TABLE 2.
Plasmids used in this study
| Plasmid | Relevant characteristic(s) | Source or reference |
|---|---|---|
| pGEM-T Easy | Cloning vector for PCR products; Apr in E. coli | Promega |
| pGEM22 | A 2,136-bp region overlapping ORF 22 was amplified with the 22F/22R primers and cloned into pGEM-T Easy | This work |
| pUC4-ΩKM2 | pUC4 carrying an Ω element with kanamycin resistance marker; Kmr in E. coli and B. anthracis | J. R. Scott |
| pGEM22Ω | A 2,258 -bp SmaI fragment from pUC4-ΩKM2 containing the Ω-km cassette was inserted instead of AfeI fragment of the amplified ORF 22 sequence in pGEM22. | This work |
| pHY304 | Contains Emr gene and strongly temperature-sensitive replicon for both E. coli and Gram-positive bacteria; Emr in both E. coli and B. anthracis | 19 |
| pHY304K | pHY304 with pXO1 fragment containing Ω-km cassette from pGEM22Ω inserted into EcoRI; Emr Kmr in both E. coli and B. anthracis | This work |
| pFLPo | Contains the Flp recombinase gene; Apr in E. coli | Addgene |
| pSW4 | Encodes promoter of anthrax protective antigen (pag) in shuttle plasmid; Apr in E. coli; Kmr in B. anthracis | 21 |
| pSW4-Flp | pSW4 with the flp gene under the control of the pagA promoter | This work |
| pFPAS | Contains the entire Flp recombinase gene under the control of the pagA promoter; pFPAS has permissive and restrictive temperatures of 30°C and 37°C for B. anthracis, respectively, and can be easily isolated from E. coli strains grown at 37°C; Spr in both E. coli and B. anthracis | This work |
| pCrePA | pHY304 containing the entire Cre recombinase gene under the control of the pagA promoter | 19 |
| pCrePAS | pCrePA with Emr replaced by Ω-sp; Spr in both E. coli and B. anthracis | 1 |
| pCrePAS2 | pCrePAS inserted into the SacI site of pUC18. pCrePAS2 has permissive and restrictive temperatures of 30°C and 37°C for B. anthracis, respectively, and can be easily isolated from E. coli strains grown at 37°C. | This work |
| pSC | Contains multiple restriction site flanked by two direct loxP sequences; pSC has permissive and restrictive temperatures of 30°C and 37°C for B. anthracis, respectively. Ampr in E. coli, Emr in both E. coli and B. anthracis | 1 |
| pMA-FRT | Contains a multiple-cloning site between two directly repeated FRT sequences, ColE1origin of replication, and Ampr gene for selection in E. coli | GeneArt |
| pSCF | pHY304 (Ecl136II) hybrid with pMA-FRT (BstZ17I). Contains multiple restriction sites flanked by two direct FRT sequences; pSCF has permissive and restrictive temperatures of 30°C and 37°C for B. anthracis, respectively, and can be easily isolated from E. coli strains grown at 37°C; Ampr in E. coli, Emr in both E. coli and B. anthracis | This work |
| pXO1 | B. anthracis Ames 35 plasmid | 19 |
| pXO1K | pXO1 with Ω-km cassette inserted instead of AfeI fragment of ORF 22 | This work |
| pXO1Δ(85–19) Δ(26–68) | pXO1K with Δ(26–68) and Δ(85–19) deletions | This work |
| pMR | pXO1 minimal replicon; Spr in both E. coli and B. anthracis | 1 |
| pMR8186 | ORF 81–86 PvuI fragment inserted into pMR | This work |
| pMR8184 | pMR8186 with HpaI small fragment deleted | This work |
| pMR6970 | pMR8184 with ORFs 69 and 70 inserted | This work |
| pMS10 | pMR6970 with ORF 23 downstream area inserted; the UTR includes stem-loop as possible transcriptional terminator for ORF 23 | This work |
| pMS10ΔBsu36I | pMS10 with Bsu36I fragment deleted | This work |
| pMS10ΔNaeI/SalI | pMS10 with NaeI/SalI fragment deleted | This work |
| pMS11 | pMS10 with 67-bp stem-loop deleted | This work |
| pMS12 | pMS10 with 45-bp stem-loop deleted | This work |
| pMS13 | pMS12 with Bsu36I/NaeI fragment deleted | This work |