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. 2018 Apr 2;217(4):1411–1429. doi: 10.1083/jcb.201707027

Table 1. Primers used for cloning of full-size or deletion mutants of SLAMF1, TRAM, TLR4, and Rab11 FIP2.

Construct For/Rev Primer sequence (5′–3′) Restriction enzyme PCR product mapped on nucleotide sequence
SLAMF1 ( NM_003037.3 )
pcDNA3.1 and pLVX-EF1α-IRES-ZsGreen1 For TTCGAATTCTGATGGGATCCCAAGGGGCTCC EcoRI 367–1,374
Rev GCAGCGGCCGCTCAGCTCTCTGGAAGTGTCA NotI
C-terminal DYKDDDDK For TTAGAATTCATGGATCCCAAGGGGCTCC EcoRI 367–1,371
Rev GGTACCTCGAGAGCTCTCTGGAAGTGTCACAC XhoI
pGEX-2TK For TATGGATCCCAGTTGAGAAGAAGAGGTAAAACG BamHI 1,141–1,374
Rev ATAGAATTCTCAGCTCTCTGGAAGTGTCAC EcoRI
SLAMF1 ( NM_003037.3 ) deletion mutants to C-terminal DYKDDDDK
All deletion mutants For TTAGAATTCATGGATCCCAAGGGGCTCC EcoRI N/a
1–265 aa Rev GGTACCTCGAGATTTACCTCTTCTTCTCAACTGTAG XhoI 367–1,161
1–326 aa Rev GTACCTCGAGAGACTGTGATGGAATTTGTTTCCTG XhoI 367–1,344
1–330 aa Rev GTACCTCGAGACACACTAGCATAGACTGTGATG XhoI 367–1,356
SLAMF1 ( NM_003037.3 ) deletion mutant to pcDNA3.1
1–265 aa For TTCGAATTCTGATGGGATCCCAAGGGGCTCC EcoRI 367–1,161
Rev TTAAGCGGCCGCTCATTTACCTCTTCTTCTCAACTG NotI
SLAMF1 ( NM_003037.3 ) mutants with amino acid substitutions to C-terminal DYKDDDDK
SLAMF1 Y281F For TTAGAATTCATGGATCCCAAGGGGCTCC EcoRI 367–1,225
Rev GTTTCTGGACTTGGGCAAAGATCGTAAGGC
For GCCTTACGATCTTTGCCCAAGTCCAGAAAC 1,196–1,371
Rev GGTACCTCGAGATTTACCTCTTCTTCTCAACTGTAG XhoI
SLAMF1 Y327F For TTAGAATTCATGGATCCCAAGGGGCTCC EcoRI 367–1,371
Rev GGTACCTCGAGAGCTCTCTGGAAGTGTCACACTAGCAAAGACTGTG XhoI
SLAMF1 TNSI/PNPT For TTAGAATTCATGGATCCCAAGGGGCTCC EcoRI 367–1,341
Rev TGTGGTGGGGTTTGGTTCCTGGACAGACTCTGG
For GAACCAAACCCCACCACAGTCTATGCTAGTGTGACACTTC 1,324–1,371a
Revb CTTGTCATCGTCGTCCTTG
TRAM/TICAM-2 ( NM_021649.7 )
C-terminal DYKDDDDK For CATGAATTCATGGGTATCGGGAAGTCTAAA EcoRI 443–1,147
Rev TTAACTCGAGCGGCAATAAATTGTCTTTGTACC XhoI
TRAM deletion mutants to C-terminal DYKDDDDK
1–68 aa For CATGAATTCATGGGTATCGGGAAGTCTAAA EcoRI 443–646
Rev TTAACTCGAGCCATCTCTTCCACGCTCTGAGC XhoI
1–79 aa For CATGAATTCATGGGTATCGGGAAGTCTAAA EcoRI 443–679
Rev TTACCTCGAGAGAGGAACACCTCTTCTTCAGC XhoI
1–90 aa For CATGAATTCATGGGTATCGGGAAGTCTAAA EcoRI 443–712
Rev TTACCTCGAGATGTGTCATCTTCTGCATGCAATATC XhoI
1–100 aa For CATGAATTCATGGGTATCGGGAAGTCTAAA EcoRI 443–742
Rev TTACCTCGAGATAGCAGATTCTGGACTCTGAGG XhoI
1–120 aa For CATGAATTCATGGGTATCGGGAAGTCTAAA EcoRI 443–802
Rev TTACCTCGAGACTGTCTGCCACATGGCATCTC XhoI
68–235 aa For CATGAATTCATGTTTGAAGAAGAAGCTGAA EcoRI 644–1,147
Rev TTAACTCGAGCGGCAATAAATTGTCTTTGTACC XhoI
68–176 aa For CATGAATTCATGTTTGAAGAAGAAGCTGAA EcoRI 644–970
Rev TTAACTCGAGCCAGGGGCCGCATGGGTATAACAG XhoI
158–235 aa For CATGAATTCATGAACTCCGTTAACAGGCAGC EcoRI 914–1,147
Rev TTAACTCGAGCGGCAATAAATTGTCTTTGTACC XhoI
TLR4 ( NM_138554.4 )
C-terminal DYKDDDDK For CGGTCGACCGAGATCTCATGATGTCTGCCTCGCGCCTGG N/a 299–2,815
Rev CTTGTAGTCGCCGGTACCGATAGATGTTGCTTCCTGCCAATTG N/a
Mus Musculus TRAM/Ticam-2 ( NM_173394.3 ) to EGFP-N1
1–232 aa For ACTAAGCTTATGGGTGTTGGGAAGTCTAAAC HindIII 477–1,175
Rev ATATGGATCCCGGGCAATGAACTGTTTCTGCGAC BamHI
M. Musculus Slamf1 ( NM_013730.4 ) from pDisplay vector to C-terminal DYKDDDDK
30–343 aa For TTGGAATTCGGCTTGGGGATATCCACCATGG EcoRI 183–1,127
Rev TATACCTCGAGAGCTCTCTGGCAGTGTCACACTG XhoI
Rab11 FIP2 ( NM_014904.2 ) and deletion mutants to N-terminal DYKDDDDK
All constructs For GCCCGAATTCGGCTGTCCGAGCAAGCCCAAAAG EcoRI
1–512 aa Rev ATAGCGGCCGCTCATTAACTGTTAGAGAATTTGCCAGC NotI 446–1,980
1–327 aa Rev ATAGCGGCCGCTCATTCGCTGCTTTCTTCAAATGG NotI 446–1,429
1–290 aa Rev ATAGCGGCCGCTTACACAATGCTGTCAGGTTGG NotI 446–1,310
1–254 aa Rev ATAGCGGCCGCTTATCCGAGAAGATGTGTTTGACC NotI 446–1,199
1–192 aa Rev ATAGCGGCCGCTTAGTGAGTACTTGGAATGATTGC NotI 446–1,016
Rab11a ( NM_004663.4 ) QL and SN mutants to pECFP-C1
1–216 aa For ATCAGTCGACATGGGCACCCGCGACGAC SalI 128–145
Rev TTAAGGATCCTTATATGTTCTGACAGCACTG BamHI 758–774

For, forward; QL, Rab11a Q70L mutant; Rev, reverse; SN, Rab11a S25N mutant.

a

Partially on vector sequence.

b

On C-terminal DYKDDDDK vector.