TABLE 2.
Primers and peptides used in this study
| Primer | Sequencea (5’-3’) | Relevant property | Restriction site |
|---|---|---|---|
| Npr-f | ACTGGTATACCGCACCCGATTCAATTCTGC | Primer pair to amplify nprB-plcR region | BstZ17I |
| Npr-r | ACTGGGATCCTTGCGAAGGCACTTTATCTTTCGCC | BamHI | |
| StuBf | AAGGAGGAAGCAGGTATGAATTATAGAAAACTTGCTAGTGCTTTCG | Primer pair to amplify nprB structural gene | |
| StuBr | GACACGCACGAGGTTTATTGTACCGCTATTACTTTATTTATTTTTTG | ||
| LoxSB | CCCCCCAGATCT CCGATCATATTCAATAACCCT | Primer pair to amplify loxP-Ω-sp-loxP | BglII |
| LoxEB | CCCCCCAGATCTCCAGTACTAGTGAACCTCTTC | BglII | |
| Sp5 | GAGGGTTTGCAACTGCGGG | Ω-sp inner primer for analysis of modifications in nprB from 5’ | |
| Bc-ext | ATTCGTAATGTAATGAAGGG | nprB external primer for analysis of modifications in nprB from 3’ | |
| R3 | CCCGGGATCCTTATTTCTTCATTTTTTTCAAAA | Used to amplify mutated B. cereus 569 plcR gene from 3’ | BamHI |
| BseqF | GAACAACTTTACCACTAACAGT | Primer pair to verify loxP inside nprB | |
| BseqR | TCAAAGTTGAAATTGCTCAG | ||
| PapRF | AAAGCAATTAGAGGCATTAC | Primer pair to verify papR replacement by loxP | |
| PapRR | GCAAACCTGTTTCTAGTAAAGA | ||
| EryF | TTAACGAGTGAAAAAGTACTCAACC | Primer pair to verify ery gene of pHY304 plasmid | |
| EryR | TGACCCATTTTGAAACAAAG | ||
| Peptide | Sequence (N-C) | Relevant property | |
| 5aa | MPFEF | All peptides are derivative of the PapR | |
| 7aa | SDMPFEF | C-terminal region of B. cereus 569 PlcR group II | |
| 8aa | ASDMPFEF | ||
| 27aa | DTALEKSKVISHDNQEVQLASDMPFEF | ||
a
Restriction enzyme recognition sites are underlined.