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. 2022 Jun 29;10(4):e01165-22. doi: 10.1128/spectrum.01165-22

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Copyright © 2022 Li et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

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FIG 3 — Deletion of an ~9-kb DNA fragment in M. maripaludis using the Cas9 editing system by one-step transformation. (A) Schematic of the plasmid designed for deleting the ~9-kb fla operon that synthesizes archaella (left). Plasmid pMEV405 encodes two sgRNAs that match the first (MMP1166) and the last (MMP1176) genes in the fla operon. The sequences upstream (U4) and downstream (D4) of the fla operon were concatenated on pMEV405 to provide the recombinant donor. (B) Ten puromycin-resistant transformants were randomly selected for PCRs using the primer pairs F4/R4, F4′/R4′, F4″/R4″, F4′/R4″, F4′/R4, and F4″/R4, which target the regions indicated in panel A. Black and red arrows indicate the PCR products amplified from the wild-type (lane CK) and mutated genomes (lanes 1-10), respectively. M, dsDNA size marker.