Constructing versatile CRISPR plasmids

pBGC^{24 (link)} was used to construct pBGA by exchanging the cat gene (chloramphenicol resistance) with aac(3)-IV (apramycin resistance) from pMDIAI^{35 (link)} by Gibson assembly (New England Biolabs, UK). pLC10-Apra was constructed by exchanging the aph(3’)-Ia gene (Kanamycin resistance) with the aac(3)-IV gene, by Gibson assembly. Plasmids pLC10-Kan/pLC10-Apra carry the Streptococcus pyogenes cas9 gene under the control of a Ptet promoter inducible by anhydrotetracycline (derived from pWJ153^{36 (link)}), cloned on a thermosensitive pSC101 plasmid backbone with a guide RNA under the control of a Ptrc promoter derived from pCas^{37 (link)} (Addgene plasmid #62225). Single guide RNA (sgRNA) targeting pOXA-48 pemK gene (Fig.1A) was introduced into pLC10-Kan by golden gate assembly^{38 (link)} (New England Biolabs, UK). pLC10-Apra was constructed by exchanging the aph(3’)-Ia gene (Kanamycin resistance) with the aac(3)-IV gene, by Gibson assembly.