Table 1. Primers, templates and vectors used for cloning of FIP2 and TRAM mutants.
| Construct | Template | Primer sequence 5’-3’ | Restriction site | Vector | |
|---|---|---|---|---|---|
| FIP2 constructs (hRab11FIP2 KIAA0941) | |||||
| FIP2 | pEGFP-FIP2 | Fwd | GCCCGAATTCGGCTGTCCGAGCAAGCCCAAAAG | EcoRI | N-terminal DYKDDDDK (Flag) |
| Rev | ATAGCGGCCGCTCATTAACTGTTAGAGAATTTGCCAGC | NotI | |||
| FIP2 1–192 | pEGFP-FIP2 | Fwd | GCCCGAATTCGGCTGTCCGAGCAAGCCCAAAAG | EcoRI | |
| Rev | ATAGCGGCCGCTTAGTGAGTACTTGGAATGATTGC | NotI | |||
| FIP2 129–512 | pEGFP-FIP2 | Fwd | GCCCGAATTCGGCGAATCAAAAACAGGGGTGAG | EcoRI | |
| Rev | ATAGCGGCCGCTCATTAACTGTTAGAGAATTTGCCAGC | NotI | |||
| FIP2 193–512 | pEGFP-FIP2 | Fwd | GCCCGAATTCGGATGCCCGATGCCAATAGTGAA | EcoRI | |
| Rev | ATAGCGGCCGCTCATTAACTGTTAGAGAATTTGCCAGC | NotI | |||
| FIP2 I481E | pEGFP-FIP2 I481E | Fwd | GCCCGAATTCGGCTGTCCGAGCAAGCCCAAAAG | EcoRI | |
| Rev | ATAGCGGCCGCTCATTAACTGTTAGAGAATTTGCCAGC | NotI | |||
| TRAM constructs (hTICAM-2 NM_021649.7) | |||||
| TRAM | TRAM-YFP | Fwd | CATGAATTCATGGGTATCGGGAAGTCTAAA | EcoRI | C-terminal DYKDDDDK (Flag) |
| Rev | TTAACTCGAGCGGCAATAAATTGTCTTTGTACC | XhoI | |||
| TRAM 1–68 | TRAM-YFP | Fwd | CATGAATTCATGGGTATCGGGAAGTCTAAA | EcoRI | |
| Rev | TTAACTCGAGCCATCTCTTCCACGCTCTGAGC | XhoI | |||
| TRAM 1–79 | TRAM-YFP | Fwd | CATGAATTCATGGGTATCGGGAAGTCTAAA | EcoRI | |
| Rev | TTACCTCGAGAGAGGAACACCTCTTCTTCAGC | XhoI | |||
| TRAM 1–90 | TRAM-YFP | Fwd | CATGAATTCATGGGTATCGGGAAGTCTAAA | EcoRI | |
| Rev | TTACCTCGAGATGTGTCATCTTCTGCATGCAATATC | XhoI | |||
| TRAM 1–100 | TRAM-YFP | Fwd | CATGAATTCATGGGTATCGGGAAGTCTAAA | EcoRI | |
| Rev | TTACCTCGAGATAGCAGATTCTGGACTCTGAGG | XhoI | |||
| TRAM 1–120 | TRAM-YFP | Fwd | CATGAATTCATGGGTATCGGGAAGTCTAAA | EcoRI | |
| Rev | TTACCTCGAGACTGTCTGCCACATGGCATCTC | XhoI | |||
| TRAM E87A/D88A/D89A | TRAM-YFP | Fwd | CATGAATTCATGGGTATCGGGAAGTCTAAA | EcoRI | |
| Rev | CATCTGTGGCAGCTGCTGCATGCAATATCACAAATTTGAG | ||||
| TRAM-YFP | Fwd | GCATGCAGCAGCTGCCACAGATGAAGCCCTCAGAGTCC | |||
| Rev | TTAACTCGAGCGGCAATAAATTGTCTTTGTACC | XhoI | |||
| TRAM D91A/E92A |
TRAM-YFP | Fwd | CATGAATTCATGGGTATCGGGAAGTCTAAA | EcoRI | |
| Rev | CTGAGGGCTGCAGCTGTGTCATCTTCTGCATGCAA | ||||
| TRAM-YFP | Fwd | GATGACACAGCTGCAGCCCTCAGAGTCCAGAATC | |||
| Rev | TTAACTCGAGCGGCAATAAATTGTCTTTGTACC | XhoI | |||
| Other constructs | |||||
| PAK1-PBD | pDONOR-PAK | Fwd | AATTGGATCCAAGAAAGAGAAAGAGCGGCCAG | pGex-2TK (GST) | |
| Rev | TATAGAATTCTCAAGCTGACTTATCTGTAAAGCTCATG | ||||
| pLVX-FIP2 | pEGFP-FIP2 | NA | EcoRI | pLVX-EF2α- IRES-ZsGreen | |