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DescriptionBack to Top

pCLIPf Vector is a mammalian expression plasmid intended for the cloning and stable or transient expression of CLIP-tag® protein fusions in mammalian cells. This plasmid encodes CLIPf, a CLIP-tag protein, which is expressed under control of the CMV promoter. The expression vector has an IRES (internal ribosome entry site) and a neomycin resistance gene downstream of the CLIPf for the efficient selection of stable transfectants. pCLIPf Vector contains two multiple cloning sites to allow cloning of the fusion partner as a fusion to the N- or C-terminus of the CLIPf.

The CLIP-tag is a novel tool for protein research, allowing the specific, covalent attachment of virtually any molecule to a protein of interest. The CLIP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT). CLIP-tag substrates are derivatives of benzylcytosine (BC). In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the reactive cysteine of CLIP-tag forming a stable thioether link.

pCLIPf contains an improved version of CLIP-tag, termed CLIPf. CLIPf displays faster kinetics in in vitrolabeling and fast, specific and efficient labeling in live and fixed cell applications, thereby rendering it a desired research tool for analysis of protein dynamics.

Although CLIP-tag is based on the same protein as SNAP-tag®, the benzylcytosine substrates form a separate class of substrates, different from the benzylguanine substrates recognized by SNAP-tag. CLIP-tag and SNAP-tag can be used for orthogonal simultaneous labeling. 

There are two steps to using this system: sub cloning and expression of the protein of interest as a CLIPf fusion, and labeling of the fusion with the CLIP-tag substrate of choice. Cloning and expression of CLIPf fusion proteins are described in this document. The labeling of the fusion proteins with CLIP-tag substrates is described in the instructions supplied with the CLIP-tag substrates.

Cloning Region of pCLIPf: Unique restriction sites in the regions flanking the CLIPfgene are displayed above the coding strand. The complete sequence for pCLIPf and the control plasmids can be downloaded here .

DNASU is a central repository for plasmid clones and collections that may also be helpful.

Advantages and FeaturesBack to Top

Features

BACKGROUND:

A plasmid map and the sequence of the cloning region can be found in this document. The complete plasmid sequence can be downloaded. This plasmid encodes the gene CLIPf which is a mutant form of the human gene for O6-alkylguanine-DNA-alkyltransferase (hAGT). The codon usage of the gene is optimized for expression in mammalian cells. In the plasmid sequence, the CLIPf gene is encoded from 969 bp to 1514 bp.

This plasmid is intended for the cloning and stable or transient expression of CLIP-tag protein fusions in mammalian cells. It is suitable for the efficient production of stable cell lines expressing CLIPf gene fusions. The plasmid contains the CMV promoter followed by the genes for CLIPf and neomycin resistance separated by the IRES of the encephalomyocarditis virus (ECMV), which permits the translation of two open reading frames from one messenger RNA; therefore after selection of stable mammalian cells for neomycin resistance, nearly all surviving colonies should stably express the CLIPffusion protein. Unless the expression experiments require a pure population of cells, the pool of resistant cells can simply be used, otherwise cell clones can be isolated and characterized using standard procedures.

The plasmid contains the β-lactamase (Ampicillin resistance) gene for maintenance in bacteria. The gene of interest can be cloned upstream or downstream of the CLIPf coding sequence, as a fusion to the N- or C-terminus of the CLIP-tag. pCLIPf Vector can also be used as an expression control plasmid, expressing CLIPf alone, in which case the CLIP-tag protein is distributed throughout the cell. The CLIPf gene can be isolated from the plasmid using PCR or direct cloning in order to subclone it into a different vector of choice.

Properties and UsageBack to Top

Materials Required but not Supplied

• Tissue culture reagents and media 
• Mammalian cell line(s) 
• Transfection reagents
• CLIP-tag substrates

Storage Temperature

-20°C

Vectors：pACT

pBIND

Contorl plasmids：pACT-MyoD

pBIND-Id

Reporter plasmid：pG5luc

Stratagene

Vectors：pCMV-BD

pCMV-AD

Contorl plasmids：pBD-p53

pBD-NFκB

pAD-SV40T

pAD-TRAF

Reporter plasmid：pFR-luc

酵母雙雜交系統

Clontech

Vectors：pGBKT7

pGADT7

Positivecontorls：pCL1

pGBKT7-53

pGADT7-T

Negativecontrol：pGBKT7-Lam

Strain：AH109

Vector：pACT2 AD

Stratagene

Vectors：pSos

pMyr

Control plasmid：pSos MAFB

Strain：

酵母單雜交系統

Clontech

Vector：pHis2

酵母三雜交系統

Clontech

Vector：pBridge

Strains：Y2H Gold Yeast strain

Y187

誘導系統：

蛻皮激素誘導表達系統

Invitrogen

Vectors：pIND

pVgRxR

LacSwith II哺乳動物誘導表達系統

Stratagene

Vectors：pOPRSVI

pOPI3CAT

pCMVLacI

GeneSwith哺乳動物誘導系統

Invitrogen

Vectors：pSwitch

pGenev5-His B

四環素誘導系統

Invitrogen

Vectors：pcDNA4/TO/Myc-HisA

pcDNA4/TO/Myc-His B

pcDNA4/TO/Myc-HisC

Contorl plasmid：pcDNA4/TO/Myc-His/LacZ

Regulatory plasmid：pcDNA6/TR

Clontech

Vectors：pTet-On

pTet-Off

pTRE2

pTRE2 hygro

pTRE2pur

pTK-hyg

pRevTet-On

pRevTet-Off

pRevTRE

pTet on advanced

pTRETight

pTRE Tight Luciferase

pCMV-Tet3G

pTRE3G

pTRE3G-Luc

TaleToolBox kit（TALEN and TALETF）

Vectors：pNI v2

pNG v2

pNN v2

pHD v2

pTALETFv2 (NN)

pTALETF v2 (NG)

pTALETF v2 (NI)

pTALETF v2 (HD)

pTALENv2 (NI)

pTALEN v2 (NG)

pTALEN v2 (NN)

pTALEN v2 (HD)

CRISPR/Cas系統

Vectors：pX334

蛋白體內分析系統

NEB

Vectors：pCLIP f

pCLIPf -NK1R

腺病毒系統：

Stratagene

Vectors：pShuttle-CMV

pShuttle

pShuttle-IRES-hrGFP2

pAdTrack-CMV

pAdTrack

pAdEasy-1

Strains：BJ5183

AdEasy-1

Invitrogen

Vector：pAd/BLOCK-iT-DEST RNAi Gateway Vector

Vectors：pDC315

pBHGloxdelE13cre

Cellbiolabs

Vectors：pacAd5 CMV

pacAd5 9.2-100

pacAd5CMV-GFP

腺相關病毒系統：

Vectors：pAAV-MCS

pAAV-RC

pHelper

pAAV-LacZ

pAAV-IRES-hrGFP

pCMV-MCS

逆病毒系統：

RNAi

Clontech

Vectors：RNAi-Ready pSIREN-RetroQ

RNAi-Ready pSIREN-RetroQ-DsRed-Express

RNAi-ReadypSIREN-RetroQ-ZsGreen

Vectors：pSilencer5.1-U6-Retro

pSilencer5.1-H1-Retro

基因表達載體

Vectors：pRetroX-IRES-ZsGreen1

pLNCX2

pQCXIH

pMSCV puro

pMSCV neo

pRetroX-IRES-DsRed Express

Vectors：pBabe puro

pSuper retro puro

 pSuper retro GFP-neo

逆病毒包裝載體

Vectors：pCMV-Gag-pol

pCMV-VSV-G

Vectors：pCL Ampho

Vectors：pVPack-GP

pCMV VSV G

Vectors：pMXs-hOct4

pMXs-hSox2

pMXs-hKlf4

pMXs-hC-Myc

慢病毒系統：

RNAi載體

Vectors：pSicoR

pSicoRpGK puro

pLentilox 3.7

pLKO.1puro

pLKO.3G

pPRIME-TET-GFP-FF3

pPRIME-TREX-GFP-FF3

pLVTH

pLVTHM

Vectors：pLVX-shRNA1

pLVX-shRNA2

Vectors：pSIH1-H1-CoGFP

Vectors：pLKO-puro TurboGFP

OpenBiosystems

Vectors：pGIPZ empty

pTRIPZempty

基因表達載體

Vectors：pLVX-DsRed-Monomer-N1

pLVX-AcGFP1-N1

pLVX-AcGFP1-C1

pLVX-IRES-ZsGreen1

pLVX-IRES-td Tomato

pLVX-IRES-mCherry

pLVX-IRES-neo

pLVX-IRES-puro

pLVX-puro

pLVX-Tight-Puro

pLVX-Tet-On-Advanced

pLVX-TRE3G

pLVX-Tet3G

pLVX-EF1a-IRES puro

Invitrogen

Vectors：pLenti4 TO/V5 DEST

pLenti6V5 DEST

pLenti6-UbC-V5-DEST

Vectors：pCDH-CMV-MCS-EF1-copGFP

pCDH-CMV-MCS-EF1-GFP+Puro

pCDH-CMV-MCS-EF1-puro

pCDH-EF1-MCS-T2A-Puro

pCDH-MCS-T2A-Puro-MSCV

Vectors：pWPXL

FUGW

慢病毒包裝載體

Invitrogen

Vectors：pLp1

pLp2

pLpVSV G

Strains：Stbl3

Stbl4

Cellbiolabs

Vector：pCgpV

Clontech

Vector：pVSV G

Addgene

Second generation：

Vectors：pCMV-dR8.91

pCMV-VSV-G

Second generation：

Vectors：pMD2.G

pSPAX2

Third generation：

Vectors：pMDLg pRRE

pRSV-rev

pCMV-VSV-G

iPS慢病毒載體

Vectors：pLentG-KOSM

Vectors：FUW-M2rtTA

TetO-FUW-OSKM

Vectors：FUW-tetO-hOCT4

FUW-tetO-hSOX2

FUW-tetO-hKLF4

FUW-tetO-hMYC

FUW-M2rtTA

Vector：LeGO-iC2

Vector：pLOX-CW-CRE

建永生化細胞系慢病毒載體

Vectors：pLOX-CWBmi1

pLOX-TERT-iresTK

pLOX-Ttag-iresTK

PiggyBac轉座子系統

SBI

Vectors：PiggyBac Dualpromoter (PB513B-1)

Super PiggyBac Transposase (PB200PA-1)

信號通路報告系統：

Clontech

Vectors：pGAS-TA-Luc

pSTAT3-TA-Luc

pISRE-TA-Luc

pTA-Luc

pIκB-EGFP

pNFAT-TA-Luc

pCaspase3-sensor

pAP1(PMA)-TA-Luc

pCRE-Luc

pGRE-Luc

pHSE-Luc

pE2F-TA-Luc

Stratagene

Vectors：p53-Luc

pAP-1-Luc

pNF-κB-Luc

pSRE-Luc

Vectors：pGL4.10

pGL4.13

pGL4.17

pGL4.19

pGL4.26

pGL4.27

pGL4.29

pGL4.30

pGL4.75

pGL4.79

Millipore

Vectors：TOPFlash

FOPFlash

Vector：SuperTopFlash

RNAi系統：

Vectors：pSilencer1.0

pSilencer 2.1-U6 hygro

pSilencer 3.1-H1 hygro

pSilencer 3.1-H1neo

pSilencer 4.1-CMV neo

pSilencer 4.1-CMV puro

Reporter plasmid：pMIR-REPORT Luciferase

Promega

Reproter plasmids：psiCHECK-2

pmir GLO

Clontech

Vector：pmR mCherry

SBI

Vector：pmiRZipanti-microRNA

Genscript

Vectors：pRNATin-H1.2/Neo

pRNATin-H1.2/Hygro

pRNATin-H1.2/Retro

pRNA-H1.1/Adeno

Clontech

Vector：pSingle-tTS-shRNA

酵母表達系統：

釀酒酵母

Invitrogen

Vectors：pYES2

pYES6/CT

pYES2/CT/α-factor

Strain：INVSc1

畢赤酵母

Invitrogen

Vectors：pGAPZαA

pGAPZαB

pGAPZαC

pPICZαA

pPICZαB

pPICZαC

pPIC9K

pPIC3.5K

pPICZ A

pPICZ B

pPICZ C

Strains：X33

KM71

KM71H

GS115

SMD1163

SMD1168

Invitrogen

Vectors：pPink HC

pPinkLC

Strains：Pichiapink-1

Pichiapink-4

LifeSensors

Vectors：pP-αSUMO3

pE-SUMO

SUMO protease 2（gene）

Gateway系統：

Invitrogen

Vectors：pENTR 1A

pENTR3C

pDONR221

pcDNA6.2-GWEmGFP-miRnegative

pDONR Zeo

Regulatory plasmid：pLenti6/TR

Strain：DB3.1

表面展示系統:

酵母表面展示

Invitrogen

Vector：pYD1

Strain：EBY100

哺乳動物表面展示

Invitrogen

Vector：pDisplay

噬菌體表面展示

Vector：pCantab 5e

Strains：HB2151

TG1

重組蛋白乳汁表達系統：

Invitrogen

Vector：pBC1

絕緣子檢測系統：

Invivogen

Vector：pWHERE

果蠅細胞表達系統：

Invitrogen

Vectors：pAc5.1 V5-His B

pCo hygro

哺乳動物表達系統：

Invitrogen

Vectors：pcDNA3.1(neo)+

pcDNA3.1(neo)-

pcDNA3.1(hyg)+

pcDNA3.1(zeo)+

pcDNA3.1/GS

pcDNA3.1(-)/myc-His A

pcDNA4/HisMax B

pcDNA6-Myc/His B

pSecTag2 A

pVAX1

pBudCE4.1

pTracerCMV2

pCEP4

pUB6/V5 His A

pUB6/V5His B

pUB6/V5 His C

pUB6/V5 His lacZ

pSFV1

pEF1/myc-HisB

pcDNA3.1(+)/Myc HisB

pEF6/Myc-HisC

pEF4/V5 HisA

pcDNA4/V5-His A

Stratagen

Vectors：pCMV-Tag 2B

pCMV-Tag 5B

pNTAP-B

pBK-CMV

pIRES-hrGFP-1a

pSG5

Promega

Vectors：pCI-neo

pCI

pSI

Clontech

Vectors：pCMV-myc

pCMV-HA

pIRES

pIRES neo

pIRES hyg3

pIRES neo3

pIRES puro3

pIRES2-EGFP

pIRES puro2

pBI CMV1

Sigma

Vectors：pFLAG CMV2

Invivogen

Vectors：pVitro2-neo-mcs

Addgene

Vectors：pCAGGS

昆蟲細胞表達系統：

Invitrogen

Vectors：pFastBac1

pFastBac1-Gus

pFastBacHT A

pFastBacHT B

pFastBacHT C

pFastBacHT-CAT

pFastBacDual

pBlueBacHis2 A

pBlueBacHis2 B

pBlueBacHis2 C

pVL1392

pVL1393

pDEST 10

Strain：DH10Bac

Vectors：pIZT/V5-His

pMIB v5-His B

MultiBac Expression System

Vectors：pFBDM

pUCDM

pBADZ-His6Cre

Strains：DH10MultiBac

BW23473

Novagen

Vectors：pIEX-Bac1

熒光蛋白報告系統：

Clontech

Vectors：pDsRed2-N1

pDsRed2-C1

 pDsRED-Monomer-N1

pmCherry-N1

pmCherry-C1, pEYFP-N1

pEYFP-C1

pEGFP-N1

pEGFP-C1

pEGFP-N3

pEGFP-1

pAcGFP1-1

pECFP-N1

pECFP-C1

pDsRED2-ER

pDsRED2-Mito

pDsRED2-Nuc

pDsRED2-Peroxi

pEYFP-Golgi

pEYFP-Mem

pEYFP-ER

pEYFP actin

pEYFP tub

pEGFP-Actin

pECFP-ER

pECFP-Mito

pLEGFP-N1

pLEGFP-C1

pDsRed2-1

pEGFP-C3

pPAmCherry-Tubulin

pZsYellow-N1

pAmCyan-N1

pDsRed-Monomer

pEBFP-N1

pEBFP-C1

pmRaspberry

pIRES2-DsRedexpress

FRET assay

Vectors：Dkeima fluorescent protein

mAmetrine

啟動子增強子報告系統：

Promega

Vectors：pGL3-basic

pGL3-control

pGL3-enhancer

pGL3-promoter

pRL-TK

pRL-CMV

pRL-SV40

NEB

Vectors：pGluc-basic

原核表達系統：

Novagen

Vectors：pET-11a(+)

pET15b(+)

pET-17b(+)

pET-19b(+)

pET-20b(+)

pET-21a(+)

pET-22b(+)

pET-26b(+)

pET-27b(+)

pET-28a(+)

pET-28b(+)

pET-29a(+)

pET-30a(+)

pET31b(+)

pET-32a(+)

pET-39b(+)

pET-40b(+)

pET41a

pET42a(+)

pET43.1a

pET48b(+)

pET49b(+)

pET50b(+)

pET52b(+)

pETduet-1

pACYCDuet-1

pRSFDuet-1

pCDFDuet-1

pCOLA duet-1

Strains：BL21

BL21(DE3)

BL21(DE3)plysS

Rosetta(DE3)

Rosetta-gami 2(DE3)pLysS

Rosetta-gamiB(DE3)pLysS

BLR(DE3)

RosettaBlue(DE3)

Rosetta(DE3)plysS

Rosetta-gami(DE3)

Origami(DE3)

OrigamiB(DE3)

Tuner（DE3）

Takara

Vectors：pCold I

pCold II

pCold III

pCold TF

NEB

Vectors：pMAL-c2x

pMAL-c4x

pMAL-p4x

Biosee

Vectors：pLLP-OmpA

pLLP-STII

pMBP-P

pMBP-C

pET-GST

pET-Trx

pET-His

pTrc-CKS

pET-DsbA

Stratagene

Strain：BL21 codonplus（DE3）RIPL

BL21gold(DE3)

BL21gold(DE3) plysS

ArcticExpress(DE3)RP

Invitrogen

Vector：pRSET A

Strain：BL21(AI)

Qiagen

Vector：pQE30

pQE32

Strain：M15

NEB

Vectors：pTWIN1

pTYB11

pMXB10

pTXB1

Strain：ER2566

Amersham

Vectors：pGEX-4T-1

pGEX-5X-2

pGEX-6P-1

pGEX-2TK

Invitrogen

Vectors：pBADHisA

pBADHis B

pBADHisC

Vector：pEcoli-6xHN-GFPuv

Lucigen

Strain：C41(DE3)

C43(DE3)

Other

Vectors：pBV220

pBluescript II SK(+)

pBluescript II KS(+)

Takara

分子伴侶系統

Vectors：pG-KJE8

pGro7

pKJE7

pG-Tf2

pTf16

乳酸菌表達系統

Vector：pMG36e

Strain：MG1363

基因載體：

Invitrogen

Vector：pOG44

Invivogen

Vectors：pORF-lacZ

pORF-HSV1tk

克隆載體：

Promega

Vectors：pUC18

pUC19

pBR322

pSP64

pSP73

NEB

pACYC184

柯斯載體：

Stratagene

Vector：SuperCos1

同源重組載體：

Vectors：pSIM5

pSIM6

細菌基因敲除系統：

Vectors：pKD3

pKD4

pKD20

pKD46

PCP20

Strain：BW25113

克隆菌株：

Strains：DH5α

JM109

JM110

TOP10

XL2-Blue MRF’

Mach1 T1

OmniMAX2 T1Phage-Resistant Cells

XL10 Gold

EZ10

DH10B

SURE

XL1blue