One-Entry Golden Seamless Cloning with a Reusable Donor Streamlines Plant Gene Functional Analysis

Molecular cloning underpins functional genomics, yet current toolboxes force a trade-off. Golden Gate frameworks handle one-pot multi-fragment assembly but overcomplicate routine single-gene cloning, whereas Gibson, In-Fusion, and Gateway systems need backbone linearization, resequencing, or proprietary recombinases that leave scars. Here we present Golden Seamless (GS), a one-entry, many-destination system in which a single PCR captures a gene once into a reusable Entry-and-Expression Clone that is never re-cloned. Circle-to-circle Golden Gate moves the same validated clone into plant, yeast, or bacterial vectors without linearization, and a ccdB counter-selection with orthogonal antibiotic markers drives background near zero. Bacterial destinations embed superfolder GFP, enabling naked-eye monitoring of recombinant proteins and an antibody-free dual-modality EMSA that resolves protein and DNA channels across a >150-nm spectral gap. GS thus consolidates capture, distribution, and validation into one low-background workflow with built-in quality control, cutting the time and cost of screening many candidates across functional assays.