TABLE 1.
Oligonucleotide primers used for site-directed mutagenesis
| Primer | Sequencea | Positionsb | Codon change | Primer used in combination | Amplicon size (bp) |
|---|---|---|---|---|---|
| Mutagenic primers | |||||
| Mutagenic for P51L | |||||
| P51L/F | 5′-GAGCTGGATCTCAACAGCGGTAAG-3′ | 135-159 | CCT→CTC | ||
| P51L/R | 5′-CTTACCGCTGTTGAGATCCAGCTC-3′ | 159-135 | |||
| Mutagenic for K104E | |||||
| K104E/F | 5′-GAATGACTTGGTTGAGTACTCACCAG-3′ | 290-316 | AAG→GAG | ||
| K104E/R | 5′-CTGGTGAGTACTCAACCAAGTCATTC-3′ | 316-290 | |||
| Mutagenic for R164S | |||||
| R164S/F | 5′-CGCCTTGATAGATGGGAACCGGA-3′ | 474-497 | AGT→AGA | ||
| R164S/R | 5′-TCCGGTTCCCATCTATCAAGGCG-3′ | 497-474 | |||
| Amplification primers | |||||
| TEM/F | 5′-CCCGGATCCATGAGTATTCAACATTTCCGTGCT-3′ | P51L/R | 159 | ||
| K104E/R | 316 | ||||
| R164S/R | 497 | ||||
| TEM/R | 861 | ||||
| TEM/R | 5′-CCCGAATTCTTACCAATGCTTAATCAGTGAGGCA-3′ | P51L/F | 727 | ||
| K104E/F | 572 | ||||
| R164S/F | 388 |
a
The mutated positions are underlined.
b
Positions according to the numbering of the blaTEM-60 coding sequence.
c
The BamHI and EcoRI restriction sites introduced immediately before the start and stop codons (in boldface type) of the blaTEM-60 coding sequence to facilitate cloning are underlined.