BackBone Builder (B3): A modular Golden Gate standard with a compatible Agrobacterium parts library

Agrobacterium-mediated transformation relies on binary vectors in which T-DNA and virulence genes are maintained on separate replicons. While Golden Gate cloning has become standard for T-DNA assembly, no modular framework exists for systematic construction of Agrobacterium vector backbones. Here, we present BackBone Builder (B3), a Golden Gate-based platform for combinatorial backbone assembly. B3 uses the Type IIS enzyme PaqCI to minimize domestication and enables one-pot assembly of nine backbone modules plus a selectable cloning cassette. The system is compatible with GreenGate and remains independent of downstream cloning strategies. We generated a library of 42 backbone components, supporting a theoretical design space exceeding 370,000 constructs. A 4 x 4 origin-of-replication (ORI) matrix combining four Escherichia coli and four Agrobacterium ORIs assembled with 100% efficiency and functioned robustly in bacterial and plant contexts. Reporter expression reflected expected ORI-dependent patterns in E. coli, Agrobacterium, and Nicotiana benthamiana. A B3-derived maize transformation backbone achieved stable transformation efficiencies comparable to established vectors. B3 establishes a standardized and extensible framework for rational engineering of Agrobacterium binary vector architecture.