3 replies to this topic

[huseler23]

In order to clone a promoter, does it require ATG start site?

[perneseblue]

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.

[huseler23]

perneseblue, on 03 January 2011 - 05:56 PM, 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?

[perneseblue]

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)