Heterologous higher order control modalities will be important tools for targeted multigene interventions in next-generation gene therapy, tissue engineering, and sophisticated gene-function studies. In this study, we present the design and rigorous quantitative analysis of a variety of different dual-regulated gene transcription control configurations combining streptogramin- and tetracycline-responsive expression systems in a one-vector format. Quantitative assessment of dual-regulated expression performance in various mammalian and human cell lines is based on two compatible secreted reporter genes, SEAP, the human placental secreted alkaline phosphatase, and the recently developed SAMY, the secreted alpha-amylase. Assembly of streptogramin-and tetracycline-responsive transgene control units in consecutive (--> -->), divergent (<-- -->), and convergent (--> <--) orientation showed excellent regulation characteristics in most genetic arrangements exemplified by neglectable interference and high transgene induction ratios in all four control settings (ON/ON, OFF/ON, ON/OFF, OFF/OFF). The overall regulation performance of divergent dual-regulated expression configurations could be substantially increased when placing noncoding stuffer fragments or insulator modules between the divergently oriented antibiotic-responsive promoters. Dual-regulated expression technology pioneers artificial higher order gene control networks that will likely enable new opportunities in multigene metabolic engineering and generate significant therapeutic impact.