TABLE 1.

Plasmids used in this study

Plasmid	Relevant characteristic(s)a	Reference or source
pBS246	Contains two directly repeated loxP sites flanking a multiple cloning site; Apr in E. coli	GIBCO-BRL
pJRS312	pUC18 carrying an Ω element with spectinomycin resistance marker aad9 (Ω-sp) from Enterococcus faecalis; Spr in E. coli and B. anthracis	J. R. Scott
pΩL	2.3-kb BamHI fragment with Ω-sp from pJRS312 inserted between two directly repeated loxP of pBS246	This work
pUTE408	Contains 2.3-kb BamHI fragment with Ω-sp from pJRS312 flanked by both 3′ and 5′ spo0A sequences; spo0A::Ω-sp; Apr in E. coli; Tcr in B. anthracis; Spr in both E. coli and B. anthracis	32
pSΩL408	pUTE408 with 2.4-kb Bg/II PCR fragment of pΩL that contains the loxP-flanked Ω-sp; the fragment was amplified with LoxSB-LoxEB primers and inserted into the BamHI site in place of existing Ω-sp; spo0A:: loxP-Ω-sp-loxP	This work
pYJ335	Hybrid of pE194 with pUC19 plasmid	12
pUE1	PvuII-fragment of pYJ335 containing both pE194 and pUC19 replicons; Apr in E. coli; Emr in B. anthracis	This work
pSΩL1	pUE1 containing PvuII fragment of pSΩL408 with spo0A, loxP; and Ω-sp sequences; spo0A::loxP-Ω-sp-loxP	This work
pHY304	Contains Emr gene and strongly temperature-sensitive pWVO1 replicon for both E. coli and gram-positive bacteria; Emr in both E. coli and B. anthracis	30
pSΩL304	pHY304 with PvuII fragment of pSΩL408 containing spo0A, loxP, and Ω-sp sequences; the fragment was inserted into the SmaI site of pHY304; spo0A::loxP-Ω-sp-loxP	This work
pCR 2.1-TOPO	Cloning vector for PCR products; Apr Kmr in E. coli	Invitrogen
pGEM-T easy	Cloning vector for PCR products; Apr in E. coli	Promega
TOPO-M4	2.5-kb PCR fragment (M4S-M4E primers) containing whole pepM gene of B. anthracis cloned into pCR2.1-TOPO	This work
TOPO-MΩL	TOPO-M4 with 2.6-kb NdeI PCR fragment of pΩL that contains the loxP-flanked Ω-sp; the fragment was amplified with LoxSN-LoxEN primers and inserted into the NdeI site of pepM, pepM::loxP-Ω-sp-loxP	This work
pMΩL1	pUE1 containing PvuII fragment of TOPO-MΩL with pepM, loxP, and Ω-sp sequences; pepM::loxP-Ω-sp-loxP	This work
pCS1	pUE1 containing pXO2 PCR fragment (CAPF-CAPE primers) disrupted by loxP-Ω-sp-loxP cassette	This work
pDS2	pUE1 containing pXO2 PCR fragment (DEPF-DEPE primers) disrupted by loxP-Ω-sp-loxP cassette; capD::loxP-Ω-sp-loxP	This work
pBS185	Contains whole Cre recombinase gene	GIBCO-BRL
pAE5	Hybrid of pUB110 with pBR322 plasmid; contains promoter of B. anthracis pagA gene; Apr in E. coli, Kmr in B. anthracis	27
pAEC5	pAE5 with 1.05-kb NdeI-HindIII PCR fragment of pBS185 that contains the entire Cre recombinase gene; the fragment was amplified with CreS-CreE primers and inserted into corresponding sites of pAE5	This work
pCrePA	pHY304 with BglII-HindIII fragment of pAEC5 containing entire Cre recombinase gene under control of pagA promoter; the fragment was inserted into BamHI-HindIII of pHY304	This work
pMrrΩL	Self-ligated pGEM-T easy with (i) mrr 5′ sequences amplified by primers mrr 5F and mrr 5R and cloned into the ApaI-SacII sites; (ii) mrr 3′ sequences amplified by mrr 3F and mrr 3R and cloned into the PstI-SalI sites; and (iii) the NotI fragment from pΩL (containing the loxP-Ω-sp-loxP cassette) cloned into the NotI site	This work
pHYMrrΩL	pHY304 with the SalI fragment from pMrrΩL consisting of mrr::loxP-Ω-sp-loxP cloned into the SalI site	This work
pMcrB3PΩL	pGEM-T easy with (i) mcrB3P sequences amplified by McrB3P For1 and McrB3P Rev, cloned by the T-A method; (ii) loxP-Ω-sp-loxP amplified by Tn5loxPaad9For and Tn5loxPaad9Rev and cloned into the BstZ17I site of mcrB giving mcrB::loxP-Ω-sp-loxP	This work
pHYMcrB3PΩL	pHY304 with the NoI fragment from pMcrB3PΩL consisting of mcrB::loxP-Ω-sp-loxP, cloned into the NotI site	This work

^aAbbreviations: Ap^r, ampicillin resistant; Em^r, erythromycin resistant; Km^r, kanamycin resistant; Tc^r, tetracycline resistant.