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[alingus]

So I've made miR30-based shRNA constructs and for reasons I won't go into I've decided to put them in front of a U6 promoter. My question is, does it really matter which orientation it goes in? That is, there is a 5' and 3' miR30 sequence flanking my shRNA, but I got some of my constructs such that they're U6 promoter-3'miR30-shRNA-5'miR30. I've also taken my constructs and folded them (http://rna.tbi.univi...bin/RNAfold.cgi) and they look somewhat different.

Other than that, I don't know if the secondary structure of the flanking stuff really matters that much. I'd be happy if it still processes well. Any thoughts?