Inserting promoters upstrem of reporter genes - (Oct/19/2006 )
Hi, I want to test a number of characterised promoters by inserting them upstream of the eGFP gene. Some of these promoters have the characteristic TATA sequences and some don't. Does anybody know if there is any specific criteria in terms of where the first ATG start codon needs to be in relation to the promoter e.g. does it have to be in frame with any particular sequence? How close should it be to the promoter sequence, etc. Thanks!
in some cases i have seen the promoter being quite close to the ATG, like 10-15 bases. and in some more than 70 bases. both work. but some people advised us to have something like 50 bases between the end of promoter and ATG.
one has to try it out to really find out if they work or not.
What I usually do is, take 2kb upstream sequence of predicted ATG and take about 20bp ahead of ATG. I make sure that ATG is in frame with GFP sequence and PCR up promoter region as well as GFP region and stich them together.
When you do this stitching stuff, reverse primer of promoter region should have hanging GFP forward primer so that there is a small region of 20bp where this stitching takes place and make sure of the frame so I usually try to take 3-4 frames after ATG frame.