pDsRed2-Peroxi

pDsRed2-Peroxi is a mammalian expression vector that encodes a fusion of Discosoma sp. red fluorescent protein (DsRed2; 1, 2) and the peroxisomal targeting signal 1 (PTS1). The PTS1 sequence is fused to the 3'-end of DsRed2, a DsRed variant engineered for faster maturation and lower non-specific aggregation. The PTS1 sequence encodes the tripeptide SKL, which targets the DsRed2-PTS1 fusion protein to the matrix of peroxisomes (3–6).

To drive expression of DsRed2-PTS1, this vector contains the immediate early promoter of cytomegalovirus (PCMV IE). SV40 polyadenylation signals downstream of the DsRed2 gene direct proper processing of the 3'-end of the DsRed2-PTS1 mRNA transcript. Because it encodes DsRed2, a gene variant that uses human-preferred codons (7), the DsRed2-PTS1 transcript is suited for efficient translation in mammalian cells. To further increase the translational efficiency of DsRed2- PTS1, upstream sequences have been converted to a Kozak consensus translation initiation site (8). The 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 singlestranded 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 (9). A bacterial promoter (P) upstream of this cassette drives expression of the gene encoding kanamycin resistance in E. coli.

载体应用

pDsRed2-Peroxi is designed for fluorescent labeling of peroxisomes. The vector can be introduced into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (9). The DsRed2-PTS1 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 DsRed2- PTS1-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 GCTAGCGCTA 
0601 CCGGTCGCCA CCATGGCCTC CTCCGAGAAC GTCATCACCG AGTTCATGCG CTTCAAGGTG 
0661 CGCATGGAGG GCACCGTGAA CGGCCACGAG TTCGAGATCG AGGGCGAGGG CGAGGGCCGC 
0721 CCCTACGAGG GCCACAACAC CGTGAAGCTG AAGGTGACCA AGGGCGGCCC CCTGCCCTTC 
0781 GCCTGGGACA TCCTGTCCCC CCAGTTCCAG TACGGCTCCA AGGTGTACGT GAAGCACCCC 
0841 GCCGACATCC CCGACTACAA GAAGCTGTCC TTCCCCGAGG GCTTCAAGTG GGAGCGCGTG 
0901 ATGAACTTCG AGGACGGCGG CGTGGCGACC GTGACCCAGG ACTCCTCCCT GCAGGACGGC 
0961 TGCTTCATCT ACAAGGTGAA GTTCATCGGC GTGAACTTCC CCTCCGACGG CCCCGTGATG 
1021 CAGAAGAAGA CCATGGGCTG GGAGGCCTCC ACCGAGCGCC TGTACCCCCG CGACGGCGTG 
1081 CTGAAGGGCG AGACCCACAA GGCCCTGAAG CTGAAGGACG GCGGCCACTA CCTGGTGGAG 
1141 TTCAAGTCCA TCTACATGGC CAAGAAGCCC GTGCAGCTGC CCGGCTACTA CTACGTGGAC 
1201 GCCAAGCTGG ACATCACCTC CCACAACGAG GACTACACCA TCGTGGAGCA GTACGAGCGC 
1261 ACCGAGGGCC GCCACCACCT GTTCCTGAGA TCGTACAAGT CCAAGCTGTA GCGGCCGCGA 
1321 CTCTAGATCA TAATCAGCCA TACCACATTT GTAGAGGTTT TACTTGCTTT AAAAAACCTC 
1381 CCACACCTCC CCCTGAACCT GAAACATAAA ATGAATGCAA TTGTTGTTGT TAACTTGTTT 
1441 ATTGCAGCTT ATAATGGTTA CAAATAAAGC AATAGCATCA CAAATTTCAC AAATAAAGCA 
1501 TTTTTTTCAC TGCATTCTAG TTGTGGTTTG TCCAAACTCA TCAATGTATC TTAAGGCGTA 
1561 AATTGTAAGC GTTAATATTT TGTTAAAATT CGCGTTAAAT TTTTGTTAAA TCAGCTCATT 
1621 TTTTAACCAA TAGGCCGAAA TCGGCAAAAT CCCTTATAAA TCAAAAGAAT AGACCGAGAT 
1681 AGGGTTGAGT GTTGTTCCAG TTTGGAACAA GAGTCCACTA TTAAAGAACG TGGACTCCAA 
1741 CGTCAAAGGG CGAAAAACCG TCTATCAGGG CGATGGCCCA CTACGTGAAC CATCACCCTA 
1801 ATCAAGTTTT TTGGGGTCGA GGTGCCGTAA AGCACTAAAT CGGAACCCTA AAGGGAGCCC 
1861 CCGATTTAGA GCTTGACGGG GAAAGCCGGC GAACGTGGCG AGAAAGGAAG GGAAGAAAGC 
1921 GAAAGGAGCG GGCGCTAGGG CGCTGGCAAG TGTAGCGGTC ACGCTGCGCG TAACCACCAC 
1981 ACCCGCCGCG CTTAATGCGC CGCTACAGGG CGCGTCAGGT GGCACTTTTC GGGGAAATGT 
2041 GCGCGGAACC CCTATTTGTT TATTTTTCTA AATACATTCA AATATGTATC CGCTCATGAG 
2101 ACAATAACCC TGATAAATGC TTCAATAATA TTGAAAAAGG AAGAGTCCTG AGGCGGAAAG 
2161 AACCAGCTGT GGAATGTGTG TCAGTTAGGG TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC 
2221 AGAAGTATGC AAAGCATGCA TCTCAATTAG TCAGCAACCA GGTGTGGAAA GTCCCCAGGC 
2281 TCCCCAGCAG GCAGAAGTAT GCAAAGCATG CATCTCAATT AGTCAGCAAC CATAGTCCCG 
2341 CCCCTAACTC CGCCCATCCC GCCCCTAACT CCGCCCAGTT CCGCCCATTC TCCGCCCCAT 
2401 GGCTGACTAA TTTTTTTTAT TTATGCAGAG GCCGAGGCCG CCTCGGCCTC TGAGCTATTC 
2461 CAGAAGTAGT GAGGAGGCTT TTTTGGAGGC CTAGGCTTTT GCAAAGATCG ATCAAGAGAC 
2521 AGGATGAGGA TCGTTTCGCA TGATTGAACA AGATGGATTG CACGCAGGTT CTCCGGCCGC 
2581 TTGGGTGGAG AGGCTATTCG GCTATGACTG GGCACAACAG ACAATCGGCT GCTCTGATGC 
2641 CGCCGTGTTC CGGCTGTCAG CGCAGGGGCG CCCGGTTCTT TTTGTCAAGA CCGACCTGTC 
2701 CGGTGCCCTG AATGAACTGC AAGACGAGGC AGCGCGGCTA TCGTGGCTGG CCACGACGGG 
2761 CGTTCCTTGC GCAGCTGTGC TCGACGTTGT CACTGAAGCG GGAAGGGACT GGCTGCTATT 
2821 GGGCGAAGTG CCGGGGCAGG ATCTCCTGTC ATCTCACCTT GCTCCTGCCG AGAAAGTATC 
2881 CATCATGGCT GATGCAATGC GGCGGCTGCA TACGCTTGAT CCGGCTACCT GCCCATTCGA 
2941 CCACCAAGCG AAACATCGCA TCGAGCGAGC ACGTACTCGG ATGGAAGCCG GTCTTGTCGA 
3001 TCAGGATGAT CTGGACGAAG AGCATCAGGG GCTCGCGCCA GCCGAACTGT TCGCCAGGCT 
3061 CAAGGCGAGC ATGCCCGACG GCGAGGATCT CGTCGTGACC CATGGCGATG CCTGCTTGCC 
3121 GAATATCATG GTGGAAAATG GCCGCTTTTC TGGATTCATC GACTGTGGCC GGCTGGGTGT 
3181 GGCGGACCGC TATCAGGACA TAGCGTTGGC TACCCGTGAT ATTGCTGAAG AGCTTGGCGG 
3241 CGAATGGGCT GACCGCTTCC TCGTGCTTTA CGGTATCGCC GCTCCCGATT CGCAGCGCAT 
3301 CGCCTTCTAT CGCCTTCTTG ACGAGTTCTT CTGAGCGGGA CTCTGGGGTT CGAAATGACC 
3361 GACCAAGCGA CGCCCAACCT GCCATCACGA GATTTCGATT CCACCGCCGC CTTCTATGAA 
3421 AGGTTGGGCT TCGGAATCGT TTTCCGGGAC GCCGGCTGGA TGATCCTCCA GCGCGGGGAT 
3481 CTCATGCTGG AGTTCTTCGC CCACCCTAGG GGGAGGCTAA CTGAAACACG GAAGGAGACA 
3541 ATACCGGAAG GAACCCGCGC TATGACGGCA ATAAAAAGAC AGAATAAAAC GCACGGTGTT 
3601 GGGTCGTTTG TTCATAAACG CGGGGTTCGG TCCCAGGGCT GGCACTCTGT CGATACCCCA 
3661 CCGAGACCCC ATTGGGGCCA ATACGCCCGC GTTTCTTCCT TTTCCCCACC CCACCCCCCA 
3721 AGTTCGGGTG AAGGCCCAGG GCTCGCAGCC AACGTCGGGG CGGCAGGCCC TGCCATAGCC 
3781 TCAGGTTACT CATATATACT TTAGATTGAT TTAAAACTTC ATTTTTAATT TAAAAGGATC 
3841 TAGGTGAAGA TCCTTTTTGA TAATCTCATG ACCAAAATCC CTTAACGTGA GTTTTCGTTC 
3901 CACTGAGCGT CAGACCCCGT AGAAAAGATC AAAGGATCTT CTTGAGATCC TTTTTTTCTG 
3961 CGCGTAATCT GCTGCTTGCA AACAAAAAAA CCACCGCTAC CAGCGGTGGT TTGTTTGCCG 
4021 GATCAAGAGC TACCAACTCT TTTTCCGAAG GTAACTGGCT TCAGCAGAGC GCAGATACCA 
4081 AATACTGTCC TTCTAGTGTA GCCGTAGTTA GGCCACCACT TCAAGAACTC TGTAGCACCG 
4141 CCTACATACC TCGCTCTGCT AATCCTGTTA CCAGTGGCTG CTGCCAGTGG CGATAAGTCG 
4201 TGTCTTACCG GGTTGGACTC AAGACGATAG TTACCGGATA AGGCGCAGCG GTCGGGCTGA 
4261 ACGGGGGGTT CGTGCACACA GCCCAGCTTG GAGCGAACGA CCTACACCGA ACTGAGATAC 
4321 CTACAGCGTG AGCTATGAGA AAGCGCCACG CTTCCCGAAG GGAGAAAGGC GGACAGGTAT 
4381 CCGGTAAGCG GCAGGGTCGG AACAGGAGAG CGCACGAGGG AGCTTCCAGG GGGAAACGCC 
4441 TGGTATCTTT ATAGTCCTGT CGGGTTTCGC CACCTCTGAC TTGAGCGTCG ATTTTTGTGA 
4501 TGCTCGTCAG GGGGGCGGAG CCTATGGAAA AACGCCAGCA ACGCGGCCTT TTTACGGTTC 
4561 CTGGCCTTTT GCTGGCCTTT TGCTCACATG TTCTTTCCTG CGTTATCCCC TGATTCTGTG 
4621 GATAACCGTA TTACCGCCAT GCAT