Hi there,

So there is a 175bp region between the 2 stop codons, is that right? If yes, the essential thing is that cassette insertion within this 175bp region won't be disrupting any ORF. About mRNA stability, of course modifying 3'UTR might have an effect but if you compare the phenotype of the mutants to the wt bearing the same cassette at the same place it should be fine. Eventually you could check the possible cassette insertion effect on transcription of the 2 ORFs by comparing Northern run on wt strains with and without cassette insertion.

If there is really only 175 bp of non-coding region between two coding regions (ORFs) facing each other, then you should carefully check the sequence in both directions and look for consensus signals. Polyadenylation signals in yeasts are not the same as in mammals and may be confined to a smaller sequence, check out the following link so that you see what I mean:

https://aghunt.wordpress.com/2008/06/29/polyadenylation-signals/

To keep the two existing genes happy, they both need to end with an intact polyadenylation signal. You have to insert your new gene inbetween the two, and your HYG ORF should be flanked by its own promoter and its own polyadenylation signal.

Also please be advised that polyadenylation should not be confused with transcription termination as we know it in prokaryotes. The RNApolymerase II in eukaryotes carries on transcribing, and termination is ill-defined and can happen more than 1 kb after the stop codon. Cleaving the nascent RNA and subsequent polyadenylation is done by other proteins, and the RNA polymerase simply continues.