TABLE 1.
PCR strategy used to amplify DNA sequences coding for regions I, II, and III of KdpD
| Amplified KdpD region (residues)a | Primer name | Sequence (5′ to 3′)b | Size of PCR fragment (bp) | Enzymes used for cloning |
|---|---|---|---|---|
| I (D375 to V403) | SpeIsense | 5′-CTCGATCAGGTACTAGTCGCGCTT-3′ | 135 | SpeI+NotI |
| NotIanti | 5′-AACGGCGCATAACGCGGCCGCCGCAAC-3′ | |||
| II (L408 to S473) | NotIsense | 5′-ATGCGTGGTTGCGGCCGC-3′ | 195 | NotI+AatII |
| AatIIanti | 5′-GTCAGCAGATATTGGACGTC-3′ | |||
| III (L479 to R526) | KpnIAatIIsense | 5′-CACGCGGTACCCTCGCCGTCTCTGACGTCCAATATCTGC-3′ | 207 | AatII+Bsu36I |
| Bsu36Ianti | 5′-TGGTGGCAGCAATATCCTGAGGACTGC-3′ |
a
Amino acid numbers refer to positions in the KdpD wild-type protein (Fig. 1). For details of the PCR method, see Materials and Methods.
b
Sites for restriction enzymes are shown in bold letters. The corresponding nucleotide positions in the kdpD gene are given in Materials and Methods.