The original attenuation of the IRES sequence was performed to allow for a greater difference between the expression levels of the upstream gene of interest and the downstream reporter gene. If the downstream reporter gene, the product of less than optimal translation efficiency, is present and detectable, it can be assumed that the upstream gene of interest is also present, to a higher degree. Please see data in our Webinar "Tips and Tricks for Successful Transfection Experiments using the Nucleofector™ Technology" The data generated by our Research department show the different expression profiles of 3 vectors in HL-60 and HUVEC cells. You can see that our pmaxGFP cloned into three different vector sites show a different expression level.