Nonviral gene therapy requires a high yield and a low cost production of eukaryotic expression vectors that meet defined criteria such as biosafety and quality of pharmaceutical grade. To fulfil these objectives, we designed a novel antibiotic-free selection system.The proposed strategy relies on the suppression of a chromosomal amber mutation by a plasmid-borne function. We first introduced a nonsense mutation into the essential Escherichia coli thyA gene, resulting in thymidine auxotrophy. The bacterial strain was optimized for the production of small and novel plasmids free of antibiotic resistance markers (pFARs) and encoding an amber suppressor t-RNA. Finally, the potentiality of pFARs as eukaryotic expression vectors was assessed by monitoring luciferase activities after electrotrans…