Primers
| Experiment type . | Vector or construct . | Forward primer . | Reverse primer . | |
|---|---|---|---|---|
| CM1 deletion | pCFD4 guide RNA vector | CFD4_CM1_1f 5′-TATATAGGAAAGATATCCGGGTGAACTTCGAACTCGCCCTTGCCGTCACAGTTTTAGAGCTAGAAATAGCAAG-3′ | CFD4_CM1_3b 5′-ATTTTAACTTGCTATTTCTAGCTCTAAAACCTCGAGCCATTTCTCATCACGACGTTAAATTGAAAATAGGTC-3′ | |
| Homology vector | CM1Del_BS_b1 5′-TAGCGATCGCTGATTTGGAACAGTCCGTAATCCCGGGATCCAATTCGCCCTATAGTG-3′ | CM1Del_BS_f1 5′-CATGACGGCGGATGCCGGGGTTGGTATCACATCTTCTCTTCAGCTTTTGTTCCCTTTAGTGAGGG-3′ | ||
| CM1Del_UHA_f1 5′-GATCCCGGGATTACGGACTGTTCCAAATCAG-3′ | CM1Del_UHA_b1 5′-CGTGATGAGAAATGGCTCGAGCTGCGCCTTGCGAGGGCAAGGGCGAGTTGCCGCCG-3′ | |||
| CM1Del_DHA_f1 5′-GCAGCTCGAGCCATTTCTCATCACG-3′ | CM1Del_DHA_b1 5′-GCTGAAGAGAAGATGTGATACCAACCCCGG-3′ | |||
| PCR screening | Amplification primer 1 5′-ATTGGATGTTGTGCTGCGAGG-3′ | Amplification primer 2 5′-TTCAGATAAGTGTCGTGCTCG-3′ | ||
| Recombinant proteins | pDEST-MBP-PreSci-his-Strep | Empty pDEST_f 5′-AATTCGATCACAAGTTTGTACAAAAAAGC-3′ | Empty pDEST_mid_b 5′-TCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATC-3′ | |
| Empty pDEST_mid_f 5′-CAAGTTTACTCATATATACTTTAGATTGATTTACCCCGGTTGATAATC-3′ | His remove_b 5′-GATTTTCATAATCTATGGTCCTTGTTGGTGAAGTGCTCGTGAAAACACCTAAACGG-3′ | |||
| His remove_f 5′-CCAACAAGGACCATAGATTATGAAAATCGAAGAAGGTAAACTGGTAATCTGGATTAACGG-3′ | Empty pDEST_MBP_b 5′-AGTCTGCGCGTCTTTCAGGGCTTCATC-3′ | |||
| PreSci-His_Insert_f 5′-CGATGAAGCCCTGAAAGACGCGCAGACTAATTCGAGCCTGGAAGTTCTGTTCCAGGGGCCCAGTGGACATCACCATCACC-3′ | PreSci_His_Strep_Insert_b 5′-GCTTTTTTGTACAAACTTGTGATCGAATTACCTCCACTTTTCTCGAACTGCGGGTGGCTCCAAGTGTGATGGTGATGGTGATGGTGATGTCCACTGGGCCCCTGG-3′ | |||
| Cnn-C-NT27E,F115A,S186D | Cnn_F115A_QC_f 5′-CCGCGCTGCGCAAGGAGAACGCCAATCTAAAGCTGCGC-3′ | Cnn_F115A_QC_b 5′-GCGCAGCTTTAGATTGGCGTTCTCCTTGCGCAGCGCGG-3′ | ||
| Cnn-C-NT27E,R101Q,E102A,S186D | CnnCM1_mut_QC_f1 5′-CCGTCACAGGGTCGCTCTGTACAGGCCTTGGAGGAGCAGATGTCC-3′ | CnnCM1_mut_QC_b1 5′-GGACATCTGCTCCTCCAAGGCCTGTACAGAGCGACCCTGTGACGG-3′ | ||
| Experiment type . | Vector or construct . | Forward primer . | Reverse primer . | |
|---|---|---|---|---|
| CM1 deletion | pCFD4 guide RNA vector | CFD4_CM1_1f 5′-TATATAGGAAAGATATCCGGGTGAACTTCGAACTCGCCCTTGCCGTCACAGTTTTAGAGCTAGAAATAGCAAG-3′ | CFD4_CM1_3b 5′-ATTTTAACTTGCTATTTCTAGCTCTAAAACCTCGAGCCATTTCTCATCACGACGTTAAATTGAAAATAGGTC-3′ | |
| Homology vector | CM1Del_BS_b1 5′-TAGCGATCGCTGATTTGGAACAGTCCGTAATCCCGGGATCCAATTCGCCCTATAGTG-3′ | CM1Del_BS_f1 5′-CATGACGGCGGATGCCGGGGTTGGTATCACATCTTCTCTTCAGCTTTTGTTCCCTTTAGTGAGGG-3′ | ||
| CM1Del_UHA_f1 5′-GATCCCGGGATTACGGACTGTTCCAAATCAG-3′ | CM1Del_UHA_b1 5′-CGTGATGAGAAATGGCTCGAGCTGCGCCTTGCGAGGGCAAGGGCGAGTTGCCGCCG-3′ | |||
| CM1Del_DHA_f1 5′-GCAGCTCGAGCCATTTCTCATCACG-3′ | CM1Del_DHA_b1 5′-GCTGAAGAGAAGATGTGATACCAACCCCGG-3′ | |||
| PCR screening | Amplification primer 1 5′-ATTGGATGTTGTGCTGCGAGG-3′ | Amplification primer 2 5′-TTCAGATAAGTGTCGTGCTCG-3′ | ||
| Recombinant proteins | pDEST-MBP-PreSci-his-Strep | Empty pDEST_f 5′-AATTCGATCACAAGTTTGTACAAAAAAGC-3′ | Empty pDEST_mid_b 5′-TCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATC-3′ | |
| Empty pDEST_mid_f 5′-CAAGTTTACTCATATATACTTTAGATTGATTTACCCCGGTTGATAATC-3′ | His remove_b 5′-GATTTTCATAATCTATGGTCCTTGTTGGTGAAGTGCTCGTGAAAACACCTAAACGG-3′ | |||
| His remove_f 5′-CCAACAAGGACCATAGATTATGAAAATCGAAGAAGGTAAACTGGTAATCTGGATTAACGG-3′ | Empty pDEST_MBP_b 5′-AGTCTGCGCGTCTTTCAGGGCTTCATC-3′ | |||
| PreSci-His_Insert_f 5′-CGATGAAGCCCTGAAAGACGCGCAGACTAATTCGAGCCTGGAAGTTCTGTTCCAGGGGCCCAGTGGACATCACCATCACC-3′ | PreSci_His_Strep_Insert_b 5′-GCTTTTTTGTACAAACTTGTGATCGAATTACCTCCACTTTTCTCGAACTGCGGGTGGCTCCAAGTGTGATGGTGATGGTGATGGTGATGTCCACTGGGCCCCTGG-3′ | |||
| Cnn-C-NT27E,F115A,S186D | Cnn_F115A_QC_f 5′-CCGCGCTGCGCAAGGAGAACGCCAATCTAAAGCTGCGC-3′ | Cnn_F115A_QC_b 5′-GCGCAGCTTTAGATTGGCGTTCTCCTTGCGCAGCGCGG-3′ | ||
| Cnn-C-NT27E,R101Q,E102A,S186D | CnnCM1_mut_QC_f1 5′-CCGTCACAGGGTCGCTCTGTACAGGCCTTGGAGGAGCAGATGTCC-3′ | CnnCM1_mut_QC_b1 5′-GGACATCTGCTCCTCCAAGGCCTGTACAGAGCGACCCTGTGACGG-3′ | ||