TABLE 2.
Oligonucleotides used for construction of specific amber mutations in orf46, orf47, orf48, orf49, and orf50 of phage TP901-1a
| Oligonucleotide | Sequence |
|---|---|
| 46am1F | 5′ CGCGGATCCGTTGTAAATACGATTCAAAGTCTTTGGGGAG |
| 46am1R | 5′ CATCATAACTCATGGCctaGGTTGGAATAAAAG |
| 46am2F | 5′ CCTTTTATTCCAACCtagGCCATGAGTTATG |
| 46am2R | 5′ ACGCGTCGACAACTCTTGTTCTTTCGTATCATTGACTGC |
| 47am1F | 5′ CGCGGATCCTGACGATAAAATATAAGCTTGAGGATAG |
| 47am1R | 5′ GTAATTTCAATctAgAAGGTAAACTCTGCACC |
| 47am2F | 5′ GCAGAGTTTACCTTcTagATTGAAATTACAGG |
| 47am2R | 5′ ACGCGTCGACAACGAATCTTGACCCCTTGAAGCGTCC |
| 48am1F | 5′ CGCGGATCCGACACAATCACAGCCCAATTTAAGCTC |
| 48am1R | 5′ CTGCAAAGCATTATCctaGGCAACATATTTC |
| 48am2F | 5′ GAAATATGTTGCCtagGATAATGCTTTGCAGTTTG |
| 48am2R | 5′ ACGCGTCGACAATGATCCCACCTCCGAGTTCTCCAGACC |
| 49am1F | 5′ CGCGGATCCATGCGGATGTCAATAGTCAAGCCATTGTTG |
| 49am1R | 5′ TGAACTACATTTCCACCctaGGTTAATTCAG |
| 49am2F | 5′ CTGAATTAACCtagGGTGGAAATGTAGTTC |
| 49am2R | 5′ ACGCGTCGACCTGTCATCTCAGTAGGAATTATAAGCGTG |
| 50am1F | 5′ CGCGGATCCATGCAATTGGCACTGAAACTGATGGATTT |
| 50am1R | 5′ AGTTTTGCATTctATTGGGCTATTAAAAGC |
| 50am2F | 5′ TTTTAATAGCCCAATagAATGCAAAACTTA |
| 50am2R | 5′ ACGCGTCGACGCTGTAAACTTTCTAAATCTGTTTGATTAG |
a
TP901-1 DNA fragments with overlapping amber mutations were amplified with oligonucleotide sets 1 and 2, and the fragments were spliced by the SOEing PCR technique. AvrII, BamHI, SalI, and XbaI restrictions sites are underlined, and mismatch nucleotides are indicated with lowercase letters.