Introduction of specific mutations into a synthetic internal ribosome entry site (IRES GTX ) derived from the GTX homeodomain protein revealed additional transcriptional activity. This novel synthetic P GTX promoter exhibited consensus core promoter modules such as the initiator (Inr) and the partial downstream promoter elements (DPE) and mediated high-level expression of a variety of transgenes including the human vascular endothelial growth factor 121 (VEGF 121 ), the human placental secreted alkaline phosphatase (SEAP), and the Bacillus stearothermophilus-derived secreted a-amylase (SAMY) in Chinese hamster ovary cells (CHO-K1) and a variety of other mammalian and human cell lines. The spacing between Inr and DPE modules was found to be critical for promoter performance since introduction of a single nucleotide (resulting in P GTX2 ) doubled the SEAP expression levels in CHO-K1. P GTX2 reached near 70% of P SV40 -driven expression levels and outperformed constitutive phosphoglycerate kinase (P PGK ) and human ubiquitin C (P hUBC ) promoters in CHO-K1. Also, P GTX2 was successfully engineered for macrolide-inducible transgene expression. Owing to its size of only 182 bp, P GTX2 is one of the smallest eukaryotic promoters. Although P GTX2 was found to be a potent promoter, it retained its IRES GTX -specific translation-initiation capacity. Synthetic DNAs, which combine multiple activities in a most compact sequence format may foster advances in therapeutic engineering of mammalian cells.