ABSTRACT Sleeping Beauty ( SB ) is a synthetic transposon that was constructed based on sequences of transpositionally inactive elements isolated from fish genomes. SB is a Tc1/ mariner superfamily transposon following a cut-and-paste transpositional reaction, during which the element-encoded transposase interacts with its binding sites in the terminal inverted repeats of the transposon, promotes the assembly of a synaptic complex, catalyzes excision of the element out of its donor site, and integrates the excised transposon into a new location in target DNA. SB transposition is dependent on cellular host factors. Transcriptional control of transposase expression is regulated by the HMG2L1 transcription factor. Synaptic complex assembly is promoted by the HMGB1 protein and regulated by chromatin structure. SB transposition is highly dependent on the nonhomologous end joining (NHEJ) pathway of double-strand DNA break repair that generates a transposon footprint at the excision site. Through its association with the Miz-1 transcription factor, the SB transposase downregulates cyclin D1 expression that results in a slowdown of the cell-cycle in the G1 phase, where NHEJ is preferentially active. Transposon integration occurs at TA dinucleotides in the target DNA, which are duplicated at the flanks of the integrated transposon. SB shows a random genome-wide insertion profile in mammalian cells when launched from episomal vectors and “local hopping” when launched from chromosomal donor sites. Some of the excised transposons undergo a self-destructive autointegration reaction, which can partially explain why longer elements transpose less efficiently. SB became an important molecular tool for transgenesis, insertional mutagenesis, and gene therapy.