phiC31 integrase-based gene delivery has been developed. However, the expression of integrated transgenes is often suppressed by a negative position effect. To improve this system, we constructed a new phiC31 integrase-based expression vector that contains attB, an expression unit placed in reverse orientation with two sea urchin-derived Ars-insulators to avoid position effects. In vitro and in vivo transfection experiments revealed that this new system produces higher levels of transgene expression as well as continued gene expression. Thus, the present gene delivery system will facilitate reverse genetics-based molecular biological studies.