Promoter distance from ATG - (Jun/17/2013 )

Hey all,

I'm planning to clone a fairly complex construct for conditional shRNA, and one step is to include a tissue specific promoter 5' of the whole thing. I've never had experience placing promoters far from the start site in transgenic constructs. How far away can one place a promoter (in this case the Gfa2 glial-specific promoter) from a downstream CDS start site?

There simply aren't many restriction sites in my vectors that would allow me to get closer than ~250bp. As it stands right now, the cleanest/most efficient way to insert the promoter sequence would leave a gap of 277 base pairs. Will this be sufficient to drive expression?

Any advice is appreciated, I'm fairly new to this.

Thanks

that is far, think of an alternative.

You could amplify it with a primer that adds a restriction site (closer to the CDS). Just don't get too close that you'd cut away the Kozak.

That's exactly what I've decided to do. Minor annoyance, but looks like it will work well.

Thanks for the input.