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As far as I know there is an optimum distance between a promoter and the gene for the best expression levels. What is that distance for common promoters like CMV, SV40? If you have a first hand experience about this (e.g. if you have cloned the gene way downstream of the promoter and didn't/did see expression) and share it I will be grateful.

If you have comprehensive knowledge for other promoters please feel free to add them in the list too; they don't need to be common. I just want to hear people's experience on the subject.

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    $\begingroup$ a 10bp linker is sufficient. From all your other posts I gather that you are trying to build a synthetic system. My personal strategy for things like these is to pick up sequences from the vectors in your lab that you know work quite well. $\endgroup$ – WYSIWYG Apr 10 '13 at 4:14
  • $\begingroup$ Eukaryote is rather general - different eukaryotes have vastly different regulatory systems. Are you talking about something like human or something like yeast? $\endgroup$ – Bitwise Apr 10 '13 at 12:41
  • $\begingroup$ Take a look at the sequences for some common/popular vectors for your target organism. As WYSIWYG pointed out, it doesn't need to be very big, and it can vary somewhat depending on where in the multiple cloning site you insert your gene of interest. $\endgroup$ – MattDMo Apr 10 '13 at 18:43
  • $\begingroup$ Thank you all for your comments. I did look to other common constructs and decided to leave around 40 bp between promoter and the initiation codon (this was the minimum distance I could leave because of the restriction site limitation). @Bitwise, I am using mammalian cells (specifically; fibroblast and MDCK cells). $\endgroup$ – Engin Yapici Apr 10 '13 at 19:23
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I used pEYFP-N1 (stock vector) where the spacing between the CMV promoter and the EYFP start codon is ~88 nt, and the expression is high.

I also cloned a protein 5' of the EYFP, only 4 nt after the CMV promoter. The expression of this fusion was also high. Because I was only using the chimeric protein in experiments, I did not try to assess differences in EYFP expression.

If you look here: https://synbiota.com/projects/19/sequences you'll see the basic pEYFP-N1 vector and the separation between the CMV promoter and the EYFP coding sequence.

On wiki: "The TATA element and BRE typically are located close to the transcriptional start site (typically within 30 to 40 base pairs)."

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