Does transcription terminate within the unc-54 3’ UTR seqeunce commonly used to flank reporters? If not is there a transcriptional termination sequence I could clone in after it?
The unc-54 3’UTR sequence in most of the Fire vectors includes the unc-54 3’UTR and even part of the next downstream
gene, aex-5. The vast number of successful reporter genes that express well (at least, zygotically) using this 3’UTR fragment
argues strongly that there is a good universal transcription termination sequence in there.
But, other sequences might work better depending on the context. In the early embryo I found the 3’UTR of another gene
resulted in slightly earlier appearance of GFP with all else equal. For germline expression the pie-1 3’UTR has been used
successfully.
If you have concerns, you could always take your gene of interest and insert GFP into it, keeping all coding regions, upstream
and downstream regions, and introns in the reporter.
There is some concern that aex-5 sequences in the unc-54 3’UTR may drive some gut expression, a commonly observed background in many transgenics. That said, vectors with that unc-54 3’UTR have been used by almost every lab that does transgenics. If you want an alternative, I think the let-858 3’UTR, used widely in the later Fire kit vectors, has been well characterized. If you’re concerned about tissue-specific expression, I’d go with let-858 3’UTR.
OK, thanks. Unfortunate it’s become known as the ‘unc-54 3’UTR’ because as Morris points out above (and a simple BLAST confirms), it’s actually a rather large 3’ flanking region which is therefore very likely going to contain all required termination signals.