Vaccinia virus, a member of the poxvirus family, is widely used as a mammalian cell expression vector. Vaccinia virus replicates in the cytoplasm and has its own transcriptional system, making it necessary to use viral promoters. Here, we describe the design, construction and use of a 40-bp synthetic, vaccinia virus promoter with largely overlapping early and late regulatory elements. Convenient plasmid transfer vectors are depicted for expression of one or two genes under control of strong early/late promoters and allowing for thymidine kinase (TK) or antibiotic selection of recombinant viruses.