pDsRed-Express-N1

pDsRed-Express-N1

pDsRed-Express-N1载体基本信息

pDsRed-Express-N1载体质粒图谱和多克隆位点信息

pDsRed-Express-N1载体简介

pDsRed-Express-N1 is a mammalian expression vector that encodes DsRed-Express, a variant of Discosoma sp. red fluorescent protein (DsRed; 1). DsRed-Express contains nine amino acid substitutions (listed on page 2), which improve the solubility of the protein and reduce the time from transfection to detection of red fluorescence (2). In addition, these substitutions reduce the level of residual green emission (2). When DsRed-Express is expressed in mammalian cell cultures, redemitting cells can be detected by either fluorescence microscopy or flow cytometry 8–12 hours after transfection (DsRed-Express excitation and emission maxima = 557 nm and 579 nm, respectively). Although DsRed-Express most likely forms the same tetrameric structure as wild-type DsRed, DsRed- Express displays a reduced tendency to aggregate (2). The DsRed-Express coding sequence is human codon-optimized for high expression in mammalian cells (3).

The multiple cloning site (MCS) in pDsRed-Express-N1 is positioned between the immediate early promoter of CMV (PCMV IE) and the DsRed-Express coding sequence. Genes cloned into the MCS are expressed as fusions to the N-terminus of DsRed-Express if they are in the same reading frame as DsRed-Express and there are no intervening stop codons. Sequences upstream of DsRed-Express have been converted to a Kozak consensus translation initiation site to increase translation efficiency in eukaryotic cells (4). SV40 polyadenylation signals downstream of the DsRed-Express gene direct proper processing of the 3' end of the DsRed-Express mRNA. The vector backbone contains an SV40 origin for replication in mammalian cells expressing the SV40 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. This cassette consists of the SV40 early promoter, the neomycin/kanamycin resistance geneof Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene.A bacterial promoter upstream of the cassette confers kanamycin resistance to E. coli.

pDsRed-Express-N1 can be used to construct fusions to the N-terminus of DsRed-Express. If a fusion construct retains the fluorescent properties of the native DsRed-Express protein, its expression can be monitored by flow cytometry and its localization in vivo can be determined by fluorescence microscopy. The target gene should be cloned into pDsRed-Express-N1 so that it is in frame with the DsRed-Express coding sequence, with no intervening in-frame stop codons. The inserted gene should include an initiating ATG codon. Recombinant pDsRed-Express-N1 can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418 (5). pDsRed-Express-N1 can also be used as a cotransfection marker; the unmodified vector will express DsRed-Express.

Propagation in E. coli

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

a host containing an F plasmid, such as the JM109 or XL1-Blue strains..

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

 E. coli replication origin: pUC

 Copy number: ≈500

 Plasmid incompatibility group: pMB1/ColE1

Excitation and emission maxima of DsRed-Express

 Excitation maximum = 557 nm

 Emission maximum = 579 nm

pDsRed-Express-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 GATCCACCGG TCGCCACCAT GGCCTCCTCC GAGGACGTCA TCAAGGAGTT CATGCGCTTC

  721 AAGGTGCGCA TGGAGGGCTC CGTGAACGGC CACGAGTTCG AGATCGAGGG CGAGGGCGAG

  781 GGCCGCCCCT ACGAGGGCAC CCAGACCGCC AAGCTGAAGG TGACCAAGGG CGGCCCCCTG

  841 CCCTTCGCCT GGGACATCCT GTCCCCCCAG TTCCAGTACG GCTCCAAGGT GTACGTGAAG

  901 CACCCCGCCG ACATCCCCGA CTACAAGAAG CTGTCCTTCC CCGAGGGCTT CAAGTGGGAG

  961 CGCGTGATGA ACTTCGAGGA CGGCGGCGTG GTGACCGTGA CCCAGGACTC CTCCCTGCAG

 1021 GACGGCTCCT TCATCTACAA GGTGAAGTTC ATCGGCGTGA ACTTCCCCTC CGACGGCCCC

 1081 GTAATGCAGA AGAAGACTAT GGGCTGGGAG GCCTCCACCG AGCGCCTGTA CCCCCGCGAC

 1141 GGCGTGCTGA AGGGCGAGAT CCACAAGGCC CTGAAGCTGA AGGACGGCGG CCACTACCTG

 1201 GTGGAGTTCA AGTCCATCTA CATGGCCAAG AAGCCCGTGC AGCTGCCCGG CTACTACTAC

 1261 GTGGACTCCA AGCTGGACAT CACCTCCCAC AACGAGGACT ACACCATCGT GGAGCAGTAC

 1321 GAGCGCGCCG AGGGCCGCCA CCACCTGTTC CTGTAGCGGC 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 TGTCCTTCTA

 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

//

其他哺乳动物表达载体：