pFLAG-CMV-3

pFLAG-CMV-3

pFLAG-CMV-3载体基本信息

pFLAG-CMV-3载体质粒图谱和多克隆位点信息

pFLAG-CMV-3载体简介

FLAG 和 3xFLAG 标签：

A Proven System for Detection and Purification of Proteins The FLAG Expression System is an established way to express, purify and detect recombinant fusion proteins. FLAG and 3xFLAG have proven utility in numerous applications such as Western blotting, immunocytochemistry, immunoprecipitation, flow cytometry, protein purification, and in the study of protein-protein interactions, cell ultrastructure and protein localization. These small hydrophilic tags facilitate superior detection and purification of recombinant fusion proteins when using our highly specific and sensitive ANTI-FLAG antibodies. Ideal Epitope Tags: Small, Hydrophilic and Cleavable The FLAG expression system utilizes a short, hydrophilic 8-amino acid peptide that is fused to the recombinant protein of interest. Because of its hydrophilic nature, the FLAG peptide is likely to be located on the surface of the fusion protein. As a result, the FLAG peptide is easily accessible for cleavage by enterokinase (Ek) and for detection with antibodies. In addition, because of the small size of the FLAG peptide tag, it is not likely to obscure other epitopes, domains, or alter function, secretion, or transport of the fusion protein. The 3xFLAG system is an improvement upon the original system by fusing 3 tandem FLAG epitopes (22 amino acids). Detection of fusion proteins containing 3xFLAG is enhanced up to 200 times more than any other system. Like the original FLAG tag, 3xFLAG is hydrophilic, contains an Ek cleavage site, and is relatively small. Therefore, the risk of altering protein function, blocking other epitopes or decreasing solubility is minimized.

3xFLAG System Expression Vectors for Ultra-Sensitive

重组蛋白的检测

The 3xFLAG system is an improvement upon the original system by fusing 3 tandem FLAG epitopes for a total of 22 amino acids. Detection of fusion proteins containing 3xFLAG is enhanced up to 200 times more than any other system. Like the original FLAG tag, 3xFLAG is hydrophilic, contains an Enterokinase cleavage site, and is relatively small. Therefore, the risk of altering protein function, blocking other epitopes or decreasing solubility is minimized. In cases of low-level expression, 3xFLAG is ideal. Our selection of CMV vectors provides options for transient or stable expression of 3xFLAG fusion proteins.

Ultrasensitive System - Detect < 10 femtomoles using ANTI-FLAG Antibodies; Ideal for cases of low-level expression in mammalian cells.

Superior Detection - 20-200x more sensitive than any other system.

Versatile - Enhanced detection for immunoprecipitation, Western blots and immunocytochemistry.

Wide Selection of Detection and Purification Products - ANTI-FLAG antibodies, resins, and affinity capture plates.

Figure 1. 3xFLAG Sequence. Removal of N-terminal 3xFLAG tags is possible using enterokinase, which cleaves following the Asp-Asp-Asp-Asp-Lys amino acid sequence at the c-terminal end of the tag.

Figure 2. Western Blot of Original FLAG Versus 3xFLAG. Western blot of purified 3xFLAG-BAP (bacterial alkaline phosphatase) and FLAG-BAP transferred onto a nitrocellulose membrane. Detection was performed with ANTI-FLAG M2 monoclonal primary antibody, anti-mouse-HRP secondary antibody, and ECL chemiluminescent substrate.

Figure 3. Comparative Sensitivity of 3xFLAG. Each protein fusion tag was cloned separately on the C terminal of GST. The purified proteins were diluted from 1 ug to 0.01 ng and analyzed. The limit of detection of each tag was determined by Western blot analysis using the recommended dilutions of each respective primary antibody and secondary anti-mouse IgG-HRP conjugate. The blot was developed using ECL.

CMV启动子：

Our mammalian expression vectors contain the strong CMV promoter for high-level constitutive expression in mammalian cells. The strong human cytomegalovirus (CMV) promoter regulatory region drives constitutive protein expression levels as high as 1 mg/L in COS cells. For less potent cell lines, protein levels are typically ~0.1 mg/L. The presence of the SV40 replication origin will result in high levels of DNA replication in SV40 replication permissive COS cells. Vectors containing the preprotrypsin leader (PPT) sequence direct secretion of FLAG fusion proteins into the culture medium for purification using ANTI-FLAG antibodies, resins, and plates.

pFLAG-CMV-3载体序列

ORIGIN

    1 CCATTCGCCA TTCAGGCTGC GCAACTGTTG GGAAGGGCGA TCGGTGCGGG CCTCTTCGCT

   61 ATTACGCCAG CTGGCGAAAG GGGGATGTGC TGCAAGGCGA TTAAGTTGGG TAACGCCAGG

  121 GTTTTCCCAG TCACGACGTT GTAAAACGAC GGCCAGTGCC AAGCTGATCT ATACATTGAA

  181 TCAATATTGG CAATTAGCCA TATTAGTCAT TGGTTATATA GCATAAATCA ATATTGGCTA

  241 TTGGCCATTG CATACGTTGT ATCTATATCA TAATATGTAC ATTTATATTG GCTCATGTCC

  301 AATATGACCG CCATGTTGAC ATTGATTATT GACTAGTTAT TAATAGTAAT CAATTACGGG

  361 GTCATTAGTT CATAGCCCAT ATATGGAGTT CCGCGTTACA TAACTTACGG TAAATGGCCC

  421 GCCTGGCTGA CCGCCCAACG ACCCCCGCCC ATTGACGTCA ATAATGACGT ATGTTCCCAT

  481 AGTAACGCCA ATAGGGACTT TCCATTGACG TCAATGGGTG GAGTATTTAC GGTAAACTGC

  541 CCACTTGGCA GTACATCAAG TGTATCATAT GCCAAGTCCG CCCCCTATTG ACGTCAATGA

  601 CGGTAAATGG CCCGCCTGGC ATTATGCCCA GTACATGACC TTACGGGACT TTCCTACTTG

  661 GCAGTACATC TACGTATTAG TCATCGCTAT TACCATGGTG ATGCGGTTTT GGCAGTACAC

  721 CAATGGGCGT GGATAGCGGT TTGACTCACG GGGATTTCCA AGTCTCCACC CCATTGACGT

  781 CAATGGGAGT TTGTTTTGGC ACCAAAATCA ACGGGACTTT CCAAAATGTC GTAATAACCC

  841 CGCCCCGTTG ACGCAAATGG GCGGTAGGCG TGTACGGTGG GAGGTCTATA TAAGCAGAGC

  901 TCGTTTAGTG AACCGTCAGA ATTAATTCAG ATCGATCTAC CATGTCTGCA CTTCTGATCC

  961 TAGCTCTTGT TGGAGCTGCA GTTGCTGACT ACAAAGACGA TGACGACAAG CTTGCGGCCG

 1021 CGAATTCATC GATAGATCTG ATATCGGTAC CAGTCGACTC TAGAGGATCC CGGGTGGCAT

 1081 CCCTGTGACC CCTCCCCAGT GCCTCTCCTG GCCCTGGAAG TTGCCACTCC AGTGCCCACC

 1141 AGCCTTGTCC TAATAAAATT AAGTTGCATC ATTTTGTCTG ACTAGGTGTC CTTCTATAAT

 1201 ATTATGGGGT GGAGGGGGGT GGTATGGAGC AAGGGGCAAG TTGGGAAGAC AACCTGTAGG

 1261 GCCTGCGGGG TCTATTGGGA ACCAAGCTGG AGTGCAGTGG CACAATCTTG GCTCACTGCA

 1321 ATCTCCGCCT CCTGGGTTCA AGCGATTCTC CTGCCTCAGC CTCCCGAGTT GTTGGGATTC

 1381 CAGGCATGCA TGACCAGGCT CAGCTAATTT TTGTTTTTTT GGTAGAGACG GGGTTTCACC

 1441 ATATTGGCCA GGCTGGTCTC CAACTCCTAA TCTCAGGTGA TCTACCCACC TTGGCCTCCC

 1501 AAATTGCTGG GATTACAGGC GTGAACCACT GCTCCCTTCC CTGTCCTTCT GATTTTAAAA

 1561 TAACTATACC AGCAGGAGGA CGTCCAGACA CAGCATAGGC TACCTGGCCA TGCCCAACCG

 1621 GTGGGACATT TGAGTTGCTT GCTTGGCACT GTCCTCTCAT GCGTTGGGTC CACTCAGTAG

 1681 ATGCCTGTTG AATTGGGTAC GCGGCCAGCT TGGCTGTGGA ATGTGTGTCA GTTAGGGTGT

 1741 GGAAAGTCCC CAGGCTCCCC AGCAGGCAGA AGTATGCAAA GCATGCATCT CAATTAGTCA

 1801 GCAACCAGGT GTGGAAAGTC CCCAGGCTCC CCAGCAGGCA GAAGTATGCA AAGCATGCAT

 1861 CTCAATTAGT CAGCAACCAT AGTCCCGCCC CTAACTCCGC CCATCCCGCC TCCGCTAACT

 1921 CCGCCCAGTT CCGCCCATTC TCCGCCCCAT GGCTGACTAA TTTTTTTTAT TTATGCAGGC

 1981 CGAGGCCGCC TCGGCCTCTG AGCTATTCCA GAAGTAGTGA GGAGGCTTTT TTGGAGGAAT

 2041 TGATCAGCTT GGGATCTGAT CAAGAGACAG GATGAGGATC GTTTCGCATG ATTGAACAAG

 2101 ATGGATTGCA CGCAGGTTCT CCGGCCGCTT GGGTGGAGAG GCTATTCGGC TATGACTGGG

 2161 CACAACAGAC AATCGGCTGC TCTGATGCCG CCGTGTTCCG GCTGTCAGCG CAGGGGCGCC

 2221 CGGTTCTTTT TGTCAAGACC GACCTGTCCG GTGCCCTGAA TGAACTGCAG GACGAGGCAG

 2281 CGCGGCTATC GTGGCTGGCC ACGACGGGCG TTCCTTGCGC AGCTGTGCTC GACGTTGTCA

 2341 CTGAAGCGGG AAGGGACTGG CTGCTATTGG GCGAAGTGCC GGGGCAGGAT CTCCTGTCAT

 2401 CTCACCTTGC TCCTGCCGAG AAAGTATCCA TCATGGCTGA TGCAATGCGG CGGCTGCATA

 2461 CGCTTGATCC GGCTACCTGC CCATTCGACC ACCAAGCGAA ACATCGCATC GAGCGAGCAC

 2521 GTACTCGGAT GGAAGCCGGT CTTGTCGATC AGGATGATCT GGACGAAGAG CATCAGGGGC

 2581 TCGCGCCAGC CGAACTGTTC GCCAGGCTCA AGGCGCGCAT GCCCGACGGC GAGGATCTCG

 2641 TCGTGACCCA TGGCGATGCC TGCTTGCCGA ATATCATGGT GGAAAATGGC CGCTTTTCTG

 2701 GATTCATCGA CTGTGGCCGG CTGGGTGTGG CGGACCGCTA TCAGGACATA GCGTTGGCTA

 2761 CCCGTGATAT TGCTGAAGAG CTTGGCGGCG AATGGGCTGA CCGCTTCCTC GTGCTTTACG

 2821 GTATCGCCGC TCCCGATTCG CAGCGCATCG CCTTCTATCG CCTTCTTGAC GAGTTCTTCT

 2881 GAGCGGGACT CTGGGGTTCG AAATGACCGA CCAAGCGACG CCCAACCTGC CATCACGAGA

 2941 TTTCGATTCC ACCGCCGCCT TCTATGAAAG GTTGGGCTTC GGAATCGTTT TCCGGGACGC

 3001 CGGCTGGATG ATCCTCCAGC GCGGGGATCT CATGCTGGAG TTCTTCGCCC ACCCCGGGCT

 3061 CGATCCCCTC GCGAGTTGGT TCAGCTGCTG CCTGAGGCTG GACGACCTCG CGGAGTTCTA

 3121 CCGGCAGTGC AAATCCGTCG GCATCCAGGA AACCAGCAGC GGCTATCCGC GCATCCATGC

 3181 CCCCGAACTG CAGGAGTGGG GAGGCACGAT GGCCGCTTTG GTCGACCCGG ACGGGACGCT

 3241 CCTGCGCCTG ATACAGAACG AATTGCTTGC AGGCATCTCA TGAGTGTGTC TTCCCGTTTT

 3301 CCGCCTGAGG TCACTGCGTG GATGGAGCGC TGGCGCCTGC TGCGCGACGG CGAGCTGCTC

 3361 ACCACCCACT CGCCAAGCTG GAACCGTAAA AAGGCCGCGT TGCTGGCGTT TTTCCATAGG

 3421 CTCCGCCGAT CATAATCAGC CATACCACAT TTGTAGAGGT TTTACTTGCT TTAAAAAACC

 3481 TCCCACACCT CCCCCTGAAC CTGAAACATA AAATGAATGC AATTGTTGTT GTTAACTTGT

 3541 TTATTGCAGC TTATAATGGT TACAAATAAA GCAATAGCAT CACAAATTTC ACAAATAAAG

 3601 CATTTTTTTC ACTGCATTCT AGTTGTGGTT TGTCCAAACT CATCAATGTA TCTTATCATG

 3661 TCTGGATCAA TTCCCTATAG TGAGTCGTAT TAAATTCGTA ATCATGTCAT AGCTGTTTCC

 3721 TGTGTGAAAT TGTTATCCGC TCACAATTCC ACACAACATA CGAGCCGGAA GCATAAAGTG

 3781 TAAAGCCTGG GGTGCCTAAT GAGTGAGCTA ACTCACATTA ATTGCGTTGC GCTCACTGCC

 3841 CGCTTTCCAG TCGGGAAACC TGTCGTGCCA GCTGCATTAA TGAATCGGCC AACGCGCGGG

 3901 GAGAGGCGGT TTGCGTATTG GGCGCTCTTC CGCTTCCTCG CTCACTGACT CGCTGCGCTC

 3961 GGTCGTTCGG CTGCGGCGAG CGGTATCAGC TCACTCAAAG GCGGTAATAC GGTTATCCAC

 4021 AGAATCAGGG GATAACGCAG GAAAGAACAT GTGAGCAAAA GGCCAGCAAA AGGCCAGGAA

 4081 CCGTAAAAAG GCCGCGTTGC TGGCGTTTTT CCATAGGCTC CGCCCCCCTG ACGAGCATCA

 4141 CAAAAATCGA CGCTCAAGTC AGAGGTGGCG AAACCCGACA GGACTATAAA GATACCAGGC

 4201 GTTTCCCCCT GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG ACCCTGCCGC TTACCGGATA

 4261 CCTGTCCGCC TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT CATAGCTCAC GCTGTAGGTA

 4321 TCTCAGTTCG GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT GTGCACGAAC CCCCCGTTCA

 4381 GCCCGACCGC TGCGCCTTAT CCGGTAACTA TCGTCTTGAG TCCAACCCGG TAAGACACGA

 4441 CTTATCGCCA CTGGCAGCAG CCACTGGTAA CAGGATTAGC AGAGCGAGGT ATGTAGGCGG

 4501 TGCTACAGAG TTCTTGAAGT GGTGGCCTAA CTACGGCTAC ACTAGAAGAA CAGTATTTGG

 4561 TATCTGCGCT CTGCTGAAGC CAGTTACCTT CGGAAAAAGA GTTGGTAGCT CTTGATCCGG

 4621 CAAACAAACC ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC AAGCAGCAGA TTACGCGCAG

 4681 AAAAAAAGGA TCTCAAGAAG ATCCTTTGAT CTTTTCTACG GGGTCTGACG CTCAGTGGAA

 4741 CGAAAACTCA CGTTAAGGGA TTTTGGTCAT GAGATTATCA AAAAGGATCT TCACCTAGAT

 4801 CCTTTTAAAT TAAAAATGAA GTTTTAAATC AATCTAAAGT ATATATGAGT AAACTTGGTC

 4861 TGACAGTTAC CAATGCTTAA TCAGTGAGGC ACCTATCTCA GCGATCTGTC TATTTCGTTC

 4921 ATCCATAGTT GCCTGACTCC CCGTCGTGTA GATAACTACG ATACGGGAGG GCTTACCATC

 4981 TGGCCCCAGT GCTGCAATGA TACCGCGAGA CCCACGCTCA CCGGCTCCAG ATTTATCAGC

 5041 AATAAACCAG CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT CCTGCAACTT TATCCGCCTC

 5101 CATCCAGTCT ATTAATTGTT GCCGGGAAGC TAGAGTAAGT AGTTCGCCAG TTAATAGTTT

 5161 GCGCAACGTT GTTGCCATTG CTACAGGCAT CGTGGTGTCA CGCTCGTCGT TTGGTATGGC

 5221 TTCATTCAGC TCCGGTTCCC AACGATCAAG GCGAGTTACA TGATCCCCCA TGTTGTGCAA

 5281 AAAAGCGGTT AGCTCCTTCG GTCCTCCGAT CGTTGTCAGA AGTAAGTTGG CCGCAGTGTT

 5341 ATCACTCATG GTTATGGCAG CACTGCATAA TTCTCTTACT GTCATGCCAT CCGTAAGATG

 5401 CTTTTCTGTG ACTGGTGAGT ACTCAACCAA GTCATTCTGA GAATAGTGTA TGCGGCGACC

 5461 GAGTTGCTCT TGCCCGGCGT CAATACGGGA TAATACCGCG CCACATAGCA GAACTTTAAA

 5521 AGTGCTCATC ATTGGAAAAC GTTCTTCGGG GCGAAAACTC TCAAGGATCT TACCGCTGTT

 5581 GAGATCCAGT TCGATGTAAC CCACTCGTGC ACCCAACTGA TCTTCAGCAT CTTTTACTTT

 5641 CACCAGCGTT TCTGGGTGAG CAAAAACAGG AAGGCAAAAT GCCGCAAAAA AGGGAATAAG

 5701 GGCGACACGG AAATGTTGAA TACTCATACT CTTCCTTTTT CAATATTATT GAAGCATTTA

 5761 TCAGGGTTAT TGTCTCATGA GCGGATACAT ATTTGAATGT ATTTAGAAAA ATAAACAAAT

 5821 AGGGGTTCCG CGCACATTTC CCCGAAAAGT GCCACCTGAC GCGCCCTGTA GCGGCGCATT

 5881 AAGCGCGGCG GGTGTGGTGG TTACGCGCAG CGTGACCGCT ACACTTGCCA GCGCCCTAGC

 5941 GCCCGCTCCT TTCGCTTTCT TCCCTTCCTT TCTCGCCACG TTCGCCGGCT TTCCCCGTCA

 6001 AGCTCTAAAT CGGGGGCTCC CTTTAGGGTT CCGATTTAGT GCTTTACGGC ACCTCGACCC

 6061 CAAAAAACTT GATTAGGGTG ATGGTTCACG TAGTGGGCCA TCGCCCTGAT AGACGGTTTT

 6121 TCGCCCTTTG ACGTTGGAGT CCACGTTCTT TAAGAGTCCA CTATTAAAGA ACGTGGACTC

 6181 TTGTTCCAAA CTGGAACAAC ACTCAACCCT ATCTCGGTCT ATTCTTTTGA TTTATAAGGG

 6241 ATTTTGCCGA TTTCGGCCTA TTGGTTAAAA AATGAGCTGA TTTAACAAAA ATTTAACGCG

 6301 AATTTTAACA AAATATTAAC GCTTACAATT T

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其他哺乳动物表达载体：