Table 1.
Primer sequences for the plasmid constructs in the present study
| Plasmid no.a | Plasmid name | Primer sequenceb |
|
|---|---|---|---|
| PCR 1 | PCR 2 | ||
| 1 | pTGFP-GPC | Fp: 5′-CTCTATCCATGGTGAGCAAGGGCGAGGAGCTG-3′ | |
| Rp: 5′-AACTATGGATCCCTTGTACAGCTCGTCCATG-3′ | |||
| 2 | pTGPC-GFP | Fp: 5′-CTCTATCCATGGTAGGGTGGGTTTGCATCTTC-3′ | Fp: 5′-AGTCGAGGATCCGTGAGCAAGGGCGAGGAGCTG-3′ |
| Rp: 5′-AGTCAAGGATCCATTAGCTTTATTTTTTCTAC-3′ | Rp: 5′-AACTATCTCGAGTTACTTGTACAGCTCGTCCATG-3′ | ||
| 3 | pTGFP-Gn | Fp: 5′-CTCTATCCATGGTGAGCAAGGGCGAGGAGCTG-3′ | Fp: 5′-CTCTACGGATCCGTAGGGTGGGTTTGCATCTTC-3′ |
| Rp: 5′-AACTATGGATCCCTTGTACAGCTCGTCCATG-3′ | Rp: 5′-AGTCAACTCGAGCTATGCACTAGCTGCCCATATGATC-3′ | ||
| 4 | pTGn-GFP | Fp: 5′-CTCTATCCATGGTAGGGTGGGTTTGCATCTTC-3′ | Fp: 5′-AGTCGAGGATCCGTGAGCAAGGGCGAGGAGCTG-3′ |
| Rp: 5′-AGTCAAGGATCCTATGATCAGTTCAGTTGTTAAAAGG-3′ | Rp: 5′-AACTATCTCGAGTTACTTGTACAGCTCGTCCATG-3′ | ||
| 5 | pTGFP-Gc | Fp: 5′-CTCTATCCATGGTGAGCAAGGGCGAGGAGCTG-3′ | Fp: 5′-CTCTACGGATCCGATACCCCCTTAATGGAGTCC-3′ |
| Rp: 5′-AACTATGGATCCCTTGTACAGCTCGTCCATG-3′ | Rp: 5′-AGTCAACTCGAGCTAATTAGCTTTATTTTTTCTAC-3′ | ||
| 6 | pTGc-GFP | Fp: 5′-CTCTATCCATGGGATACCCCCTTAATGGAGTCC-3′ | Fp: 5′-AGTCGAGGATCCGTGAGCAAGGGCGAGGAGCTG-3′ |
| Rp: 5′-AGTCAAGGATCCATTAGCTTTATTTTTTCTAC-3′ | Rp: 5′-AACTATCTCGAGTTACTTGTACAGCTCGTCCATG-3′ | ||
| 7 | pTGFP-GnΔtail | Fp: 5′-CTCTATCCATGGTGAGCAAGGGCGAGGAGCTG-3′ | Fp: 5′-CTCTACGGATCCGTAGGGTGGGTTTGCATCTTC-3′ |
| Rp: 5′-AACTATGGATCCCTTGTACAGCTCGTCCATG-3′ | Rp: 5′-AGTCAACTCGAGCTATAATATAATTAAGGTGACTG-3′ | ||
| 8 | pTGFP-GnTail | Fp: 5′-CTCTATCCATGGTGAGCAAGGGCGAGGAGCTG-3′ | Fp: 5′-CTCTACGGATCCAAGATCCTAAGGTTGCTTACT-3′ |
| Rp: 5′-AACTATGGATCCCTTGTACAGCTCGTCCATG-3′ | Rp: 5′-AGTCAACTCGAGCTATGCACTAGCTGCCCATATGATC-3′ | ||
| 9 | pT-Gn | Fp: 5′-CTCTATCCATGGTAGGGTGGGTTTGCATCTTC-3′ | |
| Rp: 5′-AGTCAACTCGAGCTATGCACTAGCTGCCCATATGATC-3′ | |||
| 10 | pT-Gc | Fp: 5′-CTCTATCCATGGGATACCCCCTTAATGGAGTCC-3′ | |
| Rp: 5′-AGTCAACTCGAGCTAATTAGCTTTATTTTTTCTAC-3′ | |||
Plasmids 1 to 8 were generated by cloning two PCR products (PCR 1 and PCR 2) between the NcoI and XhoI sites of pTriEx1.1 plasmid. PCR 1 was digested with NcoI and BamHI restriction enzymes. Similarly, PCR 2 was digested with BamHI and XhoI. The two digested PCR products were then ligated to pTriEx1.1 digested with NcoI and XhoI. In plasmids 1 to 8, except for plasmids 4 and 6, PCR 1 was generated from pT-GFP plasmid and PCR 2 was generated from a pT-GPC construct (see Materials and Methods for details). For plasmids 4 and 6, PCR 1 was generated from pT-GPC, and PCR 2 was generated from pT-GFP. Construction of pT-GPC is described in detail in Materials and Methods. Plasmids 9 and 10 were constructed by generating a PCR product (PCR 1) from the pT-GPC plasmid, using the primers mentioned in the table. The PCR product was cloned in the pTriEx1.1 backbone between NcoI and XhoI restriction sites.
Fp, forward primer; Rp, reverse primer. Restriction sites are shown in boldface.