TABLE 2.
Oligonucleotide primers used in this study
| Primer pair | Sequence (5′ to 3′)a | Product size (bp) and Tmb | Target | Comment(s) and restriction site(s) |
|---|---|---|---|---|
| Ori-F | GGCCCCAAGCTTTTGCCAAATGTTCTTCTTC | 1,991 | pMVSCS1 | Amplification for pME18; HindIII |
| Ori-R | GGCCCCAAGCTTGCGGTCGATCAAAAAAC | ori | HindIII | |
| Ori-F | GGCCCCAAGCTTTTGCCAAATGTTCTTCTTC | 1,714 | pMVSCS1 | Amplification for pME18RIA; HindIII |
| Ori-A-R | AAAACTGCAGCGTAATGATACTACTGCTCACC | ori | PstI | |
| Ori-F | GGCCCCAAGCTTTTGCCAAATGTTCTTCTTC | 1,260 | pMVSCS1 | Amplification for pME18RI; HindIII |
| Ori-I-R | GGCCCCAAGCTTGAGATTTTAACGACTACAAATT | ori | HindIII | |
| Ppck-F | CAAATTAACCGAAGATCTACATCACCTCATAAAATAAATTAAAA | 348 | pckA | Amplification for pMEx; BglII |
| Ppck-R | CGCGGATCC CGGCAATCGAGGTATTTGTATA | Promoter | BamHI | |
| TTpck-F | CCATCGATAGCCTGATAACTTCACCAACCT | 260 | pckA TTc | Amplification for pMEx; ClaI |
| TTpck-R | ATGAGGTGATGTAGATCTTCGGTTAATTTGATTCAATCCT | BglII | ||
| PfrdA-F | CCACAGCTGGACGTCTATTCTGTTGGCTAATGC | 511 | frdA | Amplification for pMS1; PvuII, AatII |
| PfrdA-R | CCCGAATTCCTCCCCAGTAGAGTTGAT | Promoter | EcoRI | |
| MsMU | CTCGACGTCGTTCTTCTTCGGTCACAG | 1,693 | pMVSCS1 | Amplification for pMS3; AatII |
| MsMD | CTCGACGTCTACTACTGCTCACCGTAG | ori | AatII | |
| FumC-F | CGGGATCCTCGGCTTTGCCCACATTAGC | 1,772 | fumC | Amplification of strain MBEL55E fumC; BamHI |
| FumC-R | CGGGATCCCATCCGCTGCGGTTTTGTAA | BamHI | ||
| GFPuv-F | GGCGAATTCATGAGTAAAGGAGAAGAAC | 652 | gfpuv | Amplification for pMS3-G; EcoRI |
| GFPuv-R | GGCTCTAGAGGATCCTTATTTGTAGAGC | XbaI-BamHI | ||
| RFP-F | AATGAATTCCATATGGAGGGCACCGTGAACG | 741 | rfp | Amplification for pMS3-R; EcoRI-NdeI |
| RFP-R | AATTCTAGAGTCGCGGCCGCTACAG | XbaI | ||
| qM-fum1 | GATATTCGTTTATTGGCATCCGG | 170 | fumC | qPCR primers; single-copy reference for Mannheimia chromosome |
| qM-fum2 | GCGAATGAAATGGTGGTATCGT | 83.0°C | ||
| qA-pck1 | TGCGGAAGCTTATTTGGTGAAC | 170 | pckA | qPCR primers; single-copy reference for Actinobacillus chromosome |
| qA-pck2 | CAACACCCGGTAATGCTTTAGG | 81.5°C | ||
| qori1 | GGATTAAACAACCGTAGGGCGT | 170 | ori | qPCR primers; detection of the copy no. of the pMEx and pMVSCS1 |
| qori2 | GTTGGTAGCTATCCCCGACCTT | 78.5°C |
a
Underlined sequences are the restriction sites.
b
Melting points (Tm) were determined based on melting curve analyses following qPCRs.
c
TT, transcription terminator.