construction and characterization of an invivo linear covalently closed dna vector production system background while safer than their viral counterparts conventional nonviral gene delivery dna vectors offer a limited safety profile they often result in the delivery of unwanted prokaryotic sequences antibiotic resistance genes and the bacterial origins of replication to the target which may lead to the stimulation of unwanted immunological responses due to their chimeric dna composition such vectors may also impart the potential for chromosomal integration thus potentiating oncogenesis we sought to engineer an in vivo system for the quick and simple production of safer dna vector alternatives that were devoid of nontransgene bacterial sequences and would lethally disrupt the host chromosome in the event of an unwanted vector integration eventresults we constructed a parent eukaryotic expression vector possessing a specialized manufactured multitarget site called super sequence and engineered e coli cells rcell that conditionally produce phagederived recombinase tel py54 teln n15 or cre p1 passage of the parent plasmid vector through rcells under optimized conditions resulted in rapid efficient and one step in vivo generation of mini lcclinear covalently closed teltelncell or mini ccccircular covalently closed crecell dna constructs separated from the backbone plasmid dna sitespecific integration of lcc plasmids into the host chromosome resulted in chromosomal disruption and 105 fold lower viability than that seen with the ccc counterpartconclusion we offer a high efficiency mini dna vector production system that confers simple rapid and scalable in vivo production of mini lcc dna vectors that possess all the benefits of minicircle dna vectors and virtually eliminate the potential for undesirable vector integration events