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. 2021 Mar 24;9(4):670. doi: 10.3390/microorganisms9040670

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© 2021 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Construction of the dual-inducible CRISPRi expression plasmid pS_dCas9. Adapted pRG_dCas9 plasmid carrying the dCas9 handle followed by the terminator from S. pyogenes amplified from plasmid piCas between the PstI and SalI restriction sites. A 20 bp sgRNA sequence can be inserted in the PstI cloning site. For multiplexing of more than one specific sgRNA, the restriction site SalI after the first sgRNA-cs can be used. It has chloramphenicol resistance. P1: tetR/tetA promotor; P2: tac promotor; T1: rrnB T1 terminator; T2: terminator from S. pyogenes; T3: lambda terminator; oriEc: p15A; oriCg: pCG1; cat: chloramphenicol resistance.