TABLE 2.
Primers and constructed plasmids used in the study
| Primers | Sequence (5′–3′) | Constructs |
|---|---|---|
| AP-MS | ||
| pcDNA3.1-N-His-Strep-F | GGCTAGCGTTTAAACTTAAGC ATG GAACACCAAATCATCAG | pcDNA3.1-N-strep |
| pcDNA3.1-N-His-Strep-R |
GCTGGATATCTGCAGAATTCTCAGTGGTGGTGGTGGTGGTGACTTGCAGGAT
CCCTTCTCAAACTGTGGATGACTCCA CAAAGCTTGGTGCTTCAGCA |
|
| Expression | ||
| LucN-eEF1A-FLAG-F | GAGGCTCTGGAGGGGAATTCATGGGAAAGGAAAAGATC | pcDNA3.1-NlucN-eEF1A-Flag |
| LucN-eEF1A-FLAG-R | TCATCCTTGTAGTCGGATCCCTTGGTCTTGGCAGC | |
| LucN-SCRV-N-FLAG-F | GAGGCTCTGGAGGGGAATTCATGGAACACCAAATC | pcDNA3.1-NlucN-N-Flag |
| LucN-SCRV-N-FLAG-R | TCATCCTTGTAGTCGGATCCCAAAGCTTGGTGCTTCAG | |
| LucN-SCRV-P-FLAG-F | GAGGCTCTGGAGGGGAATTCATGGCAAAACCAACT | pcDNA3.1-NlucN-P-Flag |
| LucN-SCRV-P-FLAG-R | TCATCCTTGTAGTCGGATCCCCGAATCACCTCGAG | |
| LucN-SCRV-M-FLAG-F | GAGGCTCTGGAGGGGAATTCATGCCTCTGTTTAAG | pcDNA3.1-NlucN-M-Flag |
| LucN-SCRV-M-FLAG-R | TCATCCTTGTAGTCGGATCCATGCCAGCTATGACC | |
| eEF1A-LucC-HA-F | GGGGCTCATCGGGGGAATTCATGGGAAAGGAAAAGATC | pcDNA3.1-eEF1A-NlucC-HA |
| eEF1A-LucC-HA-R | CACATCATAGGGGTAGGATCCCTTGGTCTTGGCAGC | |
| SCRV-N-LucC-HA-F | GGGGCTCATCGGGG GAATTCATGGAACACCAAATC | pcDNA3.1-N-NlucC-HA |
| SCRV-N-LucC-HA-R | CACATCATAGGGGTAGGATCCCAAAGCTTGGTGCTTCAG | |
| SCRV-P-LucC-HA-F | GGGGCTCATCGGGGGAATTCATGGCAAAACCAACT | pcDNA3.1-P-NlucC-HA |
| SCRV-P-LucC-HA-R | CACATCATAGGGGTAGGATCCCCGAATCACCTCGAG | |
| SCRV-M-LucC-HA-F | GGGGCTCATCGGGGGAATTCATGCCTCTGTTTAAG | pcDNA3.1-M-NlucC-HA |
| SCRV-M-LucC-HA-R | CACATCATAGGGGTAGGATCCATGCCAGCTATGACC | |
| SCRV-G-LucC-HA-F | GGGAGACCCAAGCTGGCTAGC ATGGGAATGTACCCACTGTTTGTTCCG | pcDNA3.1-G-NlucC-HA |
| SCRV-G-LucC-HA-R | GGAGCCGCTGTTTCCGGATCC GGGAACAAATTGATACTGCTGCAAAGGG | |
| eEF1A-His-F | GATATCTGCAGAATTCCACCATGGGAAAGGAAAAGATCCAC | pcDNA3.1-eEF1A-His |
| eEF1A-His-R | CCTCTTCTGAGATGAGTTTTTGTTCCTTGGTCTTGGCAGCCTTCTGTGCA | |
| eEF1A truncated mutant | ||
| eEF1A-D1-F | GAGGCTCTGGAGGGGAATTCATGGGAAAGGAAAAGATCCACA | eEF1A-I |
| eEF1A-D1-R | TCATCCTTGTAGTCGGATCCGGGCAAGATGGCATCCAGGGCA | |
| eEF1A-D2-F | GAGGCTCTGGAGGG GAATTCCCAAGCCGTCCCACCGACAA | eEF1A-II |
| eEF1A-D2-R | TCATCCTTGTAGTCGGATCCTGGGTCGTTCTTGCTGTCTC | |
| eEF1A-D3-F | GAGGCTCTGGAGGG GAATTCGCCCCAGTGCTGGACTGCCACACT | eEF1A-III |
| eEF1A-D3-R | TCATCCTTGTAGTCGGATCCCTTGGTCTTGGCAGCCTTCTGT | |
| eEF1A-D1 + D2 F | GAGGCTCTGGAGGGGAATTCATGGGAAAGGAAAAGATCCACA | eEF1A-I + II |
| eEF1A-D1 + D2 R | TCATCCTTGTAGTCGGATCCTGGGTCGTTCTTGCTGTCTCCA | |
| eEF1A-D2 + D3 F | GAGGCTCTGGAGGGGAATTCCCAAGCCGTCCCACCGACAA | eEF1A-II + III |
| eEF1A-D2 + D3 R | TCATCCTTGTAGTCGGATCCCTTGGTCTTGGCAGCCTTCTGT | |
| eEF1A-D1 + D2△200aa-F | GAGGCTCTGGAGGGGAATTCATGCTGGAGGCCAGCTCAAA | eEF1A-I + IIΔ200 |
| eEF1A-D1 + D2△200aa-R | TCATCCTTGTAGTCGGATCCTGGGTCGTTCTTGCTGTCTC | |
| eEF1A-D1-200aa-F | GAGGCTCTGGAGGGGAATTCATGGGAAAGGAAAAGATCCACA | eEF1A-I200 |
| eEF1A-D1-200aa-R | TCATCCTTGTAGTCGGATCCGTTGTCTCCATGCCATCCAG | |
| eEF1A-D1 + D3-His-F | GATATCTGCAGAATTCCACC ATG GGAAAGGAAAAGATCCAC | eEF1A-I + III |
| eEF1A-D1 + D3-His-R | CCTCTTCTGAGATGAGTTTTTGTTC CTTGGTCTTGGCAGCCTTCT | |
| N truncated mutant | ||
| SCRV-N-1-F | GAGGCTCTGGAGGG GAATTC ATGGAACACCAAATCATCAGGAGAG | N1–200 |
| SCRV-N-1-R | TCATCCTTGTAGTCGGATCC AATTGCTGCCACCATTCTGAGA | |
| SCRV-N-2-F | GAGGCTCTGGAGGG GAATTC GACATGTTCTTTTACAGATTCAAGG | N201–240 |
| SCRV-N-2-R | TCATCCTTGTAGTCGGATCC AGTGAATGATGCGACATGAGCA | |
| SCRV-N-3-F | GAGGCTCTGGAGGG GAATTC GGATTAACCCTCAGTGGAGTGCTGG | N241–430 |
| SCRV-N-3-R | TCATCCTTGTAGTCGGATCC CAAAGCTTGGTGCTTCAGCAGATC | |
| M lysine point mutant | ||
| M-K5R-F | CTCTGTTTCGAAAGAGCAACAAGAAGTCGACTATTAAG | M-K5R |
| M-K5R-R | GCTCTTTCGAAACAGAGGCATGCTAGCCAGCT | |
| M-K6R-F | CTCTGTTTAAGCGAAGCAACAAGAAGTCGACTATTAAGCC | M-K6R |
| M-K6R-R | GCTTCGCTTAAACAGAGGCATGCTAGCCAGCT | |
| M-K9R-F | GAGCAACCGAAAGTCGACTATTAAGCCATATCAAGC | M-K9R |
| M-K9R-R | TCGACTTTCGGTTGCTCTTCTTAAACAGAGGCATG | |
| M-K10R-F | CAACAAGCGATCGACTATTAAGCCATATCAAGCAC | M-K10R |
| M-K10R-R | TAGTCGATCGCTTGTTGCTCTTCTTAAACAGAGGC | |
| M-K166R-F | TTTCGAGCGAATGTTCCACGGTGTGTTAGTGCA | M-K166R |
| M-K166R-R | GGAACATTCGCTCGAAAGATGATCCTGTCATCC | |
| Dual-Luciferase reporter | ||
| pGL-ppeEF1A-F | TTTCTCTATCGATAGGTACCTGTATCACGATTTCCACAAACATATT | pGL-ppeEF1A |
| pGL-ppeEF1A-R | TTGGCGTCTTCCATGGTGGCTTGATTAGTTTCTGTACAAGGAAGGAA | |
| Vectors linearization | ||
| pcDNA3.1-His-strep-F | GCTTAAGTTTAAACGCTAGCC | |
| pcDNA3.1-His-strep-R | GAATTCTGCAGATATCCAGC | |
| NlucN-Flag-F | GGATCCGACTACAAGGATGA | |
| NlucN-Flag-R | GAATTCCCCTCCAGAGCCTC | |
| NlucC-HA-F | GCTAGCCAGCTTGGGTCTCCC | |
| NlucC-HA-R | GGATCCGGAAACAGCGGCTCC | |
| pcDNA3.1-His-F | GGTGGAATTCTGCAGATATC | |
| pcDNA3.1-His-R | GAACAAAAACTCATCTCAGAAGAGG | |
| pGL-F | GCCACCATGGAAGACGCCAA | |
| pGL-R | GGTACCTATCGATAGAGAAA | |
| RT-qPCR | ||
| FHM-actin-F | GAATCCCAAAGCCAACAG | |
| FHM-actin-R | GGAAGAGCATAACCCTCATAG | |
| eEF1A-F | CAAGGAAGTCAGCGCCTACA | |
| eEF1A-R | CATCCCTTGAACCAGCCCAT | |
| SCRV-qRT-F | GGGCTGGATGATAGACGATTG | |
| SCRV-qRT-R | TGGCGGAGGTGCTTGATATGG | |