TABLE 1.
Oligonucleotides used for randomization and cloning of blaSME gene
| Primer name | Sequence | Use |
|---|---|---|
| SME-Sac | 5′-GGGGCGGAGCTCAACTCATTCAACACTCGG-3′a | Cloning |
| SME-BamHI | 5′-GGGGCGGGATCCGCGTCAAGGCCACAGTCAGCTCTAACGC-3′a | Cloning |
| SME-Cys69-Bot | 5′-CCTTTAAATGAACTSNNTAAAGGGAACCGCTCATC-3′b | Randomizing |
| SME-Cys69-Top | 5′-GATGAGCGGTTCCCTTTANNSAGTTCATTTAAAGG-3′b | Randomizing |
| SME-Cys238-Bot | 5′-CGCAGTACCTATAGCCCCSNNGCTCCCAGTTTTGT-3′b | Randomizing |
| SME-Cys238-Top | 5′-ACAAAACTGGGAGCNNSGGGGCTATAGGTACTGCG-3′b | Randomizing |
a
The SacI and BamHI restriction sites (underlined) were used to clone the PCR products into pTP123.
b
The randomizing primers were used to replace the target codon by NNS randomization, where N represents any nucleotide and S represents guanine or cytosine.