to be joined to was cut with BglII. These two were then able to ligate together, which produced a novel 6bp sequence that is half-BamHI site, half-BglII site, and as a result is a recognizable cut site to neither. The same procedure was used to join the protein coding sequence and the terminator.
Source
The promoter and terminator in this cassette were amplified from the yeast genome, using primers that added extra overhangs with novel restriction enzyme cut sites on either end: the BioBrick prefix (for the promoter) or suffix (for the terminator) on one side, and an overhang with a cut site for ligating the part to the protein coding sequence on the other side. The protein coding domain was synthesized in G-blocks and constructed via Gibson assembly. None of these components came from their own BioBricks, which is why we've entered the composite as a single "Basic" part.