Stop codon before GFP - Stop codon before GFP (Aug/03/2006 )

hi guys...i'll be cloning a gene upstream of GFP...wanted to know if the presence of a stop codon in my gene...will inhibit GFP production...please let me know.

it shouldn't be a problem and would be ideal for your gene of interest. You need to make sure the promoter for the GFP remains intact.

If its a second expression cassette, it shouldnt b a problem. But if one of the promoter is stronger than the other, there might b some problems with expression of the one with weaker promoter (expression could b weaker than predicted).

No one can actually predict it. One has to try the constructs and check expression of both proteins.

it needs be removed if you want a fusion protein .......

I'm with aussieUK and was wondering the same thing myself...if you are using the GFP to monitor expression of your GOI, then the stop codon must go because they will be expressed independently

my vector has an IRES...so i should be okay even with the stop codon...right?

is the ires in between your GOI and the GFP?
if not, then yep, get rid of that stop codon!

aussie gets the point.
I assume you have your first ORF with the STOPcodon, IRES and then the GFP.
f so, that's an appropriate construction.