To quickly find an optimal expression system for recombinant protein production, a set Of vectors with the same restriction sites were constructed for parallel cloning of a target gene and recombinant protein production in prokaryotic and eukaryotic expression systems, simultaneously . These vectors include nucleotide sequences encoding protein tags and protease recognition sites for tag removal, followed by the cloning sites 5'-EcoRI/3'-Xhol identical in these vectors for ligating with the sticky-end PCR product of a target gene. Our vectors allow parallel gene cloning and protein production in multiple expression systems with minimal cloning effort. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 1582-1586, 2009