Antibiotic resistance markers, typically kanamycin resistance (kanR), allow selective retention of plasmid DNA during bacterial fermentation and are the most commonly utilized selectable markers. However, to ensure safety, regulatory agencies recommend elimination of antibiotic resistance markers from therapeutic and vaccine plasmid DNA vectors. The presence of an antibiotic resistance gene in the plasmid backbone is considered undesirable by regulatory agencies, due to: 1) the potential transfer of antibiotic resistance to endogenous microbial flora; and 2) the potential activation and transcription of the genes from mammalian promoters after cellular incorporation into the genome. Here, we describe the development and application of a novel antibiotic-free (AF) selection system. Vectors with this selection system incorporate and express a 150 bp RNA-OUT antisense RNA. RNA-OUT represses expression of a counter-selectable marker (SacB) from the host chromosome. SacB encodes a levansucrase, which is toxic in the presence of sucrose. Sucrose selectable DNA vaccine vectors combine antibiotic-free selection with highly productive fermentation manufacturing (\u3e1 g/L plasmid DNA yields), while improving in vivo expression of encoded proteins. The RNA-OUT selectable marker can be used to retrofit existing kanR DNA vaccine plasmids into antibiotic-free vectors. Interestingly, a minimum vector size for high yield plasmid production was identified; strategies to ensure high yield production of small plasmids are reported. These vectors are safer, more potent alternatives for DNA therapy or vaccination
