Table 1. Primers used for cloning of... | Rockefeller University Press

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		1,324–1,371^{^a}
	Rev^{^b}	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

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		1,324–1,371^a
	Rev^b	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.

Partially on vector sequence.

On C-terminal DYKDDDDK vector.

[ViewLarge]

Sharing Unavailable