Cloning a Promoter - (Jan/03/2011 )
In order to clone a promoter, does it require ATG start site?
The ORF of your gene starts with an ATG codon (most of the time).
Your promoter can start with any sequence. A promoter contains transcription factor binding site, the TATA box and kozak sequence.
perneseblue on Tue Jan 4 01:56:25 2011 said:
The ORF of your gene starts with an ATG codon (most of the time).
Your promoter can start with any sequence. A promoter contains transcription factor binding site, the TATA box and kozak sequence.
For an experimental sample, how far upstream must the promoter be placed?
In an experimental system?
As close as you want to. However there are a few papers that report the spacing between promoter and ATG sequence can influence gene expression. The paper reports an optimum distance. Unfortunately I can not remember the details. I usually just stick the promoter immediately adjacent to the gene of interest.
If you are expressing a gene in eukaryotic systems, you need to include an intron for efficient export of the mRNA into the cytoplasm. Some synthetic promoters have the intron included. (ie CAG promoter)