pDsRed-Express2-N1

pDsRed-Express2-N1载体基本信息

pDsRed-Express2-N1载体质粒图谱和多克隆位点信息

pDsRed-Express2-N1载体描述

pDsRed-Express2-N1 is a mammalian expression vector designed to express a protein of interest fused to the N-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-N1 is positioned upstream of the DsRed-Express2 coding sequence. A Kozak consensus sequence (4), located between the MCS and the DsRed-Express2 coding sequence, enhances the translational efficiency of the unfused DsRed-Express2 protein in eukaryotic cells. SV40 polyadenylation signals downstream of the DsRed-Express2 coding sequence direct proper processing of the 3' ends of the DsRed-Express2 and fusion gene 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-N1 so that it is in-frame with the DsRed-Express2 coding sequence; the gene must include an initiation codon (ATG), and lack in-frame stop codons. pDsRed-Express2-N1 can also be used as a cotransfection marker, as the unmodified vector will express DsRed-Express2 in mammalian cells.

pDsRed-Express2-N1 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-N1载体含有以下元件：

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

 MCS (multiple cloning site): 591–671

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

Kozak consensus translation initiation site: 672–682

Start codon (ATG): 679–681; Stop codon: 1354-1356

 SV40 early polyA+ signals: 1508–1513 & 1537–1542; mRNA 3' ends: 1546 & 1558

 f1 origin of replication: 1605–2060 (complementary)

 SV40 origin of replication: 2401–2539

 Kanr/Neor (kanamycin/neomycin resistance gene)

Neomycin phosphotransferase coding sequences:

Start codon (ATG): 2585–2587; stop codon: 3377–3379

 pUC origin of replication: 3964–4607

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-N1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 ATCTCGAGCT CAAGCTTCGA ATTCTGCAGT CGACGGTACC GCGGGCCCGG

  661 GATCCACTAG TCGCCACCAT GGATAGCACT GAGAACGTCA TCAAGCCCTT CATGCGCTTC

  721 AAGGTGCACA TGGAGGGCTC CGTGAACGGC CACGAGTTCG AGATCGAGGG CGAGGGCGAG

  781 GGCAAGCCCT ACGAGGGCAC CCAGACCGCC AAGCTGCAGG TGACCAAGGG CGGCCCCCTG

  841 CCCTTCGCCT GGGACATCCT GTCCCCCCAG TTCCAGTACG GCTCCAAGGT GTACGTGAAG

  901 CACCCCGCCG ACATCCCCGA CTACAAGAAG CTGTCCTTCC CCGAGGGCTT CAAGTGGGAG

  961 CGCGTGATGA ACTTCGAGGA CGGCGGCGTG GTGACCGTGA CCCAGGACTC CTCCCTGCAG

 1021 GACGGCACCT TCATCTACCA CGTGAAGTTC ATCGGCGTGA ACTTCCCCTC CGACGGCCCC

 1081 GTAATGCAGA AGAAGACTCT GGGCTGGGAG CCCTCCACCG AGCGCCTGTA CCCCCGCGAC

 1141 GGCGTGCTGA AGGGCGAGAT CCACAAGGCG CTGAAGCTGA AGGGCGGCGG CCACTACCTG

 1201 GTGGAGTTCA AGTCAATCTA CATGGCCAAG AAGCCCGTGA AGCTGCCCGG CTACTACTAC

 1261 GTGGACTCCA AGCTGGACAT CACCTCCCAC AACGAGGACT ACACCGTGGT GGAGCAGTAC

 1321 GAGCGCGCCG AGGCCCGCCA CCACCTGTTC CAGTAGCGGC CGCGACTCTA GATCATAATC

 1381 AGCCATACCA CATTTGTAGA GGTTTTACTT GCTTTAAAAA ACCTCCCACA CCTCCCCCTG

 1441 AACCTGAAAC ATAAAATGAA TGCAATTGTT GTTGTTAACT TGTTTATTGC AGCTTATAAT

 1501 GGTTACAAAT AAAGCAATAG CATCACAAAT TTCACAAATA AAGCATTTTT TTCACTGCAT

 1561 TCTAGTTGTG GTTTGTCCAA ACTCATCAAT GTATCTTAAG GCGTAAATTG TAAGCGTTAA

 1621 TATTTTGTTA AAATTCGCGT TAAATTTTTG TTAAATCAGC TCATTTTTTA ACCAATAGGC

 1681 CGAAATCGGC AAAATCCCTT ATAAATCAAA AGAATAGACC GAGATAGGGT TGAGTGTTGT

 1741 TCCAGTTTGG AACAAGAGTC CACTATTAAA GAACGTGGAC TCCAACGTCA AAGGGCGAAA

 1801 AACCGTCTAT CAGGGCGATG GCCCACTACG TGAACCATCA CCCTAATCAA GTTTTTTGGG

 1861 GTCGAGGTGC CGTAAAGCAC TAAATCGGAA CCCTAAAGGG AGCCCCCGAT TTAGAGCTTG

 1921 ACGGGGAAAG CCGGCGAACG TGGCGAGAAA GGAAGGGAAG AAAGCGAAAG GAGCGGGCGC

 1981 TAGGGCGCTG GCAAGTGTAG CGGTCACGCT GCGCGTAACC ACCACACCCG CCGCGCTTAA

 2041 TGCGCCGCTA CAGGGCGCGT CAGGTGGCAC TTTTCGGGGA AATGTGCGCG GAACCCCTAT

 2101 TTGTTTATTT TTCTAAATAC ATTCAAATAT GTATCCGCTC ATGAGACAAT AACCCTGATA

 2161 AATGCTTCAA TAATATTGAA AAAGGAAGAG TCCTGAGGCG GAAAGAACCA GCTGTGGAAT

 2221 GTGTGTCAGT TAGGGTGTGG AAAGTCCCCA GGCTCCCCAG CAGGCAGAAG TATGCAAAGC

 2281 ATGCATCTCA ATTAGTCAGC AACCAGGTGT GGAAAGTCCC CAGGCTCCCC AGCAGGCAGA

 2341 AGTATGCAAA GCATGCATCT CAATTAGTCA GCAACCATAG TCCCGCCCCT AACTCCGCCC

 2401 ATCCCGCCCC TAACTCCGCC CAGTTCCGCC CATTCTCCGC CCCATGGCTG ACTAATTTTT

 2461 TTTATTTATG CAGAGGCCGA GGCCGCCTCG GCCTCTGAGC TATTCCAGAA GTAGTGAGGA

 2521 GGCTTTTTTG GAGGCCTAGG CTTTTGCAAA GATCGATCAA GAGACAGGAT GAGGATCGTT

 2581 TCGCATGATT GAACAAGATG GATTGCACGC AGGTTCTCCG GCCGCTTGGG TGGAGAGGCT

 2641 ATTCGGCTAT GACTGGGCAC AACAGACAAT CGGCTGCTCT GATGCCGCCG TGTTCCGGCT

 2701 GTCAGCGCAG GGGCGCCCGG TTCTTTTTGT CAAGACCGAC CTGTCCGGTG CCCTGAATGA

 2761 ACTGCAAGAC GAGGCAGCGC GGCTATCGTG GCTGGCCACG ACGGGCGTTC CTTGCGCAGC

 2821 TGTGCTCGAC GTTGTCACTG AAGCGGGAAG GGACTGGCTG CTATTGGGCG AAGTGCCGGG

 2881 GCAGGATCTC CTGTCATCTC ACCTTGCTCC TGCCGAGAAA GTATCCATCA TGGCTGATGC

 2941 AATGCGGCGG CTGCATACGC TTGATCCGGC TACCTGCCCA TTCGACCACC AAGCGAAACA

 3001 TCGCATCGAG CGAGCACGTA CTCGGATGGA AGCCGGTCTT GTCGATCAGG ATGATCTGGA

 3061 CGAAGAGCAT CAGGGGCTCG CGCCAGCCGA ACTGTTCGCC AGGCTCAAGG CGAGCATGCC

 3121 CGACGGCGAG GATCTCGTCG TGACCCATGG CGATGCCTGC TTGCCGAATA TCATGGTGGA

 3181 AAATGGCCGC TTTTCTGGAT TCATCGACTG TGGCCGGCTG GGTGTGGCGG ACCGCTATCA

 3241 GGACATAGCG TTGGCTACCC GTGATATTGC TGAAGAGCTT GGCGGCGAAT GGGCTGACCG

 3301 CTTCCTCGTG CTTTACGGTA TCGCCGCTCC CGATTCGCAG CGCATCGCCT TCTATCGCCT

 3361 TCTTGACGAG TTCTTCTGAG CGGGACTCTG GGGTTCGAAA TGACCGACCA AGCGACGCCC

 3421 AACCTGCCAT CACGAGATTT CGATTCCACC GCCGCCTTCT ATGAAAGGTT GGGCTTCGGA

 3481 ATCGTTTTCC GGGACGCCGG CTGGATGATC CTCCAGCGCG GGGATCTCAT GCTGGAGTTC

 3541 TTCGCCCACC CTAGGGGGAG GCTAACTGAA ACACGGAAGG AGACAATACC GGAAGGAACC

 3601 CGCGCTATGA CGGCAATAAA AAGACAGAAT AAAACGCACG GTGTTGGGTC GTTTGTTCAT

 3661 AAACGCGGGG TTCGGTCCCA GGGCTGGCAC TCTGTCGATA CCCCACCGAG ACCCCATTGG

 3721 GGCCAATACG CCCGCGTTTC TTCCTTTTCC CCACCCCACC CCCCAAGTTC GGGTGAAGGC

 3781 CCAGGGCTCG CAGCCAACGT CGGGGCGGCA GGCCCTGCCA TAGCCTCAGG TTACTCATAT

 3841 ATACTTTAGA TTGATTTAAA ACTTCATTTT TAATTTAAAA GGATCTAGGT GAAGATCCTT

 3901 TTTGATAATC TCATGACCAA AATCCCTTAA CGTGAGTTTT CGTTCCACTG AGCGTCAGAC

 3961 CCCGTAGAAA AGATCAAAGG ATCTTCTTGA GATCCTTTTT TTCTGCGCGT AATCTGCTGC

 4021 TTGCAAACAA AAAAACCACC GCTACCAGCG GTGGTTTGTT TGCCGGATCA AGAGCTACCA

 4081 ACTCTTTTTC CGAAGGTAAC TGGCTTCAGC AGAGCGCAGA TACCAAATAC TGTTCTTCTA

 4141 GTGTAGCCGT AGTTAGGCCA CCACTTCAAG AACTCTGTAG CACCGCCTAC ATACCTCGCT

 4201 CTGCTAATCC TGTTACCAGT GGCTGCTGCC AGTGGCGATA AGTCGTGTCT TACCGGGTTG

 4261 GACTCAAGAC GATAGTTACC GGATAAGGCG CAGCGGTCGG GCTGAACGGG GGGTTCGTGC

 4321 ACACAGCCCA GCTTGGAGCG AACGACCTAC ACCGAACTGA GATACCTACA GCGTGAGCTA

 4381 TGAGAAAGCG CCACGCTTCC CGAAGGGAGA AAGGCGGACA GGTATCCGGT AAGCGGCAGG

 4441 GTCGGAACAG GAGAGCGCAC GAGGGAGCTT CCAGGGGGAA ACGCCTGGTA TCTTTATAGT

 4501 CCTGTCGGGT TTCGCCACCT CTGACTTGAG CGTCGATTTT TGTGATGCTC GTCAGGGGGG

 4561 CGGAGCCTAT GGAAAAACGC CAGCAACGCG GCCTTTTTAC GGTTCCTGGC CTTTTGCTGG

 4621 CCTTTTGCTC ACATGTTCTT TCCTGCGTTA TCCCCTGATT CTGTGGATAA CCGTATTACC

 4681 GCCATGCAT

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