Many mammalian gene expression vectors express the transferred genes under the control of the cytomegalovirus (CMV) major immediate-early promoter (MIEP). The human MIEP has been known as the strongest promoter in mammalian cells and utilized widely in mammalian expression systems. There are four NF-kappa B binding sites (named as KBs) in the human MIEP. In this study, we have constructed multiple mutated MIEPs by changing the natural kappa Bs in the human MIEP into the high-affinity artificial sequences that were in vitro selected by using systematic evolution of ligands by exponential enrichment (SELEX) and predicted by bioinformatics. With various transcriptional activity evaluations, we found three mutated MIEPs with the transcriptional activity higher than the wild-type MIEP, which should be useful and widely applicable in many mammalian transgene expression fields such as gene engineering, gene therapy and gene editing. This study provides a useful approach for promoter engineering in biotechnology. This study also produced a series of mutated MIEPs with various transcriptional activities, which may be used for the fine control of gene expression output in the future synthetic biology. (C) 2017 Elsevier Inc. All rights reserved.