TABLE 7.
Primers used for the generation of hemagglutinin-tagged Pteropus alecto and Myotis macropus tetherin expression constructs through a two-step PCR process
| Tetherin | Construct | Two-step PCR usage | Primer |
Direction | Encoded RE site | Encoded epitope | |
|---|---|---|---|---|---|---|---|
| Name | Sequence (5′ > 3′) | ||||||
| Pteropus alecto | |||||||
| Isoform A | pD-PaTAH1 | Step 1, 5′ half | TethXF_JH_353 | CTCTCTCGAGAGCTTCTTCTCCTGACTCC | Forward | Xho I | -a |
| TethAB-HA1_JH_355 | CGTATGGGTACCCGCTGGCATGCTCTTTCCTTAGCTG | Reverse | - | HA | |||
| Step 1, 3′ half | TethAB-HA2_JH_356 | CAGCGGGTACCCATACGATGTTCCAGATTACGCTGGCAGCTCTGGCGAGAAAAATGG | Forward | - | HA | ||
| TethXR_JH_354 | TTTTTCTAGAATGTTTCTCCACCCCTAAGGC | Reverse | Xba I | - | |||
| Step 2 | TethXF_JH_353 | CTCTCTCGAGAGCTTCTTCTCCTGACTCC | Forward | Xho I | - | ||
| TethXR_JH_354 | TTTTTCTAGAATGTTTCTCCACCCCTAAGGC | Reverse | Xba I | - | |||
| Isoform B | pD-PaTBH1 | Step 1, 5′ half | TethXF_JH_353 | CTCTCTCGAGAGCTTCTTCTCCTGACTCC | Forward | Xho I | - |
| TethAB-HA1_JH_355 | CGTATGGGTACCCGCTGGCATGCTCTTTCCTTAGCTG | Reverse | - | HA | |||
| Step 1, 3′ half | TethAB-HA2_JH_356 | CAGCGGGTACCCATACGATGTTCCAGATTACGCTGGCAGCTCTGGCGAGAAAAATGG | Forward | - | HA | ||
| TethXR_JH_354 | TTTTTCTAGAATGTTTCTCCACCCCTAAGGC | Reverse | Xba I | - | |||
| Step 2 | TethXF_JH_353 | CTCTCTCGAGAGCTTCTTCTCCTGACTCC | Forward | Xho I | - | ||
| TethXR_JH_354 | TTTTTCTAGAATGTTTCTCCACCCCTAAGGC | Reverse | Xba I | - | |||
| Isoform C | pD-PaTCH1 | Step 1, 5′ half | TethXF_JH_353 | CTCTCTCGAGAGCTTCTTCTCCTGACTCC | Forward | Xho I | - |
| TethC-HA1_JH_359 | TATGGGTACCCGCTCCTTAGCTGTTCCGGCTCCG | Reverse | - | HA | |||
| Step 1, 3′ half | TethC-HA2_JH_360 | AGCGGGTACCCATACGATGTTCCAGATTACGCTGGCAGCCCACAACCTGGACTGGTCC | Forward | - | HA | ||
| TethXR_JH_354 | TTTTTCTAGAATGTTTCTCCACCCCTAAGGC | Reverse | Xba I | - | |||
| Step 2 | TethXF_JH_353 | CTCTCTCGAGAGCTTCTTCTCCTGACTCC | Forward | Xho I | - | ||
| TethXR_JH_354 | TTTTTCTAGAATGTTTCTCCACCCCTAAGGC | Reverse | Xba I | - | |||
| Myotis macropus | |||||||
| Tetherin A | pD-MmTAH1 | Step 1, 5′ half | MmacXF1_JH_510 | CTGCAGAATTCGCCCTTATG | Forward | Eco RI | - |
| MmacAH1_JH_512 | GGGACGTCGTATGGGTATGGGTATGGCCGGCCGACCAAGGCCTCATTCTC | Reverse | - | HA | |||
| Step 1, 3′ half | MmacAH2_JH_513 | TACCCATACGACGTCCCAGACTACGCTGCTAGCTCTGCCAAGGGTCCCC | Forward | - | HA | ||
| MmacXR1_JH_511 | CAGTGTGCTGGAATTCGCC | Reverse | Eco RI | - | |||
| Step 2 | MmacXF1_JH_510 | CTGCAGAATTCGCCCTTATG | Forward | Eco RI | - | ||
| MmacXR1_JH_511 | CAGTGTGCTGGAATTCGCC | Reverse | Eco RI | - | |||
| Tetherin B | pD-MmTBH1 | Step 1, 5′ half | MmacXF2_JH_516 | CTGCAGAATTCGCCCTTTCC | Forward | Eco RI | - |
| MmacBH1_JH_518 | CTGGGACGTCGTATGGGTATGGGTATGGCCGGCCGTTGGGGTCCTTGCCGAACAC | Reverse | - | HA | |||
| Step 1, 3′ half | MmacBH2_JH_519 | CCCATACGACGTCCCAGACTACGCTGCTAGCAATGGCAAGGGCTTCCCTAAC | Forward | - | HA | ||
| MmacXR2_JH_517 | CAGTGTGCTGGAATTCGCC | Reverse | Eco RI | - | |||
| Step 2 | MmacXF2_JH_516 | CTGCAGAATTCGCCCTTTCC | Forward | Eco RI | - | ||
| MmacXR2_JH_517 | CAGTGTGCTGGAATTCGCC | Reverse | Eco RI | - | |||
a
The dashes indicate that either there is no encoded RE site, or no encoded epitope within the primer sequence referenced by that row.