1 |
NRP-2 KpnI insertion after Cys592
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NRP-2 |
5′-GAGGTGCTGGGCTGTGGTACCATGGACTGGACAG-ACTCC-3′ |
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5′-GGAGTCTGTCCAGTCCATGGTACCACAGCCCAGC-ACCTC-3′ |
2 |
NRP-2 signal sequence amplification including HindIII and KpnI sites |
NRP-2 |
5′-GTTAAGCTTATGGATATGTTTCCTCTCACCTGGG-3′ |
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5′-AATGGTACCGCCTCTCACTTGGTGTCTTGA-3′ |
3 |
MAM domain amplification with KpnI and XbaI sites |
NRP-2 |
5′-CGGGGTACCTCGGGATTCAATTGCAACTTC-3′ |
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5′-CCTTCTAGAGCTGCCTCGGAGCAGCACTTT-3′ |
4 |
NRP-2 EcoRV insertion between Pro641 and Ser642
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NRP-2 |
5′-GCAGCTCCCTGATATCTCGGGATTCAATTG-3′ |
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5′-CGTCGAGGGACTATAGAGCCCTAAGTTAAC-3′ |
5 |
Nrp-2 NheI insertion between Glu802 and Pro803
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NRP-2 |
5′-GAACTGCATGGAAGCTAGCCCCATCTCGGC-3′ |
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5′-CTTGACGTACCTTCGATCGGGGTAGAGCCG-3′ |
6 |
Nrp-1 EcoRV insertion between Pro644 and Thr645
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NRP-1 |
5′-CAATCAGAGTTTCCAGATATCACATATGGTTTT-AAC-3′ |
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5′-GTTAGTCTCAAAGGTCTATAGTGTATACCAAAA-TTG-3′ |
7 |
Nrp-1 NheI insertion between Lys811 and Pro812
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NRP-1 |
5′-GAAGATTGTGCAAAAGCTAGCCCAGCAGACCTG-3′ |
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5′-CTTCTAACACGTTTTCGATCGGGTCGTCTGGAC-3′ |
8 |
NRP-1Δ2 remove EcoRV |
NRP-1Δ2 + restriction sites |
5′-CAATCAGAGTTTCCATCGGGATTCAATTG-3′ |
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5′-GTTAGTCTCAAAGGTAGCCCTAAGTTAAC-3′ |
9 |
NRP-1Δ2 remove NheI |
NRP-1Δ2 + restriction sites |
5′-GAACTGCATGGAACCAGCAGACCTG-3′ |
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5′-CTTGACGTACCTTGGTCGTCTGGAC-3′ |
10 |
NRP-2Δ1 remove EcoRV |
NRP-2Δ1 + restriction sites |
5′-GCAGCTCCCTACATATGGTTTTAAC-3′ |
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5′-CGTCGAGGGATGTATACCAAAATTG-3′ |
11 |
NRP-2Δ1 remove NheI |
NRP-2Δ1 + restriction sites |
5′--GAAGATTGTGCAAAACCCATCTGCGC--3′ |
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5′-CTTCTAACACGTTTTGGGTAGACGCG-3′ |
12 |
NRP-2Δ1 (to remove stop codon inserted during MAM swap at Glu622) |
NRP-2Δ1 |
5′-CCACCGAAGAGGAGGCCACAGAGTG-3′ |
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5′-GGTGGCTTCTCCTCCGGTGTCTCAC-3′ |
13 |
Amplification of ectodomains to make NRP-Fc constructs |
NRP-1 and NRP-1Δ2 |
5′-AGACCCAAGCTGGCCATGGAGAGGGGGCTGCCGCTCCTC-3′ |
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5′-AGACACCCTCCCTCCGGGGTCTAAGGTCTTCAACACATT-3′ |
14 |
Amplification of ectodomains to make NRP-Fc constructs |
NRP-2Δ1 |
5′-GAGACCCAAGCTGGCCATGGATATGTTTCCTCTCACCTGG-3′ |
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5′-AGACACCCTCCCTCCGGGATCCAGGGTGTACAGCCAGCTC-3′ |
15 |
Amplification of pcDNA4-hFc vector for In-Fusion |
pcDNA4-NRP-2-hFc |
5′-GCTCTAGAGGGCCCGCGGTTCGAAGGTA-3′ |
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5′-AGTCTAGAGGGCCCGCGGTTC-3′ |
16 |
Insertion of splice consensus sequence |
NRP-2-Fc and NRP-2Δ1-Fc |
5′-CACCCTGGATCCCACAGGTAAGTGGAGGGAG-GGTGT-3′ |
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5′-ACACCCTCCCTCCACTTACCTGTGGGATCCAGGGTG-3′ |
17 |
Insertion of splice consensus sequence |
NRP-1-Fc and NRP-1Δ2-Fc |
5′-AGACCTTAGACCCCACAGGTAAGTGGAGGGAGGGTGTC-3′ |
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5′-GACACCCTCCCTCCACTTACCTGTGGGGTCTAAGGTCT-3′ |
18 |
Amplification of NRP-1 linker region through the cytoplasmic tail for NRP-2ΔLM1 construct |
NRP-1 |
5′-GAGGTGCTGGGCTGTGAAGTGGAAGCCCCTACAGCTGGA-3′ |
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5′-CGGGCCCTCTAGAGCTGCCTCCGAATAAGTACTCTGTG-3′ |
19 |
Amplification of NRP-2 linker region through the cytoplasmic tail for NRP-1ΔLM2 construct |
NRP-2 |
5′-GAGCTGCTGGGCTGTGACTGGACAGACTCCAAGCCCACG-3′ |
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5′-CGGGCCCTCTAGAGCTGCCTCGGAGCAGCACTTTTGGT-3′ |
20 |
Amplification of NRP-2 sequences except the linker region through the cytoplasmic tail sequences in pcDNA3.1/V5-His B vector |
NRP-2 |
5′-ACAGCCCAGCACCTCCAGCCGCATCCCA-3′ |
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5′-GCTCTAGAGGGCCCGCGGTTCGAAGGTA-3′ |
21 |
Amplification of NRP-1 sequences except the linker region through the cytoplasmic tail in pcDNA3.1/V5-His B vector |
NRP-1 |
5′-ACAGCCCAGCAGCTCCATTCTGAGCCCCA-3′ |
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5′-GCTCTAGAGGGCCCGCGGTTCGAAGGTA-3′ |
22 |
Deletion of extra cytosine base |
NRP-1ΔLM2 |
5′-GACAACCACCCCCTACCCCACCGAAG-3′ |
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5′-CTTCGGTGGGGTAGGGGGTGGTTGTC-3′ |
23 |
Amplification of ectodomains to make NRP-Fc constructs |
NRP-2ΔLM1 |
5′-GAGACCCAAGCTGGCCATGGATATGTTTCCTCT-CACCTGG-3′ |
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5′-GACACCCTCCCTCCACTTACCTGTGGGGTCTAAGGTCT-3′ |
24 |
Amplification of ectodomains to make NRP-Fc constructs |
NRP-1ΔLM2 |
5′-AGACCCAAGCTGGCCATGGAGAGGGGGCTGCCGCTCCTC-3′ |
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5′-ACACCCTCCCTCCACTTACCTGTGGGATCCAGGGTG-3′ |
25 |
Inverse PCR of NRP-2-Fc to eliminate MAM domain |
NRP-2-Fc |
5′-CCTCCACTTACCTGTAGGGAGCTGCAAATCTTTGTCATCCTCA-3′ |
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5′-GGAGGGAGGGTGTCTGCTGGAA-3′ |
26 |
NRP-2 E652A |
NRP-2-Fc |
5′-CTTCGATTTCCTCGCGGAGCCCTGTGG-3′ |
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5′-GAAGCTAAAGGAGCGCCTCGGGACACC-3′ |
27 |
NRP-2 E653A |
NRP-2-Fc |
5′-CGATTTCCTCGAGGCGCCCTGTGGTTGG-3′ |
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5′-GCTAAAGGAGCTCCGCGGGACACCAACC-3′ |
28 |
NRP-2 E652A/E653A |
NRP-2-Fc |
5′-CTTCGATTTCGCGGCGCCCTGTGGTTGG-3′ |
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5′-GAAGCTAAAGCGCCGCGGGACACCAACC-3′ |
29 |
NRP-2 D683A |
NRP-2-Fc |
5′-GACGTTTCCAGATGCCAGGAATTTCTTGCG-3′ |
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5′-CTGCAAAGGTCTACGGTCCTTAAAGAACGC-3′ |