Since you wish to express two gene under the same promoter, you can use an IRES sequence, or other such elements, between the genes to create a bicistronic gene. Alternatively, you can use a vector containing a bidirectional promoter, such as the pBI backbone. In this case you clone each gene into the backbone separately.

I have worked with IRES sequences and I have found that using a viral 2A sequence usually works better.  The problem with using IRES (at least the EMCV IRES) is that the expression from the second gene is usually much lower than the upstream gene. Using a 2A sequence, the two proteins are produced at similar levels.  The only downside is that there is a small (~20 aa) addition to the C-terminus of the upstream gene.  I have never had this be a problem, but it is something you can check out. 

We routinely use the P2A sequence (see below link for more information).

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0018556

Good luck

We can do it. In my case, I clone them into different fluorescence color (CFP, GFP, mCherry). They are expressed well but in some case, one protein will be dominant compared to others. 

I would concur with Jonathan.

I have never worked with the 2A sequence, only with IRES which worked excellent in plasmids I introduced into the avian embryo. Yet, I have heard from people who used the 2A that it is better.

Dear Ramachandra,

The GAL10-GAL1 promoter is bidirectionnal. So using a yeast vector with this natural promoter allows you to express two different coding sequences under the control of a single promoter. Please have a look at the attached paper.

Best wishes

Prof Philippe Urban

The answer given by Jonathan looks so interesting. I just wanted to know is that P2a works in yeast system?