Hi!

Yes, you can add two, three, or four genes to a single construct (although the size might be your limiting factor - how big are these genes?), the genes can be driven by the same promoter, or by a different one. If you are thinking of using two promoters (driving two genes each), these can be either in the same direction or facing each other. If you put them in the same direction, the genes driven by the second promoter will probably be less transcribed, which could be useful if the protein they encode is toxic or if you don't require a high protein expression from them. I use two promoters facing each other to hopefully reduce cross-talk and also avoid a lesser expression on one of the constructs, but that is not always the case as this can always vary depending on promoter and genes, etc.

The sequences you want to add in between the promoters to avoid interference are called insulators, and they are great not only to avoid interference if placed in between the promoters, but you can also place them flanking your construct to avoid position effects or heterochromatinization once integrated into the genome. There is a lot of literature on them.

If you are going to have the promoters facing each other remember to use the complementary antiparallel sequences of your genes and promoter etc for the second one that will read "backward" (so that it's on the bottom strand)

Good luck!

Bruno Salomone Gonzalez de Castejon thanks a lot for your answer. So, in my case I would need 2 different promoters, each of them drives one gene. In particular, the gene of the second promoter would be the transcription factor binding the first promoter, that's why I need to keep their expression separate. From your answer, I think I could design my construct like this: Insulator - promoter 1 - gene 1 - insulator - promoter 2 - gene 2 - insulator. Is it correct? As an insulator, I already have a 77-bp element, FII/BEAD-A (FB), which contains the minimal enhancer blocking components of the chicken β-globin 5′HS4 insulator and a homologous region from the human T-cell receptor α/δ BEAD-1 insulator (copied and pasted from literature). Do you think it could be ok? Thanks again!

Hi Alessio,

Yes, that sequence should work!!

Good luck :)

There should be a reasoanble spacer between two genes, alongwith all the ingredients required (Promoter etc) for each genes independent from each other. It is equally laborious to go with same construct keeping it independent.