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Promoter distance from ATG

Promoter expression construct

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3 replies to this topic

#1 miST32

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Posted 17 June 2013 - 04:11 PM

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

#2 Curtis

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Posted 17 June 2013 - 07:50 PM

that is far, think of an alternative.

#3 Glynn

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Posted 19 June 2013 - 05:39 AM

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.

#4 miST32

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Posted 20 June 2013 - 04:58 PM

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

Thanks for the input.





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