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To facilitate development of synthetic biology tools for genetic engineering of cyanobacterial strains, we constructed pANS-derived self-replicating shuttle vectors that are based on the minimal replication element of the Synechococcus elongatus strain PCC 7942 plasmid pANS. To remove the possibility of homologous recombination events between the shuttle plasmids and the native pANS plasmid, the endogenous pANS was cured through plasmid incompatibility-mediated spontaneous loss. A heterologous toxin–antitoxin cassette was incorporated into the shuttle vectors for stable plasmid maintenance in the absence of antibiotic selection. The pANS-based shuttle vectors were shown to be able to carry a large 20 kb DNA fragment containing a gene cluster for biosynthesis of the omega-3 fatty acid eicosapentaenoic acid. Based on quantitative PCR analysis, there are about 10 copies of pANS and 3 copies of the …