Next-generation sequencing of pIJ101-based Streptomyces-E. coli shuttle vectors

High-copy number plasmids are indispensable tools for gene overexpression studies in prokaryotes to engineer pathways or probe phenotypes of interest. Industrially relevant actinomycetes are interested in producing keratolytic enzymes and various biologically active natural products. Within the Actinomycetota class, Streptomyces-Escherichia coli shuttle vectors based on the SCP2* and pIJ101 incompatibility groups are widely employed for molecular cloning and gene expression studies. Here, the sequences of two commonly used pIJ101-based Streptomyces-E. coli shuttle vectors, pEM4, and pUWL201, were determined using next-generation sequencing and mapping. These plasmids drive the expression of heterologous genes using the constitutive ermE*p promoter. pEM4 was found to be 8.3 kbp long, containing genes encoding β-lactamase, thiostrepton resistance, the lacZɑ fragment, an E. coli pUC19 origin of replication, and the Streptomyces pIJ101 origin of replication. pUWL201 was found to be 6.78 kbp long, containing genes encoding β-lactamase, thiostrepton resistance, the lacZɑ fragment, an E. coli pUC19 origin of replication, and the Streptomyces pIJ101 origin of replication. Interestingly, the sequences for both pEM4 and pUWL201 exceed their previously reported size by 1.1 kbp and 0.4 kbp, respectively. This report updates the literature with the corrected sequences for these expression vectors, ensuring their compatibility with modern synthetic biology cloning methodologies.