Plasmid size is one of the factors affecting transfection efficacy in most of the molecular genetic research studies. One effective approach for reducing plasmid size is to replace relatively large, conventional antibiotic resistance genes with the short-size gene. The successful construct of a series of based tool plasmids and their functional validation, via comparison with original plasmids, suggest that is a potent drug resistance selection marker. The antibiotic trimethoprim offers convenient usage comparable to that of ampicillin or kanamycin. Additionally, fluorescence analysis has demonstrated the compatibility of TMP with protein expression in various host cells. Based on these findings, TMP- could be an alternative choice for future use in molecular genetic research studies that require miniature plasmids to achieve optimal results.