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Species: Synthetic
Genetic Insert: T7 sigma fragment
Vector Backbone Description: Vector Backbone:pSB1C3; Vector Types:Bacterial Expression; Bacterial Resistance:Chloramphenicol
Defining Citation: PMID:25080493
Proper citation: RRID:Addgene_59966 Copy
Vector Backbone Description: Backbone Marker:Mel Simon lab; Backbone Size:7507; Vector Backbone:pBeloBAC11; Vector Types:Bacterial Expression, Cre/Lox; Bacterial Resistance:Chloramphenicol
Defining Citation: PMID:28754135
Comments: GenBank Accession # U51113 for vector backbone pBeloBAC11
Proper citation: RRID:Addgene_60342 Copy
Species: Synthetic
Genetic Insert: Reporter_9
Vector Backbone Description: Vector Backbone:unknown; Vector Types:Bacterial Expression; Bacterial Resistance:Chloramphenicol
Defining Citation: PMID:25344638
Comments: This reporter plasmid contains the attB and attP sites flanking a green fluorescent protein reporter gene (gfp). A strong constitutive promoter (BBa_J23119) is placed upstream of the attB site, which transcribes in the opposite orientation as gfp. After the integrase is expressed, the orientation of gfp is inverted and it is transcribed. After inversion, recombined attB and attP sites result in the formation of attL and attR. The attL site is located on the 5'UTR and could impact gfp expression. To insulate against this effect, we included a spacer and the ribozyme RiboJ.
Also contains mRFP in order to aid the gating of cells.
Proper citation: RRID:Addgene_60569 Copy
Species: Synthetic
Genetic Insert: Reporter_8
Vector Backbone Description: Vector Backbone:unknown; Vector Types:Bacterial Expression; Bacterial Resistance:Chloramphenicol
Defining Citation: PMID:25344638
Comments: This reporter plasmid contains the attB and attP sites flanking a green fluorescent protein reporter gene (gfp). A strong constitutive promoter (BBa_J23119) is placed upstream of the attB site, which transcribes in the opposite orientation as gfp. After the integrase is expressed, the orientation of gfp is inverted and it is transcribed. After inversion, recombined attB and attP sites result in the formation of attL and attR. The attL site is located on the 5'UTR and could impact gfp expression. To insulate against this effect, we included a spacer and the ribozyme RiboJ.
Also contains mRFP in order to aid the gating of cells.
Proper citation: RRID:Addgene_60568 Copy
Species: Synthetic
Genetic Insert: Reporter_7
Vector Backbone Description: Vector Backbone:unknown; Vector Types:Bacterial Expression; Bacterial Resistance:Chloramphenicol
Defining Citation: PMID:25344638
Comments: This reporter plasmid contains the attB and attP sites flanking a green fluorescent protein reporter gene (gfp). A strong constitutive promoter (BBa_J23119) is placed upstream of the attB site, which transcribes in the opposite orientation as gfp. After the integrase is expressed, the orientation of gfp is inverted and it is transcribed. After inversion, recombined attB and attP sites result in the formation of attL and attR. The attL site is located on the 5'UTR and could impact gfp expression. To insulate against this effect, we included a spacer and the ribozyme RiboJ.
Also contains mRFP in order to aid the gating of cells.
Proper citation: RRID:Addgene_60567 Copy
Species: Synthetic
Genetic Insert: Reporter_12
Vector Backbone Description: Vector Backbone:unknown; Vector Types:Bacterial Expression; Bacterial Resistance:Chloramphenicol
Defining Citation: PMID:25344638
Comments: This reporter plasmid contains the attB and attP sites flanking a green fluorescent protein reporter gene (gfp). A strong constitutive promoter (BBa_J23119) is placed upstream of the attB site, which transcribes in the opposite orientation as gfp. After the integrase is expressed, the orientation of gfp is inverted and it is transcribed. After inversion, recombined attB and attP sites result in the formation of attL and attR. The attL site is located on the 5'UTR and could impact gfp expression. To insulate against this effect, we included a spacer and the ribozyme RiboJ.
Also contains mRFP in order to aid the gating of cells.
Proper citation: RRID:Addgene_60572 Copy
Species: Synthetic
Genetic Insert: MemoryArray
Vector Backbone Description: Vector Backbone:unknown; Vector Types:Bacterial Expression; Bacterial Resistance:Chloramphenicol
Defining Citation: PMID:25344638
Comments: An array was constructed by concatenating the attB/P sites of the 11 phage integrases to form a linear 2kb piece of DNA. Random spacers (50 bp) with 50% GC content are included between the att sites.
Proper citation: RRID:Addgene_60585 Copy
Species: Synthetic
Genetic Insert: Cas_5+7_gfp
Vector Backbone Description: Vector Backbone:unknown; Vector Types:Bacterial Expression; Bacterial Resistance:Chloramphenicol
Defining Citation: PMID:25344638
Proper citation: RRID:Addgene_60595 Copy
Species: Other
Genetic Insert: T7 RNAP
Vector Backbone Description: Backbone Size:5500; Vector Backbone:pBAD33; Vector Types:Bacterial Expression, Synthetic Biology; Bacterial Resistance:Chloramphenicol
Defining Citation: PMID:23479654
Proper citation: RRID:Addgene_60717 Copy
Species: Synthetic
Genetic Insert: N Terminal fragment of T7 RNAP split betwen 179-180
Vector Backbone Description: Backbone Size:5500; Vector Backbone:pBAD33; Vector Types:Bacterial Expression; Bacterial Resistance:Chloramphenicol
Defining Citation: PMID:23479654
Proper citation: RRID:Addgene_60720 Copy
Species: Synthetic
Genetic Insert: Residues 1-179 of T7 RNAP
Vector Backbone Description: Backbone Size:5500; Vector Backbone:pBAD33; Vector Types:Bacterial Expression, Synthetic Biology; Bacterial Resistance:Chloramphenicol
Defining Citation: PMID:23479654
Proper citation: RRID:Addgene_60721 Copy
Species: Synthetic
Genetic Insert: Residues 1-179 of split T7 RNAP
Vector Backbone Description: Backbone Size:5500; Vector Backbone:pBAD33; Vector Types:Bacterial Expression, Synthetic Biology; Bacterial Resistance:Chloramphenicol
Defining Citation: PMID:23479654
Proper citation: RRID:Addgene_60728 Copy
Species: Synthetic
Genetic Insert: Residues 1-179 of split T7 RNAP
Vector Backbone Description: Backbone Size:5500; Vector Backbone:pBAD33; Vector Types:Bacterial Expression, Synthetic Biology; Bacterial Resistance:Chloramphenicol
Defining Citation: PMID:23479654
Proper citation: RRID:Addgene_60727 Copy
Species: Synthetic
Genetic Insert: Residues 1-179 of split T7 RNAP
Vector Backbone Description: Backbone Size:5500; Vector Backbone:pBAD33; Vector Types:Bacterial Expression, Synthetic Biology; Bacterial Resistance:Chloramphenicol
Defining Citation: PMID:23479654
Proper citation: RRID:Addgene_60730 Copy
Species: Streptococcus pyogenes
Genetic Insert: Cas9, tracrRNA, crRNA
Vector Backbone Description: Vector Backbone:Custom pR1162-CmR; Vector Types:Bacterial Expression, CRISPR; Bacterial Resistance:Chloramphenicol
Defining Citation: PMID:25240928
Proper citation: RRID:Addgene_61271 Copy
Species: E. coli
Genetic Insert: SCRIBE_kanR(ON)
Vector Backbone Description: Backbone Marker:Expressys; Vector Backbone:pZE32; Vector Types:Synthetic Biology; Bacterial Resistance:Chloramphenicol
Defining Citation: PMID:25395541
Proper citation: RRID:Addgene_61450 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
Vector Backbone Description: Backbone Size:3691; Vector Backbone:pBTBXh-3; Vector Types:Bacterial Expression; Bacterial Resistance:Chloramphenicol
Defining Citation: PMID:20148414
Comments: There is an extra G in the middle of the alignment between Addgene's quality control sequence and the author's sequence. This discrepancy is in a non-coding region and does affect function.
Proper citation: RRID:Addgene_26079 Copy
Vector Backbone Description: Backbone Size:4922; Vector Backbone:pBMTBX-3; Vector Types:Bacterial Expression; Bacterial Resistance:Chloramphenicol
Defining Citation: PMID:20148414
Proper citation: RRID:Addgene_26074 Copy
Species: Aequorea victoria
Genetic Insert: destabilized GFP
Vector Backbone Description: Backbone Size:0; Vector Backbone:pLuxI-tet8 + pRKM-102; Vector Types:Bacterial Expression; Bacterial Resistance:Chloramphenicol
Defining Citation: PMID:15096621
Comments: A novel hybrid promoter luxPRcI-OR1 (wild-type luxPR promoter with a CI OR1 operator site inserted at the +1 transcription start) drives expression of GFP(LVA). The promoter was constructed by encoding OR1 on PCR primers and inserting it after luxPR
Proper citation: RRID:Addgene_26580 Copy
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