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Plasmids, which are currently used in interleukin 12 (IL-12) gene electrotransfer (GET) clinical trials in the USA, contain antibiotic resistance genes and are thus, according to the safety recommendation of the European Medicines Agency (EMA), not suitable for clinical trials in the EU. In the current study, our aim was to prepare an IL-12 plasmid without an antibiotic resistance gene and test its functionality and toxicity after GET in a preclinical B16F10 mouse melanoma model. The antibiotic resistance-free plasmid encoding the human IL-12 fusion gene linked to the p21 promoter, i.e., p21-hIL-12-ORT, was constructed using operator-repressor titration (ORT) technology. Next, the expression profile of the plasmid after GET was determined in B16F10 cells and tumors. Additionally, blood chemistry, hematological and histological changes, and antitumor response were evaluated after GET of the plasmid in melanoma tumors. The results demonstrated a good expression and safety profile of the p21-hIL-12-ORT GET and indications of efficacy. We hope that the obtained results will help to accelerate the transfer of this promising treatment from preclinical studies to clinical application in the EU.