pCMV-DsRed-Express2

pCMV-DsRed-Express2载体基本信息

pCMV-DsRed-Express2载体质粒图谱和多克隆位点信息

pCMV-DsRed-Express2载体描述

pCMV DsRed-Express2 is a mammalian expression vector designed to be used for whole cell labeling or as a marker of cotransfection. In mammalian cells, the vector constitutively expresses DsRed-Express2, a variant of the Discosoma sp. red fluorescent protein, DsRed (1). DsRed-Express2 retains the fast maturation and high photostability characteristic of its predecessor, DsRed-Express (2), and has been engineered (through additional amino acid substitutions) for increased solubility (3). Although it most likely forms the same tetrameric structure as wild-type DsRed, DsRed-Express2 displays a greatly reduced tendency to aggregate, resulting in reduced cyto- and phototoxicity, and making DsRed-Express2 much better suited for in vivo applications involving sensitive cells, such as primary or stem cells. In fact, DsRed-Express2 has been shown to be the best red fluorescent protein for whole cell labeling applications (3). DsRed-Express2 also exhibits extremely low residual green fluorescence, which allows cells expressing the protein to be effectively separated from other fluorescently labeled cell populations by flow cytometry. The DsRed-Express2 gene is positioned just downstream of the constitutively active human cytomegalovirus immediate early promoter (PCMV IE). As a result, mammalian cells transfected with this vector will constitutively express the red fluorescent protein. A Kozak consensus sequence (4) has been placed immediately upstream of the DsRed-Express2 coding sequence to enhance translational efficiency in eukaryotic cells. SV40 polyadenylation signals downstream of the DsRed-Express2 gene direct proper processing of the 3' end of the DsRed-Express2 mRNA. The vector backbone contains an SV40 origin for replication in mammalian cells expressing the SV40 large T antigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded DNA production. A neomycin resistance cassette (Neor) allows stably transfected eukaryotic cells to be selected using G418 (5). This cassette consists of the SV40 early promoter, a Tn5 kanamycin/neomycin resistance gene, and herpes simplex virus thymidine kinase (HSV TK) polyadenylation signals. A bacterial promoter upstream of the cassette expresses kanamycin resistance in E. coli.

pCMV DsRed-Express2 can be used for whole cell labeling or as a cotransfection marker. The vector can be transfected into mammalian cells using any standard transfection method. Cells expressing DsRed-Express2 (excitation and emission maxima: 554 nm and 591 nm, respectively) can be detected by fluorescence microscopy or flow cytometry 8-12 hours after transfection. If required, stable transfectants can be selected using G418. After cotransfection with pCMV DsRed-Express2 and an expression construct of interest, cells can also be sorted by flow cytometry to enrich for transfected cells.

Propagation in E. coli

 Recommended host strain: DH5α, HB101, and other general purpose strains. Single-stranded DNA production

requires a host containing an F plasmid such as JM109 or XL1-Blue.

 Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) in E. coli hosts.

 E. coli replication origin: pUC

 Copy number: high

 Plasmid incompatibility group: pMB1/ColE1

Excitation and emission maxima of DsRed-Express2

 Excitation maximum = 554 nm

 Emission maximum = 591 nm

References

1. Matz, M. V. et al. (1999) Nat. Biotechnol. 17(10):969-973.

2. Bevis, B. J. & Glick, B. S. (2002) Nat. Biotechnol. 20(1):83–87. Erratum in Nat. Biotechnol. (2002) 20(11):1159

3. Strack, R. L. et al. (2008) Nat. Methods 5(11):955–957.

4. Kozak, M. (1987) Nucleic Acids Res. 15(20): 8125-8148

5. Gorman, C. (1985) In DNA Cloning: A Practical Approach, Vol. II. Ed. D. M. Glover. (IRL Press, Oxford, U.K.), pp. 143–190.

pCMV-DsRed-Express2载体序列

ORIGIN

        1 TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG

       61 CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT

      121 GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC ATTGACGTCA

      181 ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT ATCATATGCC

      241 AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA

      301 CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC

      361 CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG ACTCACGGGG

      421 ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC AAAATCAACG

      481 GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT

      541 ACGGTGGGAG GTCTATATAA GCAGAGCTGG TTTAGTGAAC CGTCAGATCC GCTAGCGCTA

      601 CCGGACTCAG ATCCACCGGT CGCCACCATG GATAGCACTG AGAACGTCAT CAAGCCCTTC

      661 ATGCGCTTCA AGGTGCACAT GGAGGGCTCC GTGAACGGCC ACGAGTTCGA GATCGAGGGC

      721 GAGGGCGAGG GCAAGCCCTA CGAGGGCACC CAGACCGCCA AGCTGCAGGT GACCAAGGGC

      781 GGCCCCCTGC CCTTCGCCTG GGACATCCTG TCCCCCCAGT TCCAGTACGG CTCCAAGGTG

      841 TACGTGAAGC ACCCCGCCGA CATCCCCGAC TACAAGAAGC TGTCCTTCCC CGAGGGCTTC

      901 AAGTGGGAGC GCGTGATGAA CTTCGAGGAC GGCGGCGTGG TGACCGTGAC CCAGGACTCC

      961 TCCCTGCAGG ACGGCACCTT CATCTACCAC GTGAAGTTCA TCGGCGTGAA CTTCCCCTCC

     1021 GACGGCCCCG TAATGCAGAA GAAGACTCTG GGCTGGGAGC CCTCCACCGA GCGCCTGTAC

     1081 CCCCGCGACG GCGTGCTGAA GGGCGAGATC CACAAGGCGC TGAAGCTGAA GGGCGGCGGC

     1141 CACTACCTGG TGGAGTTCAA GTCAATCTAC ATGGCCAAGA AGCCCGTGAA GCTGCCCGGC

     1201 TACTACTACG TGGACTCCAA GCTGGACATC ACCTCCCACA ACGAGGACTA CACCGTGGTG

     1261 GAGCAGTACG AGCGCGCCGA GGCCCGCCAC CACCTGTTCC AGTAGCGGCC GCGACTCTAG

     1321 ATCATAATCA GCCATACCAC ATTTGTAGAG GTTTTACTTG CTTTAAAAAA CCTCCCACAC

     1381 CTCCCCCTGA ACCTGAAACA TAAAATGAAT GCAATTGTTG TTGTTAACTT GTTTATTGCA

     1441 GCTTATAATG GTTACAAATA AAGCAATAGC ATCACAAATT TCACAAATAA AGCATTTTTT

     1501 TCACTGCATT CTAGTTGTGG TTTGTCCAAA CTCATCAATG TATCTTAAGG CGTAAATTGT

     1561 AAGCGTTAAT ATTTTGTTAA AATTCGCGTT AAATTTTTGT TAAATCAGCT CATTTTTTAA

     1621 CCAATAGGCC GAAATCGGCA AAATCCCTTA TAAATCAAAA GAATAGACCG AGATAGGGTT

     1681 GAGTGTTGTT CCAGTTTGGA ACAAGAGTCC ACTATTAAAG AACGTGGACT CCAACGTCAA

     1741 AGGGCGAAAA ACCGTCTATC AGGGCGATGG CCCACTACGT GAACCATCAC CCTAATCAAG

     1801 TTTTTTGGGG TCGAGGTGCC GTAAAGCACT AAATCGGAAC CCTAAAGGGA GCCCCCGATT

     1861 TAGAGCTTGA CGGGGAAAGC CGGCGAACGT GGCGAGAAAG GAAGGGAAGA AAGCGAAAGG

     1921 AGCGGGCGCT AGGGCGCTGG CAAGTGTAGC GGTCACGCTG CGCGTAACCA CCACACCCGC

     1981 CGCGCTTAAT GCGCCGCTAC AGGGCGCGTC AGGTGGCACT TTTCGGGGAA ATGTGCGCGG

     2041 AACCCCTATT TGTTTATTTT TCTAAATACA TTCAAATATG TATCCGCTCA TGAGACAATA

     2101 ACCCTGATAA ATGCTTCAAT AATATTGAAA AAGGAAGAGT CCTGAGGCGG AAAGAACCAG

     2161 CTGTGGAATG TGTGTCAGTT AGGGTGTGGA AAGTCCCCAG GCTCCCCAGC AGGCAGAAGT

     2221 ATGCAAAGCA TGCATCTCAA TTAGTCAGCA ACCAGGTGTG GAAAGTCCCC AGGCTCCCCA

     2281 GCAGGCAGAA GTATGCAAAG CATGCATCTC AATTAGTCAG CAACCATAGT CCCGCCCCTA

     2341 ACTCCGCCCA TCCCGCCCCT AACTCCGCCC AGTTCCGCCC ATTCTCCGCC CCATGGCTGA

     2401 CTAATTTTTT TTATTTATGC AGAGGCCGAG GCCGCCTCGG CCTCTGAGCT ATTCCAGAAG

     2461 TAGTGAGGAG GCTTTTTTGG AGGCCTAGGC TTTTGCAAAG ATCGATCAAG AGACAGGATG

     2521 AGGATCGTTT CGCATGATTG AACAAGATGG ATTGCACGCA GGTTCTCCGG CCGCTTGGGT

     2581 GGAGAGGCTA TTCGGCTATG ACTGGGCACA ACAGACAATC GGCTGCTCTG ATGCCGCCGT

     2641 GTTCCGGCTG TCAGCGCAGG GGCGCCCGGT TCTTTTTGTC AAGACCGACC TGTCCGGTGC

     2701 CCTGAATGAA CTGCAAGACG AGGCAGCGCG GCTATCGTGG CTGGCCACGA CGGGCGTTCC

     2761 TTGCGCAGCT GTGCTCGACG TTGTCACTGA AGCGGGAAGG GACTGGCTGC TATTGGGCGA

     2821 AGTGCCGGGG CAGGATCTCC TGTCATCTCA CCTTGCTCCT GCCGAGAAAG TATCCATCAT

     2881 GGCTGATGCA ATGCGGCGGC TGCATACGCT TGATCCGGCT ACCTGCCCAT TCGACCACCA

     2941 AGCGAAACAT CGCATCGAGC GAGCACGTAC TCGGATGGAA GCCGGTCTTG TCGATCAGGA

     3001 TGATCTGGAC GAAGAGCATC AGGGGCTCGC GCCAGCCGAA CTGTTCGCCA GGCTCAAGGC

     3061 GAGCATGCCC GACGGCGAGG ATCTCGTCGT GACCCATGGC GATGCCTGCT TGCCGAATAT

     3121 CATGGTGGAA AATGGCCGCT TTTCTGGATT CATCGACTGT GGCCGGCTGG GTGTGGCGGA

     3181 CCGCTATCAG GACATAGCGT TGGCTACCCG TGATATTGCT GAAGAGCTTG GCGGCGAATG

     3241 GGCTGACCGC TTCCTCGTGC TTTACGGTAT CGCCGCTCCC GATTCGCAGC GCATCGCCTT

     3301 CTATCGCCTT CTTGACGAGT TCTTCTGAGC GGGACTCTGG GGTTCGAAAT GACCGACCAA

     3361 GCGACGCCCA ACCTGCCATC ACGAGATTTC GATTCCACCG CCGCCTTCTA TGAAAGGTTG

     3421 GGCTTCGGAA TCGTTTTCCG GGACGCCGGC TGGATGATCC TCCAGCGCGG GGATCTCATG

     3481 CTGGAGTTCT TCGCCCACCC TAGGGGGAGG CTAACTGAAA CACGGAAGGA GACAATACCG

     3541 GAAGGAACCC GCGCTATGAC GGCAATAAAA AGACAGAATA AAACGCACGG TGTTGGGTCG

     3601 TTTGTTCATA AACGCGGGGT TCGGTCCCAG GGCTGGCACT CTGTCGATAC CCCACCGAGA

     3661 CCCCATTGGG GCCAATACGC CCGCGTTTCT TCCTTTTCCC CACCCCACCC CCCAAGTTCG

     3721 GGTGAAGGCC CAGGGCTCGC AGCCAACGTC GGGGCGGCAG GCCCTGCCAT AGCCTCAGGT

     3781 TACTCATATA TACTTTAGAT TGATTTAAAA CTTCATTTTT AATTTAAAAG GATCTAGGTG

     3841 AAGATCCTTT TTGATAATCT CATGACCAAA ATCCCTTAAC GTGAGTTTTC GTTCCACTGA

     3901 GCGTCAGACC CCGTAGAAAA GATCAAAGGA TCTTCTTGAG ATCCTTTTTT TCTGCGCGTA

     3961 ATCTGCTGCT TGCAAACAAA AAAACCACCG CTACCAGCGG TGGTTTGTTT GCCGGATCAA

     4021 GAGCTACCAA CTCTTTTTCC GAAGGTAACT GGCTTCAGCA GAGCGCAGAT ACCAAATACT

     4081 GTTCTTCTAG TGTAGCCGTA GTTAGGCCAC CACTTCAAGA ACTCTGTAGC ACCGCCTACA

     4141 TACCTCGCTC TGCTAATCCT GTTACCAGTG GCTGCTGCCA GTGGCGATAA GTCGTGTCTT

     4201 ACCGGGTTGG ACTCAAGACG ATAGTTACCG GATAAGGCGC AGCGGTCGGG CTGAACGGGG

     4261 GGTTCGTGCA CACAGCCCAG CTTGGAGCGA ACGACCTACA CCGAACTGAG ATACCTACAG

     4321 CGTGAGCTAT GAGAAAGCGC CACGCTTCCC GAAGGGAGAA AGGCGGACAG GTATCCGGTA

     4381 AGCGGCAGGG TCGGAACAGG AGAGCGCACG AGGGAGCTTC CAGGGGGAAA CGCCTGGTAT

     4441 CTTTATAGTC CTGTCGGGTT TCGCCACCTC TGACTTGAGC GTCGATTTTT GTGATGCTCG

     4501 TCAGGGGGGC GGAGCCTATG GAAAAACGCC AGCAACGCGG CCTTTTTACG GTTCCTGGCC

     4561 TTTTGCTGGC CTTTTGCTCA CATGTTCTTT CCTGCGTTAT CCCCTGATTC TGTGGATAAC

     4621 CGTATTACCG CCATGCAT

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