Title: Engineering Plasmids with Synthetic Origins of Replication

Plasmids remain by far the most common medium for delivering engineered DNA to microorganisms. However, the reliance on natural plasmid replication mechanisms limits their tunability, compatibility, and modularity. Here we refactor the natural pMB1 origin and create plasmids with customizable copy numbers by tuning refactored components. We then create compatible origins that use synthetic RNA regulators to implement independent copy control. We further demonstrate that the synthetic origin of replication (SynORI) can be engineered modularly to respond to various signals, allowing for multiplexed copy-based reporting of environmental signals. Lastly, a library of 6 orthogonal SynORI plasmids is created and co-maintained in E. coli for a week. This work establishes the feasibility of creating plasmids with SynORI that can serve as a new biotechnology for synthetic biology.