Standardised Type IIS DNA assembly methods are becoming essential for biological engineering and research. Although a common syntax has been proposed to enable higher interoperability between DNA libraries, Golden Gate (GG) -based assembly systems remain specific to target organisms. Furthermore, these GG assembly systems become laborious and unnecessarily complicated beyond the assembly of 4 transcriptional units. Here, we describe 'universal Loop' (uLoop) assembly, a simple system based on Loop assembly that enables hierarchical fabrication of large DNA constructs (> 30 kb) for any organism of choice. uLoop comprises two sets of four plasmids that are iteratively used as odd and even levels to compile DNA elements in an exponential manner (4n-1). The elements required for transformation/maintenance in target organisms are also assembled as standardised parts, enabling customisation of host-specific plasmids. Thus, this species-agnostic method decouples efficiency of assembly from the stability of vectors in the target organism. As a proof-of-concept, we show the engineering of multi-gene expression vectors in diatoms, yeast, plants and bacteria. These resources will become available through the OpenMTA for unrestricted sharing and open-access.

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