pDsRed-Monomer-N1

pDsRed-Monomer-N1 is a mammalian expression vector that encodes DsRed-Monomer (DsRed.M1), a monomeric mutant derived from the tetrameric Discosoma sp. red fluorescent protein DsRed (1). DsRed-Monomer contains forty-five amino acid substitutions (listed on page 2). When DsRed-Monomer is expressed in mammalian cell cultures, red fluorescent cells can be detected by either fluorescence microscopy or flow cytometry 12–16 hr after transfection (DsRed-Monomer excitation and emission maxima = 557 nm and 592 nm, respectively). The DsRed-Monomer coding sequence is human codon-optimized for high expression in mammalian cells (2).

DsRed-Monomer is well suited for use as a fusion tag. The multiple cloning site (MCS) in pDsRed-Monomer-N1 is positioned between the immediate early promoter of CMV (PCMV IE) and the DsRed-Monomer coding sequence. Genes cloned into the MCS are expressed as fusions to the N-terminus of DsRed-Monomer if they are in the same reading frame as DsRed- Monomer and there are no intervening stop codons. A Kozak consensus sequence is located immediately upstream of the DsRed-Monomer gene to enhance translational efficiency in eukaryotic systems (3). SV40 polyadenylation signals downstream of the DsRed-Monomer gene direct proper processing of the 3' end of the DsRed-Monomer 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 in E. coli.

载体应用

pDsRed-Monomer-N1 can be used to construct fusions to the N-terminus of DsRed-Monomer. If a fusion construct retains the fluorescent properties of the native DsRed-Monomer protein, its expression can be monitored by flow cytometry and its localization in vivo can be determined by fluorescence microscopy. The target gene must be cloned into pDsRed-Monomer-N1 so that it is in frame with the DsRed-Monomer coding sequence, with no intervening in-frame stop codons. The inserted gene must include an initiating ATG codon. Recombinant pDsRed-Monomer-N1 can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418 (4). pDsRed-Monomer-N1 can also be used as a cotransfection marker; the unmodified vector will express DsRed-Monomer.

The DsRed1-N Sequencing Primer (Cat. No. 632387) can be used to sequence genes cloned adjacent to the 5' end of the DsRed-Monomer coding region.

For Western blotting, the Living Colors? DsRed Polyclonal Antibody (Cat. No. 632496) can be used to recognize the DsRed-Monomer protein. However, to generate optimal results it may be necessary to use a higher concentration of antibody than recommended on the DsRed Polyclonal Antibody Certificate of Analysis.

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 GGACAACACC GAGGACGTCA TCAAGGAGTT CATGCAGTTC 
0721 AAGGTGCGCA TGGAGGGCTC CGTGAACGGC CACTACTTCG AGATCGAGGG CGAGGGCGAG 
0781 GGCAAGCCCT ACGAGGGCAC CCAGACCGCC AAGCTGCAGG TGACCAAGGG CGGCCCCCTG 
0841 CCCTTCGCCT GGGACATCCT GTCCCCCCAG TTCCAGTACG GCTCCAAGGC CTACGTGAAG 
0901 CACCCCGCCG ACATCCCCGA CTACATGAAG CTGTCCTTCC CCGAGGGCTT CACCTGGGAG 
0961 CGCTCCATGA ACTTCGAGGA CGGCGGCGTG GTGGAGGTGC AGCAGGACTC CTCCCTGCAG 
1021 GACGGCACCT TCATCTACAA GGTGAAGTTC AAGGGCGTGA ACTTCCCCGC CGACGGCCCC 
1081 GTAATGCAGA AGAAGACTGC CGGCTGGGAG CCCTCCACCG AGAAGCTGTA CCCCCAGGAC 
1141 GGCGTGCTGA AGGGCGAGAT CTCCCACGCC CTGAAGCTGA AGGACGGCGG CCACTACACC 
1201 TGCGACTTCA AGACCGTGTA CAAGGCCAAG AAGCCCGTGC AGCTGCCCGG CAACCACTAC 
1261 GTGGACTCCA AGCTGGACAT CACCAACCAC AACGAGGACT ACACCGTGGT GGAGCAGTAC 
1321 GAGCACGCCG AGGCCCGCCA CTCCGGCTCC CAGTAGAGCG GCCGCGACTC TAGATCATAA 
1381 TCAGCCATAC CACATTTGTA GAGGTTTTAC TTGCTTTAAA AAACCTCCCA CACCTCCCCC 
1441 TGAACCTGAA ACATAAAATG AATGCAATTG TTGTTGTTAA CTTGTTTATT GCAGCTTATA 
1501 ATGGTTACAA ATAAAGCAAT AGCATCACAA ATTTCACAAA TAAAGCATTT TTTTCACTGC 
1561 ATTCTAGTTG TGGTTTGTCC AAACTCATCA ATGTATCTTA AGGCGTAAAT TGTAAGCGTT 
1621 AATATTTTGT TAAAATTCGC GTTAAATTTT TGTTAAATCA GCTCATTTTT TAACCAATAG 
1681 GCCGAAATCG GCAAAATCCC TTATAAATCA AAAGAATAGA CCGAGATAGG GTTGAGTGTT 
1741 GTTCCAGTTT GGAACAAGAG TCCACTATTA AAGAACGTGG ACTCCAACGT CAAAGGGCGA 
1801 AAAACCGTCT ATCAGGGCGA TGGCCCACTA CGTGAACCAT CACCCTAATC AAGTTTTTTG 
1861 GGGTCGAGGT GCCGTAAAGC ACTAAATCGG AACCCTAAAG GGAGCCCCCG ATTTAGAGCT 
1921 TGACGGGGAA AGCCGGCGAA CGTGGCGAGA AAGGAAGGGA AGAAAGCGAA AGGAGCGGGC 
1981 GCTAGGGCGC TGGCAAGTGT AGCGGTCACG CTGCGCGTAA CCACCACACC CGCCGCGCTT 
2041 AATGCGCCGC TACAGGGCGC GTCAGGTGGC ACTTTTCGGG GAAATGTGCG CGGAACCCCT 
2101 ATTTGTTTAT TTTTCTAAAT ACATTCAAAT ATGTATCCGC TCATGAGACA ATAACCCTGA 
2161 TAAATGCTTC AATAATATTG AAAAAGGAAG AGTCCTGAGG CGGAAAGAAC CAGCTGTGGA 
2221 ATGTGTGTCA GTTAGGGTGT GGAAAGTCCC CAGGCTCCCC AGCAGGCAGA AGTATGCAAA 
2281 GCATGCATCT CAATTAGTCA GCAACCAGGT GTGGAAAGTC CCCAGGCTCC CCAGCAGGCA 
2341 GAAGTATGCA AAGCATGCAT CTCAATTAGT CAGCAACCAT AGTCCCGCCC CTAACTCCGC 
2401 CCATCCCGCC CCTAACTCCG CCCAGTTCCG CCCATTCTCC GCCCCATGGC TGACTAATTT 
2461 TTTTTATTTA TGCAGAGGCC GAGGCCGCCT CGGCCTCTGA GCTATTCCAG AAGTAGTGAG 
2521 GAGGCTTTTT TGGAGGCCTA GGCTTTTGCA AAGATCGATC AAGAGACAGG ATGAGGATCG 
2581 TTTCGCATGA TTGAACAAGA TGGATTGCAC GCAGGTTCTC CGGCCGCTTG GGTGGAGAGG 
2641 CTATTCGGCT ATGACTGGGC ACAACAGACA ATCGGCTGCT CTGATGCCGC CGTGTTCCGG 
2701 CTGTCAGCGC AGGGGCGCCC GGTTCTTTTT GTCAAGACCG ACCTGTCCGG TGCCCTGAAT 
2761 GAACTGCAAG ACGAGGCAGC GCGGCTATCG TGGCTGGCCA CGACGGGCGT TCCTTGCGCA 
2821 GCTGTGCTCG ACGTTGTCAC TGAAGCGGGA AGGGACTGGC TGCTATTGGG CGAAGTGCCG 
2881 GGGCAGGATC TCCTGTCATC TCACCTTGCT CCTGCCGAGA AAGTATCCAT CATGGCTGAT 
2941 GCAATGCGGC GGCTGCATAC GCTTGATCCG GCTACCTGCC CATTCGACCA CCAAGCGAAA 
3001 CATCGCATCG AGCGAGCACG TACTCGGATG GAAGCCGGTC TTGTCGATCA GGATGATCTG 
3061 GACGAAGAGC ATCAGGGGCT CGCGCCAGCC GAACTGTTCG CCAGGCTCAA GGCGAGCATG 
3121 CCCGACGGCG AGGATCTCGT CGTGACCCAT GGCGATGCCT GCTTGCCGAA TATCATGGTG 
3181 GAAAATGGCC GCTTTTCTGG ATTCATCGAC TGTGGCCGGC TGGGTGTGGC GGACCGCTAT 
3241 CAGGACATAG CGTTGGCTAC CCGTGATATT GCTGAAGAGC TTGGCGGCGA ATGGGCTGAC 
3301 CGCTTCCTCG TGCTTTACGG TATCGCCGCT CCCGATTCGC AGCGCATCGC CTTCTATCGC 
3361 CTTCTTGACG AGTTCTTCTG AGCGGGACTC TGGGGTTCGA AATGACCGAC CAAGCGACGC 
3421 CCAACCTGCC ATCACGAGAT TTCGATTCCA CCGCCGCCTT CTATGAAAGG TTGGGCTTCG 
3481 GAATCGTTTT CCGGGACGCC GGCTGGATGA TCCTCCAGCG CGGGGATCTC ATGCTGGAGT 
3541 TCTTCGCCCA CCCTAGGGGG AGGCTAACTG AAACACGGAA GGAGACAATA CCGGAAGGAA 
3601 CCCGCGCTAT GACGGCAATA AAAAGACAGA ATAAAACGCA CGGTGTTGGG TCGTTTGTTC 
3661 ATAAACGCGG GGTTCGGTCC CAGGGCTGGC ACTCTGTCGA TACCCCACCG AGACCCCATT 
3721 GGGGCCAATA CGCCCGCGTT TCTTCCTTTT CCCCACCCCA CCCCCCAAGT TCGGGTGAAG 
3781 GCCCAGGGCT CGCAGCCAAC GTCGGGGCGG CAGGCCCTGC CATAGCCTCA GGTTACTCAT 
3841 ATATACTTTA GATTGATTTA AAACTTCATT TTTAATTTAA AAGGATCTAG GTGAAGATCC 
3901 TTTTTGATAA TCTCATGACC AAAATCCCTT AACGTGAGTT TTCGTTCCAC TGAGCGTCAG 
3961 ACCCCGTAGA AAAGATCAAA GGATCTTCTT GAGATCCTTT TTTTCTGCGC GTAATCTGCT 
4021 GCTTGCAAAC AAAAAAACCA CCGCTACCAG CGGTGGTTTG TTTGCCGGAT CAAGAGCTAC 
4081 CAACTCTTTT TCCGAAGGTA ACTGGCTTCA GCAGAGCGCA GATACCAAAT ACTGTCCTTC 
4141 TAGTGTAGCC GTAGTTAGGC CACCACTTCA AGAACTCTGT AGCACCGCCT ACATACCTCG 
4201 CTCTGCTAAT CCTGTTACCA GTGGCTGCTG CCAGTGGCGA TAAGTCGTGT CTTACCGGGT 
4261 TGGACTCAAG ACGATAGTTA CCGGATAAGG CGCAGCGGTC GGGCTGAACG GGGGGTTCGT 
4321 GCACACAGCC CAGCTTGGAG CGAACGACCT ACACCGAACT GAGATACCTA CAGCGTGAGC 
4381 TATGAGAAAG CGCCACGCTT CCCGAAGGGA GAAAGGCGGA CAGGTATCCG GTAAGCGGCA 
4441 GGGTCGGAAC AGGAGAGCGC ACGAGGGAGC TTCCAGGGGG AAACGCCTGG TATCTTTATA 
4501 GTCCTGTCGG GTTTCGCCAC CTCTGACTTG AGCGTCGATT TTTGTGATGC TCGTCAGGGG 
4561 GGCGGAGCCT ATGGAAAAAC GCCAGCAACG CGGCCTTTTT ACGGTTCCTG GCCTTTTGCT 
4621 GGCCTTTTGC TCACATGTTC TTTCCTGCGT TATCCCCTGA TTCTGTGGAT AACCGTATTA 
4681 CCGCCATGCA T