Technically speaking it could work. Because the new proximal promoter will be present pretty much in the same vicinity as the old one. However, if the transcription start site is disturbed by your new cloning you may end up with a mutated GFP product. If you look at the parent vector (pFPV25) MCS it starts with EcoRI and ends with XbaI. These sites are still present in the same order in pFPV25.1. The best bet would be to excise the constituive promoter using these restriction enzymes and clone your new promoter in these sites if the sequence allows for it.

Technically speaking it could work. Because the new proximal promoter will be present pretty much in the same vicinity as the old one. However, if the transcription start site is disturbed by your new cloning you may end up with a mutated GFP product. If you look at the parent vector (pFPV25) MCS it starts with EcoRI and ends with XbaI. These sites are still present in the same order in pFPV25.1. The best bet would be to excise the constituive promoter using these restriction enzymes and clone your new promoter in these sites if the sequence allows for it.

Hello Adam,

your question is a bit unusual, since promoters normally don't come with MCSs within... They might have some unique sites that cut within, but they just happen to be there.

Now, concerning your specific plan: the pFPV25 vector is a promoter trap vector for bacteria. I'm not familiar with the specific vector, but since it is designed to express the eGFP in bacteria, it is probably designed so to insert the promoter with the transcription start site before the Shine-Dalgarno sequence, in order to obtain optimal eGFP translation.

Now, you have the pFPV25.1 plasmid, which if I get it right contains already the Salmonella rpsM promoter cloned EcoRI/XbaI. If you insert a new promoter somewhere in the middle of this rpsM promoter, you should at least check that you don't generate an in frame ATG before the eGFP proper ATG, because this might compete partially (probably will not have a SD sequence in front) for translation and decrease your eGFP yield. But, honestly, it would be a much cleaner approach to remove the rpsM promoter with and EcoRI/XbaI digestion and clone the new promoter in its place...

Good luck.

In my opinion you must remove with restriction enzyme the old promotor and insert the new promotor .

Otherwise if you need the old promotor in your plasmid, you can add a stop codon by pcr in your primer together with the restriction enzyme and the begining of you interest promotor for clonning, in order to not disturb the old promotor. I mean STOPcodon sequence+restriction enzyme for cloning+sequence of new promotor. The primer must not  be necessary 25-30 nt it can be larger or you can use mutagenesis kit  NEB in order to insert, delete etc.

Good look

O performance the strategies before described by colegues