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. 1999 Jul;67(7):3357–3366. doi: 10.1128/iai.67.7.3357-3366.1999

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Copyright © 1999, American Society for Microbiology

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FIG. 1 — Construction of pΔHBL for insertional mutagenesis of B. cereus hemolysin BL. Relevant restriction sites are shown. (A) A 3.3-kb PCR fragment of hemolysin BL was ligated into the multicloning region of pKRX, creating pKRX-hbl. The ermAM gene of pUC-ermAM was ligated to the 4.7-kb Eco47III/PstI fragment of pKRX-hbl to create pKRX-hbl::erm^R. (B) The multicloning region of p3ERM was ligated to an inverse PCR fragment of pTV1OK containing the temperature-sensitive replicon from pWVO1 and a Kan^r marker, to create pCASPER. (C) The EcoRI/PstI fragment of pKRX-hbl::erm^R containing the disrupted hemolysin BL operon was ligated into the multicloning region of pCASPER to create pΔHBL.

FIG. 1 — Construction of pΔHBL for insertional mutagenesis of B. cereus hemolysin BL. Relevant restriction sites are shown. (A) A 3.3-kb PCR fragment of hemolysin BL was ligated into the multicloning region of pKRX, creating pKRX-hbl. The ermAM gene of pUC-ermAM was ligated to the 4.7-kb Eco47III/PstI fragment of pKRX-hbl to create pKRX-hbl::erm^R. (B) The multicloning region of p3ERM was ligated to an inverse PCR fragment of pTV1OK containing the temperature-sensitive replicon from pWVO1 and a Kan^r marker, to create pCASPER. (C) The EcoRI/PstI fragment of pKRX-hbl::erm^R containing the disrupted hemolysin BL operon was ligated into the multicloning region of pCASPER to create pΔHBL.