Although there are several inducible expression systems for mammalian cells, the most reliable one is the tetracycline-regulated expression system. This system is well-established and widely used by many researchers. Although Clontech provides several types of cells that stably express reverse tetracycline transactivator (rtTA), the cells that are not provided can be generated with pTet-On-Advanced by first integrating this plasmid into the require type of cell and then introducing the genes of interest. These processes are experimental bottlenecks. To improve this situation, we synthesized an all-in-one vector, termed pMAK17, which enables constitutive expression of puromycin N-acetyltransferase, modified Discosoma red fluorescent protein, and rtTA, as well as P(Tight)-driven enhanced green fluorescent protein (EGFP). The pMAK17-transfected cells could be successfully induced to express EGFP, were selectable by fluorescence-activated cell sorting, and displayed puromycin resistance.