We tested the effectiveness of a novel 13-bp hepatitis B virus (HBV)-derived cis-acting element (CAE) (ACCTCGACAAGGC), called the DT2 CAE, in augmenting transgene expression delivered by plasmid vectors in eukaryotic cells. The addition of the DT2 CAE just upstream of the start codon of several different target proteins (luciferase, EGFP, LHB, HBsAg, and MIF) in DNA plasmid constructs enhanced their translation in a posttranscriptional manner, irrespective of cell type (cell lines or primary cells) or promoter (CMV or HBV preS1 promoters), suggesting its feasibility for enhanced protein production in eukaryotic cell systems. In conclusion, a novel HBV-derived DT2 CAE could be used effectively for enhanced protein production in eukaryotic cell culture systems. (C) 2017 Elsevier Inc. All rights reserved.