Part:BBa_K4249002:Design
The expression cassette carrying an ADH1 promoter, NcEgt1 coding sequence and an CYC1 terminator
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1461
Illegal EcoRI site found at 2788
Illegal XbaI site found at 2145
Illegal XbaI site found at 3375 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1461
Illegal EcoRI site found at 2788
Illegal NotI site found at 3362 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1461
Illegal EcoRI site found at 2788
Illegal BglII site found at 1323
Illegal BglII site found at 1809
Illegal BglII site found at 2142
Illegal BglII site found at 2811 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1461
Illegal EcoRI site found at 2788
Illegal XbaI site found at 2145
Illegal XbaI site found at 3375 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1461
Illegal EcoRI site found at 2788
Illegal XbaI site found at 2145
Illegal XbaI site found at 3375 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 90
Design Notes
During the experiment, we found that some regions of the promoter sequence templates are rich in GC residues, which tend to fold into complex secondary structures and might not melt during the annealing phase of the PCR cycle. Also, the primers used to amplify GC-rich regions often have a high capacity to form self- and cross-dimers and a strong tendency to fold into stem-loop structures that can impede the progress of the DNA polymerase along the template molecule. Therefore, instead of PCR method, we obtained the gene expression cassettes by enzyme digestion.
Source
These two gene were codon-optimized for S. cerevisiae, the genes were then ordered as synthetic gene strings from Sangon Biotech (Shanghai). For the acquisition of the gene expression cassettes, taking Ba_K4249001(pTEF1-CpEgt2-tCYC) as an example, the complete coding sequence was amplified by primer pairs, and the obtained fragment was inserted into the plasmid pTEF2, which was double digested by BamHI and EcoRI. Then, the complete gene expression cassette was gained by enzyme digestion.