Bicistronic expression vectors have been widely used for co-expression studies since the initial discovery of the internal ribosome entry site (IRES) about 25 years ago. IRES sequences allow the 5' cap-independent initiation of translation of multiple genes on a single messenger RNA strand. Using a commercially available mammalian expression vector containing an IRES sequence with a 3' green fluorescent protein fluorescent marker, we found that sequence length of the gene of interest expressed 5' of the IRES site influences both expression of the 3' fluorescent marker and overall transfection efficiency of the vector construct. Furthermore, we generated a novel construct expressing two distinct fluorescent markers and found that high expression of one gene can lower expression of the other. Observations from this study indicate that caution is warranted in the design of experiments utilizing an IRES system with a short 5' gene of interest sequence (<300 bp), selection of single cells based on the expression profile of the 3' optogenetic fluorescent marker, and assumptions made during data analysis.