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 gene of 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.

載體應(yīng)用

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.

0001 TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG 
0061 CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT 
0121 GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC ATTGACGTCA 
0181 ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT ATCATATGCC 
0241 AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA 
0301 CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC 
0361 CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG ACTCACGGGG 
0421 ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC AAAATCAACG 
0481 GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT 
0541 ACGGTGGGAG GTCTATATAA GCAGAGCTGG TTTAGTGAAC CGTCAGATCC GCTAGCGCTA 
0601 CCGGACTCAG ATCTCGAGCT CAAGCTTCGA ATTCTGCAGT CGACGGTACC GCGGGCCCGG 
0661 GATCCACCGG TCGCCACCAT GGCCTCCTCC GAGGACGTCA TCAAGGAGTT CATGCGCTTC 
0721 AAGGTGCGCA TGGAGGGCTC CGTGAACGGC CACGAGTTCG AGATCGAGGG CGAGGGCGAG 
0781 GGCCGCCCCT ACGAGGGCAC CCAGACCGCC AAGCTGAAGG TGACCAAGGG CGGCCCCCTG 
0841 CCCTTCGCCT GGGACATCCT GTCCCCCCAG TTCCAGTACG GCTCCAAGGT GTACGTGAAG 
0901 CACCCCGCCG ACATCCCCGA CTACAAGAAG CTGTCCTTCC CCGAGGGCTT CAAGTGGGAG 
0961 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