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Updated: Jul 15, 2026
Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning
Published on: February 5, 2021
Construction of plasmid vectors for screening replicons from gram-positive bacteria and their use as shuttle cloning
Abdellah Benachour1, Yanick Auffray, Axel Hartke
1Laboratoire de Microbiologie de l'Environnement, EA956-USC INRA 2017, Université de Caen, Esplanade de la Paix, 14032 Caen Cedex, France. abdellah.benachour@unicaen.fr
Abstract:
Plasmids play a central role in engineering recombinant bacteria because they are the primary vehicles used to manipulate targeted sequences. In some cases, bacteria of interest are poorly provided with suitable tools for these molecular or genetic manipulations. In this context, we constructed from two shuttle cloning vectors, pUCB2871 and pUCB2872, the basic vectors pUCB30 and pUCB31, which could represent suitable tools to isolate replicons from Gram-positive bacteria. These plasmid vectors are characterized by the following after-features: (a) the pUC origin of replication is unable to replicate in Gram-positive bacteria; (b) an erythromycin-resistance encoding gene that is functional in both Gram-negative and -positive bacteria; (c) the pUC19 multiple cloning site (MCS) within the lacZalpha reporter gene; and (4) an additional multiple cloning site (MCS). Cloning replicons from Gram-positive bacteria in this additional MCS would allow the derivative vectors to function directly as shuttle cloning vectors.
