The manipulation of recombinant DNA has been an integral step in molecular biology to date. A number of strategies have been developed over the years, as traditional cloning methods are time consuming, have high backgrounds and low efficiency and are often limited by the number of suitable restriction sites available. Here, we constructed a series of new positive-selection-based cloning vectors that overcome most of the above mentioned drawbacks and can be applied in both eukaryotic and prokaryotic systems. This strategy is based on the extreme toxicity of Dpnl in wild-type E. coli and the inactivation of this lethality by the introduction of target gene within multiple cloning sites. There are no rapid approaches for identifying soluble proteins for high-throughput screening. In this study, we combined this highly efficient cloning strategy with rapid identification of soluble proteins to construct vectors with multiple fusion tags, such as MBP, GST, CBD, NusA, and Sumo, to generate enzymes with potential diagnostic, industrial or therapeutic applications. Thus, this versatile positive-selection-based technology is appropriate for routine cloning, DNA library construction, and high-throughput screening for the expression of proteins of interest. (C) 2018 Elsevier Inc. All rights reserved.