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I currently have a plasmid (pFPV25.1) which contains a promoter region with multiple cloning sights in it. I would like to place a new promoter I have isolated, and I was wondering whether I could insert the new promoter into the middle of the old promoter; would that result in the new promoter controlling the gene of interest?

What I understand is that the RNA sequence that would be transcribed will change, but the start codon for translation would be kept consistent by doing this method, resulting in the gene being controlled by the new promoter.

Thanks.

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  • $\begingroup$ If you're trying to study the new promoter, you should probably remove the old one completely. There should be restriction sites outside of the MCS that you could use to cut the old one out and insert the new one. $\endgroup$ – canadianer Sep 19 '17 at 16:52
  • $\begingroup$ If you do choose to insert the new one inside of the old one, you should find a way to convince yourself (and the reviewers) that the old one has in fact been inactivated by the insertion. $\endgroup$ – canadianer Sep 19 '17 at 16:55
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    $\begingroup$ Ok, I will sequence the plasmid would this be enough? What protocol would you recommend. $\endgroup$ – Adam Radek Martinez Sep 19 '17 at 17:12
  • $\begingroup$ I'm not saying that you need to confirm that the insertion has taken place but rather confirm that the insertion completely abrogates activity of the old promoter. I'm entirely unfamiliar with the plasmid and promoter that you are using, so it may be that this has already been done and reported in literature. However, if this is something new you are trying, you need to show that expression is controlled entirely by the new promoter and not some residual activity of the old promoter, or at least rationalize that that is the case based on the nature of the old promoter and the insertion site. $\endgroup$ – canadianer Sep 19 '17 at 17:32
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    $\begingroup$ That would be quite convincing. However, can you be sure that elements of the old promoter don't interact with the new promoter? Again, I don't know anything about the promoter and so this may not be an issue. I'm just trying to think of potential problems. $\endgroup$ – canadianer Sep 19 '17 at 17:48

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