||The power of synthetic biology has enabled the expression of heterologous pathways in cells as well genome-scale synthesis projects. Together these can lead to valuable industrial applications and simultaneously build a deeper fundamental understanding of biological systems. The complexity of biological networks makes rational de novo design a grand challenge. Introducing features that confer genetic flexibility in biosynthetic pathways or genomes is a powerful strategy for downstream engineering while minimizing the number of defined design decisions made up front. Here we develop an in vitro method of DNA library construction to accomplish this goal. The in vitro SCRaMbLE system uses recombinant Cre recombinase mixed in a test tube with purified DNA encoding multiple loxPsym sites. Using a Î²-carotene pathway designed for expression in yeast as an example, we demonstrate top-down and bottom-up in vitro SCRaMbLE, enabling optimization of biosynthetic pathway flux via rearrangement of relevant transcription units. We show that our system provides a straightforward way to correlate phenotype and genotype. In vitro SCRaMbLE is a unique method for generating DNA libraries based on structural variation and is potentially amenable to biochemical optimization in ways that the in vivo system cannot achieve.