pDsRed-Express2-C1

pDsRed-Express2-C1

pDsRed-Express2-C1载体基本信息

pDsRed-Express2-C1载体质粒图谱和多克隆位点信息

pDsRed-Express2-C1载体描述

pDsRed-Express2-C1 is a mammalian expression vector designed to express a protein of interest fused to the C-terminus of 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. (Please note: Because DsRed-Express2 likely forms tetramers, its suitability as a fusion partner will largely depend on how its tetramerization affects the function of the protein to which it is fused.) 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 multiple cloning site (MCS) in pDsRed-Express2-C1 is positioned downstream of the DsRed-Express2 coding sequence. A Kozak consensus sequence (4), located immediately upstream of DsRed-Express2, enhances translational efficiency in eukaryotic cells. SV40 polyA signals downstream of the MCS direct proper processing of the 3' end of mRNA transcripts.

The vector backbone also 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. This vector also has a neomycin-resistance cassette (Neor) that allows G418 selection of stably transfected eukaryotic cells (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 this cassette allows kanamycin resistance in E. coli.

To construct a fusion protein, the gene of interest must be cloned into pDsRed-Express2-C1 so that it is in-frame with the DsRed-Express2 coding sequence; it should also contain a proper stop codon at the 3' end of its coding region. pDsRed-Express2-C1 can also be used as a cotransfection marker, as the unmodified vector will express DsRed-Express2 in mammalian cells.

pDsRed-Express2-C1 can be transfected into mammalian cells using any standard transfection method. Fusions that retain the fluorescence properties of the native DsRed-Express2 protein (excitation and emission maxima: 541 and 591, respectively) can be monitored by flow cytometry and localized by fluorescence microscopy. If required, stable transfectants can be selected using G418.

For Western analysis, DsRed-Express2 can be detected with either the Living Colors DsRed Polyclonal Anti-body (Cat. No. 632496) or the Living Colors DsRed Monoclonal Antibody (Cat. Nos. 632392 and 632393)

pDsRed-Express2-C1载体含有以下元件：

 PCMV IE (human cytomegalovirus immediate early promoter): 1–589

 DsRed-Express2 (Discosoma sp. red fluorescent protein variant)

Kozak consensus translation initiation site: 606–616

Start codon (ATG): 613–615; Last amino acid: 1285-1287

 MCS (multiple cloning site): 1288–1344

 SV40 early polyA+ signals: 1499–1504 & 1528–1533; mRNA 3' ends: 1537 & 1549

 f1 origin of replication: 1596–2051 (complementary)

 SV40 origin of replication: 2392–2530

 Kanr/Neor (kanamycin/neomycin resistance gene)

Neomycin phosphotransferase coding sequences:

Start codon (ATG): 2576–2578; stop codon: 3368–3370

 pUC origin of replication: 3955–4598

Propagation in E. coli

 Recommended host strain: DH5α, HB101, and other general purpose strains.

 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

pDsRed-Express2-C1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 CCGGTCGCCA CCATGGATAG CACTGAGAAC GTCATCAAGC CCTTCATGCG CTTCAAGGTG

  661 CACATGGAGG GCTCCGTGAA CGGCCACGAG TTCGAGATCG AGGGCGAGGG CGAGGGCAAG

  721 CCCTACGAGG GCACCCAGAC CGCCAAGCTG CAGGTGACCA AGGGCGGCCC CCTGCCCTTC

  781 GCCTGGGACA TCCTGTCCCC CCAGTTCCAG TACGGCTCCA AGGTGTACGT GAAGCACCCC

  841 GCCGACATCC CCGACTACAA GAAGCTGTCC TTCCCCGAGG GCTTCAAGTG GGAGCGCGTG

  901 ATGAACTTCG AGGACGGCGG CGTGGTGACC GTGACCCAGG ACTCCTCCCT GCAGGACGGC

  961 ACCTTCATCT ACCACGTGAA GTTCATCGGC GTGAACTTCC CCTCCGACGG CCCCGTAATG

 1021 CAGAAGAAGA CTCTGGGCTG GGAGCCCTCC ACCGAGCGCC TGTACCCCCG CGACGGCGTG

 1081 CTGAAGGGCG AGATCCACAA GGCGCTGAAG CTGAAGGGCG GCGGCCACTA CCTGGTGGAG

 1141 TTCAAGTCAA TCTACATGGC CAAGAAGCCC GTGAAGCTGC CCGGCTACTA CTACGTGGAC

 1201 TCCAAGCTGG ACATCACCTC CCACAACGAG GACTACACCG TGGTGGAGCA GTACGAGCGC

 1261 GCCGAGGCCC GCCACCACCT GTTCCAGAGA TCTCGAGCTC AAGCTTCGAA TTCTGCAGTC

 1321 GACGGTACCG CGGGCCCGGG ATCCACCGGA TCTAGATAAC TGATCATAAT CAGCCATACC

 1381 ACATTTGTAG AGGTTTTACT TGCTTTAAAA AACCTCCCAC ACCTCCCCCT GAACCTGAAA

 1441 CATAAAATGA ATGCAATTGT TGTTGTTAAC TTGTTTATTG CAGCTTATAA TGGTTACAAA

 1501 TAAAGCAATA GCATCACAAA TTTCACAAAT AAAGCATTTT TTTCACTGCA TTCTAGTTGT

 1561 GGTTTGTCCA AACTCATCAA TGTATCTTAA CGCGTAAATT GTAAGCGTTA ATATTTTGTT

 1621 AAAATTCGCG TTAAATTTTT GTTAAATCAG CTCATTTTTT AACCAATAGG CCGAAATCGG

 1681 CAAAATCCCT TATAAATCAA AAGAATAGAC CGAGATAGGG TTGAGTGTTG TTCCAGTTTG

 1741 GAACAAGAGT CCACTATTAA AGAACGTGGA CTCCAACGTC AAAGGGCGAA AAACCGTCTA

 1801 TCAGGGCGAT GGCCCACTAC GTGAACCATC ACCCTAATCA AGTTTTTTGG GGTCGAGGTG

 1861 CCGTAAAGCA CTAAATCGGA ACCCTAAAGG GAGCCCCCGA TTTAGAGCTT GACGGGGAAA

 1921 GCCGGCGAAC GTGGCGAGAA AGGAAGGGAA GAAAGCGAAA GGAGCGGGCG CTAGGGCGCT

 1981 GGCAAGTGTA GCGGTCACGC TGCGCGTAAC CACCACACCC GCCGCGCTTA ATGCGCCGCT

 2041 ACAGGGCGCG TCAGGTGGCA CTTTTCGGGG AAATGTGCGC GGAACCCCTA TTTGTTTATT

 2101 TTTCTAAATA CATTCAAATA TGTATCCGCT CATGAGACAA TAACCCTGAT AAATGCTTCA

 2161 ATAATATTGA AAAAGGAAGA GTCCTGAGGC GGAAAGAACC AGCTGTGGAA TGTGTGTCAG

 2221 TTAGGGTGTG GAAAGTCCCC AGGCTCCCCA GCAGGCAGAA GTATGCAAAG CATGCATCTC

 2281 AATTAGTCAG CAACCAGGTG TGGAAAGTCC CCAGGCTCCC CAGCAGGCAG AAGTATGCAA

 2341 AGCATGCATC TCAATTAGTC AGCAACCATA GTCCCGCCCC TAACTCCGCC CATCCCGCCC

 2401 CTAACTCCGC CCAGTTCCGC CCATTCTCCG CCCCATGGCT GACTAATTTT TTTTATTTAT

 2461 GCAGAGGCCG AGGCCGCCTC GGCCTCTGAG CTATTCCAGA AGTAGTGAGG AGGCTTTTTT

 2521 GGAGGCCTAG GCTTTTGCAA AGATCGATCA AGAGACAGGA TGAGGATCGT TTCGCATGAT

 2581 TGAACAAGAT GGATTGCACG CAGGTTCTCC GGCCGCTTGG GTGGAGAGGC TATTCGGCTA

 2641 TGACTGGGCA CAACAGACAA TCGGCTGCTC TGATGCCGCC GTGTTCCGGC TGTCAGCGCA

 2701 GGGGCGCCCG GTTCTTTTTG TCAAGACCGA CCTGTCCGGT GCCCTGAATG AACTGCAAGA

 2761 CGAGGCAGCG CGGCTATCGT GGCTGGCCAC GACGGGCGTT CCTTGCGCAG CTGTGCTCGA

 2821 CGTTGTCACT GAAGCGGGAA GGGACTGGCT GCTATTGGGC GAAGTGCCGG GGCAGGATCT

 2881 CCTGTCATCT CACCTTGCTC CTGCCGAGAA AGTATCCATC ATGGCTGATG CAATGCGGCG

 2941 GCTGCATACG CTTGATCCGG CTACCTGCCC ATTCGACCAC CAAGCGAAAC ATCGCATCGA

 3001 GCGAGCACGT ACTCGGATGG AAGCCGGTCT TGTCGATCAG GATGATCTGG ACGAAGAGCA

 3061 TCAGGGGCTC GCGCCAGCCG AACTGTTCGC CAGGCTCAAG GCGAGCATGC CCGACGGCGA

 3121 GGATCTCGTC GTGACCCATG GCGATGCCTG CTTGCCGAAT ATCATGGTGG AAAATGGCCG

 3181 CTTTTCTGGA TTCATCGACT GTGGCCGGCT GGGTGTGGCG GACCGCTATC AGGACATAGC

 3241 GTTGGCTACC CGTGATATTG CTGAAGAGCT TGGCGGCGAA TGGGCTGACC GCTTCCTCGT

 3301 GCTTTACGGT ATCGCCGCTC CCGATTCGCA GCGCATCGCC TTCTATCGCC TTCTTGACGA

 3361 GTTCTTCTGA GCGGGACTCT GGGGTTCGAA ATGACCGACC AAGCGACGCC CAACCTGCCA

 3421 TCACGAGATT TCGATTCCAC CGCCGCCTTC TATGAAAGGT TGGGCTTCGG AATCGTTTTC

 3481 CGGGACGCCG GCTGGATGAT CCTCCAGCGC GGGGATCTCA TGCTGGAGTT CTTCGCCCAC

 3541 CCTAGGGGGA GGCTAACTGA AACACGGAAG GAGACAATAC CGGAAGGAAC CCGCGCTATG

 3601 ACGGCAATAA AAAGACAGAA TAAAACGCAC GGTGTTGGGT CGTTTGTTCA TAAACGCGGG

 3661 GTTCGGTCCC AGGGCTGGCA CTCTGTCGAT ACCCCACCGA GACCCCATTG GGGCCAATAC

 3721 GCCCGCGTTT CTTCCTTTTC CCCACCCCAC CCCCCAAGTT CGGGTGAAGG CCCAGGGCTC

 3781 GCAGCCAACG TCGGGGCGGC AGGCCCTGCC ATAGCCTCAG GTTACTCATA TATACTTTAG

 3841 ATTGATTTAA AACTTCATTT TTAATTTAAA AGGATCTAGG TGAAGATCCT TTTTGATAAT

 3901 CTCATGACCA AAATCCCTTA ACGTGAGTTT TCGTTCCACT GAGCGTCAGA CCCCGTAGAA

 3961 AAGATCAAAG GATCTTCTTG AGATCCTTTT TTTCTGCGCG TAATCTGCTG CTTGCAAACA

 4021 AAAAAACCAC CGCTACCAGC GGTGGTTTGT TTGCCGGATC AAGAGCTACC AACTCTTTTT

 4081 CCGAAGGTAA CTGGCTTCAG CAGAGCGCAG ATACCAAATA CTGTTCTTCT AGTGTAGCCG

 4141 TAGTTAGGCC ACCACTTCAA GAACTCTGTA GCACCGCCTA CATACCTCGC TCTGCTAATC

 4201 CTGTTACCAG TGGCTGCTGC CAGTGGCGAT AAGTCGTGTC TTACCGGGTT GGACTCAAGA

 4261 CGATAGTTAC CGGATAAGGC GCAGCGGTCG GGCTGAACGG GGGGTTCGTG CACACAGCCC

 4321 AGCTTGGAGC GAACGACCTA CACCGAACTG AGATACCTAC AGCGTGAGCT ATGAGAAAGC

 4381 GCCACGCTTC CCGAAGGGAG AAAGGCGGAC AGGTATCCGG TAAGCGGCAG GGTCGGAACA

 4441 GGAGAGCGCA CGAGGGAGCT TCCAGGGGGA AACGCCTGGT ATCTTTATAG TCCTGTCGGG

 4501 TTTCGCCACC TCTGACTTGA GCGTCGATTT TTGTGATGCT CGTCAGGGGG GCGGAGCCTA

 4561 TGGAAAAACG CCAGCAACGC GGCCTTTTTA CGGTTCCTGG CCTTTTGCTG GCCTTTTGCT

 4621 CACATGTTCT TTCCTGCGTT ATCCCCTGAT TCTGTGGATA ACCGTATTAC CGCCATGCAT

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