Many vectors expressing shRNA or microRNA bear the U6 promoter, but what is the exact transcription start site? I have heard people put an additional G before their small RNA insert, is this G the start site? Can anyone provide an example to show how this is defined?
The "G" that you mentioned is in fact the "preferred" transcription start site, however, this can be replaced by any other nucleotide. It's just that U6 prefers "G" as the first transcribed nucleotide and that makes the transcription more efficient. You will find many examples of this in vectors available for transcription of shRNA or gRNA (for CRISPR) system. Just look for a restriction enzyme after U6 that is used for cloning a short RNA gene. You'll figure out the exact position of the transcription start site.
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