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http://www.addgene.org/43939

Species: Homo sapiens
Genetic Insert: ubiquitin-conjugating enzyme E2N
Vector Backbone Description: Backbone Size:5000; Vector Backbone:pCR3; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:17135271

Proper citation: RRID:Addgene_43939 Copy   

•   Source: Addgene

http://www.addgene.org/43894

Species: Homo sapiens
Genetic Insert: USP44
Vector Backbone Description: Backbone Size:4700; Vector Backbone:p142; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:17443180

Proper citation: RRID:Addgene_43894 Copy   

•   Source: Addgene

http://www.addgene.org/43896

Species: Homo sapiens
Genetic Insert: USP44
Vector Backbone Description: Backbone Size:7556; Vector Backbone:MSCV; Vector Types:Mammalian Expression, Retroviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:17443180

Proper citation: RRID:Addgene_43896 Copy   

•   Source: Addgene

http://www.addgene.org/43930

Genetic Insert: TNF Receptor-Associated Factor 6
Vector Backbone Description: Backbone Marker:GE Healthcare; Backbone Size:4910; Vector Backbone:pGEX4T1; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:17135271

Proper citation: RRID:Addgene_43930 Copy   

•   Source: Addgene

http://www.addgene.org/44062

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_44062 Copy   

•   Source: Addgene

http://www.addgene.org/43920

Species: Homo sapiens
Genetic Insert: AILR-LplA36-NRX3b
Vector Backbone Description: Backbone Size:4247; Vector Backbone:pCAG; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:23457442

Proper citation: RRID:Addgene_43920 Copy   

•   Source: Addgene

http://www.addgene.org/43921

Species: Homo sapiens
Genetic Insert: BirA272-NRX3b
Vector Backbone Description: Backbone Size:6299; Vector Backbone:pXL-CAG; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:23457442
Comments: 272 indicates the amino acid position that the BirA is inserted in for NRX3b. There is a single alanine deletion in BirA, which is a non-dimerizing mutation. The BirA and AILR insert regions of the plasmid is codon optimized.

Proper citation: RRID:Addgene_43921 Copy   

•   Source: Addgene

http://www.addgene.org/43922

Species: Homo sapiens
Genetic Insert: BirA272-NRX3b
Vector Backbone Description: Backbone Size:6299; Vector Backbone:pXL-CAG; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:23457442

Proper citation: RRID:Addgene_43922 Copy   

•   Source: Addgene

http://www.addgene.org/43923

Species: Rattus norvegicus
Genetic Insert: 3XAP-NLG1
Vector Backbone Description: Backbone Size:6390; Vector Backbone:pCAG; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:23457442

Proper citation: RRID:Addgene_43923 Copy   

•   Source: Addgene

http://www.addgene.org/43926

Species: Mus musculus
Genetic Insert: TNF Receptor-Associated Factor 6
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5000; Vector Backbone:pCR3; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:17135271

Proper citation: RRID:Addgene_43926 Copy   

•   Source: Addgene

http://www.addgene.org/43917

Species: Synthetic
Genetic Insert: GCaMP3
Vector Backbone Description: Backbone Marker:Addgene plasmid 43915; Backbone Size:9600; Vector Backbone:FU-MAP2-Gateway; Vector Types:Mammalian Expression, Lentiviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:22174877
Comments: The MAP2-GFP lentiviral reporter plasmid pGZ-hMAP2 was purchased from System Biosciences (SR10047PA-1). The MAP2 promoter sequence from this plasmid was then cloned along with the Gateway cassette of pEF-DEST51 into the backbone of FUdeltaGW-rtTA (Addgene plasmid 19780) following removal of the ubiquitin promoter-rtTA sequence to produce FU-MAP2-Gateway (Addgene plasmid #43915). This destination vector was subsequently used to generate MAP2-GCaMP3 (GCaMP3 cloned from G-CaMP3, Addgene plasmid 22692). The V5 and His tags present in the vector backbone are not expressed with the GCaMP3 insert.

Proper citation: RRID:Addgene_43917 Copy   

•   Source: Addgene

http://www.addgene.org/43910

Species: Saccharomyces cerevisiae
Genetic Insert: DIF1
Vector Backbone Description: Backbone Marker:Stratagene; Backbone Size:4898; Vector Backbone:pRS416; Vector Types:Yeast Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:18851834

Proper citation: RRID:Addgene_43910 Copy   

•   Source: Addgene

http://www.addgene.org/43911

Species: Saccharomyces cerevisiae
Genetic Insert: DIF1
Vector Backbone Description: Backbone Marker:Stratagene; Backbone Size:4898; Vector Backbone:pRS416; Vector Types:Yeast Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:18851834

Proper citation: RRID:Addgene_43911 Copy   

•   Source: Addgene

http://www.addgene.org/43912

Species: Saccharomyces cerevisiae
Genetic Insert: DIF1
Vector Backbone Description: Backbone Marker:Stratagene; Backbone Size:4898; Vector Backbone:pRS416; Vector Types:Yeast Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:18851834

Proper citation: RRID:Addgene_43912 Copy   

•   Source: Addgene

http://www.addgene.org/43915

Vector Backbone Description: Backbone Marker:Addgene plasmid 19780; Backbone Size:9600; Vector Backbone:FUdeltaGW-rtTA; Vector Types:Mammalian Expression, Lentiviral, Gateway Cloning; Bacterial Resistance:Chloramphenicol and Ampicillin
Defining Citation: PMID:22174877
Comments: The MAP2-GFP lentiviral reporter plasmid pGZ-hMAP2 was purchased from System Biosciences (SR10047PA-1). The MAP2 promoter sequence from this plasmid was then cloned along with the Gateway cassette of pEF-DEST51 into the backbone of FUdeltaGW-rtTA (Addgene plasmid 19780) following removal of the ubiquitin promoter-rtTA sequence to produce FU-MAP2-Gateway.

Proper citation: RRID:Addgene_43915 Copy   

•   Source: Addgene

http://www.addgene.org/43990

Vector Backbone Description: Backbone Marker:Novagen, EMBL; Backbone Size:6657; Vector Backbone:pET, pET28M-Sumo3; 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_43990 Copy   

•   Source: Addgene

http://www.addgene.org/43991

Vector Backbone Description: Backbone Marker:Novagen; Backbone Size:6669; Vector Backbone:pET, pCoofy6; 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_43991 Copy   

•   Source: Addgene

http://www.addgene.org/43992

Vector Backbone Description: Backbone Marker:Novagen; Backbone Size:5764; Vector Backbone:pET, pCoofy7; 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_43992 Copy   

•   Source: Addgene

http://www.addgene.org/43993

Vector Backbone Description: Backbone Marker:Novagen; Backbone Size:5808; Vector Backbone:pET, pCoofy12; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:23410102
Comments: C-terminal Tag has 6 His residues, LP2 primer needs to be adapted to 6His 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_43993 Copy   

•   Source: Addgene

http://www.addgene.org/43994

Vector Backbone Description: Backbone Marker:Novagen; Backbone Size:5845; Vector Backbone:pET, pCoofy34; 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_43994 Copy   

•   Source: Addgene