How base pairs upstream of gene to include for the promoter? - (Jan/26/2013 )
I want to amplify and clone my gene of interest along with its native promoter. My question is how many base pairs should be included to constitute the promoter. A lot of papers I read include 1500 or 2000 bp upstream. However, I noticed for my gene there is another gene located immediately upstream. From the stop codon of the upstream gene to the start codon of my gene of interest is only 303 bp. If the 3'UTR of the upstream gene is considered, there is only 92 bp between them. So how much should I include if I want the promoter? 303 bp? Could I just include 1500 or even 2000 bp even though it is part of another gene? Thanks for the help.
Are you working with a bacterium or with a eukaryotic species? There is a huge difference in the size and complexity of promoter sequences. If you are working with bacteria (likely, given your distance) then the 300 bp should be enough.
This is hard to decide, depending on the purpose of your study. If you want the promoter to have similar activity as the endogenous one, I'd say you need 3-5kb. if you just need some basal promoter activity, 1 kb should be good.
Have any one characterized the promoter in published paper? If yes, the study should give your some idea how big the core promoter is.
I'm working in a eukaryotic species. In plants. I have a mutant in the gene so I want to transform the plant with the promoter and gene to rescue the mutant phenotype. So it would be nice if it had similar activity as the endogenous gene.
Nothing published on the promoter or even on the gene in general.