Autonomous and self-sustained oscillator circuits mediating the periodic induction of specific target genes are minimal genetic time-keeping devices found in the central and peripheral circadian clocks. They have attracted significant attention because of their intriguing dynamics and their importance in controlling critical repair, metabolic and signalling pathways. The precise molecular mechanism and expression dynamics of this mammalian circadian clock are still not fully understood. Here we describe a synthetic mammalian oscillator based on an auto-regulated sense-antisense transcription control circuit encoding a positive and a time-delayed negative feedback loop, enabling autonomous, self-sustained and tunable oscillatory gene expression. After detailed systems design with experimental analyses and mathematical modelling, we monitored oscillating concentrations of green fluorescent protein with tunable frequency and amplitude by time-lapse microscopy in real time in individual Chinese hamster ovary cells. The synthetic mammalian clock may provide an insight into the dynamics of natural periodic processes and foster advances in the design of prosthetic networks in future gene and cell therapies.