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http://www.addgene.org/44106

Species: Homo sapiens
Genetic Insert: TRAF2 S102A
Vector Backbone Description: Vector Backbone:pEBB; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:23007157

Proper citation: RRID:Addgene_44106 Copy   

•   Source: Addgene

http://www.addgene.org/44105

Species: Homo sapiens
Genetic Insert: TRAF2 S11A
Vector Backbone Description: Vector Backbone:pEBB; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:23007157

Proper citation: RRID:Addgene_44105 Copy   

•   Source: Addgene

http://www.addgene.org/44183

Vector Backbone Description: Backbone Marker:http://www.cambia.org/daisy/cambia/2045/version/1/part/4/data/pCAMBIA1300.pdf?branch=main&language=default; Backbone Size:8958; Vector Backbone:pCambia1300; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:23098881
Comments: Note- the SpeI site located within the Kanamycin resistance cassette was destroyed (tested by digest). The fact that the plasmid is growing on Kanamycin indicates that the loss of the SpeI site does not affect the plasmid's function.

Proper citation: RRID:Addgene_44183 Copy   

•   Source: Addgene

http://www.addgene.org/44065

Vector Backbone Description: Backbone Marker:Mark Hochstrasser lab (Yale Univeristy, New Haven, CT) (PMID: 19243080); Backbone Size:4142; Vector Backbone:pFA6a-6xGLY-3xFLAG-HIS3MX6 (Addgene plasmid #20753); 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_44065 Copy   

•   Source: Addgene

http://www.addgene.org/44064

Vector Backbone Description: Backbone Marker:Mark Hochstrasser lab (Yale Univeristy, New Haven, CT) (PMID: 19243080); Backbone Size:4142; Vector Backbone:pFA6a-6xGLY-3xFLAG-HIS3MX6 (Addgene plasmid #20753); 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_44064 Copy   

•   Source: Addgene

http://www.addgene.org/44176

Species: Saccharomyces cerevisiae
Genetic Insert: Gal4/DBD (1-147)
Vector Backbone Description: Backbone Size:8600; Vector Backbone:N103; Vector Types:Lentiviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:22704655

Proper citation: RRID:Addgene_44176 Copy   

•   Source: Addgene

http://www.addgene.org/44170

Species: Influenza virus (A/chicken/Rostock/8/34)
Genetic Insert: Influenza A virus M2 matrix gene
Vector Backbone Description: Backbone Marker:Promega Corporation; Backbone Size:4000; Vector Backbone:pCI; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:23319179

Proper citation: RRID:Addgene_44170 Copy   

•   Source: Addgene

http://www.addgene.org/44172

Species: E. coli
Genetic Insert: LacZ
Vector Backbone Description: Backbone Marker:Promega Corporation; Backbone Size:2700; Vector Backbone:pCI; Vector Types:Lentiviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:16397505

Proper citation: RRID:Addgene_44172 Copy   

•   Source: Addgene

http://www.addgene.org/44175

Species: Saccharomyces cerevisiae
Genetic Insert: Atg17
Vector Backbone Description: Backbone Size:5756; Vector Backbone:pJK59; Vector Types:Yeast Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:23219485
Comments: Constructed by amplifying Atg17 with 300bp 5' of the start site.

Proper citation: RRID:Addgene_44175 Copy   

•   Source: Addgene

http://www.addgene.org/44167

Species: E. coli
Genetic Insert: LacZ
Vector Backbone Description: Backbone Marker:Promega Corporation; Backbone Size:2700; Vector Backbone:pCI; Vector Types:Lentiviral, Luciferase; Bacterial Resistance:Ampicillin
Defining Citation: PMID:23319179

Proper citation: RRID:Addgene_44167 Copy   

•   Source: Addgene

http://www.addgene.org/44169

Species: Influenza A/FPV/Rostock/1934, subtype H7
Genetic Insert: hemagglutinin gene
Vector Backbone Description: Backbone Marker:InvivoGen; Backbone Size:6321; Vector Backbone:pVITRO2-mcs; Vector Types:Mammalian Expression; Bacterial Resistance:Hygromycin
Defining Citation: PMID:23319179
Comments: Please note that a T55A mutation was identified in the N1 sequence.

Proper citation: RRID:Addgene_44169 Copy   

•   Source: Addgene

http://www.addgene.org/44160

Species: Mus musculus
Genetic Insert: Tak1
Vector Backbone Description: Vector Backbone:pRK6-HA; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:16260783

Proper citation: RRID:Addgene_44160 Copy   

•   Source: Addgene

http://www.addgene.org/44163

Genetic Insert: firefly luciferase
Vector Backbone Description: Backbone Marker:Promega Corporation; Backbone Size:2700; Vector Backbone:pCI; Vector Types:Lentiviral, Luciferase; Bacterial Resistance:Ampicillin
Defining Citation: PMID:23319179

Proper citation: RRID:Addgene_44163 Copy   

•   Source: Addgene

http://www.addgene.org/44317

Species: Yarrowia lipolytica
Genetic Insert: UAS1B9-Leum promoter
Vector Backbone Description: Backbone Size:2623; Vector Backbone:pUC19; Vector Types:Bacterial Expression, Synthetic Biology; Bacterial Resistance:Ampicillin
Defining Citation: PMID:21926196

Proper citation: RRID:Addgene_44317 Copy   

•   Source: Addgene

http://www.addgene.org/44318

Species: Yarrowia lipolytica
Genetic Insert: UAS1B10-Leum promoter
Vector Backbone Description: Backbone Size:2623; Vector Backbone:pUC19; Vector Types:Bacterial Expression, Synthetic Biology; Bacterial Resistance:Ampicillin
Defining Citation: PMID:21926196

Proper citation: RRID:Addgene_44318 Copy   

•   Source: Addgene

http://www.addgene.org/44275

Species: Synthetic
Genetic Insert: TagRFP675
Vector Backbone Description: Backbone Marker:Clontech; Backbone Size:3998; Vector Backbone:pN1; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:23677204

Proper citation: RRID:Addgene_44275 Copy   

•   Source: Addgene

http://www.addgene.org/44278

Species: Synthetic
Genetic Insert: TagRFP675
Vector Backbone Description: Backbone Marker:Clontech; Backbone Size:6640; Vector Backbone:pN1; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:23677204

Proper citation: RRID:Addgene_44278 Copy   

•   Source: Addgene

http://www.addgene.org/44311

Species: Yarrowia lipolytica
Genetic Insert: UAS1B3-Leum promoter
Vector Backbone Description: Backbone Size:2623; Vector Backbone:pUC19; Vector Types:Bacterial Expression, Synthetic Biology; Bacterial Resistance:Ampicillin
Defining Citation: PMID:21926196
Comments: Discrepancies between the Addgene QC sequence and the depositor's sequence should not affect plasmid function.

Proper citation: RRID:Addgene_44311 Copy   

•   Source: Addgene

http://www.addgene.org/44277

Species: Synthetic
Genetic Insert: TagRFP675
Vector Backbone Description: Backbone Marker:Clontech; Backbone Size:5072; Vector Backbone:pN1; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:23677204

Proper citation: RRID:Addgene_44277 Copy   

•   Source: Addgene

http://www.addgene.org/44310

Species: Yarrowia lipolytica
Genetic Insert: UAS1B2-Leum promoter
Vector Backbone Description: Backbone Size:2623; Vector Backbone:pUC19; Vector Types:Bacterial Expression, Synthetic Biology; Bacterial Resistance:Ampicillin
Defining Citation: PMID:21926196
Comments: Discrepancies between the Addgene QC sequence and the depositor's sequence should not affect plasmid function.

Proper citation: RRID:Addgene_44310 Copy   

•   Source: Addgene