TABLE 2.
Oligonucleotidea | Restriction enzyme | Comment |
---|---|---|
F, 5′-GCTTCCGGAATGGTGAGCAAGGGCGAGG | BspEI | YFP cloning (PCR) |
R, 5′-GCTGGATCCTTACTTGTACAGCTCGTCCATG | BamHI | |
5′-CTTTGCCCGTTATCCCGATCATATGAAACGG | YFP A206K mutantb | |
5′-CCGTTTCATATGATCGGGATAACGGGCAAAG | ||
F, 5′-GCTACTAGTTGACAATTAATCATCGGCTCG | SpeI | p15A/Ptac cloning (PCR), cMyc-YFP and RepA-WH1 WT, A31V, or ΔN37 variant with YFP tag |
R, 5′-GCTGGATCCTTACTTGTACAGCTCGTCCATG | BamHI | |
F, 5′-GCTACTAGTTGACAATTAATCATCGGCTCG | SpeI | pRK2/Ptac cloning (PCR), His6-RepA-WH1 WT, A31V, or ΔN37 variant with mCherry tag |
R, 5′-AGTGAATTCGAGCTCGGTAC | EcoRI | |
F, 5′-GCTCGGTACCTCGATCCTCTACGCCGGAC | KpnI | lacIq cloning (PCR), pRK2/p15A |
R, 5′-CGAGGAATTCTCATGCCCCGCGCCCAC | EcoRI | lacIq cloning (PCR), pRK2 |
R, 5′-CGAGGAGCTCTCATGCCCCGCGCCCAC | SacI | lacIq cloning (PCR), p15A |
Restriction sites are underlined; mutations are in boldface. F, forward; R, reverse.
QuikChange kit (Stratagene).