Table 1.
Plasmids and primers used in this study.
| Plasmid or oligonucleotide | Relevant features or sequence | Source, reference or purpose |
|---|---|---|
| Plasmids | ||
| pET20b | Expression vector, pelB leader sequence for potential periplasmic localization, AmpR | Laboratory stock |
| pOrf8H | orf8 in pET20b, expression for Orf8 (8×His-tagged) fused to pelB leader sequence, AmpR | This study |
| pOrf8F | orf8 in pET20b, expression for Orf8 (FLAG-tagged) fused to pelB leader sequence, AmpR | This study |
| pOrf16H | orf8 in pET20b, expression for Orf8 (8×His-tagged) fused to pelB leader sequence, AmpR | This study |
| pOrf16F | orf8 in pET20b, expression for Orf8 (FLAG-tagged) fused to pelB leader sequence, AmpR | This study |
| pOrf8H2 | Plasmid which removed pelB leader sequence from pOrf8H, AmpR | This study |
| pOrf8F2 | Plasmid which removed pelB leader sequence from pOrf8F, AmpR | This study |
| pOrf16H2 | Plasmid which removed pelB leader sequence from pOrf16H, AmpR | This study |
| pOrf16F2 | Plasmid which removed pelB leader sequence from pOrf16F, AmpR | This study |
| pBBR1MCS2 | Cloning vector, KmR | Kovach et al. (1995) |
| pIR1 | IR1 in pBBR1MCS2 at SacI and Asp718 sites, KmR | This study |
| pIR2 | IR2 in pBBR1MCS2 at SacI and Asp718 sites, KmR | This study |
| pIR3 | IR3 in pBBR1MCS2 at SacI and Asp718 sites, KmR | This study |
| pIR4 | IR4 in pBBR1MCS2 at SacI and Asp718 sites, KmR | This study |
| Oligonucleotides | ||
| 652R | CGCTAAAGTTGAAAGGCAGA | Gap closure |
| 5036F | ATATCCCTCCTTTGGTTTGG | Gap closure |
| 7489R | TGGTTACTCTCGGTTTGCTC | Gap closure |
| 14243F | GGGACCCCTATAAATCCTCA | Gap closure |
| 20bf | CATGGCCATCGCCGGCTGGG | Linearization of pET20b |
| 20br-8His | CACCACCACCACCACCACCACCACTGAGAT | Linearization of pET20b |
| 20br-FLAG | GATTACAAGGATGACGACGATAAGTGAGATCCGGCTGCTAACAAA | Linearization of pET20b |
| orf8f(-327) | CATCCGTCTGTGGGAGGTAG | Amplification of orf8 |
| orf8r1692 | GCTGACATCGTTATAGACAACACA | Amplification of orf8 |
| orf16f(-233) | ATTGCTGCTCGTCTCCAGTT | Amplification of orf16 |
| orf16r1549 | CGGGACCCCTATAAATCCTC | Amplification of orf16 |
| orf8F | CCGGCGATGGCCATGGCAGGAGTAGCAAAAATCACG | Amplification for In-Fusion substrate |
| orf8-16RHis | GTGGTGGTGGTGGTGTGACTTTTCCTCACCCTTATTAATAGACTT | Amplification for In-Fusion substrate |
| orf16F | CCGGCGATGGCCATGGCAGGAGTAGCACGGATTAC | Amplification for In-Fusion substrate |
| orf8-16RFlag | GTCATCCTTGTAATCTGACTTTTCCTCACCCTTATTAATAGACTT | Amplification for In-Fusion substrate |
| orf8Fpeldel | ATGGCAGGAGTAGCAAAAATCAC | Removal of pelB leader sequence |
| orf16Fpeldel | ATGGCAGGAGTAGCACGG | Removal of pelB leader sequence |
| orf8-16Rpeldel | ATGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTC | Removal of pelB leader sequence |
| orf8(-476)f | CGCGCTGGAGAGATTAAGG | Amplification of orf8 upstream region |
| orf16(-570)f | GGGGTGCTATATGTCGAGGA | Amplification of orf16 upstream region |
| orf8-16(71)r | TTACCCTCTTGTAACAACTTTTCTG | Amplification of orf8 and orf16 upstream regions |
| IR1f | CATGAAAAAATCATG | Creation of IR1 |
| IR1r | GTACCATGATTTTTTCATGAGCT | Creation of IR1 |
| IR2f | CATATTGTAGTATG | Creation of IR2 |
| IR2r | GTACCATACTACAATATGAGCT | Creation of IR2 |
| IR3f | CATACTACATATTACATATTACATACTACATACTACAATATGGAGGGTG | Creation of IR3 |
| IR3r | GTACCACCCTCCATATTGTAGTATGTAGTATGTAATATGTAATATGTAGTATGAGCT | Creation of IR3 |
| IR4f | CATACTACATACAATATACAACATACTACAATATGGAGGGTG | Creation of IR4 |
| IR4r | GTACCACCCTCCATATTGTAGTATGTTGTATATTGTATGTAGTATGAGCT | Creation of IR4 |
| FITC-UnivF | (FITC)-CGCCAGGGTTTTCCCAGTCACGAC | Preparation of FITC-labeled fragment |
| UnivR | TCACACAGGAAACAGCTATGAC | Preparation of FITC-labeled fragment |