Are poly-A sequences (SV40 or BGH) necessary at the end of genes inserted into vectors?
Usually yes. If you don't have them, you're basically going to continue transcribing until you hit the next termination signal, which could be a long time. That will in many cases destabilize the mRNA and cause decreased protein expression.
The main reason you'd want to avoid them is if you work with a retrovirus or lentivirus, whose RNA genome would be interrupted by the polyA element. With these viruses, you should rely on the terminators embedded in their ITRs.
Yes. The stop codon terminates the amino acid sequence. The polyA terminates the mRNA sequence. You'll need both of those.
So if I insert a gene with a stop codon at the end of the sequence, upstream of a His-tag and the SV40 poly-a sequence, will the poly-a still be transcribed or should I remove the stop codon?
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