Table 3.
Plasmids.
| Plasmid | Relevant Properties | Derivation |
|---|---|---|
| pTA95 | Integrative plasmid for trpA gene deletion | (Allers et al. 2004) |
| pTA131 | Integrative plasmid based on pBluescript II, with pyrE2+ marker | (Allers et al. 2004) |
| pTA298 | pUC19 with trpA+ marker flanked by BamHI sites | (Lestini et al. 2010) |
| pTA333 | pUC19 with SacI-NspI chromosomal fragment containing orc4 gene | This study |
| pTA415 | pBluescript II SK+ with MluI chromosomal fragment containing hel308 helicase gene | This study |
| pTA416 | pBluescript II with SacI chromosomal fragment containing orc5 and oriC2 | (Norais et al. 2007) |
| pTA419 | pTA131 with NheI-EcoRI fragment of pTA416 containing orc5 and oriC2 | This study |
| pTA1100 | pBluescript II with AciI chromosomal fragment containing orc2 and oriC3 | (Hawkins, Malla, et al. 2013) |
| pTA1329 | pTA131 with Δori-pHV4 construct | (Hawkins, Malla, et al. 2013) |
| pTA1343 | pTA131 with p.tnaA-radA+:: hdrB+ construct flanked by upstream and downstream radA regions | (Hawkins, Malla, et al. 2013) |
| pTA1370 | pBluescript II SK+ with HindIII-KpnI chromosomal fragment containing orc1 gene and oriC1 origin | This study |
| pTA1371 | pBluescript II SK+ with BstBI chromosomal fragment containing orc3 gene | This study |
| pTA1373 | pTA131 with Δorc3 construct, comprising ClaI-BamHI fragment of upstream flanking region of orc3 and BamHI-XbaI fragment of downstream flanking region of orc3, PCR amplified from pTA1371 | This study |
| pTA1375 | pTA131 with Δorc5 construct, comprising KpnI-BamHI fragment of downstream flanking region of orc5 and BamHI-XbaI fragment of upstream flanking region of orc5, PCR amplified from pTA416 | This study |
| pTA1379 | pTA131 with Δorc2 construct, comprising KpnI-BamHI upstream flanking region of orc2 and BamHI-XbaI fragment of downstream flanking region of orc2, PCR amplified from pTA1100 | This study |
| pTA1431 | pTA131 with inactivation of unique BamHI site in MCS by filling-in with Klenow | This study |
| pTA1432 | pBluescript II SK+ with NotI chromosomal fragment containing orc9 gene | This study |
| pTA1433 | pTA1431 with Δorc9 construct, comprising XbaI-BstXI upstream flanking region of orc9 and XbaI-BstXI fragment of downstream flanking region of orc9, PCR amplified from pTA1432 | This study |
| pTA1610 | pTA131 with Δorc1 construct, comprising KpnI-BamHI upstream flanking region of orc1 and BamHI-XhoI fragment of downstream flanking region of orc1, PCR amplified from pTA1370 | This study |
| pTA1631 | Δorc3 Δori-pHV4 construct, where orc3 upstream region of pTA1373 was replaced by KpnI-BamHI fragment of ori-pHV4 upstream region from pTA1329 | This study |
| pTA1632 | pTA1379 with insertion of BamHI trpA+ fragment from pTA298 | This study |
| pTA1633 | pTA1375 with insertion of BamHI trpA+ fragment from pTA298 | This study |
| pTA1691 | pTA131 with Δorc1 ΔoriC1 construct, comprising StuI-BamHI upstream flanking region of oriC1 and BamHI-XbaI fragment of downstream flanking region of orc1, PCR amplified from pTA1370 | This study |
| pTA1692 | pTA131 with Δorc2 ΔoriC3 construct, comprising AatII-BamHI upstream flanking region of oriC3 and BamHI-KpnI fragment of downstream flanking region of orc2, PCR amplified from pTA1100 | This study |
| pTA1712 | pTA131 with Δorc5 ΔoriC2 construct, comprising XbaI-BamHI upstream flanking region of oriC2 and BamHI-XbaI fragment of downstream flanking region of orc5, PCR amplified from pTA416 | This study |
| pTA1837 | pTA131 with p.tnaA-radA+ construct. XbaI-BamHI fragment of hdrB+ marker was removed from pTA1343, and 890 bp EcoRV-PvuII fragment of radA upstream flanking region (PCR amplified from H26 genomic DNA) was used to replace 315 bp EcoRV-PvuII fragment of radA upstream flanking region in pTA1343 | This study |
| pID19T-HVO_2042 | pTA131 with Δorc4:: trpA+ construct, comprising XhoI-HindIII fragment of upstream flanking region of orc4 and BamHI-XbaI fragment of downstream flanking region of orc4, PCR amplified from H26 genomic DNA, joined using HindIII-BamHI trpA+fragment | Jerry Eichler |