I am trying to design a plasmid to be synthesised with two genes in the MCS, and appropriate RE’s. I will then subclone this into a number of other vectors pET and one other.
The question I have is – do i have to include a specific sequence between the two genes as a transcription initiation of gene 2? Or can i go straight from the stop codon of gene 1 to the start codon of gene 2?
in what organism will you transcribe it? Bacteria (but probably every organism) should be able to do transcriptional fusion, so that two genes would be transcribed from one promoter, but the second gene would have lower expression. If you want the same level of expression, you should probably use two promoters.
Or use a novagen Duet plasmid. They are designed for co-expression study and each gene has an independant initiation sequence, with two MCS and everything to make sure that it is easy to have your 2 proteins expressed at the same level.