pKK-BI16-ORF1-3C-FLAG_ORF2-TEV-mClover3
(Plasmid
#105803)
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Purpose(Empty Backbone) Concomitant expression of two proteins. One protein expressed with FLAG, cleavable by 3C protease; second protein expressed with mClover3, cleavable by TEV protease; tags positions: C-termini.
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Depositing Lab
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Sequence Information
Ordering
Item | Catalog # | Description | Quantity | Price (USD) | |
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Plasmid | 105803 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $85 |
Backbone
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Vector backboneBI-16 (constructed based on pcDNA5/FRT/TO from Invitrogen)
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Backbone manufacturerSammarco & Grabczyk (PMID: 16212928)
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Vector typeMammalian Expression ; Flp-In competent
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Selectable markersHygromycin
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Tag
/ Fusion Protein
- ORF1: 3C-FLAG; ORF2: TEV-mClover3 (C terminal on backbone)
Growth in Bacteria
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Bacterial Resistance(s)Ampicillin, 100 μg/mL
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Growth Temperature37°C
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Growth Strain(s)DH5alpha
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Copy numberHigh Copy
Gene/Insert
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Gene/Insert nameNone
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Tag
/ Fusion Protein
- ORF1: 3C-FLAG; ORF2: TEV-mClover3 (C terminal on backbone)
Resource Information
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Supplemental Documents
Terms and Licenses
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Academic/Nonprofit Terms
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Industry Terms
- Not Available to Industry
Trademarks:
- Zeocin® is an InvivoGen trademark.
Depositor Comments
This vector is a member of the pKK-BI16 series which belongs to the pKK vector family. It enables concomitant expression of two genes, which transcription is driven by a tetracycline responsive bidirectional CMV promoter. This vector is a derivate of the BI16 plasmid (PMID: 16212928) and is suitable for stable cell line generation using the Flp-In system. ORF2 is cloned with the universal SLIC protocol (just three universal PCR primers are required to clone a given coding sequence into all vectors from pKK family by a ligation-independent DNA cloning method), ORF1 can be cloned using SLIC with specifically designed primers (applicable enzymes: Bsp120I, ApaI, MluI, NotI, BspTI, Eco47III, MunI, Psp5II) or conventional restriction-ligase based cloning (see Szczesny RJ et al. for detailed description and protocols; bioRxiv, doi: https://doi.org/10.1101/160101) mClover3 is a brighter derivative of mEGFP (PMID: 26879144). FLAG is a short tag, less likely to interfere with protein function (PMID: 11694294).
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
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For your Materials & Methods section:
pKK-BI16-ORF1-3C-FLAG_ORF2-TEV-mClover3 was a gift from Andrzej Dziembowski (Addgene plasmid # 105803 ; http://n2t.net/addgene:105803 ; RRID:Addgene_105803) -
For your References section:
Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system. Szczesny RJ, Kowalska K, Klosowska-Kosicka K, Chlebowski A, Owczarek EP, Warkocki Z, Kulinski TM, Adamska D, Affek K, Jedroszkowiak A, Kotrys AV, Tomecki R, Krawczyk PS, Borowski LS, Dziembowski A. PLoS One. 2018 Mar 28;13(3):e0194887. doi: 10.1371/journal.pone.0194887. eCollection 2018. 10.1371/journal.pone.0194887 PubMed 29590189