TABLE 1.
Primers used for amplification or mutagenesis of HCoV-NL63 sequences
| Purpose | Primer | Oligonucleotide sequencea | Nucleotide no.b | Polarity or mutation |
|---|---|---|---|---|
| Generation of GST-R3 fusion protein | R3-1 | 5′ CCGGAA TTC GGT AAA ATA TCT TTT TCT GAT GAT G 3′ (EcoRI) | 2981-3005 | Forward |
| R3-2 | 5′ CCGCTC GAG ACC ATT ACT ATC ATT ACT AAT AGG 3′ (XhoI) | 3257-3280 | Reverse | |
| Generation of GST-R4 fusion protein | R4-1 | 5′ CCGGAA TTC GAG CTT TTA CCT AAT GTT TTT AAG 3′ (EcoRI) | 8774-8797 | Forward |
| R4-2 | 5′ CCG CTC GAG TTG TAA GGT GGA ATT GTA GCT AAT AG 3′ (XhoI) | 9078-9103 | Reverse | |
| Expression of six-His-tagged PLP2 in E. coli | ZC1 | 5′ CGC GGA TCCGGT TGT AGA GAG TAA TGT TAT GG 3′ | 4979-5000 | Forward |
| ZC2 | 5′ CTA TTG CTC AGC ACC AGT ATC AAG TTT ATC CAT AAC 3′ | 5950-5968 | Reverse | |
| Cloning of pNL-1 | ZC3 | 5′ CCG GAA TTC ACC ATG GCT GTT GCA AGT GAT TCG 3′ (EcoRI) | 311-328 | Forward |
| ZC4 | 5′ AAG CTC GAGCGA CAG TCG TTA ACA TCC ATA ACA TTA C 3′ (XhoI) | 4989-5014 | Reverse | |
| Amplification of PLP2 for cloning into pNL-1 to make pNL-2 | ZC7 | 5′ C CGC TCG AGT TTT AAG AAT GAT AAT GTA GTT TTG 3′ (XhoI) | 5015-5038 | Forward |
| ZC8 | 5′ CG CGG GCC CTC ACT TTT AGA ACA AGC TAC ACA GTC 3′ (ApaI) | 6689-6712 | Reverse | |
| Amplification of nsp123 to make pNL3 by addition of PLP2 to C terminus | ZC5 | 5′ CCG GAA TTC ACC ATG GCT GTT GCA AGT GAT TCG 3′ (EcoRI) | 311-328 | Forward |
| ZC6 | 5′ AA CT CGA GCG TTG CAA CTG TAC AAG TGT GG 3′ (XhoI) | 3483-3502 | Reverse | |
| Cloning of 5′ Flag | ZC9 | 5′ CGG GGT ACC ATG GAC TAC AAA GAC CAT GAC GGT G 3′ (KpnI) | Forward | |
| ZC10 | 5′ CCG GAA TTC CAT ATT CAT ATT CAT CAT CAT GGC CGC AAG CTT GTC ATC GTC ATC 3′ (EcoRI) | Reverse | ||
| Cloning of pNL-4 | ZC11 | 5′ CCG GAA TTC ACC ATG GAT ATT TCA CAC CTT GTA AAC TGT G 3′ (EcoRI) | 4100-4124 | Forward |
| ZC12 | 5′ AAG CTC GAG CGT TGT AAG GTG GAA TTG TAG CTA ATA G 3′ (XhoI) | 9078-9103 | Reverse | |
| Cloning of PLP2 | ZC13 | 5′ C CGG AAT TC ACC ATG GTT GTA GAG AGT AAT GTT ATG 3′ (EcoRI) | 4979-4999 | Forward |
| ZC14 | 5′ CC GCT CGA GCG ACC AGT ATC AAG TTT ATC C 3′ (XhoI) | 5950-5968 | Reverse | |
| Cloning of pNL-5 | ZC15 | 5′ C CGC TCG AGT GCT AGA CTT AAA CGT GTA CCA CTT C 3′ (XhoI) | 6713-6737 | Forward |
| ZC16 | 5′ CG CGG GCC CTC TTG TAA GGT GGA ATT GTA GCT AAT AG 3′ (ApaI) | 9078-9103 | Reverse | |
| Cloning of pNL-6 | ZC17 | 5′ C CGC TCG AGT ACA ACT AAA GCA AAG GGT TTG AC 3′ (XhoI) | 7574-7596 | Forward |
| ZC18 | 5′ CG CGG GCC CTC TTG TAA GGT GGA ATT GTA GCT AAT AG 3′ (ApaI) | 9078-9103 | Reverse | |
| Site-directed mutagenesis | ||||
| PLP1 C1062A | ZC19 | 5′ ATA ATG CTT GGA TTA GTA CCA CAC TTG TAC AGT TG 3′ | 3465-3499 | C1062A |
| ZC20 | 5′ TCC AAG CAT TAT TAT CAG ATT GAT CTA AAA CAC G 3′ | 3443-3476 | ||
| PLP2 C1678A | ZC21 | 5′ CCA CCG ACA ATA ATGCTT GGG TTA ATG CAA C 3′ | 5304-5334 | C1678A |
| ZC22 | 5′ GTT GCA TTA ACC CAAGCA TTA TTG TCG GTG G 3′ | 5304-5334 | ||
| PLP2 C1684A | ZC23 | 5′ GTT GGG TTA ATG CAA CTGCTA TAA TTT TAC AGT ATC 3′ | 5319-5354 | C1684A |
| ZC24 | 5′ GAT ACT GTA AAA TTA TAGCAG TTG CAT TAA CCC AAC 3′ | 5319-5354 | ||
| PLP2 H1836A | ZC25 | 5′ GGT TCT TTT GAT AAC GGT GCC TAT GTA GTT TAT GAT GC 3′ | 5774-5811 | H1836A |
| ZC26 | 5′ GCA TCA TAA ACT ACA TAGGCA CCG TTA TCA AAA GAA CC 3′ | 5774-5811 | ||
| CS3 G2461A | ZC27 | 5′ GTA TAG TTG CAA AAC AGGCTG CTG GTT TTA AAC GTA C 3′ | 7650-7686 | G2461A |
| ZC28 | 5′ GTA CGT TTA AAA CCA GCAGCC TGT TTT GCA ACT ATA C 3′ | 7650-7686 | ||
| CS3 A2462N | ZC29 | 5′ GTT GCA AAA CAG GGT AAC GGT TTT AAA CGT ACT 3′ | 7655-7687 | A2462N |
| ZC30 | 5′ AGT ACG TTT AAA ACC GTT ACC CTG TTT TGC AAC 3′ | 7655-7687 | ||
| CS3 LXGG | ZC31 | 5′ TT GCA CTA CAG GGT GGT GGT TTT AAA CGT ACT TAT AA 3′ | 7656-7692 | LXGG |
| ZC32 | 5′ A ACC ACC ACC CTG TAG TGC AAC TAT ACT AGT AG 3′ | 7644-7676 | ||
| CS2 L896A | ZC33 | 5′ ACA AAA GCA GCT GGT GGT AAA ATA TCT TTT TCT GAT G 3′ | 2966-3002 | L896A |
| ZC34 | 5′ ACC AGC TGC TTT TGT AAA AGC AAC AGG CAA TAC ACC 3′ | 2945-2980 | ||
| CS2 A897N | ZC35 | 5′ AAA TTA AAT GGT GGT AAA ATA TCT TTT TCT GAT GAT G 3′ | 2969-3005 | A897N |
| ZC36 | 5′ ACC ACC ATT TAA TTT TGT AAA AGC AAC AGG CAA TAC 3′ | 2948-2983 | ||
| CS2 G898A | ZC37 | 5′ TTA GCT GCT GGT AAA ATA TCT TTT TCT GAT GAT GTT 3′ | 2972-3007 | G898A |
| ZC38 | 5′ TTT ACC AGC AGC TAA TTT TGT AAA AGC AAC AGG CAA 3′ | 2951-2986 | ||
| CS2 G899A | ZC39 | 5′ GCT GGT GCT AAA ATA TCT TTT TCT GAT GAT GTT ATA G 3′ | 2975-3011 | G899A |
| ZC40 | 5′ TAT TTT AGC ACC AGC TAA TTT TGT AAA AGC AAC AGG 3′ | 2954-2989 | ||
| CS1 A109N | ZC41 | 5′ CAT GGT AAC GGA AGT GTG GTT TTT GTG GAT AAG TAC 3′ | 605-640 | A109N |
| ZC42 | 5′ ACT TCC GTT ACC ATG GCC AAA AAC AAC ATC AAA GTC 3′ | 584-619 | ||
| CS1 G110A | ZC43 | 5′ GGT GCA GCA AGT GTG GTT TTT GTG GAT AAG TAC ATG 3′ | 608-643 | G110A |
| ZC44 | 5′ CAC ACT TGC TGC ACC ATG GCC AAA AAC AAC ATC AAA G 3′ | 586-622 |
a
Underlined nucleotides were added for cloning purposes or mutated sequences. Restriction enzymes are indicated in parentheses.
b
All nucleotide numbers are based on the NL63 genome sequence (GenBank accession no. NC_005831).