Directed enzyme evolution is now a routine approach to improve desirable biocatalytic properties. When only a low-throughput screen is available to detect improved variants from a mutant gene library, it is imperative that cloning efficiency be maximized during library synthesis to avoid wasting effort screening empty plasmids. To achieve this we developed pUCXKT, a gain-of-function positive selection expression vector. Insertion of genes amplified using a specialized downstream PCR primer restores key regulatory and genetic elements necessary for co-expression of a kanamycin resistance marker adjacent to the pUCXKT cloning region. We show that pUCXKT enables 100 % cloning efficiency as well as high-level expression of inserted genes. Unlike previous positive selection expression plasmids, the strategy we used to design pUCXKT is readily adaptable to different vector backbones, antibiotic marker genes, and multiple cloning regions.