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Species: Homo sapiens
Genetic Insert: SAT
Vector Backbone Description: Backbone Marker:SGC; Backbone Size:7328; Vector Backbone:pET28a-LIC; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Comments: PDB: 2FXF http://www.thesgc.org/structures/2FXF/
Proper citation: RRID:Addgene_25493 Copy
Species: Mus musculus
Genetic Insert: mMCL1(T144D)
Vector Backbone Description: Backbone Size:5169; Vector Backbone:pBabe IRES puro; Vector Types:Mammalian Expression, Retroviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:19433446
Proper citation: RRID:Addgene_25388 Copy
Species: Homo sapiens
Genetic Insert: CHKA
Vector Backbone Description: Backbone Marker:SGC; Backbone Size:7328; Vector Backbone:pET28a-LIC; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Comments: PDB: 2I7Q http://www.thesgc.org/structures/2I7Q/
PDB: 3G15 http://www.thesgc.org/structures/3G15/
PDB: 3F2R http://www.thesgc.org/structures/3F2R/
Proper citation: RRID:Addgene_25515 Copy
Species: Homo sapiens
Genetic Insert: CDC34
Vector Backbone Description: Backbone Marker:SGC; Backbone Size:7328; Vector Backbone:pET28a-LIC; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Comments: PDB: 2OB4 http://www.thesgc.org/structures/2OB4/
Proper citation: RRID:Addgene_25598 Copy
Species: Homo sapiens
Genetic Insert: HK2
Vector Backbone Description: Backbone Marker:SGC; Backbone Size:7328; Vector Backbone:pET28a-LIC; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Comments: PDB: 2NZT http://www.thesgc.org/structures/2NZT/
Proper citation: RRID:Addgene_25529 Copy
Species: Homo sapiens
Genetic Insert: NEDD4L-C2
Vector Backbone Description: Backbone Marker:SGC; Backbone Size:7325; Vector Backbone:pET28-MHL; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Comments: PDB: 2NSQ http://www.thesgc.org/structures/2NSQ/
Proper citation: RRID:Addgene_25615 Copy
Species: Homo sapiens
Genetic Insert: PPIC
Vector Backbone Description: Backbone Marker:SGC; Backbone Size:7328; Vector Backbone:pET28a-LIC; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Comments: PDB: 2ESL http://www.thesgc.org/structures/2ESL/
Proper citation: RRID:Addgene_25606 Copy
Species: Homo sapiens
Genetic Insert: KIF2
Vector Backbone Description: Backbone Marker:SGC; Backbone Size:7328; Vector Backbone:pET28a-LIC; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Comments: PDB: 2GRY http://www.thesgc.org/structures/2GRY/
Proper citation: RRID:Addgene_25548 Copy
Vector Backbone Description: Backbone Marker:ATCC 10326362; Backbone Size:4218; Vector Backbone:pENTR1A-Gent; Vector Types:Gateway Cloning; Bacterial Resistance:Gentamicin
Defining Citation: PMID:16945906
Comments: shRNA expression; miR30-based topology; TRE-TIGHT Pmin promoter + DsRed2 spacer; Entry vector backbone; +ccdB in parent; Spe1 d/s of TRE-TIGHT
Proper citation: RRID:Addgene_25755 Copy
Species: Homo sapiens
Genetic Insert: p53-shRNA-941
Vector Backbone Description: Backbone Size:7032; Vector Backbone:pLKO.1 puro; Vector Types:Mammalian Expression, Lentiviral, RNAi; Bacterial Resistance:Ampicillin
Defining Citation: PMID:17060456
Proper citation: RRID:Addgene_25637 Copy
Species: Homo sapiens
Genetic Insert: p53-shRNA-427
Vector Backbone Description: Backbone Size:7032; Vector Backbone:pLKO.1 puro; Vector Types:Mammalian Expression, Lentiviral, RNAi; Bacterial Resistance:Ampicillin
Defining Citation: PMID:17060456
Proper citation: RRID:Addgene_25636 Copy
Vector Backbone Description: Backbone Marker:ATCC 10326362; Backbone Size:4422; Vector Backbone:pENTR1A-Gent; Vector Types:Gateway Cloning; Bacterial Resistance:Gentamicin
Defining Citation: PMID:16945906
Comments: shRNA expression; miR30-based topology; TRE-TIGHT Pmin promoter; Entry vector backbone; +ccdB in parent; Spe1 d/s of TRE-TIGHT
Proper citation: RRID:Addgene_25752 Copy
Species: Mus musculus
Genetic Insert: Nampt
Vector Backbone Description: Backbone Marker:Novagen; Backbone Size:5369; Vector Backbone:PET28a; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:16783373
Proper citation: RRID:Addgene_25630 Copy
Vector Backbone Description: Backbone Marker:ATCC 10326362; Backbone Size:4347; Vector Backbone:pENTR1A-Gent; Vector Types:Gateway Cloning; Bacterial Resistance:Gentamicin
Defining Citation: PMID:16945906
Comments: TRE Pmin promoter; Entry vector backbone; +ccdB in parent; Multiple cloning sites d/s of TRE
Proper citation: RRID:Addgene_25751 Copy
Vector Backbone Description: Backbone Marker:ATCC 10326362; Backbone Size:4655; Vector Backbone:pENTR1A-Gent; Vector Types:Gateway Cloning; Bacterial Resistance:Gentamicin
Defining Citation: PMID:16945906
Comments: Entry vector with TRE-driven GFP
Proper citation: RRID:Addgene_25767 Copy
Genetic Insert: 0
Vector Backbone Description: Backbone Size:5020; Vector Backbone:pAAV (Strategene); Vector Types:Mammalian Expression, AAV; Bacterial Resistance:Ampicillin
Defining Citation: PMID:18784188
Comments: Please note that an 11bp deletion was found in the 3' ITR ("AAV2 alternate). The depositing lab was not able to confirm whether this was present in their original stock.
Proper citation: RRID:Addgene_25648 Copy
Vector Backbone Description: Backbone Marker:N/A; Backbone Size:8673; Vector Backbone:pMAK700, pMAK705, pBS-TS; Vector Types:Bacterial Expression; Bacterial Resistance:Chloramphenicol
Defining Citation: PMID:9335267
Comments: pKOV is identical to the pKO3 vector described in J. Bacteriology 179: 6228-6237, except for the addition of a 3kb stuffer sequence in the multiple cloning site. This stuffer permits (i) directional cloning using NotI and BamHI and (ii) clean separation of doubly cut vector from singly cut contaminants when using this pair of enzymes. The pKOV cloning site is: 5' - SmaI - NotI - SmaI- stuffer - BamHI - SalI - 3'. BamHI and SalI are not used together, nor is SmaI used with NotI. BglII & BclI cut ends are compatible with BamHI. PmeI & SwaI are compatible with SmaI.
NOTE: pKOV has a temperature sensitive pSC101 replication origin. To recover the plasmid, strains harboring the plasmid must be grown at 30 deg C under chloramphenicol selection.
Gene replacement: Mutant alleles cloned into the pKOV gene replacement vector are electroporated into recombination proficient strains (eg. EMG2) and allowed to recover for 1 h at 30 deg C. The cells are plated on prewarmed chloramphenicol/LB plates and incubated at 42 deg C. To measure the integration frequency, the electroporated cells are also plated on chloramphenicol/LB plates at 30 deg C. From the 42 deg C plate, 1-5 colonies are picked into 1 ml of LB broth, serially diluted, and immediately plated at 30°ree;C on either 5% w/v sucrose or 5% sucrose+antibiotic plates. The 5% sucrose plates are replica plated to chloramphenicol plates at 30 deg C to test for loss of the replacement vector (cms). The gene replacement is confirmed by either PCR using primers flanking the targeted open reading frame or by genomic Southern's.
Note from depositor: If sucrose plates do not select correctly, pKOV should be used in liquid selection on a plate reader with a LB + Cm control growth and a LB + Cm + sucrose growth. pKOV can sometimes still allow survival in the presence of sucrose, but significantly decreases fitness, so tracking the kinetic growth can be helpful.
The sacB gene loses its efficacy,, sacB is toxic in E. coli even in the absence of sucrose.
Proper citation: RRID:Addgene_25769 Copy
Species: Homo sapiens
Genetic Insert: RB1-shRNA 19
Vector Backbone Description: Backbone Size:7032; Vector Backbone:pLKO.1 puro; Vector Types:Mammalian Expression, Lentiviral, RNAi; Bacterial Resistance:Ampicillin
Defining Citation: PMID:20354191
Proper citation: RRID:Addgene_25640 Copy
Species: Homo sapiens
Genetic Insert: UEV1
Vector Backbone Description: Backbone Marker:SGC; Backbone Size:7328; Vector Backbone:pET28a-LIC; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Comments: PDB: 2A4D http://www.thesgc.org/structures/2A4D/
Proper citation: RRID:Addgene_25619 Copy
Genetic Insert: None
Vector Backbone Description: Backbone Marker:Iain Fraser; Backbone Size:13170; Vector Backbone:pSLIK; Vector Types:Mammalian Expression, Lentiviral, RNAi; Bacterial Resistance:Chloramphenicol and Ampicillin
Defining Citation: PMID:16945906
Comments: Parent lentivector, co-expression of Venus selection gene
Proper citation: RRID:Addgene_25734 Copy
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