pDsRed-Express2-1

pDsRed-Express2-1

pDsRed-Express2-1载体基本信息：

pDsRed-Express2-1载体质粒图谱和多克隆位点信息

pDsRed-Express2-1载体描述：

pDsRed-Express2-1 is a promoterless reporter vector that allows the functional analysis of promoters and promoter/enhancer combinations cloned into its multiple cloning site (MCS). The vector encodes 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. 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 sequence immediately upstream of DsRed-Express2 has been converted to a Kozak consensus sequence (4) to enhance translational efficiency in eukaryotic cells. SV40 polyadenylation signals down-stream 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.

pDsRed-Express2-1 is designed to be used as an in vivo reporter of gene expression. Promoters should be cloned into the pDsRed-Express2-1 MCS, located upstream of the DsRed-Express2 coding sequence. Without the addition of a functional promoter, this vector will not express DsRed-Express2.

The pDsRed-Express2-1 vector can be transfected into mammalian cells using any standard transfection method. Mammalian cells expressing DsRed-Express2 (excitation and emission maxima: 554 and 591 nm, respectively) can be detected by either fluorescence microscopy or flow cytometry 8–12 hours after transfection. If required, stable transfectants can be selected using G418.

pDsRed-Express2-1载体含有以下元件：

 MCS (multiple cloning site): 12–89

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

Kozak consensus translation initiation site: 90–100

Start codon (ATG): 97–99; Stop codon: 772–774

 SV40 early polyA+ signals

Polyadenlyation signals: 926–931 & 955–960; mRNA 3' ends: 964 & 976

 f1 origin of replication (for packaging the noncoding strand of DsRed-Express2): 1023–1478

 SV40 origin of replication: 1819–1957

 Kanr/Neor (kanamycin/neomycin resistance gene)Neomycin phosphotransferase coding sequences: 2003–2797

 pUC origin of replication: 3382–4015

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

pDsRed-Express2-1载体序列：

ORIGIN

    1 TAGTTATTAC TAGCGCTACC GGACTCAGAT CTCGAGCTCA AGCTTCGAAT TCTGCAGTCG

   61 ACGGTACCGC GGGCCCGGGA TCCACCGGTC GCCACCATGG ATAGCACTGA GAACGTCATC

  121 AAGCCCTTCA TGCGCTTCAA GGTGCACATG GAGGGCTCCG TGAACGGCCA CGAGTTCGAG

  181 ATCGAGGGCG AGGGCGAGGG CAAGCCCTAC GAGGGCACCC AGACCGCCAA GCTGCAGGTG

  241 ACCAAGGGCG GCCCCCTGCC CTTCGCCTGG GACATCCTGT CCCCCCAGTT CCAGTACGGC

  301 TCCAAGGTGT ACGTGAAGCA CCCCGCCGAC ATCCCCGACT ACAAGAAGCT GTCCTTCCCC

  361 GAGGGCTTCA AGTGGGAGCG CGTGATGAAC TTCGAGGACG GCGGCGTGGT GACCGTGACC

  421 CAGGACTCCT CCCTGCAGGA CGGCACCTTC ATCTACCACG TGAAGTTCAT CGGCGTGAAC

  481 TTCCCCTCCG ACGGCCCCGT AATGCAGAAG AAGACTCTGG GCTGGGAGCC CTCCACCGAG

  541 CGCCTGTACC CCCGCGACGG CGTGCTGAAG GGCGAGATCC ACAAGGCGCT GAAGCTGAAG

  601 GGCGGCGGCC ACTACCTGGT GGAGTTCAAG TCAATCTACA TGGCCAAGAA GCCCGTGAAG

  661 CTGCCCGGCT ACTACTACGT GGACTCCAAG CTGGACATCA CCTCCCACAA CGAGGACTAC

  721 ACCGTGGTGG AGCAGTACGA GCGCGCCGAG GCCCGCCACC ACCTGTTCCA GTAGCGGCCG

  781 CGACTCTAGA TCATAATCAG CCATACCACA TTTGTAGAGG TTTTACTTGC TTTAAAAAAC

  841 CTCCCACACC TCCCCCTGAA CCTGAAACAT AAAATGAATG CAATTGTTGT TGTTAACTTG

  901 TTTATTGCAG CTTATAATGG TTACAAATAA AGCAATAGCA TCACAAATTT CACAAATAAA

  961 GCATTTTTTT CACTGCATTC TAGTTGTGGT TTGTCCAAAC TCATCAATGT ATCTTAAGGC

 1021 GTAAATTGTA AGCGTTAATA TTTTGTTAAA ATTCGCGTTA AATTTTTGTT AAATCAGCTC

 1081 ATTTTTTAAC CAATAGGCCG AAATCGGCAA AATCCCTTAT AAATCAAAAG AATAGACCGA

 1141 GATAGGGTTG AGTGTTGTTC CAGTTTGGAA CAAGAGTCCA CTATTAAAGA ACGTGGACTC

 1201 CAACGTCAAA GGGCGAAAAA CCGTCTATCA GGGCGATGGC CCACTACGTG AACCATCACC

 1261 CTAATCAAGT TTTTTGGGGT CGAGGTGCCG TAAAGCACTA AATCGGAACC CTAAAGGGAG

 1321 CCCCCGATTT AGAGCTTGAC GGGGAAAGCC GGCGAACGTG GCGAGAAAGG AAGGGAAGAA

 1381 AGCGAAAGGA GCGGGCGCTA GGGCGCTGGC AAGTGTAGCG GTCACGCTGC GCGTAACCAC

 1441 CACACCCGCC GCGCTTAATG CGCCGCTACA GGGCGCGTCA GGTGGCACTT TTCGGGGAAA

 1501 TGTGCGCGGA ACCCCTATTT GTTTATTTTT CTAAATACAT TCAAATATGT ATCCGCTCAT

 1561 GAGACAATAA CCCTGATAAA TGCTTCAATA ATATTGAAAA AGGAAGAGTC CTGAGGCGGA

 1621 AAGAACCAGC TGTGGAATGT GTGTCAGTTA GGGTGTGGAA AGTCCCCAGG CTCCCCAGCA

 1681 GGCAGAAGTA TGCAAAGCAT GCATCTCAAT TAGTCAGCAA CCAGGTGTGG AAAGTCCCCA

 1741 GGCTCCCCAG CAGGCAGAAG TATGCAAAGC ATGCATCTCA ATTAGTCAGC AACCATAGTC

 1801 CCGCCCCTAA CTCCGCCCAT CCCGCCCCTA ACTCCGCCCA GTTCCGCCCA TTCTCCGCCC

 1861 CATGGCTGAC TAATTTTTTT TATTTATGCA GAGGCCGAGG CCGCCTCGGC CTCTGAGCTA

 1921 TTCCAGAAGT AGTGAGGAGG CTTTTTTGGA GGCCTAGGCT TTTGCAAAGA TCGATCAAGA

 1981 GACAGGATGA GGATCGTTTC GCATGATTGA ACAAGATGGA TTGCACGCAG GTTCTCCGGC

 2041 CGCTTGGGTG GAGAGGCTAT TCGGCTATGA CTGGGCACAA CAGACAATCG GCTGCTCTGA

 2101 TGCCGCCGTG TTCCGGCTGT CAGCGCAGGG GCGCCCGGTT CTTTTTGTCA AGACCGACCT

 2161 GTCCGGTGCC CTGAATGAAC TGCAAGACGA GGCAGCGCGG CTATCGTGGC TGGCCACGAC

 2221 GGGCGTTCCT TGCGCAGCTG TGCTCGACGT TGTCACTGAA GCGGGAAGGG ACTGGCTGCT

 2281 ATTGGGCGAA GTGCCGGGGC AGGATCTCCT GTCATCTCAC CTTGCTCCTG CCGAGAAAGT

 2341 ATCCATCATG GCTGATGCAA TGCGGCGGCT GCATACGCTT GATCCGGCTA CCTGCCCATT

 2401 CGACCACCAA GCGAAACATC GCATCGAGCG AGCACGTACT CGGATGGAAG CCGGTCTTGT

 2461 CGATCAGGAT GATCTGGACG AAGAGCATCA GGGGCTCGCG CCAGCCGAAC TGTTCGCCAG

 2521 GCTCAAGGCG AGCATGCCCG ACGGCGAGGA TCTCGTCGTG ACCCATGGCG ATGCCTGCTT

 2581 GCCGAATATC ATGGTGGAAA ATGGCCGCTT TTCTGGATTC ATCGACTGTG GCCGGCTGGG

 2641 TGTGGCGGAC CGCTATCAGG ACATAGCGTT GGCTACCCGT GATATTGCTG AAGAGCTTGG

 2701 CGGCGAATGG GCTGACCGCT TCCTCGTGCT TTACGGTATC GCCGCTCCCG ATTCGCAGCG

 2761 CATCGCCTTC TATCGCCTTC TTGACGAGTT CTTCTGAGCG GGACTCTGGG GTTCGAAATG

 2821 ACCGACCAAG CGACGCCCAA CCTGCCATCA CGAGATTTCG ATTCCACCGC CGCCTTCTAT

 2881 GAAAGGTTGG GCTTCGGAAT CGTTTTCCGG GACGCCGGCT GGATGATCCT CCAGCGCGGG

 2941 GATCTCATGC TGGAGTTCTT CGCCCACCCT AGGGGGAGGC TAACTGAAAC ACGGAAGGAG

 3001 ACAATACCGG AAGGAACCCG CGCTATGACG GCAATAAAAA GACAGAATAA AACGCACGGT

 3061 GTTGGGTCGT TTGTTCATAA ACGCGGGGTT CGGTCCCAGG GCTGGCACTC TGTCGATACC

 3121 CCACCGAGAC CCCATTGGGG CCAATACGCC CGCGTTTCTT CCTTTTCCCC ACCCCACCCC

 3181 CCAAGTTCGG GTGAAGGCCC AGGGCTCGCA GCCAACGTCG GGGCGGCAGG CCCTGCCATA

 3241 GCCTCAGGTT ACTCATATAT ACTTTAGATT GATTTAAAAC TTCATTTTTA ATTTAAAAGG

 3301 ATCTAGGTGA AGATCCTTTT TGATAATCTC ATGACCAAAA TCCCTTAACG TGAGTTTTCG

 3361 TTCCACTGAG CGTCAGACCC CGTAGAAAAG ATCAAAGGAT CTTCTTGAGA TCCTTTTTTT

 3421 CTGCGCGTAA TCTGCTGCTT GCAAACAAAA AAACCACCGC TACCAGCGGT GGTTTGTTTG

 3481 CCGGATCAAG AGCTACCAAC TCTTTTTCCG AAGGTAACTG GCTTCAGCAG AGCGCAGATA

 3541 CCAAATACTG TTCTTCTAGT GTAGCCGTAG TTAGGCCACC ACTTCAAGAA CTCTGTAGCA

 3601 CCGCCTACAT ACCTCGCTCT GCTAATCCTG TTACCAGTGG CTGCTGCCAG TGGCGATAAG

 3661 TCGTGTCTTA CCGGGTTGGA CTCAAGACGA TAGTTACCGG ATAAGGCGCA GCGGTCGGGC

 3721 TGAACGGGGG GTTCGTGCAC ACAGCCCAGC TTGGAGCGAA CGACCTACAC CGAACTGAGA

 3781 TACCTACAGC GTGAGCTATG AGAAAGCGCC ACGCTTCCCG AAGGGAGAAA GGCGGACAGG

 3841 TATCCGGTAA GCGGCAGGGT CGGAACAGGA GAGCGCACGA GGGAGCTTCC AGGGGGAAAC

 3901 GCCTGGTATC TTTATAGTCC TGTCGGGTTT CGCCACCTCT GACTTGAGCG TCGATTTTTG

 3961 TGATGCTCGT CAGGGGGGCG GAGCCTATGG AAAAACGCCA GCAACGCGGC CTTTTTACGG

 4021 TTCCTGGCCT TTTGCTGGCC TTTTGCTCAC ATGTTCTTTC CTGCGTTATC CCCTGATTCT

 4081 GTGGATAACC GTATTACCGC CATGCAT

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