The duration of transgene expression from plasmid DNAs is important for gene therapy with nonviral vectors. In the present study, various cytosine-phosphate-guanine (CpG)-free and CpG-containing transcription regulatory sequences were introduced into plasmid DNAs with a CpG-free backbone. The transgenes encoding mouse secreted alkaline phosphatase and Gaussia princeps luciferase, which are both apparently non-immunogenic, were used as reporters. The plasmid DNAs were injected by the hydrodynamics-based method, and the expression was monitored for 28 d. All transcription regulatory sequences achieved long-term expression, with different expression levels depending on the sequences themselves. These results suggested that durable transgene expression at the proper level can be achieved with plasmid DNAs containing the CpG-free backbone.