pDsRed1-C1 encodes a novel red fluorescent protein (DsRed1; 1) that has been optimized for high expression in mammalian cells (excitation maximum = 558 nm; emission maximum = 583 nm.). Red fluorescent protein was originally isolated from an IndoPacific sea anemone relative, Discosoma sp.DsRed1’s coding sequence contains 144 silent base pair changes, which correspond to human codon-usage preferences for high expression in mammalian cells. A nucleotide sequence upstream of DsRed1 has been converted to a Kozak consensus translation initiation site to further increase the translation efficiency in eukaryotic cells. The multiple cloning site (MCS) in pDsRed1-C1 is positioned between the DsRed1 coding sequence and the SV40 polyadenylation signal (SV40 poly A). Genes cloned into the MCS will be expressed as fusions to the C-terminus of DsRed1 if they are in the same reading frame as DsRed1 and there are no intervening stop codons. SV40 poly A signals downstream of the MCS direct proper processing of the 3' end of mRNA transcripts. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 T-antigen. A neomycin resistance cassette (Neor), consisting 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, allows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of this cassette expresses kanamycin resistance in E. coli. The pDsRed1-C1 backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

載體應(yīng)用

pDsRed1-C1 can be used to construct fusions to the C-terminus of DsRed1. If a fusion construct retains the fluorescent properties of the native DsRed1 protein, its expression and localization in vivo can be monitored by fluorescence microscopy or flow cytometry. The target gene should be cloned into pDsRed1-C1 so that it is in frame with the DsRed1 coding sequences, with no intervening inframe stop codons. The recombinant DsRed1 vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (4). pDsRed1-C1 can also be used simply to express DsRed1 in a cell line of interest (e.g., as a cotransfection marker).

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 CCATGGTGCG CTCCTCCAAG AACGTCATCA AGGAGTTCAT GCGCTTCAAG 
0661 GTGCGCATGG AGGGCACCGT GAACGGCCAC GAGTTCGAGA TCGAGGGCGA GGGCGAGGGC 
0721 CGCCCCTACG AGGGCCACAA CACCGTGAAG CTGAAGGTGA CCAAGGGCGG CCCCCTGCCC 
0781 TTCGCCTGGG ACATCCTGTC CCCCCAGTTC CAGTACGGCT CCAAGGTGTA CGTGAAGCAC 
0841 CCCGCCGACA TCCCCGACTA CAAGAAGCTG TCCTTCCCCG AGGGCTTCAA GTGGGAGCGC 
0901 GTGATGAACT TCGAGGACGG CGGCGTGGTG ACCGTGACCC AGGACTCCTC CCTGCAGGAC 
0961 GGCTGCTTCA TCTACAAGGT GAAGTTCATC GGCGTGAACT TCCCCTCCGA CGGCCCCGTA 
1021 ATGCAGAAGA AGACCATGGG CTGGGAGGCC TCCACCGAGC GCCTGTACCC CCGCGACGGC 
1081 GTGCTGAAGG GCGAGATCCA CAAGGCCCTG AAGCTGAAGG ACGGCGGCCA CTACCTGGTG 
1141 GAGTTCAAGT CCATCTACAT GGCCAAGAAG CCCGTGCAGC TGCCCGGCTA CTACTACGTG 
1201 GACTCCAAGC TGGACATCAC CTCCCACAAC GAGGACTACA CCATCGTGGA GCAGTACGAG 
1261 CGCACCGAGG GCCGCCACCA CCTGTTCCTG CTCAGATCTC GAGCTCAAGC TTCGAATTCT 
1321 GCAGTCGACG GTACCGCGGG CCCGGGATCC ACCGGATCTA GATAACTGAT CATAATCAGC 
1381 CATACCACAT TTGTAGAGGT TTTACTTGCT TTAAAAAACC TCCCACACCT CCCCCTGAAC 
1441 CTGAAACATA AAATGAATGC AATTGTTGTT GTTAACTTGT TTATTGCAGC TTATAATGGT 
1501 TACAAATAAA GCAATAGCAT CACAAATTTC ACAAATAAAG CATTTTTTTC ACTGCATTCT 
1561 AGTTGTGGTT TGTCCAAACT CATCAATGTA TCTTAACGCG TAAATTGTAA GCGTTAATAT 
1621 TTTGTTAAAA TTCGCGTTAA ATTTTTGTTA AATCAGCTCA TTTTTTAACC AATAGGCCGA 
1681 AATCGGCAAA ATCCCTTATA AATCAAAAGA ATAGACCGAG ATAGGGTTGA GTGTTGTTCC 
1741 AGTTTGGAAC AAGAGTCCAC TATTAAAGAA CGTGGACTCC AACGTCAAAG GGCGAAAAAC 
1801 CGTCTATCAG GGCGATGGCC CACTACGTGA ACCATCACCC TAATCAAGTT TTTTGGGGTC 
1861 GAGGTGCCGT AAAGCACTAA ATCGGAACCC TAAAGGGAGC CCCCGATTTA GAGCTTGACG 
1921 GGGAAAGCCG GCGAACGTGG CGAGAAAGGA AGGGAAGAAA GCGAAAGGAG CGGGCGCTAG 
1981 GGCGCTGGCA AGTGTAGCGG TCACGCTGCG CGTAACCACC ACACCCGCCG CGCTTAATGC 
2041 GCCGCTACAG GGCGCGTCAG GTGGCACTTT TCGGGGAAAT GTGCGCGGAA CCCCTATTTG 
2101 TTTATTTTTC TAAATACATT CAAATATGTA TCCGCTCATG AGACAATAAC CCTGATAAAT 
2161 GCTTCAATAA TATTGAAAAA GGAAGAGTCC TGAGGCGGAA AGAACCAGCT GTGGAATGTG 
2221 TGTCAGTTAG GGTGTGGAAA GTCCCCAGGC TCCCCAGCAG GCAGAAGTAT GCAAAGCATG 
2281 CATCTCAATT AGTCAGCAAC CAGGTGTGGA AAGTCCCCAG GCTCCCCAGC AGGCAGAAGT 
2341 ATGCAAAGCA TGCATCTCAA TTAGTCAGCA ACCATAGTCC CGCCCCTAAC TCCGCCCATC 
2401 CCGCCCCTAA CTCCGCCCAG TTCCGCCCAT TCTCCGCCCC ATGGCTGACT AATTTTTTTT 
2461 ATTTATGCAG AGGCCGAGGC CGCCTCGGCC TCTGAGCTAT TCCAGAAGTA GTGAGGAGGC 
2521 TTTTTTGGAG GCCTAGGCTT TTGCAAAGAT CGATCAAGAG ACAGGATGAG GATCGTTTCG 
2581 CATGATTGAA CAAGATGGAT TGCACGCAGG TTCTCCGGCC GCTTGGGTGG AGAGGCTATT 
2641 CGGCTATGAC TGGGCACAAC AGACAATCGG CTGCTCTGAT GCCGCCGTGT TCCGGCTGTC 
2701 AGCGCAGGGG CGCCCGGTTC TTTTTGTCAA GACCGACCTG TCCGGTGCCC TGAATGAACT 
2761 GCAAGACGAG GCAGCGCGGC TATCGTGGCT GGCCACGACG GGCGTTCCTT GCGCAGCTGT 
2821 GCTCGACGTT GTCACTGAAG CGGGAAGGGA CTGGCTGCTA TTGGGCGAAG TGCCGGGGCA 
2881 GGATCTCCTG TCATCTCACC TTGCTCCTGC CGAGAAAGTA TCCATCATGG CTGATGCAAT 
2941 GCGGCGGCTG CATACGCTTG ATCCGGCTAC CTGCCCATTC GACCACCAAG CGAAACATCG 
3001 CATCGAGCGA GCACGTACTC GGATGGAAGC CGGTCTTGTC GATCAGGATG ATCTGGACGA 
3061 AGAGCATCAG GGGCTCGCGC CAGCCGAACT GTTCGCCAGG CTCAAGGCGA GCATGCCCGA 
3121 CGGCGAGGAT CTCGTCGTGA CCCATGGCGA TGCCTGCTTG CCGAATATCA TGGTGGAAAA 
3181 TGGCCGCTTT TCTGGATTCA TCGACTGTGG CCGGCTGGGT GTGGCGGACC GCTATCAGGA 
3241 CATAGCGTTG GCTACCCGTG ATATTGCTGA AGAGCTTGGC GGCGAATGGG CTGACCGCTT 
3301 CCTCGTGCTT TACGGTATCG CCGCTCCCGA TTCGCAGCGC ATCGCCTTCT ATCGCCTTCT 
3361 TGACGAGTTC TTCTGAGCGG GACTCTGGGG TTCGAAATGA CCGACCAAGC GACGCCCAAC 
3421 CTGCCATCAC GAGATTTCGA TTCCACCGCC GCCTTCTATG AAAGGTTGGG CTTCGGAATC 
3481 GTTTTCCGGG ACGCCGGCTG GATGATCCTC CAGCGCGGGG ATCTCATGCT GGAGTTCTTC 
3541 GCCCACCCTA GGGGGAGGCT AACTGAAACA CGGAAGGAGA CAATACCGGA AGGAACCCGC 
3601 GCTATGACGG CAATAAAAAG ACAGAATAAA ACGCACGGTG TTGGGTCGTT TGTTCATAAA 
3661 CGCGGGGTTC GGTCCCAGGG CTGGCACTCT GTCGATACCC CACCGAGACC CCATTGGGGC 
3721 CAATACGCCC GCGTTTCTTC CTTTTCCCCA CCCCACCCCC CAAGTTCGGG TGAAGGCCCA 
3781 GGGCTCGCAG CCAACGTCGG GGCGGCAGGC CCTGCCATAG CCTCAGGTTA CTCATATATA 
3841 CTTTAGATTG ATTTAAAACT TCATTTTTAA TTTAAAAGGA TCTAGGTGAA GATCCTTTTT 
3901 GATAATCTCA TGACCAAAAT CCCTTAACGT GAGTTTTCGT TCCACTGAGC GTCAGACCCC 
3961 GTAGAAAAGA TCAAAGGATC TTCTTGAGAT CCTTTTTTTC TGCGCGTAAT CTGCTGCTTG 
4021 CAAACAAAAA AACCACCGCT ACCAGCGGTG GTTTGTTTGC CGGATCAAGA GCTACCAACT 
4081 CTTTTTCCGA AGGTAACTGG CTTCAGCAGA GCGCAGATAC CAAATACTGT CCTTCTAGTG 
4141 TAGCCGTAGT TAGGCCACCA CTTCAAGAAC TCTGTAGCAC CGCCTACATA CCTCGCTCTG 
4201 CTAATCCTGT TACCAGTGGC TGCTGCCAGT GGCGATAAGT CGTGTCTTAC CGGGTTGGAC 
4261 TCAAGACGAT AGTTACCGGA TAAGGCGCAG CGGTCGGGCT GAACGGGGGG TTCGTGCACA 
4321 CAGCCCAGCT TGGAGCGAAC GACCTACACC GAACTGAGAT ACCTACAGCG TGAGCTATGA 
4381 GAAAGCGCCA CGCTTCCCGA AGGGAGAAAG GCGGACAGGT ATCCGGTAAG CGGCAGGGTC 
4441 GGAACAGGAG AGCGCACGAG GGAGCTTCCA GGGGGAAACG CCTGGTATCT TTATAGTCCT 
4501 GTCGGGTTTC GCCACCTCTG ACTTGAGCGT CGATTTTTGT GATGCTCGTC AGGGGGGCGG 
4561 AGCCTATGGA AAAACGCCAG CAACGCGGCC TTTTTACGGT TCCTGGCCTT TTGCTGGCCT 
4621 TTTGCTCACA TGTTCTTTCC TGCGTTATCC CCTGATTCTG TGGATAACCG TATTACCGCC 
4681 ATGCAT