Table 2.
Primers used to make the minor structural protein knockout mutants
ORF(gene) | Primer sequence (5′ to 3′) | |
---|---|---|
ORF2a′ (GP2′) | F | CCCTCTCCTCTGcTGAGTTTCTGTCCAGG |
R | CCTGGACAGAAACTCAgCAGAGGAGAGGG | |
ORF2b′ (E′) | F | CACTTCTCCTCCTCCTTCAAGTACTAcGATCACACCCACATTCATACTG |
R | CAGTATGAATGTGGGTGTGATCgTAGTACTTGAAGGAGGAGGAGAAGTG | |
ORF3′ (GP3′) | F | GAAGGAGAGTTCTGTACCcTGGTTTCAACTTGGTTTC |
R | GAAACCAGATTGAAACCAgGGTACAGAACTCTCCTTC | |
ORF4′ (GP4′) | F | CCTAACAATCAAGCAATCcTGGAACGAGACAATGATG |
R | CATCATTGTCTCGTTCCAgGATTGCTTGATTGTTAGG | |
ORF2a (E) | F | GCCTTCCACCAcGCTGTCCACGAGCTCC |
R | GGAGCTCGTGGACAGCgTGGTGGAAGGC | |
ORF2b (GP2) | F | CGATCTTTAGAGCATTCTATTCTTAgTGGGTTCTATACTCACCCACATC |
R | GATGTGGGTGAGTATAGAACCCAcTAAGAATAGAATGCTCTAAAGATCG | |
ORF3 (GP3) | F | CAGTTATCTCACTAACCCcTGGATGTCCGTGG |
R | CCACGGACATCCAgGGGTTAGTGAGATAACTG | |
ORF4 (GP4) | R | CCAGTTTGATAACGAGACATTcTGCTACATTGCAAACCTTAC |
F | GTAAGGTTTGCAATGTAGCAgAATGTCTCGTTATCAAACTGG | |
Effects of the mutations on an overlapping ORF | ||
CTCTGcTGAGT (ORF2a′): ATG to CTG (knock out start codon) | ||
TACTAcGATCA (ORF2b′): ATG to ACG (knock out start codon) | ||
TACTAcGATCA (ORF2a′): TAT to TAC (Tyrosine to Tyrosine) | ||
GTACCcTGGTT (ORF3′): ATG to CTG (knock out start codon) | ||
GTACCcTGGTT (ORF2a′): CCA to CCC (Proline to Proline) | ||
CAATCcTGGAA (ORF4′): ATG to CTG (knock out start codon) | ||
CAATCcTGGAA (ORF3′): TCA to TCC (Serine to Serine) | ||
CACCAcGCTGT (ORF2a): ATG to ACG (knock out start codon) | ||
CACCAcGCTGT (ORF2b): CAT to CAC (Histidine to Histidine) | ||
TCTTAgTGGGT (ORF2b): ATG to GTG (knock out start codon) | ||
TCTTAgTGGGT (ORF4′): TAA to TAG (STOP codon to STOP codon) | ||
AACCCcTGGAT (ORF3): ATG to CTG (knock out start codon) | ||
AACCCcTGGAT (ORF2): CCA to CCC (Proline to Proline) | ||
ACATTcTGCTA (ORF4): ATG to CTG (knock out start codon) | ||
ACATTcTGCTA (ORF3): TTA to TTC (Leucine to Leucine) |