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Background: Similar to retro−/lenti- virus system, DNA transposons are useful tools for stable expression of exogenous genes in mammalian cells. Sleeping Beauty (SB) transposon has adopted for integrating genes into host genomes in recent studies. However, SB-derived vector system for proteins purifying/tracking and gene knockout are still not available. Results: In this study, we generated a series of vectors (termed as pSB vectors) containing Sleeping Beauty IRDR-L/R that can be transposed by SB transposase. Gateway cassette was combined to the pSB vectors to facilitate the cloning. Vectors with various tags, Flag, Myc, HA, V5 and SFB, were generated for multiple options. Moreover, we incorporated the CRISPR-Cas9 cassette into the pSB plasmids for gene knockout. Indeed, using one of these vectors (pSB-SFB-GFP), we performed Tandem Affinity Purification and identified that NFATc1 is a novel binding partner of FBW7. We also knocked out RCC2 and BRD7 using pSB-CRISPR vector respectively, and revealed the novel roles of these two proteins in mitosis. Conclusion: Our study demonstrated that the pSB series vectors are convenient and powerful tools for gene overexpression and knockout in mammalian cells, providing a new alternative approach for molecular cell biology research. [ABSTRACT FROM AUTHOR]