Table 1.
Primers used for amplification and construction of IBV genomic cDNA
| Primers | Primer sequence (5′–3′) | Position in genome (length) | Purpose |
|---|---|---|---|
| f1-F | agctcggtacccggggatccCTTGTTTTGCCGTGTCTC | 192–6282 bp (6091 bp) | To amplify F1, F2, F3, and F4 fragments carrying homology arm sequences for ligation into BAC vector, respectively. |
| f1-R | tcaagcttgcatgcctcgagATGCCTGTTACTGCGTGC | ||
| F2-F | agctcggtacccggggatccGGTTTCTATTTCTGGCTCT | 6172–13,192 bp (7021 bp) | |
| F2-R | tcaagcttgcatgcctcgagTCACTACAGTCACGGCAG | ||
| F3-F | agctcggtacccggggatccTTATGATCTCCTCAAGTATG | 13,085–20,019 bp (6935 bp) | |
| F3-R | tcaagcttgcatgcctcgagAATCCTGTGCAATGTCATA | ||
| f4-F | agctcggtacccggggatccGATTATGTTTCTGACGCAC | 19,752–27,397 bp (7646 bp) | |
| f4-R | tcaagcttgcatgcctcgagAGACAGATTAGACATTTCCC | ||
| f1A5472C-F | TATTGTTGGCTCCAGTGTTGTTACTACA | / | To introduce a silent mutation site 5472 A → C. |
| f1A5472C-R | AACACTGGAGCCAACAATAGCTTTCTTA | ||
| 5’UTR-F | agctcggtacccggggatccACTTAAGATAGATATTAATATA | 1–293 bp (293 bp) | To fuse the 5′UTR to the F1 subcloning vector. |
| 5’UTR-R | ACCAGAGGTCCTTCGCACT | ||
| 3’UTR-F | GAATCAAGGAAGATAGGC | 27,328–27,652 bp (325 bp) | To fuse the 3′UTR to the F4 subcloning vector. |
| 3’UTR-R | tcaagcttgcatgcctcgagTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCTCTAACTCT | ||
| CMV-F | agctcggtacccggggatcc TTAATTAACTTGACATTG | 610 bp | To add the CMV promoter to the 5′ end of F1 fragment |
| CMV-R | tatttatatcacacttaagt ACGGTTCACTAAACGAGC | ||
| HB-F | tagagcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa GGCCGGCATGGTCCCAGC | 304 bp | To add the sequence of BGH and HDVR to the 3′ end of F4 fragment |
| HB-R | tcaagcttgcatgcctcgag CCACCGCATCCCCAGCAT | ||
| (F1-F2)-F | GGTTTCTATTTCTGGCTCT | 6172–13,192 bp (7021 bp) | To fuse F2 to the (CMV) F1 subcloning vector via the F1 homology arm. |
| (F1-F2)-R | tcaagcttgcatgcctcgagTCACTACAGTCACGGCAG | ||
| (F3-F4)-F | GATTATGTTTCTGACGCAC | 7901 bp | To fuse F4 (HB) to the F3 subcloning vector via the F3 homology arm. |
| (F3-F4)-R | tcaagcttgcatgcctcgagTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCTCTAACTCT | ||
| (F34-F12)-F | GATTATGTTTCTGACGCAC | (14,872 bp) | To fuse F34 (HB) to the (CMV) F12 subcloning vector via the F2 homology arm. |
| (F34-F12)-R | tcaagcttgcatgcctcgag CCACCGCATCCCCAGCAT | ||
| H120-F4-△S1-F | GTTTTAGACGTTCTATTA | 13,518 bp | To prepare the linearized vector by deleting H120 S1 from pBeloBAC11-H120F4 (HB) |
| H120-F4-△S1-R | CTCTTACCAGTAACTTAC | ||
| SCS1-F | TGGTAAGTTACTGGTAAGAGATGTTGGGGAAGTCACTGTT | 20,296–21,931 bp (1635 bp) | To amplify the sequence of SCS1 |
| SCS1-R | AGTAATAGAACGTCTAAAACGACGTGTCTCTTTAGTGAGT | ||
| RM-F | AAAAGCGCCAGTCTACTACCC | 621 bp | To detect the mutant oligonucleotide sites (rescue marker) |
| RM-R | GGACCACATAAAGAACCCTCA | ||
| H120/SC-S-F | ACTGGTAAGAGATGTTGGTAA | 3937 bp | To amplify the S gene sequence of rH120/SCS1 |
| H120/SC-S-R | GAAAACTTCCATTCTCCTCTA |
Sequences in 5′ end with lowercase letters are the sequences of homology arms