p3XFLAG-CMV-9

p3XFLAG-CMV-9载体基本信息

p3XFLAG-CMV-9载体质粒图谱和多克隆位点信息

p3XFLAG-CMV-9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.

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.

p3XFLAG-CMV-9载体介绍：

The p3XFLAG-CMV-9 Expression Vector is a 6.4 kb derivative of pCMV5 used to establish transient or stable expression of secreted N-terminal 3XFLAG fusion proteins in mammalian cells. The vector encodes three adjacent FLAG epitopes (Asp-Tyr-Lys-Xaa-Xaa- Asp) upstream of the multiple cloning region. This results in increased detection sensitivity using ANTIFLAG M2 antibody. The third epitope includes the enterokinase recognition sequence, allowing cleavage of the 3XFLAG peptide from the purified fusion protein.

p3XFLAG-CMV-9 expression vector is a shuttle vector for E. coli and mammalian cells. Efficiency of replication and genomic integration is optimal when using an SV40 T antigen expressing host, such as COS cells. A related vector, p3XFLAG-CMV-3, has been used for stable transfection of HEK 293 cells. Efficiency of replication and genomic integration is optimal when using an SV40 T antigen-expressing host.

The p3XFLAG-CMV-7-BAP Control Plasmid is a 6.2 kb derivative of pCMV5 used for transient intracellular expression of N-terminal 3XFLAG bacterial alkaline phosphatase fusion protein in mammalian cells. The vector encodes three adjacent FLAG epitopes (Asp-Tyr-Lys-Xaa-Xaa-Asp) upstream of the multiple cloning region. This results in increased detection sensitivity using ANTI-FLAG M2 antibody. The third FLAG epitope includes the enterokinase recognition sequence, allowing cleavage of the 3XFLAG peptide from the purified fusion protein.

The promoter-regulatory region of the human cytomegalovirus drives transcription of FLAG-fusion constructs. The preprotrypsin leader sequence precedes the FLAG sequence. The aminoglycoside phosphotransferase II gene (Neo-r) confers resistance to aminoglycosides such as G418 allowing for selection of stable transfectants.

p3XFLAG-CMV-9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 ACAAAGACCA TGACGGTGAT TATAAAGATC

 1021 ATGACATCGA TTACAAGGAT GACGATGACA AGCTTGCGGC CGCGAATTCA TCGATAGATC

 1081 TGATATCGGT ACCAGTCGAC TCTAGAGGAT CCCGGGTGGC ATCCCTGTGA CCCCTCCCCA

 1141 GTGCCTCTCC TGGCCCTGGA AGTTGCCACT CCAGTGCCCA CCAGCCTTGT CCTAATAAAA

 1201 TTAAGTTGCA TCATTTTGTC TGACTAGGTG TCCTTCTATA ATATTATGGG GTGGAGGGGG

 1261 GTGGTATGGA GCAAGGGGCA AGTTGGGAAG ACAACCTGTA GGGCCTGCGG GGTCTATTGG

 1321 GAACCAAGCT GGAGTGCAGT GGCACAATCT TGGCTCACTG CAATCTCCGC CTCCTGGGTT

 1381 CAAGCGATTC TCCTGCCTCA GCCTCCCGAG TTGTTGGGAT TCCAGGCATG CATGACCAGG

 1441 CTCAGCTAAT TTTTGTTTTT TTGGTAGAGA CGGGGTTTCA CCATATTGGC CAGGCTGGTC

 1501 TCCAACTCCT AATCTCAGGT GATCTACCCA CCTTGGCCTC CCAAATTGCT GGGATTACAG

 1561 GCGTGAACCA CTGCTCCCTT CCCTGTCCTT CTGATTTTAA AATAACTATA CCAGCAGGAG

 1621 GACGTCCAGA CACAGCATAG GCTACCTGGC CATGCCCAAC CGGTGGGACA TTTGAGTTGC

 1681 TTGCTTGGCA CTGTCCTCTC ATGCGTTGGG TCCACTCAGT AGATGCCTGT TGAATTGGGT

 1741 ACGCGGCCAG CTTGGCTGTG GAATGTGTGT CAGTTAGGGT GTGGAAAGTC CCCAGGCTCC

 1801 CCAGCAGGCA GAAGTATGCA AAGCATGCAT CTCAATTAGT CAGCAACCAG GTGTGGAAAG

 1861 TCCCCAGGCT CCCCAGCAGG CAGAAGTATG CAAAGCATGC ATCTCAATTA GTCAGCAACC

 1921 ATAGTCCCGC CCCTAACTCC GCCCATCCCG CCTCCGCTAA CTCCGCCCAG TTCCGCCCAT

 1981 TCTCCGCCCC ATGGCTGACT AATTTTTTTT ATTTATGCAG GCCGAGGCCG CCTCGGCCTC

 2041 TGAGCTATTC CAGAAGTAGT GAGGAGGCTT TTTTGGAGGA ATTGATCAGC TTGGGATCTG

 2101 ATCAAGAGAC AGGATGAGGA TCGTTTCGCA TGATTGAACA AGATGGATTG CACGCAGGTT

 2161 CTCCGGCCGC TTGGGTGGAG AGGCTATTCG GCTATGACTG GGCACAACAG ACAATCGGCT

 2221 GCTCTGATGC CGCCGTGTTC CGGCTGTCAG CGCAGGGGCG CCCGGTTCTT TTTGTCAAGA

 2281 CCGACCTGTC CGGTGCCCTG AATGAACTGC AGGACGAGGC AGCGCGGCTA TCGTGGCTGG

 2341 CCACGACGGG CGTTCCTTGC GCAGCTGTGC TCGACGTTGT CACTGAAGCG GGAAGGGACT

 2401 GGCTGCTATT GGGCGAAGTG CCGGGGCAGG ATCTCCTGTC ATCTCACCTT GCTCCTGCCG

 2461 AGAAAGTATC CATCATGGCT GATGCAATGC GGCGGCTGCA TACGCTTGAT CCGGCTACCT

 2521 GCCCATTCGA CCACCAAGCG AAACATCGCA TCGAGCGAGC ACGTACTCGG ATGGAAGCCG

 2581 GTCTTGTCGA TCAGGATGAT CTGGACGAAG AGCATCAGGG GCTCGCGCCA GCCGAACTGT

 2641 TCGCCAGGCT CAAGGCGCGC ATGCCCGACG GCGAGGATCT CGTCGTGACC CATGGCGATG

 2701 CCTGCTTGCC GAATATCATG GTGGAAAATG GCCGCTTTTC TGGATTCATC GACTGTGGCC

 2761 GGCTGGGTGT GGCGGACCGC TATCAGGACA TAGCGTTGGC TACCCGTGAT ATTGCTGAAG

 2821 AGCTTGGCGG CGAATGGGCT GACCGCTTCC TCGTGCTTTA CGGTATCGCC GCTCCCGATT

 2881 CGCAGCGCAT CGCCTTCTAT CGCCTTCTTG ACGAGTTCTT CTGAGCGGGA CTCTGGGGTT

 2941 CGAAATGACC GACCAAGCGA CGCCCAACCT GCCATCACGA GATTTCGATT CCACCGCCGC

 3001 CTTCTATGAA AGGTTGGGCT TCGGAATCGT TTTCCGGGAC GCCGGCTGGA TGATCCTCCA

 3061 GCGCGGGGAT CTCATGCTGG AGTTCTTCGC CCACCCCGGG CTCGATCCCC TCGCGAGTTG

 3121 GTTCAGCTGC TGCCTGAGGC TGGACGACCT CGCGGAGTTC TACCGGCAGT GCAAATCCGT

 3181 CGGCATCCAG GAAACCAGCA GCGGCTATCC GCGCATCCAT GCCCCCGAAC TGCAGGAGTG

 3241 GGGAGGCACG ATGGCCGCTT TGGTCGACCC GGACGGGACG CTCCTGCGCC TGATACAGAA

 3301 CGAATTGCTT GCAGGCATCT CATGAGTGTG TCTTCCCGTT TTCCGCCTGA GGTCACTGCG

 3361 TGGATGGAGC GCTGGCGCCT GCTGCGCGAC GGCGAGCTGC TCACCACCCA CTCGCCAAGC

 3421 TGGAACCGTA AAAAGGCCGC GTTGCTGGCG TTTTTCCATA GGCTCCGCCG ATCATAATCA

 3481 GCCATACCAC ATTTGTAGAG GTTTTACTTG CTTTAAAAAA CCTCCCACAC CTCCCCCTGA

 3541 ACCTGAAACA TAAAATGAAT GCAATTGTTG TTGTTAACTT GTTTATTGCA GCTTATAATG

 3601 GTTACAAATA AAGCAATAGC ATCACAAATT TCACAAATAA AGCATTTTTT TCACTGCATT

 3661 CTAGTTGTGG TTTGTCCAAA CTCATCAATG TATCTTATCA TGTCTGGATC AATTCCCTAT

 3721 AGTGAGTCGT ATTAAATTCG TAATCATGTC ATAGCTGTTT CCTGTGTGAA ATTGTTATCC

 3781 GCTCACAATT CCACACAACA TACGAGCCGG AAGCATAAAG TGTAAAGCCT GGGGTGCCTA

 3841 ATGAGTGAGC TAACTCACAT TAATTGCGTT GCGCTCACTG CCCGCTTTCC AGTCGGGAAA

 3901 CCTGTCGTGC CAGCTGCATT AATGAATCGG CCAACGCGCG GGGAGAGGCG GTTTGCGTAT

 3961 TGGGCGCTCT TCCGCTTCCT CGCTCACTGA CTCGCTGCGC TCGGTCGTTC GGCTGCGGCG

 4021 AGCGGTATCA GCTCACTCAA AGGCGGTAAT ACGGTTATCC ACAGAATCAG GGGATAACGC

 4081 AGGAAAGAAC ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG AACCGTAAAA AGGCCGCGTT

 4141 GCTGGCGTTT TTCCATAGGC TCCGCCCCCC TGACGAGCAT CACAAAAATC GACGCTCAAG

 4201 TCAGAGGTGG CGAAACCCGA CAGGACTATA AAGATACCAG GCGTTTCCCC CTGGAAGCTC

 4261 CCTCGTGCGC TCTCCTGTTC CGACCCTGCC GCTTACCGGA TACCTGTCCG CCTTTCTCCC

 4321 TTCGGGAAGC GTGGCGCTTT CTCATAGCTC ACGCTGTAGG TATCTCAGTT CGGTGTAGGT

 4381 CGTTCGCTCC AAGCTGGGCT GTGTGCACGA ACCCCCCGTT CAGCCCGACC GCTGCGCCTT

 4441 ATCCGGTAAC TATCGTCTTG AGTCCAACCC GGTAAGACAC GACTTATCGC CACTGGCAGC

 4501 AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG AGTTCTTGAA

 4561 GTGGTGGCCT AACTACGGCT ACACTAGAAG AACAGTATTT GGTATCTGCG CTCTGCTGAA

 4621 GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA CCACCGCTGG

 4681 TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG GATCTCAAGA

 4741 AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT CACGTTAAGG

 4801 GATTTTGGTC ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA ATTAAAAATG

 4861 AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT ACCAATGCTT

 4921 AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG TTGCCTGACT

 4981 CCCCGTCGTG TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA GTGCTGCAAT

 5041 GATACCGCGA GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC AGCCAGCCGG

 5101 AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT CTATTAATTG

 5161 TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG TTGTTGCCAT

 5221 TGCTACAGGC ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA GCTCCGGTTC

 5281 CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG TTAGCTCCTT

 5341 CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA TGGTTATGGC

 5401 AGCACTGCAT AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG TGACTGGTGA

 5461 GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT CTTGCCCGGC

 5521 GTCAATACGG GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA TCATTGGAAA

 5581 ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA GTTCGATGTA

 5641 ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG TTTCTGGGTG

 5701 AGCAAAAACA GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC GGAAATGTTG

 5761 AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT ATTGTCTCAT

 5821 GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC CGCGCACATT

 5881 TCCCCGAAAA GTGCCACCTG ACGCGCCCTG TAGCGGCGCA TTAAGCGCGG CGGGTGTGGT

 5941 GGTTACGCGC AGCGTGACCG CTACACTTGC CAGCGCCCTA GCGCCCGCTC CTTTCGCTTT

 6001 CTTCCCTTCC TTTCTCGCCA CGTTCGCCGG CTTTCCCCGT CAAGCTCTAA ATCGGGGGCT

 6061 CCCTTTAGGG TTCCGATTTA GTGCTTTACG GCACCTCGAC CCCAAAAAAC TTGATTAGGG

 6121 TGATGGTTCA CGTAGTGGGC CATCGCCCTG ATAGACGGTT TTTCGCCCTT TGACGTTGGA

 6181 GTCCACGTTC TTTAAGAGTC CACTATTAAA GAACGTGGAC TCTTGTTCCA AACTGGAACA

 6241 ACACTCAACC CTATCTCGGT CTATTCTTTT GATTTATAAG GGATTTTGCC GATTTCGGCC

 6301 TATTGGTTAA AAAATGAGCT GATTTAACAA AAATTTAACG CGAATTTTAA CAAAATATTA

 6361 ACGCTTACAA TTT

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其他哺乳动物表达载体：