<span class="paragraphSection"><div class="boxTitle">Abstract</div>Site-specific non-long terminal repeat (non-LTR) retrotransposon-mediated gene therapy has the potential to revolutionize medicine by allowing insertion of large gene cargos at specific sites in a genome. Despite decades of effort, the reaction sequence of these elements remains to be fully elucidated, limiting the ability to engineer improved activity for gene insertion. One major question concerns whether the element-encoded protein is capable of synthesizing the second strand of the integration product or whether host factors are indispensable in this role. Here, we provide a kinetic and mechanistic framework for R2 non-LTR retrotransposition. Single turnover kinetic analysis with global data fitting defined the rate constants for each step in the pathway involving first-strand DNA cleavage to provide the first DNA primer, reverse transcription to copy the provided RNA, second-strand DNA cleavage to provide the second primer, and second-strand synthesis to make duplex cDNA, inserted between the two cleavage sites in the targeted DNA. Sequencing analysis of the final products confirms replication of a 1400-nucleotide RNA used in this study. This represents a complete analysis of the reaction sequence and the first observation of second-strand synthesis <span style="font-style: italic;">in vitro</span>. The findings provide a kinetic framework to understand non-LTR retrotransposition and guidelines to engineer improved activity.</span>