pDsRed2-1

pDsRed2-1 encodes DsRed2, a DsRed variant engineered for faster maturation and lower non-specific aggregation. Derived from the Discosoma sp. red fluorescent protein (drFP583; 1), DsRed2, like its progenitor DsRed1, contains a series of silent base-pair changes that correspond to human codon-usage preferences for high expression in mammalian cells (2). In addition to these changes, DsRed2 contains six amino acid substitutions: V105A, I161T, and S197A, which result in the more rapid appearance of red fluorescence in transfected cell lines; and R2A, K5E, and K9T, which prevent the protein from aggregating. (DsRed2 may, however, form the same tetrameric structure as DsRed1 [3].) In mammalian cell cultures when DsRed2 is expressed constitutively, red-emitting cells can be detected by fluorescence microscopy within 24 hours of transfection. Large insoluble aggregates of protein, often observed in bacterial and mammalian cell systems expressing DsRed1, are dramatically reduced in cells expressing DsRed2. The faster-maturing, more soluble red fluorescent protein is also well tolerated by host cells; mammalian cell cultures transfected with DsRed2 show no obvious signs of reduced viability—in those cell lines tested, cells expressing DsRed2 display the same morphology (e.g., adherence, light-refraction) and growth characteristics as non-transfected controls.

pDsRed2-1 is a promoterless DsRed2 vector that can be used to monitor transcription from different promoters and promoter/enhancer combinations inserted into the multiple cloning site (MCS). Sequences upstream of DsRed2 have been converted to a Kozak consensus translation initiation site (4) to increase translation efficiency in eukaryotic cells. SV40 polyadenylation signals downstream of the DsRed2 gene direct proper processing of the 3' end of the DsRed2 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 expresses kanamycin resistance in E. coli.

载体应用

DsRed2 can be used as an in vivo reporter of gene expression. Promoters should be cloned into the pDsRed2-1 MCS upstream from the DsRed2 coding sequence. Without addition of a functional promoter, this vector will not express DsRed2. The recombinant DsRed2 vector can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418 (5).

0001 TAGTTATTAC TAGCGCTACC GGACTCAGAT CTCGAGCTCA AGCTTCGAAT TCTGCAGTCG 
0061 ACGGTACCGC GGGCCCGGGA TCCACCGGTC GCCACCATGG CCTCCTCCGA GAACGTCATC 
0121 ACCGAGTTCA TGCGCTTCAA GGTGCGCATG GAGGGCACCG TGAACGGCCA CGAGTTCGAG 
0181 ATCGAGGGCG AGGGCGAGGG CCGCCCCTAC GAGGGCCACA ACACCGTGAA GCTGAAGGTG 
0241 ACCAAGGGCG GCCCCCTGCC CTTCGCCTGG GACATCCTGT CCCCCCAGTT CCAGTACGGC 
0301 TCCAAGGTGT ACGTGAAGCA CCCCGCCGAC ATCCCCGACT ACAAGAAGCT GTCCTTCCCC 
0361 GAGGGCTTCA AGTGGGAGCG CGTGATGAAC TTCGAGGACG GCGGCGTGGC GACCGTGACC 
0421 CAGGACTCCT CCCTGCAGGA CGGCTGCTTC ATCTACAAGG TGAAGTTCAT CGGCGTGAAC 
0481 TTCCCCTCCG ACGGCCCCGT GATGCAGAAG AAGACCATGG GCTGGGAGGC CTCCACCGAG 
0541 CGCCTGTACC CCCGCGACGG CGTGCTGAAG GGCGAGACCC ACAAGGCCCT GAAGCTGAAG 
0601 GACGGCGGCC ACTACCTGGT GGAGTTCAAG TCCATCTACA TGGCCAAGAA GCCCGTGCAG 
0661 CTGCCCGGCT ACTACTACGT GGACGCCAAG CTGGACATCA CCTCCCACAA CGAGGACTAC 
0721 ACCATCGTGG AGCAGTACGA GCGCACCGAG GGCCGCCACC ACCTGTTCCT GTAGCGGCCG 
0781 CGACTCTAGA TCATAATCAG CCATACCACA TTTGTAGAGG TTTTACTTGC TTTAAAAAAC 
0841 CTCCCACACC TCCCCCTGAA CCTGAAACAT AAAATGAATG CAATTGTTGT TGTTAACTTG 
0901 TTTATTGCAG CTTATAATGG TTACAAATAA AGCAATAGCA TCACAAATTT CACAAATAAA 
0961 GCATTTTTTT CACTGCATTC TAGTTGTGGT TTGTCCAAAC TCATCAATGT ATCTTAAGGC 
1021 GTAAATTGTA AGCGTTAATA TTTTGTTAAA ATTCGCGTTA AATTTTTGTT AAATCAGCTC 
1081 ATTTTTTAAC CAATAGGCCG AAATCGGCAA AATCCCTTAT AAATCAAAAG AATAGACCGA 
1141 GATAGGGTTG AGTGTTGTTC CAGTTTGGAA CAAGAGTCCA CTATTAAAGA ACGTGGACTC 
1201 CAACGTCAAA GGGCGAAAAA CCGTCTATCA GGGCGATGGC CCACTACGTG AACCATCACC 
1261 CTAATCAAGT TTTTTGGGGT CGAGGTGCCG TAAAGCACTA AATCGGAACC CTAAAGGGAG 
1321 CCCCCGATTT AGAGCTTGAC GGGGAAAGCC GGCGAACGTG GCGAGAAAGG AAGGGAAGAA 
1381 AGCGAAAGGA GCGGGCGCTA GGGCGCTGGC AAGTGTAGCG GTCACGCTGC GCGTAACCAC 
1441 CACACCCGCC GCGCTTAATG CGCCGCTACA GGGCGCGTCA GGTGGCACTT TTCGGGGAAA 
1501 TGTGCGCGGA ACCCCTATTT GTTTATTTTT CTAAATACAT TCAAATATGT ATCCGCTCAT 
1561 GAGACAATAA CCCTGATAAA TGCTTCAATA ATATTGAAAA AGGAAGAGTC CTGAGGCGGA 
1621 AAGAACCAGC TGTGGAATGT GTGTCAGTTA GGGTGTGGAA AGTCCCCAGG CTCCCCAGCA 
1681 GGCAGAAGTA TGCAAAGCAT GCATCTCAAT TAGTCAGCAA CCAGGTGTGG AAAGTCCCCA 
1741 GGCTCCCCAG CAGGCAGAAG TATGCAAAGC ATGCATCTCA ATTAGTCAGC AACCATAGTC 
1801 CCGCCCCTAA CTCCGCCCAT CCCGCCCCTA ACTCCGCCCA GTTCCGCCCA TTCTCCGCCC 
1861 CATGGCTGAC TAATTTTTTT TATTTATGCA GAGGCCGAGG CCGCCTCGGC CTCTGAGCTA 
1921 TTCCAGAAGT AGTGAGGAGG CTTTTTTGGA GGCCTAGGCT TTTGCAAAGA TCGATCAAGA 
1981 GACAGGATGA GGATCGTTTC GCATGATTGA ACAAGATGGA TTGCACGCAG GTTCTCCGGC 
2041 CGCTTGGGTG GAGAGGCTAT TCGGCTATGA CTGGGCACAA CAGACAATCG GCTGCTCTGA 
2101 TGCCGCCGTG TTCCGGCTGT CAGCGCAGGG GCGCCCGGTT CTTTTTGTCA AGACCGACCT 
2161 GTCCGGTGCC CTGAATGAAC TGCAAGACGA GGCAGCGCGG CTATCGTGGC TGGCCACGAC 
2221 GGGCGTTCCT TGCGCAGCTG TGCTCGACGT TGTCACTGAA GCGGGAAGGG ACTGGCTGCT 
2281 ATTGGGCGAA GTGCCGGGGC AGGATCTCCT GTCATCTCAC CTTGCTCCTG CCGAGAAAGT 
2341 ATCCATCATG GCTGATGCAA TGCGGCGGCT GCATACGCTT GATCCGGCTA CCTGCCCATT 
2401 CGACCACCAA GCGAAACATC GCATCGAGCG AGCACGTACT CGGATGGAAG CCGGTCTTGT 
2461 CGATCAGGAT GATCTGGACG AAGAGCATCA GGGGCTCGCG CCAGCCGAAC TGTTCGCCAG 
2521 GCTCAAGGCG AGCATGCCCG ACGGCGAGGA TCTCGTCGTG ACCCATGGCG ATGCCTGCTT 
2581 GCCGAATATC ATGGTGGAAA ATGGCCGCTT TTCTGGATTC ATCGACTGTG GCCGGCTGGG 
2641 TGTGGCGGAC CGCTATCAGG ACATAGCGTT GGCTACCCGT GATATTGCTG AAGAGCTTGG 
2701 CGGCGAATGG GCTGACCGCT TCCTCGTGCT TTACGGTATC GCCGCTCCCG ATTCGCAGCG 
2761 CATCGCCTTC TATCGCCTTC TTGACGAGTT CTTCTGAGCG GGACTCTGGG GTTCGAAATG 
2821 ACCGACCAAG CGACGCCCAA CCTGCCATCA CGAGATTTCG ATTCCACCGC CGCCTTCTAT 
2881 GAAAGGTTGG GCTTCGGAAT CGTTTTCCGG GACGCCGGCT GGATGATCCT CCAGCGCGGG 
2941 GATCTCATGC TGGAGTTCTT CGCCCACCCT AGGGGGAGGC TAACTGAAAC ACGGAAGGAG 
3001 ACAATACCGG AAGGAACCCG CGCTATGACG GCAATAAAAA GACAGAATAA AACGCACGGT 
3061 GTTGGGTCGT TTGTTCATAA ACGCGGGGTT CGGTCCCAGG GCTGGCACTC TGTCGATACC 
3121 CCACCGAGAC CCCATTGGGG CCAATACGCC CGCGTTTCTT CCTTTTCCCC ACCCCACCCC 
3181 CCAAGTTCGG GTGAAGGCCC AGGGCTCGCA GCCAACGTCG GGGCGGCAGG CCCTGCCATA 
3241 GCCTCAGGTT ACTCATATAT ACTTTAGATT GATTTAAAAC TTCATTTTTA ATTTAAAAGG 
3301 ATCTAGGTGA AGATCCTTTT TGATAATCTC ATGACCAAAA TCCCTTAACG TGAGTTTTCG 
3361 TTCCACTGAG CGTCAGACCC CGTAGAAAAG ATCAAAGGAT CTTCTTGAGA TCCTTTTTTT 
3421 CTGCGCGTAA TCTGCTGCTT GCAAACAAAA AAACCACCGC TACCAGCGGT GGTTTGTTTG 
3481 CCGGATCAAG AGCTACCAAC TCTTTTTCCG AAGGTAACTG GCTTCAGCAG AGCGCAGATA 
3541 CCAAATACTG TCCTTCTAGT GTAGCCGTAG TTAGGCCACC ACTTCAAGAA CTCTGTAGCA 
3601 CCGCCTACAT ACCTCGCTCT GCTAATCCTG TTACCAGTGG CTGCTGCCAG TGGCGATAAG 
3661 TCGTGTCTTA CCGGGTTGGA CTCAAGACGA TAGTTACCGG ATAAGGCGCA GCGGTCGGGC 
3721 TGAACGGGGG GTTCGTGCAC ACAGCCCAGC TTGGAGCGAA CGACCTACAC CGAACTGAGA 
3781 TACCTACAGC GTGAGCTATG AGAAAGCGCC ACGCTTCCCG AAGGGAGAAA GGCGGACAGG 
3841 TATCCGGTAA GCGGCAGGGT CGGAACAGGA GAGCGCACGA GGGAGCTTCC AGGGGGAAAC 
3901 GCCTGGTATC TTTATAGTCC TGTCGGGTTT CGCCACCTCT GACTTGAGCG TCGATTTTTG 
3961 TGATGCTCGT CAGGGGGGCG GAGCCTATGG AAAAACGCCA GCAACGCGGC CTTTTTACGG 
4021 TTCCTGGCCT TTTGCTGGCC TTTTGCTCAC ATGTTCTTTC CTGCGTTATC CCCTGATTCT 
4081 GTGGATAACC GTATTACCGC CATGCAT