| Expression in Dictyostelium cells . | ||
|---|---|---|
| Usage . | Plasmid backbone . | Sequence, 5′-3′ . |
| GFP-Leep1 | pDM317 | F: GGGGATCCATGTCATCCGAATTATCTACTGGTG |
| R: GGTCTAGAATATTCTGGAGGTGGTGGCATTCTA | ||
| GFP-Leep11-855 | pDM317 | F: CTACTAGAATGTCATCCGAATTATCTACTGGT |
| R: CTAGTCGACTTACATTCTATTTTCGTCTTCATATTG | ||
| GFP-Leep11-764 | pDM317 | F: CTACTAGAATGTCATCCGAATTATCTACTGGT |
| R: CTAGTCGACTTAACTATAGTCATAAGTTTCTTGTGG | ||
| GFP-Leep11-751 | pDM317 | F: CTACTAGAATGTCATCCGAATTATCTACTGGT |
| R: CTAGTCGACTTATTGTTGATAATGATTTGCATAAACATC | ||
| GFP-Leep11-736 | pDM317 | F: CTACTAGAATGTCATCCGAATTATCTACTGGT |
| R: CTAGTCGACTTAAGCATAAGTTGAAGGTGATGGTGA | ||
| GFP-Leep11-672 | pDM317 | F: CTACTAGAATGTCATCCGAATTATCTACTGGT |
| R: CTAGTCGACTTGAGAGACAAGTTTGAACATCAG | ||
| GFP-Leep11-623 | pDM317 | F: CTACTAGAATGTCATCCGAATTATCTACTGGT |
| R: CTAGTCGACTTAGGTTGTGAAAGTTCTTTTCATAGC | ||
| GFP-Leep1N | pDM317 | F: GGGGATCCATGTCATCCGAATTATCTACTGGTG |
| R: GGTCTAGATTGGTTCTCTTTCAAGGCGTCAC | ||
| GFP-Leep1M | pDM317 | F: GGGGATCCTTGGCCGACGTTTTCCATCATAAT |
| R: GGTCTAGAAAGGGTACGATTGGTTGTGAAAG | ||
| GFP-Leep1C | pDM317 | F: GGGGATCCCGTACCCTTATCGATATACCAGTAC |
| R: GGTCTAGAATATTCTGGAGGTGGTGGCATTCTA | ||
| GFP-Leep1K28AR39A | pDM317 | Insert 1 F: CTATCTAGAATGTCATCCGAATTATCTACTGGT |
| Insert 1 R: GCATCTTGAACAACATCTTTTTTATTTGTTTTTGC | ||
| Insert 2 F: GATGTTGTTCAAGATGCTCTTTTTG | ||
| Insert 2 R: CTAGTCGACATATTCTGGAGGTGGTGGCATTCTATTTTC | ||
| RFP-Leep1 | pDM449 | F: GGGGATCCATGTCATCCGAATTATCTACTGGTG |
| R: GGTCTAGAATATTCTGGAGGTGGTGGCATTCTA | ||
| GFP-Carmil | pDM317 | F: GGGGATCCATGTCAGAAGAAATATCACCAAATG |
| R: GGACTAGTATTTTCGGTCGGTGGTCTTGGTCTTC | ||
| GFP-PHPkgE | pDM317 | F: CCTCTAGAATGGCAGATAAACAAGGATTTTTAATT |
| R: CCGTCGACTGTAGATTCTTTAAGTTTATTTAATT | ||
| GFP-ArpC4 | pDM317 | F: GGGGTACCATGTCCACAGCTCAAGTTCCATATTTAAATTG |
| R: CGAGCGGCCGCAGCAAAGTTCTTTAAGTATTCAGAAGCAAC | ||
| TAPP1-GFP | pDM323 | F: CCGGAGCTCATGCCTTATGTGGATCGTCAG |
| R: CTAGCTAGCCACGTCACTGACCGGAAGGC | ||
| TAPP1-RFP | pDM451 | F: CGGGAGCTCATGCCTTATGTGGATCGTCAGAATC |
| R: CTAGCTAGCCACGTCACTGACCGGAAGGCTCGC | ||
| PHGRP1-GFP | pDM323 | F: CTATCTAGAATGTATGAAAGTATCAAGAATGAGCCG |
| R: CTAGTCGACTTTCTTATTGGCAATCCTCCTTTTC | ||
| Generation of knockout cells | ||
| leep1 knockout | pBluescript-BSR | Insert 1 F: GGGGTACCATGTCATCCGAATTATCTACTGGTG |
| Insert 1 R: GCGTCGACAACTAATTCTATGAAATGCATTAATTA | ||
| Insert 2 F: CGGGGATCCGATCAACAACAATATGAAGACG | ||
| Insert 2 R: CCGGCGGCCGCGTTGTGAAACATGAATTGTACC | ||
| Dd5P4 knockout | pBluescript-BSR | Insert 1 F: CACGGTACCGATTAAACAAAATGAAACGCAACTTTTC |
| Insert 1 R: GAGAAGCTTCTGTATTTTGAATATCACCCATTTTG | ||
| Insert 2 F: CGCGGATCCGATGCTACAACTGTTAAAAAGAAAGCTG | ||
| Insert 2 R: CCGGCGGCCGCGTTGTAAAAAAGACATTAATTGGTTTCTC | ||
| Expression in Dictyostelium cells . | ||
|---|---|---|
| Usage . | Plasmid backbone . | Sequence, 5′-3′ . |
| GFP-Leep1 | pDM317 | F: GGGGATCCATGTCATCCGAATTATCTACTGGTG |
| R: GGTCTAGAATATTCTGGAGGTGGTGGCATTCTA | ||
| GFP-Leep11-855 | pDM317 | F: CTACTAGAATGTCATCCGAATTATCTACTGGT |
| R: CTAGTCGACTTACATTCTATTTTCGTCTTCATATTG | ||
| GFP-Leep11-764 | pDM317 | F: CTACTAGAATGTCATCCGAATTATCTACTGGT |
| R: CTAGTCGACTTAACTATAGTCATAAGTTTCTTGTGG | ||
| GFP-Leep11-751 | pDM317 | F: CTACTAGAATGTCATCCGAATTATCTACTGGT |
| R: CTAGTCGACTTATTGTTGATAATGATTTGCATAAACATC | ||
| GFP-Leep11-736 | pDM317 | F: CTACTAGAATGTCATCCGAATTATCTACTGGT |
| R: CTAGTCGACTTAAGCATAAGTTGAAGGTGATGGTGA | ||
| GFP-Leep11-672 | pDM317 | F: CTACTAGAATGTCATCCGAATTATCTACTGGT |
| R: CTAGTCGACTTGAGAGACAAGTTTGAACATCAG | ||
| GFP-Leep11-623 | pDM317 | F: CTACTAGAATGTCATCCGAATTATCTACTGGT |
| R: CTAGTCGACTTAGGTTGTGAAAGTTCTTTTCATAGC | ||
| GFP-Leep1N | pDM317 | F: GGGGATCCATGTCATCCGAATTATCTACTGGTG |
| R: GGTCTAGATTGGTTCTCTTTCAAGGCGTCAC | ||
| GFP-Leep1M | pDM317 | F: GGGGATCCTTGGCCGACGTTTTCCATCATAAT |
| R: GGTCTAGAAAGGGTACGATTGGTTGTGAAAG | ||
| GFP-Leep1C | pDM317 | F: GGGGATCCCGTACCCTTATCGATATACCAGTAC |
| R: GGTCTAGAATATTCTGGAGGTGGTGGCATTCTA | ||
| GFP-Leep1K28AR39A | pDM317 | Insert 1 F: CTATCTAGAATGTCATCCGAATTATCTACTGGT |
| Insert 1 R: GCATCTTGAACAACATCTTTTTTATTTGTTTTTGC | ||
| Insert 2 F: GATGTTGTTCAAGATGCTCTTTTTG | ||
| Insert 2 R: CTAGTCGACATATTCTGGAGGTGGTGGCATTCTATTTTC | ||
| RFP-Leep1 | pDM449 | F: GGGGATCCATGTCATCCGAATTATCTACTGGTG |
| R: GGTCTAGAATATTCTGGAGGTGGTGGCATTCTA | ||
| GFP-Carmil | pDM317 | F: GGGGATCCATGTCAGAAGAAATATCACCAAATG |
| R: GGACTAGTATTTTCGGTCGGTGGTCTTGGTCTTC | ||
| GFP-PHPkgE | pDM317 | F: CCTCTAGAATGGCAGATAAACAAGGATTTTTAATT |
| R: CCGTCGACTGTAGATTCTTTAAGTTTATTTAATT | ||
| GFP-ArpC4 | pDM317 | F: GGGGTACCATGTCCACAGCTCAAGTTCCATATTTAAATTG |
| R: CGAGCGGCCGCAGCAAAGTTCTTTAAGTATTCAGAAGCAAC | ||
| TAPP1-GFP | pDM323 | F: CCGGAGCTCATGCCTTATGTGGATCGTCAG |
| R: CTAGCTAGCCACGTCACTGACCGGAAGGC | ||
| TAPP1-RFP | pDM451 | F: CGGGAGCTCATGCCTTATGTGGATCGTCAGAATC |
| R: CTAGCTAGCCACGTCACTGACCGGAAGGCTCGC | ||
| PHGRP1-GFP | pDM323 | F: CTATCTAGAATGTATGAAAGTATCAAGAATGAGCCG |
| R: CTAGTCGACTTTCTTATTGGCAATCCTCCTTTTC | ||
| Generation of knockout cells | ||
| leep1 knockout | pBluescript-BSR | Insert 1 F: GGGGTACCATGTCATCCGAATTATCTACTGGTG |
| Insert 1 R: GCGTCGACAACTAATTCTATGAAATGCATTAATTA | ||
| Insert 2 F: CGGGGATCCGATCAACAACAATATGAAGACG | ||
| Insert 2 R: CCGGCGGCCGCGTTGTGAAACATGAATTGTACC | ||
| Dd5P4 knockout | pBluescript-BSR | Insert 1 F: CACGGTACCGATTAAACAAAATGAAACGCAACTTTTC |
| Insert 1 R: GAGAAGCTTCTGTATTTTGAATATCACCCATTTTG | ||
| Insert 2 F: CGCGGATCCGATGCTACAACTGTTAAAAAGAAAGCTG | ||
| Insert 2 R: CCGGCGGCCGCGTTGTAAAAAAGACATTAATTGGTTTCTC | ||
All primer sequences are given in 5′ to 3′ direction, and each primer is designated as forward (F) or reverse (R).