TABLE 4.
Nucleotide sequences of the primers used to create the knockout mutants
| Genetic region | Primera | Sequence (5′-3′)b |
|---|---|---|
| Kan cassette | Kan 3F | gtcatgatggcctaagtggccaacCTGCAGGAACAGTGAATTGGAGTT |
| Kan 4R | cgatgcaaatttaaatgccggctagCTGCAGCGTTGCGGATGTACTTCA | |
| Erm cassette | Erm 3F | gtcatgatggcctaagtggccaacTCGATACAAATTCCCCGTAGGCGC |
| Erm 4R | cgatgcaaatttaaatgccggctagGAAACAGCAAAGAATGGCGGAAAC | |
| rlrA PI-1 | rlrA 1F | aattgtcgacTATAATCTCCACAGTGGGATTTAC |
| rlrA 2R | gttggccacttaggccatcatgacCAGATGTAAACTTAATAAAGTCCA | |
| rlrA 5F | ctagccggcatttaaatttgcatcgCAGGGATTCGCTCAGTGATTGCTG | |
| rlrA 6R | tttagcggccgcACAAAGAGCCGGAAAAAGGAACAG | |
| PI-2 | pilus-2 1F | aattgtcgacCATAAAATAGAATACAAAACTTC |
| pilus-2 2R | gttggccacttaggccatcatgacTCCAGTGACAACTCATACTGGTCT | |
| pilus-2 5F | ctagccggcatttaaatttgcatcgTTCTATCTACTACAATGGTCGAGA | |
| pilus-2 6R | tttagcggccgcTATTATTTTCTCTATCCATTCTAT | |
| pitA | pitA 1F | aattgtcgacCATAAAATAGAATACAAAACTTC |
| pitA 2R | gttggccacttaggccatcatgacTCCAGTGACAACTCATACTGGTCT | |
| pitA 5F | ctagccggcatttaaatttgcatcgATCGTATAGTGCAATAGTAAATAAT | |
| pitA 6R | tttagcggccgcGGCTAGTACAAATCCAACCATCAG | |
| sipA | sipA 1F | aattgtcgacATCGTATAGTGCAATAGTAAATAAT |
| sipA 2R | gttggccacttaggccatcatgacGGCTAGTACAAATCCAACCATCAG | |
| sipA 5F | ctagccggcatttaaatttgcatcgGACAATCGAACAACGGCTGTAGAC | |
| sipA 6R | tttagcggccgcAGCATGGCCATAAGCAACTCCCGA | |
| pitB | pitB 1F | aattgtcgacAATGACGATGGCATGAAGCCCGCC |
| pitB 2R | gttggccacttaggccatcatgacTGAATACGGACAAAGCGGTTATAG | |
| pitB 5F | ctagccggcatttaaatttgcatcgCTCTTGATTGATAACCTTCCATTC | |
| pitB 6R | tttagcggccgcTGGTGCCCGTAGATAATGGTATTA | |
| srtG1 | srtG1 1F | aattgtcgacTCCAATTCCATATCAAGATTCAAC |
| srtG1 2R | gttggccacttaggccatcatgacATGAGCAGCAAGTGATTGATCACC | |
| srtG1 5F | ctagccggcatttaaatttgcatcgCCTCTACTTGATCCTACGTCGTAG | |
| srtG1 6R | tttagcggccgcAGAGTGTCCGTAGATGATCGTATT | |
| srtG2 | srtG2 1F | aattgtcgacCTATCTAGACCACCTGCTCTCAGT |
| srtG2 2R | gttggccacttaggccatcatgacCACAGAAGAATACCAGAAATACGT | |
| srtG2 5F | ctagccggcatttaaatttgcatcgTGTTTCCTTAACATAACTAATGGA | |
| srtG2 6R | tttagcggccgcTGGTCTTGGGGTCTAACTCCACGA |
a
Primers F3 and R4 were used to amplify the antibiotic cassettes while F1-R2 and F5-R6 were used to amplify the regions upstream and downstream of the gene of interest.
b
Uppercase letters represent bases complementary to S. pneumoniae sequence. Lowercase letters represent bases added to facilitate mutagenesis.