Table 2.
Primers used for BAC recombineering and sequencing
Primers 1–6 were used to introduce galK (homology arm is italicized and sequence recognizing galK is underlined), and primers 7–12 were used to remove galK (homology arm is italicized, complementary sequence is underlined, and glycine + HA tag is bold). The deletion of galK, US28, or UL33 was confirmed using primers 13–18. Primers 16 and 18–22 were employed for sequencing of the recombineering products. The oligonucleotides were purchased from Eurofins Genomics.
| Code | Name | Sequence |
|---|---|---|
| 1 | US28->galK F | 5′-GTGCGTGGACCAGACGGCGTCCATGCACCGAGGGCAGAACTGGTGCTATCCCTGTTGACAATTAATCATCGGCA-3′ |
| 2 | US28->galK R | 5′-ATCCATAACTTCGTATAATGTATGCTATACGAAGTTATAGCGCTTTTTTATCAGCACTGTCCTGCTCCTT-3′ |
| 3 | Ul33->galK F | 5′-CGGAAGCGTCGTCGCCCCGGACTGCGCCCGCGGTCTGCTATTCGTCCACGCCTGTTGACAATTAATCATCGGCA-3′ |
| 4 | Ul33->galK R | 5′-GGGAAATGGCGACGGGTTCTGGTGCTTTCTGAATAAAGTAACAGGAAAGCTCAGCACTGTCCTGCTCCTT-3′ |
| 5 | UL33galK F | 5′-CAAAAATCCCCCATCGACTCTCACAATCGCATCATAACCTCAGCGGGGTACCTGTTGACAATTAATCATCGGCA-3′ |
| 6 | UL33galK R | 5′-AAATGGCGACGGGTTCTGGTGCTTTCTGAATAAAGTAACAGGAAAGCTCATCAGCACTGTCCTGCTCCTT-3′ |
| 7 | galK->ΔUS28 F | 5′-CAGTCTCTCGGTGCGTGGACCAGACGGCGTCCATGCACCGAGGGCAGAACTGGTGCTATCTAAAAAAGCG-3′ |
| 8 | galK->ΔUS28 R | 5′-TTAATTAAGGATCCATAACTTCGTATAATGTATGCTATACGAAGTTATAGCGCTTTTTTAGATAGCACCA-3′ |
| 9 | galK->ΔUl33 F | 5′-CTCCAGAACCCGGAAGCGTCGTCGCCCCGGACTGCGCCCGCGGTCTGCTATTCGTCCACGGCTTTCCTGT-3′ |
| 10 | galK->ΔUl33 R | 5′-CGTATATGAGGGGAAATGGCGACGGGTTCTGGTGCTTTCTGAATAAAGTAACAGGAAAGCCGTGGACGAA-3′ |
| 11 | UL33galK->UL33HA F | 5′-CAAAAATCCCCCATCGACTCTCACAATCGCATCATAACCTCAGCGGGGTAGGCTACCCGTACGACGTCCCAGACTACGCC-3′ |
| 12 | UL33galK->UL33HA R | 5′-AAATGGCGACGGGTTCTGGTGCTTTCTGAATAAAGTAACAGGAAAGCTCAGGCGTAGTCTGGGACGTCGTACGGGTAGCC-3′ |
| 13 | galK internal F | 5′-CTCTGTTTGCCAACGCATTTGG-3′ |
| 14 | galK internal R | 5′-CGAAATCATCGCGCATAGAGG-3′ |
| 15 | US28 internal F | 5′-CGCATTTCCAGAATCGTTGC-3′ |
| 16 | Downstream US28 R | 5′-CCCTGAACATGTCCATCAGG-3′ |
| 17 | UL33 internal F | 5′-GCGTGTTACTCATCTCCTTCG-3′ |
| 18 | Downstream UL33 R | 5′-GGACGTCGTTTTTATCGTACCG-3′ |
| 19 | Upstream US28 F | 5′-GTAATTCGATCCTCTCTCACGC-3′ |
| 20 | Downstream US28 R | 5′-CTTTGCATACTTCTGCCTGC-3′ |
| 21 | Upstream UL33 F | 5′-CGCCGCATTTTTTCAGGATCTTGG-3′ |
| 22 | Downstream UL33 R | 5′-CGGTACGATAAAAACGACGTCC-3′ |