RESUMEN
Versatile DNA assembly standards and compatible, well-characterized part libraries are essential tools for creating effective designs in synthetic biology. However, to date, vector standards for Gram-positive hosts have limited flexibility. As a result, users often revert to PCR-based methods for building the desired genetic constructs. These methods are inherently prone to introducing mutations, which is problematic considering vector backbone parts are often left unsequenced in cloning workflows. To circumvent this, we present the ProUSER2.0 toolbox: a standardized vector platform for building both integrative and replicative shuttle vectors forBacillus subtilis. The ProUSER2.0 vectors consist of a ProUSER cassette for easy and efficient insertion of cargo sequences and six exchangeable modules. Furthermore, the standard is semicompatible with several previously developed standards, allowing the user to utilize the parts developed for these. To provide parts for the toolbox, seven novel integration sites and six promoters were thoroughly characterized in B. subtilis. Finally, the capacity of the ProUSER2.0 system was demonstrated through the construction of signal peptide libraries for two industrially relevant proteins. Altogether, the ProUSER2.0 toolbox is a powerful and flexible framework for use in B. subtilis.