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Vector Backbone Description: Backbone Marker:Novagen, EMBL; Backbone Size:6070; Vector Backbone:pET, pCoofy14; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:23410102
Comments: Test each preparation of the plasmid by transforming it into non-resistant cells (ex. DH5a) to ensure negative selection by ccdB kills all the transformed cells as expected.
Proper citation: RRID:Addgene_43996 Copy
Species: HPV16
Genetic Insert: HPV16E6
Vector Backbone Description: Backbone Size:8100; Vector Backbone:MSCV-IP N-FlagHA; Vector Types:Mammalian Expression, Retroviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:23015706
Proper citation: RRID:Addgene_44154 Copy
Species: Homo sapiens
Genetic Insert: MEKK3 (K391A)
Vector Backbone Description: Vector Backbone:pcDNA3.1(+); Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:15572679
Comments: Alternative plasmid name: pcDNA3.1 MEKK3 (K391A). MEKK3 K391A, kinase-dead mutant, variant of Addgene plasmid #44156. Mutant of MEKK3 was generated by overlap PCR.
Proper citation: RRID:Addgene_44157 Copy
Species: Homo sapiens
Genetic Insert: HSPA5 promoter
Vector Backbone Description: Backbone Marker:Promega; Backbone Size:3000; Vector Backbone:pGEM; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:22577136
Comments: a fragment containing 429 bp of the human HSPA5 promoter
(-282 to +147) relative to the transcriptional start site) was amplified from human genomic DNA by PCR and cloned into pGEM-T Easy Vector (Promega) to generate pGEM-HSPA5P
Proper citation: RRID:Addgene_44150 Copy
Species: HPV45
Genetic Insert: HPV45E6
Vector Backbone Description: Backbone Size:8100; Vector Backbone:MSCV-IP N-HAonly; Vector Types:Mammalian Expression, Retroviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:23015706
Comments: DKFZ is the co-provider of this plasmid.
Proper citation: RRID:Addgene_44146 Copy
Species: HPV76
Genetic Insert: HPV76E6
Vector Backbone Description: Backbone Size:8100; Vector Backbone:MSCV-IP N-HAonly; Vector Types:Mammalian Expression, Retroviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:23015706
Comments: DKFZ is the co-provider of this plasmid.
Proper citation: RRID:Addgene_44145 Copy
Species: HPV92
Genetic Insert: HPV92E6
Vector Backbone Description: Backbone Size:8100; Vector Backbone:MSCV-IP N-HAonly; Vector Types:Mammalian Expression, Retroviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:23015706
Proper citation: RRID:Addgene_44147 Copy
Species: HPV57
Genetic Insert: HPV57E6
Vector Backbone Description: Backbone Size:8100; Vector Backbone:MSCV-IP N-HAonly; Vector Types:Mammalian Expression, Retroviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:23015706
Comments: DKFZ is the co-provider of this plasmid.
Proper citation: RRID:Addgene_44141 Copy
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:6612; Vector Backbone:pcDNA4-TO; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:22474084
Proper citation: RRID:Addgene_44099 Copy
Species: Homo sapiens
Genetic Insert: TRAF2 S102A
Vector Backbone Description: Vector Backbone:pWZL-Blasticidin; Vector Types:Retroviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:23007157
Proper citation: RRID:Addgene_44135 Copy
Species: HPV18
Genetic Insert: HPV18 E6
Vector Backbone Description: Backbone Size:8100; Vector Backbone:MSCV-IP N-HAonly; Vector Types:Mammalian Expression, Retroviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:23015706
Proper citation: RRID:Addgene_44137 Copy
Species: HPV6b
Genetic Insert: HPV6b E6
Vector Backbone Description: Backbone Size:8100; Vector Backbone:MSCV-IP N-HAonly; Vector Types:Mammalian Expression, Retroviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:23015706
Comments: DKFZ is the co-provider of this plasmid.
Proper citation: RRID:Addgene_44139 Copy
Species: HPV45
Genetic Insert: HPV45 E6
Vector Backbone Description: Backbone Size:8100; Vector Backbone:MSCV-IP N-HAonly; Vector Types:Mammalian Expression, Retroviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:23015706
Comments: DKFZ is the co-provider of this plasmid.
Proper citation: RRID:Addgene_44138 Copy
Vector Backbone Description: Backbone Marker:Mark Hochstrasser lab (Yale Univeristy, New Haven, CT) (PMID: 19243080); Backbone Size:4255; Vector Backbone:pFA6a-6xGLY-3xFLAG-kanMX6 (Addgene plasmid # 20754); Vector Types:PCR-based yeast C-terminal tagging; Bacterial Resistance:Ampicillin
Comments: The depositing lab has constructed and deposited here 36 plasmids that can be used to generate linear PCR products to fuse to the 3' end of an ORF in the yeast genome and add (TEV or PreScission protease site) - 6X Gly linker - (12X His, 2X Strep, 3X FLAG, Protein A, or V5) - ADH1 transcription terminator - (KanMX, HphMX, or His3MX).
Six plasmids (Addgene plasmids 44062, 44068, 44074, 44080, 44086, and 44092) have no epitope/purification tag and so can be used to add an alternate tag by standard cloning into unique XhoI-KpnI-XbaI sites, if desired.
The other 30 plasmids are all of the possible combinations of the protease site - tag - marker options listed above. After fusion, the modified protein can then be followed with the epitope or purified using the tag and released from the matrix with the protease site.
The constructs were designed with one of two different protease sites to allow a complex with two different tags to be purified in two successive steps, using each protease to release the complex from each purification matrix.
The schematic diagram (see attached above) represents the format of the constructs and shows the proper scheme for producing the desired PCR products (see also below). If only epitope tagging is needed, the upstream primer can be adjusted to skip the protease site (see example below).
Construction:
DS5421 (pFA6a-6xGLY-3XFLAG-HIS3MX6 (Addgene Plasmid #20753), DS5422 (pFA6a-6xGLY-3XFLAG-KanMX) (Addgene Plasmid #20754), or DS5423 (pFA6a-6xGLY-3XFLAG-HphMX) (Addgene Plasmid #20755) (corresponding to each selection marker) was digested with AscI and PacI to release 3XFlag and replace it with TEV or PreScision sites followed by 6xGly and an XhoI-KpnI-XbaI linker.
Affinity tags were constructed by PCR or by annealing oligos to fit into the XhoI-XbaI gap. This includes 12xHis, 2xStrep II, 3xFLAG, PrtA (2 repeats as found in pBS1479), and V5. Each tag was inserted into each base vector, making 30 templates with all combinations of protease site, tag, and selectable marker.
PCR amplification:
These two primers (along with 50 nt of homology to the targets at the 5' ends) will amplify the desired product from any of the constructs (the reading frame for P1 is important – alternating codons bracketed).
P1 5’-(homology)...[CGA]CGG[ATC]CCC[GGG]TTA[ATT]AAC-3’
P2 5’-(homology)...GATATCATCGATGAATTCGAGCTCGTTT-3’
If only epitope tagging is needed, the upstream primer can be adjusted to skip the protease site. As an example, for the V5-tag-containing constructs, usage of the following primer (5’-(homology)... GGTGGAGGCTCTAGAGGTAAGCCAATTCCA-3’) in place of P1 will omit the protease cleavage site and add only 3xGly and V5 (GGGSR-V5-Stop).
Proper citation: RRID:Addgene_44094 Copy
Vector Backbone Description: Backbone Marker:Mark Hochstrasser lab (Yale Univeristy, New Haven, CT) (PMID: 19243080); Backbone Size:4255; Vector Backbone:pFA6a-6xGLY-3xFLAG-kanMX6 (Addgene plasmid # 20754); Vector Types:PCR-based yeast C-terminal tagging; Bacterial Resistance:Ampicillin
Comments: The depositing lab has constructed and deposited here 36 plasmids that can be used to generate linear PCR products to fuse to the 3' end of an ORF in the yeast genome and add (TEV or PreScission protease site) - 6X Gly linker - (12X His, 2X Strep, 3X FLAG, Protein A, or V5) - ADH1 transcription terminator - (KanMX, HphMX, or His3MX).
Six plasmids (Addgene plasmids 44062, 44068, 44074, 44080, 44086, and 44092) have no epitope/purification tag and so can be used to add an alternate tag by standard cloning into unique XhoI-KpnI-XbaI sites, if desired.
The other 30 plasmids are all of the possible combinations of the protease site - tag - marker options listed above. After fusion, the modified protein can then be followed with the epitope or purified using the tag and released from the matrix with the protease site.
The constructs were designed with one of two different protease sites to allow a complex with two different tags to be purified in two successive steps, using each protease to release the complex from each purification matrix.
The schematic diagram (see attached above) represents the format of the constructs and shows the proper scheme for producing the desired PCR products (see also below). If only epitope tagging is needed, the upstream primer can be adjusted to skip the protease site (see example below).
Construction:
DS5421 (pFA6a-6xGLY-3XFLAG-HIS3MX6 (Addgene Plasmid #20753), DS5422 (pFA6a-6xGLY-3XFLAG-KanMX) (Addgene Plasmid #20754), or DS5423 (pFA6a-6xGLY-3XFLAG-HphMX) (Addgene Plasmid #20755) (corresponding to each selection marker) was digested with AscI and PacI to release 3XFlag and replace it with TEV or PreScision sites followed by 6xGly and an XhoI-KpnI-XbaI linker.
Affinity tags were constructed by PCR or by annealing oligos to fit into the XhoI-XbaI gap. This includes 12xHis, 2xStrep II, 3xFLAG, PrtA (2 repeats as found in pBS1479), and V5. Each tag was inserted into each base vector, making 30 templates with all combinations of protease site, tag, and selectable marker.
PCR amplification:
These two primers (along with 50 nt of homology to the targets at the 5' ends) will amplify the desired product from any of the constructs (the reading frame for P1 is important – alternating codons bracketed).
P1 5’-(homology)...[CGA]CGG[ATC]CCC[GGG]TTA[ATT]AAC-3’
P2 5’-(homology)...GATATCATCGATGAATTCGAGCTCGTTT-3’
If only epitope tagging is needed, the upstream primer can be adjusted to skip the protease site. As an example, for the V5-tag-containing constructs, usage of the following primer (5’-(homology)... GGTGGAGGCTCTAGAGGTAAGCCAATTCCA-3’) in place of P1 will omit the protease cleavage site and add only 3xGly and V5 (GGGSR-V5-Stop).
Proper citation: RRID:Addgene_44093 Copy
Vector Backbone Description: Backbone Marker:Mark Hochstrasser lab (Yale Univeristy, New Haven, CT) (PMID: 19243080); Backbone Size:4255; Vector Backbone:pFA6a-6xGLY-3xFLAG-kanMX6 (Addgene plasmid # 20754); Vector Types:PCR-based yeast C-terminal tagging; Bacterial Resistance:Ampicillin
Comments: The depositing lab has constructed and deposited here 36 plasmids that can be used to generate linear PCR products to fuse to the 3' end of an ORF in the yeast genome and add (TEV or PreScission protease site) - 6X Gly linker - (12X His, 2X Strep, 3X FLAG, Protein A, or V5) - ADH1 transcription terminator - (KanMX, HphMX, or His3MX).
Six plasmids (Addgene plasmids 44062, 44068, 44074, 44080, 44086, and 44092) have no epitope/purification tag and so can be used to add an alternate tag by standard cloning into unique XhoI-KpnI-XbaI sites, if desired.
The other 30 plasmids are all of the possible combinations of the protease site - tag - marker options listed above. After fusion, the modified protein can then be followed with the epitope or purified using the tag and released from the matrix with the protease site.
The constructs were designed with one of two different protease sites to allow a complex with two different tags to be purified in two successive steps, using each protease to release the complex from each purification matrix.
The schematic diagram (see attached above) represents the format of the constructs and shows the proper scheme for producing the desired PCR products (see also below). If only epitope tagging is needed, the upstream primer can be adjusted to skip the protease site (see example below).
Construction:
DS5421 (pFA6a-6xGLY-3XFLAG-HIS3MX6 (Addgene Plasmid #20753), DS5422 (pFA6a-6xGLY-3XFLAG-KanMX) (Addgene Plasmid #20754), or DS5423 (pFA6a-6xGLY-3XFLAG-HphMX) (Addgene Plasmid #20755) (corresponding to each selection marker) was digested with AscI and PacI to release 3XFlag and replace it with TEV or PreScision sites followed by 6xGly and an XhoI-KpnI-XbaI linker.
Affinity tags were constructed by PCR or by annealing oligos to fit into the XhoI-XbaI gap. This includes 12xHis, 2xStrep II, 3xFLAG, PrtA (2 repeats as found in pBS1479), and V5. Each tag was inserted into each base vector, making 30 templates with all combinations of protease site, tag, and selectable marker.
PCR amplification:
These two primers (along with 50 nt of homology to the targets at the 5' ends) will amplify the desired product from any of the constructs (the reading frame for P1 is important – alternating codons bracketed).
P1 5’-(homology)...[CGA]CGG[ATC]CCC[GGG]TTA[ATT]AAC-3’
P2 5’-(homology)...GATATCATCGATGAATTCGAGCTCGTTT-3’
If only epitope tagging is needed, the upstream primer can be adjusted to skip the protease site. As an example, for the V5-tag-containing constructs, usage of the following primer (5’-(homology)... GGTGGAGGCTCTAGAGGTAAGCCAATTCCA-3’) in place of P1 will omit the protease cleavage site and add only 3xGly and V5 (GGGSR-V5-Stop).
Proper citation: RRID:Addgene_44096 Copy
Species: Homo sapiens
Genetic Insert: human tumor necrosis factor receptor 1
Vector Backbone Description: Backbone Marker:Nolan Lab; Vector Backbone:pBMN-IN; Vector Types:Retroviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:20511226
Proper citation: RRID:Addgene_44098 Copy
Species: Homo sapiens
Genetic Insert: c-IAP1
Vector Backbone Description: Vector Backbone:pLKO; Vector Types:Mammalian Expression, Lentiviral, RNAi; Bacterial Resistance:Ampicillin
Defining Citation: PMID:23007157
Comments: TRC clone ID: TRC0000003782
Proper citation: RRID:Addgene_44131 Copy
Species: Homo sapiens
Genetic Insert: CIAP2
Vector Backbone Description: Vector Backbone:pLKO; Vector Types:Mammalian Expression, Lentiviral, RNAi; Bacterial Resistance:Ampicillin
Defining Citation: PMID:23007157
Comments: TRC clone ID: TRC0000003778
Proper citation: RRID:Addgene_44130 Copy
Vector Backbone Description: Backbone Marker:Mark Hochstrasser lab (Yale Univeristy, New Haven, CT) (PMID: 19243080); Backbone Size:4255; Vector Backbone:pFA6a-6xGLY-3xFLAG-kanMX6 (Addgene plasmid # 20754); Vector Types:PCR-based yeast C-terminal tagging; Bacterial Resistance:Ampicillin
Comments: The depositing lab has constructed and deposited here 36 plasmids that can be used to generate linear PCR products to fuse to the 3' end of an ORF in the yeast genome and add (TEV or PreScission protease site) - 6X Gly linker - (12X His, 2X Strep, 3X FLAG, Protein A, or V5) - ADH1 transcription terminator - (KanMX, HphMX, or His3MX).
Six plasmids (Addgene plasmids 44062, 44068, 44074, 44080, 44086, and 44092) have no epitope/purification tag and so can be used to add an alternate tag by standard cloning into unique XhoI-KpnI-XbaI sites, if desired.
The other 30 plasmids are all of the possible combinations of the protease site - tag - marker options listed above. After fusion, the modified protein can then be followed with the epitope or purified using the tag and released from the matrix with the protease site.
The constructs were designed with one of two different protease sites to allow a complex with two different tags to be purified in two successive steps, using each protease to release the complex from each purification matrix.
The schematic diagram (see attached above) represents the format of the constructs and shows the proper scheme for producing the desired PCR products (see also below). If only epitope tagging is needed, the upstream primer can be adjusted to skip the protease site (see example below).
Construction:
DS5421 (pFA6a-6xGLY-3XFLAG-HIS3MX6 (Addgene Plasmid #20753), DS5422 (pFA6a-6xGLY-3XFLAG-KanMX) (Addgene Plasmid #20754), or DS5423 (pFA6a-6xGLY-3XFLAG-HphMX) (Addgene Plasmid #20755) (corresponding to each selection marker) was digested with AscI and PacI to release 3XFlag and replace it with TEV or PreScision sites followed by 6xGly and an XhoI-KpnI-XbaI linker.
Affinity tags were constructed by PCR or by annealing oligos to fit into the XhoI-XbaI gap. This includes 12xHis, 2xStrep II, 3xFLAG, PrtA (2 repeats as found in pBS1479), and V5. Each tag was inserted into each base vector, making 30 templates with all combinations of protease site, tag, and selectable marker.
PCR amplification:
These two primers (along with 50 nt of homology to the targets at the 5' ends) will amplify the desired product from any of the constructs (the reading frame for P1 is important – alternating codons bracketed).
P1 5’-(homology)...[CGA]CGG[ATC]CCC[GGG]TTA[ATT]AAC-3’
P2 5’-(homology)...GATATCATCGATGAATTCGAGCTCGTTT-3’
If only epitope tagging is needed, the upstream primer can be adjusted to skip the protease site. As an example, for the V5-tag-containing constructs, usage of the following primer (5’-(homology)... GGTGGAGGCTCTAGAGGTAAGCCAATTCCA-3’) in place of P1 will omit the protease cleavage site and add only 3xGly and V5 (GGGSR-V5-Stop).
Proper citation: RRID:Addgene_44089 Copy
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