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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. PubMed Article

Plasmids from Article

ID Plasmid Purpose
105767pKK-NoTagControl vector for pKK series (encodes only SLIC arms (TEV-L and TEV-R)); expression without tag. Useful to design other vectors; any tag can be added.
105768pKK-FLAG-TEVExpression of your protein of interest in fusion with FLAG at the N-terminus. The tag is cleavable by TEV protease or enterokinase.
105769pKK-HA-TEVExpression of your protein of interest in fusion with HA at the N-terminus. The tag is cleavable by TEV protease.
105770pKK-FLAG-BirA-TEVExpression of your protein of interest in fusion with FLAG-BirA. FLAG-tagged at the N-terminus (cleavable by TEV). Search for protein partners using the BioID (PMID: 15707685, 22412018).
105771pKK-MBP-TEVExpression of your protein of interest in fusion with MBP at the N-terminus (cleavable by TEV). The maltose binding protein tag allows convenient purification of proteins (PMID: 22442034, 19935684).
105772pKK-mTagBFP-TEVExpression of your protein of interest in fusion with blue fluorescent protein at the N-terminus (cleavable by TEV) (PMID: 18940671, 25378625). Useful for multi-channel live cell imaging.
105773pKK-mCerulean-TEVExpression of your protein of interest in fusion with cyan fluorescent protein at the N-terminus (cleavable by TEV). Useful for FRET experiments (PMID: 14990965, 17040988, 22264545).
105774pKK-mVenus-TEVExpression of your protein of interest in fusion with yellow fluorescent protein at the N-terminus (cleavable by TEV). Useful for FRET experiments (PMID: 11753368, 17040988, 22264545).
105775pKK-mAmber-TEVExpression of your protein of interest in fusion with non-absorbing, non-emitting Venus mutant at the N-terminus (cleavable by TEV). Negative control for FRET measurements (PMID: 17040988, 22264545).
105776pKK-EGFP-TEV Expression of your protein of interest in fusion with green fluorescent protein at the N-terminus (cleavable by TEV), (PMID: 8885998, 8805248). EGFP is not truly monomeric (PMID: 11988576, 22869113)
105777pKK-mEGFP-TEVExpression of your protein of interest in fusion with green fluorescent protein at the N-terminus (cleavable by TEV). mEGFP is an EGFP A206K mutant with reduced dimerization (PMID: 11988576,22869113).
105778pKK-mClover3-TEVExpression of your protein of interest in fusion with green fluorescent protein at the N-terminus (cleavable by TEV). mClover3 is a brighter derivative of mEGFP (PMID: 26879144).
105779pKK-mCherry-TEVExpression of your protein of interest in fusion with red fluorescent protein at the N-terminus (cleavable by TEV), (PMID: 15558047). mCherry descends from the first truly monomeric red FP mRFP1.
105780pKK-mRuby3-TEVExpression of your protein of interest in fusion with red fluorescent protein at the N-terminus (cleavable by TEV), (PMID: 26879144). mRuby3 is brighter than mCherry and has shifted ex and em spectra.
105781pKK-mCardinal-TEVExpression of your protein of interest in fusion with red fluorescent protein at the N-terminus (cleavable by TEV), (PMID: 24633408). mCardinal is suitable for deep-tissue imaging.
105782pKK-CyOFP1-TEVExpression of your protein of interest in fusion with orange fluorescent protein at the N-terminus (cleavable by TEV) (PMID: 27240196). CyOFP1 is useful for multi-channel imaging.
105783pKK-TEV-FLAGExpression of your protein of interest in fusion with FLAG at the C-terminus. The tag is cleavable by TEV protease.
105784pKK-TEV-3XFLAGExpression of your protein of interest in fusion with 3xFLAG at the C-terminus. The tag is cleavable by TEV protease.
105785pKK-TEV-HAExpression of your protein of interest in fusion with HA at the C-terminus. The tag is cleavable by TEV protease.
105786pKK-TEV-BirA-FLAGExpression of your protein of interest in fusion with BirA-FLAG at the C-terminus (cleavable by TEV). Search for protein partners using the BioID (PMID: 15707685, 22412018).
105787pKK-TEV-MBPExpression of your protein of interest in fusion with MBP at the C-terminus (cleavable by TEV). The maltose binding protein tag allows convenient purification of proteins (PMID: 22442034, 19935684).
105788pKK-TEV-ProteinAExpression of your protein of interest in fusion with two IgG binding domains of Staphylococcus aureus protein A at the C-terminus (cleavable by TEV) (PMID: 3507693, 10504710). Protein purification.
105789pKK-TEV-mTagBFPExpression of your protein of interest in fusion with blue fluorescent protein at the C-terminus (cleavable by TEV) (PMID: 18940671, 25378625). Useful for multi-channel live cell imaging.
105790pKK-TEV-mCeruleanExpression of your protein of interest in fusion with cyan fluorescent protein at the C-terminus (cleavable by TEV). Useful for FRET experiments (PMID: 14990965, 17040988, 22264545).
105791pKK-TEV-mVenusExpression of your protein of interest in fusion with yellow fluorescent protein at the C-terminus (cleavable by TEV). Useful for FRET experiments (PMID: 11753368, 17040988, 22264545).
105792pKK-TEV-mAmberExpression of your protein of interest in fusion with non-absorbing, non-emitting Venus mutant at the C-terminus (cleavable by TEV). Negative control for FRET measurements (PMID: 17040988, 22264545).
105793pKK-TEV-EGFPExpression of your protein of interest in fusion with green fluorescent protein at the C-terminus (cleavable by TEV), (PMID: 8885998, 8805248). EGFP is not truly monomeric (PMID: 11988576, 22869113)
105794pKK-TEV-mEGFPExpression of your protein of interest in fusion with green fluorescent protein at the C-terminus (cleavable by TEV). mEGFP is an EGFP A206K mutant with reduced dimerization (PMID: 11988576,22869113).
105795pKK-TEV-mClover3Expression of your protein of interest in fusion with green fluorescent protein at the C-terminus (cleavable by TEV). mClover3 is a brighter derivative of mEGFP (PMID: 26879144).
105796pKK-TEV-mCherryExpression of your protein of interest in fusion with red fluorescent protein at the C-terminus (cleavable by TEV), (PMID: 15558047). mCherry descends from the first truly monomeric red FP mRFP1.
105797pKK-TEV-mRuby3Expression of your protein of interest in fusion with red fluorescent protein at the C-terminus (cleavable by TEV) (PMID: 26879144). mRuby3 is brighter than mCherry and has shifted ex and em spectra.
105798pKK-TEV-mCardinalExpression of your protein of interest in fusion with red fluorescent protein at the C-terminus (cleavable by TEV), (PMID: 24633408). mCardinal is suitable for deep-tissue imaging.
105799pKK-TEV-CyOFP1Expression of your protein of interest in fusion with orange fluorescent protein at the C-terminus (cleavable by TEV) (PMID: 27240196). CyOFP1 is useful for multi-channel imaging.
105800pKK-BI16-TEV-mCherryConcomitant expression of two proteins. One protein is expressed with mCherry fusion tag (at the C-terminus), cleavable by TEV protease; second tag introduced by user.
105801pKK-BI16-FLAG-3C-ORF1_mClover3-TEV-ORF2Concomitant expression of two proteins. One protein expressed with FLAG, cleavable by 3C or enterokinase; second protein expressed with mClover3, cleavable by TEV protease; tags positions: N-termini.
105802pKK-BI16-ORF1-3C-mRuby3_ORF2-TEV-mClover3Concomitant expression of two proteins. One protein expressed with mRuby3, cleavable by 3C; second protein expressed with mClover3, cleavable by TEV protease; tags positions: C-termini.
105803pKK-BI16-ORF1-3C-FLAG_ORF2-TEV-mClover3Concomitant 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.
105804pKK-BiFC-VenusConcomitant expression of two proteins in fusion with fragments of Venus protein. Protein interactions studies by bimolecular fluorescence complementation (BiFC), including BiFC-FRET assays.
105805pKK-FRET-ORF1-3C-Cerulean_ORF2-TEV-VenusConcomitant expression of two proteins. One protein expressed with Cerulean, cleavable by 3C; second protein expressed with Venus, cleavable by TEV. Useful for protein interaction analysis by FRET.
105806pKK-FRET-ORF1-3C-Cerulean_ORF2-TEV-AmberConcomitant expression of two proteins. One protein expressed with Cerulean, cleavable by 3C; second protein expressed with Amber, cleavable by TEV. Control for protein interaction analysis by FRET.
105807pKK-RNAi-nucEGFPmiR-FLAG-TEVFunctional studies using RNAi; rescue experiments; substituting protein for its different form. miRNA cassette with nuclear EGFP expression marker; your protein of interest with N-terminal FLAG tag.
105808pKK-RNAi-nucEGFPmiR-mCherry-TEVFunctional studies using RNAi; rescue experiments; substituting protein for its different form. miRNA cassette with nuclear EGFP expression marker; your protein of interest with N-terminal mCherry.
105809pKK-RNAi-nucCHERRYmiR-FLAG-TEVFunctional studies using RNAi; rescue experiments; substituting protein for its different form. miRNA cassette with nuclear mCherry expression marker; your protein of interest with N-terminal FLAG.
105810pKK-RNAi-nucCHERRYmiR-EGFP-TEVFunctional studies using RNAi; rescue experiments; substituting protein for its different form. miRNA cassette with nuclear mCherry expression marker; your protein of interest with N-terminal EGFP.
105811pKK-RNAi-nucEGFPmiR-TEV-FLAGFunctional studies using RNAi; rescue experiments; substituting protein for its different form. miRNA cassette with nuclear EGFP expression marker; your protein of interest with C-terminal FLAG.
105812pKK-RNAi-nucEGFPmiR-TEV-mCherryFunctional studies using RNAi; rescue experiments; substituting protein for its different form. miRNA cassette with nuclear EGFP expression marker; your protein of interest with C-terminal mCherry.
105813pKK-RNAi-nucCHERRYmiR-TEV-FLAGFunctional studies using RNAi; rescue experiments; substituting protein for its different form. miRNA cassette with nuclear mCherry expression marker; your protein of interest with C-terminal FLAG.
105814pKK-RNAi-nucCHERRYmiR-TEV-EGFPFunctional studies using RNAi; rescue experiments; substituting protein for its different form. miRNA cassette with nuclear mCherry expression marker; your protein of interest with C-terminal EGFP.

Antibodies from Article