pDsRed2-Mito is a mammalian expression vector that encodes a fusion of Discosoma sp. red fluorescent protein (DsRed2; 1, 2) and the mitochondrial targeting sequence from subunit VIII of human cytochrome c oxidase (Mito; 3, 4). The Mito sequence is fused to the 5'-end of DsRed2, a human codon-optimized DsRed variant that is engineered for faster maturation and lower nonspecific aggregation (1, 5). The Mito sequence targets the Mito-DsRed2 fusion protein to the host cell’s mitochondria.

To drive expression of Mito-DsRed2, this vector contains the immmediate early promoter of cytomegalovirus (PCMV IE ). SV40 polyadenylation signals downstream of the DsRed2 gene direct proper processing of the 3'-end of the Mito-DsRed2 mRNA. This vector also contains an SV40 origin for replication in any mammalian cell line that expresses 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—consisting of the SV40 early promoter (PSV40e), the neomycin/kanamycin resistance gene of Tn5 (Neor/Kanr), and polyadenylation signals from the herpes simplex virus thymidine kinase (HSV TK poly A) gene—allow stably transfected eukaryotic cells to be selected using G418 (6). A bacterial promoter (P) upstream of this cassette drives expression of the gene encoding kanamycin resistance in E. coli.

用途

pDsRed2-Mito is designed for fluorescent labeling of mitochondria. The vector can be introduced into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (6). The Mito-DsRed2 fusion (excitation/emission maxima: 558 nm/ 583 nm) can be detected by fluorescence microscopy and by flow cytometry. Filter sets optimized for detecting DsRed by microscopy are available from Chroma Technology Corporation and Omega Optical Inc. Please see their websites (www.chroma.com and www.omegafilters.com) and the Living Colors? Vol. II User Manual, provided with this vector, for more information. To detect Mito- DsRed2-expressing cells by flow cytometry, use the instrument’s argon-ion laser to excite the fluorophore at 488 nm and the FL-2 channel to detect the fluorophore’s emission at 583 nm.

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 GCTAGCATGT 
0601 CCGTCCTGAC GCCGCTGCTG CTGCGGGGCT TGACAGGCTC GGCCCGGCGG CTCCCAGTGC 
0661 CGCGCGCCAA GATCCATTCG TTGGGGGATC CACCGGTCGC CACCATGGCC TCCTCCGAGA 
0721 ACGTCATCAC CGAGTTCATG CGCTTCAAGG TGCGCATGGA GGGCACCGTG AACGGCCACG 
0781 AGTTCGAGAT CGAGGGCGAG GGCGAGGGCC GCCCCTACGA GGGCCACAAC ACCGTGAAGC 
0841 TGAAGGTGAC CAAGGGCGGC CCCCTGCCCT TCGCCTGGGA CATCCTGTCC CCCCAGTTCC 
0901 AGTACGGCTC CAAGGTGTAC GTGAAGCACC CCGCCGACAT CCCCGACTAC AAGAAGCTGT 
0961 CCTTCCCCGA GGGCTTCAAG TGGGAGCGCG TGATGAACTT CGAGGACGGC GGCGTGGCGA 
1021 CCGTGACCCA GGACTCCTCC CTGCAGGACG GCTGCTTCAT CTACAAGGTG AAGTTCATCG 
1081 GCGTGAACTT CCCCTCCGAC GGCCCCGTGA TGCAGAAGAA GACCATGGGC TGGGAGGCCT 
1141 CCACCGAGCG CCTGTACCCC CGCGACGGCG TGCTGAAGGG CGAGACCCAC AAGGCCCTGA 
1201 AGCTGAAGGA CGGCGGCCAC TACCTGGTGG AGTTCAAGTC CATCTACATG GCCAAGAAGC 
1261 CCGTGCAGCT GCCCGGCTAC TACTACGTGG ACGCCAAGCT GGACATCACC TCCCACAACG 
1321 AGGACTACAC CATCGTGGAG CAGTACGAGC GCACCGAGGG CCGCCACCAC CTGTTCCTGT 
1381 AGCGGCCGCG ACTCTAGATC ATAATCAGCC ATACCACATT TGTAGAGGTT TTACTTGCTT 
1441 TAAAAAACCT CCCACACCTC CCCCTGAACC TGAAACATAA AATGAATGCA ATTGTTGTTG 
1501 TTAACTTGTT TATTGCAGCT TATAATGGTT ACAAATAAAG CAATAGCATC ACAAATTTCA 
1561 CAAATAAAGC ATTTTTTTCA CTGCATTCTA GTTGTGGTTT GTCCAAACTC ATCAATGTAT 
1621 CTTAAGGCGT AAATTGTAAG CGTTAATATT TTGTTAAAAT TCGCGTTAAA TTTTTGTTAA 
1681 ATCAGCTCAT TTTTTAACCA ATAGGCCGAA ATCGGCAAAA TCCCTTATAA ATCAAAAGAA 
1741 TAGACCGAGA TAGGGTTGAG TGTTGTTCCA GTTTGGAACA AGAGTCCACT ATTAAAGAAC 
1801 GTGGACTCCA ACGTCAAAGG GCGAAAAACC GTCTATCAGG GCGATGGCCC ACTACGTGAA 
1861 CCATCACCCT AATCAAGTTT TTTGGGGTCG AGGTGCCGTA AAGCACTAAA TCGGAACCCT 
1921 AAAGGGAGCC CCCGATTTAG AGCTTGACGG GGAAAGCCGG CGAACGTGGC GAGAAAGGAA 
1981 GGGAAGAAAG CGAAAGGAGC GGGCGCTAGG GCGCTGGCAA GTGTAGCGGT CACGCTGCGC 
2041 GTAACCACCA CACCCGCCGC GCTTAATGCG CCGCTACAGG GCGCGTCAGG TGGCACTTTT 
2101 CGGGGAAATG TGCGCGGAAC CCCTATTTGT TTATTTTTCT AAATACATTC AAATATGTAT 
2161 CCGCTCATGA GACAATAACC CTGATAAATG CTTCAATAAT ATTGAAAAAG GAAGAGTCCT 
2221 GAGGCGGAAA GAACCAGCTG TGGAATGTGT GTCAGTTAGG GTGTGGAAAG TCCCCAGGCT 
2281 CCCCAGCAGG CAGAAGTATG CAAAGCATGC ATCTCAATTA GTCAGCAACC AGGTGTGGAA 
2341 AGTCCCCAGG CTCCCCAGCA GGCAGAAGTA TGCAAAGCAT GCATCTCAAT TAGTCAGCAA 
2401 CCATAGTCCC GCCCCTAACT CCGCCCATCC CGCCCCTAAC TCCGCCCAGT TCCGCCCATT 
2461 CTCCGCCCCA TGGCTGACTA ATTTTTTTTA TTTATGCAGA GGCCGAGGCC GCCTCGGCCT 
2521 CTGAGCTATT CCAGAAGTAG TGAGGAGGCT TTTTTGGAGG CCTAGGCTTT TGCAAAGATC 
2581 GATCAAGAGA CAGGATGAGG ATCGTTTCGC ATGATTGAAC AAGATGGATT GCACGCAGGT 
2641 TCTCCGGCCG CTTGGGTGGA GAGGCTATTC GGCTATGACT GGGCACAACA GACAATCGGC 
2701 TGCTCTGATG CCGCCGTGTT CCGGCTGTCA GCGCAGGGGC GCCCGGTTCT TTTTGTCAAG 
2761 ACCGACCTGT CCGGTGCCCT GAATGAACTG CAAGACGAGG CAGCGCGGCT ATCGTGGCTG 
2821 GCCACGACGG GCGTTCCTTG CGCAGCTGTG CTCGACGTTG TCACTGAAGC GGGAAGGGAC 
2881 TGGCTGCTAT TGGGCGAAGT GCCGGGGCAG GATCTCCTGT CATCTCACCT TGCTCCTGCC 
2941 GAGAAAGTAT CCATCATGGC TGATGCAATG CGGCGGCTGC ATACGCTTGA TCCGGCTACC 
3001 TGCCCATTCG ACCACCAAGC GAAACATCGC ATCGAGCGAG CACGTACTCG GATGGAAGCC 
3061 GGTCTTGTCG ATCAGGATGA TCTGGACGAA GAGCATCAGG GGCTCGCGCC AGCCGAACTG 
3121 TTCGCCAGGC TCAAGGCGAG CATGCCCGAC GGCGAGGATC TCGTCGTGAC CCATGGCGAT 
3181 GCCTGCTTGC CGAATATCAT GGTGGAAAAT GGCCGCTTTT CTGGATTCAT CGACTGTGGC 
3241 CGGCTGGGTG TGGCGGACCG CTATCAGGAC ATAGCGTTGG CTACCCGTGA TATTGCTGAA 
3301 GAGCTTGGCG GCGAATGGGC TGACCGCTTC CTCGTGCTTT ACGGTATCGC CGCTCCCGAT 
3361 TCGCAGCGCA TCGCCTTCTA TCGCCTTCTT GACGAGTTCT TCTGAGCGGG ACTCTGGGGT 
3421 TCGAAATGAC CGACCAAGCG ACGCCCAACC TGCCATCACG AGATTTCGAT TCCACCGCCG 
3481 CCTTCTATGA AAGGTTGGGC TTCGGAATCG TTTTCCGGGA CGCCGGCTGG ATGATCCTCC 
3541 AGCGCGGGGA TCTCATGCTG GAGTTCTTCG CCCACCCTAG GGGGAGGCTA ACTGAAACAC 
3601 GGAAGGAGAC AATACCGGAA GGAACCCGCG CTATGACGGC AATAAAAAGA CAGAATAAAA 
3661 CGCACGGTGT TGGGTCGTTT GTTCATAAAC GCGGGGTTCG GTCCCAGGGC TGGCACTCTG 
3721 TCGATACCCC ACCGAGACCC CATTGGGGCC AATACGCCCG CGTTTCTTCC TTTTCCCCAC 
3781 CCCACCCCCC AAGTTCGGGT GAAGGCCCAG GGCTCGCAGC CAACGTCGGG GCGGCAGGCC 
3841 CTGCCATAGC CTCAGGTTAC TCATATATAC TTTAGATTGA TTTAAAACTT CATTTTTAAT 
3901 TTAAAAGGAT CTAGGTGAAG ATCCTTTTTG ATAATCTCAT GACCAAAATC CCTTAACGTG 
3961 AGTTTTCGTT CCACTGAGCG TCAGACCCCG TAGAAAAGAT CAAAGGATCT TCTTGAGATC 
4021 CTTTTTTTCT GCGCGTAATC TGCTGCTTGC AAACAAAAAA ACCACCGCTA CCAGCGGTGG 
4081 TTTGTTTGCC GGATCAAGAG CTACCAACTC TTTTTCCGAA GGTAACTGGC TTCAGCAGAG 
4141 CGCAGATACC AAATACTGTC CTTCTAGTGT AGCCGTAGTT AGGCCACCAC TTCAAGAACT 
4201 CTGTAGCACC GCCTACATAC CTCGCTCTGC TAATCCTGTT ACCAGTGGCT GCTGCCAGTG 
4261 GCGATAAGTC GTGTCTTACC GGGTTGGACT CAAGACGATA GTTACCGGAT AAGGCGCAGC 
4321 GGTCGGGCTG AACGGGGGGT TCGTGCACAC AGCCCAGCTT GGAGCGAACG ACCTACACCG 
4381 AACTGAGATA CCTACAGCGT GAGCTATGAG AAAGCGCCAC GCTTCCCGAA GGGAGAAAGG 
4441 CGGACAGGTA TCCGGTAAGC GGCAGGGTCG GAACAGGAGA GCGCACGAGG GAGCTTCCAG 
4501 GATTTTTGTG ATGCTCGTCA GGGGGGCGGA GCCTATGGAA AAACGCCAGC AACGCGGCCT 
4561 TTTTACGGTT CCTGGCCTTT TGCTGGCCTT TTGCTCACAT GTTCTTTCCT GCGTTATCCC 
4621 CTGATTCTGT GGATAACCGT ATTACCGCCA TGCAT