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. 2019 Feb 14;13(1):199–209. doi: 10.1111/1751-7915.13374

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© 2019 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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Design of homology arms (HAs) and construction of donor DNAs.

A. Scheme of designing 100‐bp HAs and primers for the construction of linear donor dsDNAs.

B. Scheme of designing ~1‐kb HAs and primers for the construction of donor plasmids for gene knockout and adaptor plasmids for cloning genes to be integrated. Dashed lines in (A) and (B) indicate sequence homologies and directionality among different constructs. Names of primers mentioned in the protocols (Table S3) are presented next to the primers with some fixed sequences, if available. bla, ampicillin resistance gene; tetA(C), tetracycline resistance gene; HA_L & HA_R, HAs placed left and right to the tetA(C) gene; MCS, multiple cloning site. pUC, pUC origin of replication; p15A, p15A origin of replication. The primer sequences are shown again in Table S3 for better visibility.