Current approaches to generate lentiviral vectors, which have been used extensively for gene therapy, are time consuming and require a large expenditure. Here, we directly clone the full length myosin light chain kinase cDNA into enhanced green fluorescence protein (EGFP)-fused pLenti6/V5 expression vector in just one step with the use of Red-mediated recombination system, allowing for rapid and effective cloning of lentiviral expression vectors. In addition, the simultaneous expression of EGFP reporter provides a convenient monitoring mean for host cell infection and for localization of the target proteins.