Vectors with a CMV promoter [e.g. pmaxCloning™, Lonza, Cat. No. VDC-1040] typically work fine for transient protein expression in mammalian cells, like HEK293, CHO or NSO. For insect cells, like Sf9 or Sf21, vectors with insect promoters are required. In HEK293E (EBNA) cells, the use of vectors containing EBV oriP can further boost the transient expression yield. In HEK293T cells the use of vectors containing SV40 ori can also boost the transient expression yield. We do not recommend use of IRES sequences, since the gene encoded 3' of the IRES sequence is usually expressed to a lesser extent than the upstream gene, and in some experimental conditions may not be expressed at all. As alternatives we suggest: either co-transfecting two (or more) plasmids, using one plasmid with each gene under the control of its own promoter, or making a fusion protein.