pDsRed1-N1

pDsRed1-N1 encodes a novel red fluorescent protein (RFP; 1) that has been optimized for high expression in mammalian cells (excitation maximum = 558 nm; emission maximum = 583 nm). RFP was 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 (2). Sequences upstream of DsRed1 have been converted to a Kozak consensus translation initiation site (3) to increase translation efficiency in eukaryotic cells. The MCS is between the immediate early promoter of CMV (PCMV IE) and the DsRed1 coding sequence. Genes cloned into the MCS as described below are expressed as fusions to the N-terminus of DsRed1. SV40 polyadenylation signals downstream of the DsRed1 gene direct proper processing of the 3' end of the DsRed1 mRNA. The vector backbone contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen. 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 to E. coli. The pDsRed1-N1 backbone also has a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

载体应用

Fusions to the N terminus of DsRed1 typically do not alter the fluorescence properties of native DsRed1, allowing in vivo localization of the fusion protein.The target gene should be cloned into pDsRed1-N1 in frame with the DsRed1 coding sequence, with no intervening in-frame stop codons. The inserted gene should include an initiating ATG codon. Recombinant pDsRed1-N1 can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418 (4). Unmodified pDsRed1-N1 can also be used to express DsRed1 in a cell line of interest (e.g., for use as a transfection 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 CCGGACTCAG ATCTCGAGCT CAAGCTTCGA ATTCTGCAGT CGACGGTACC GCGGGCCCGG 
0661 GATCCACCGG TCGCCACCAT GGTGCGCTCC TCCAAGAACG TCATCAAGGA GTTCATGCGC 
0721 TTCAAGGTGC GCATGGAGGG CACCGTGAAC GGCCACGAGT TCGAGATCGA GGGCGAGGGC 
0781 GAGGGCCGCC CCTACGAGGG CCACAACACC GTGAAGCTGA AGGTGACCAA GGGCGGCCCC 
0841 CTGCCCTTCG CCTGGGACAT CCTGTCCCCC CAGTTCCAGT ACGGCTCCAA GGTGTACGTG 
0901 AAGCACCCCG CCGACATCCC CGACTACAAG AAGCTGTCCT TCCCCGAGGG CTTCAAGTGG 
0961 GAGCGCGTGA TGAACTTCGA GGACGGCGGC GTGGTGACCG TGACCCAGGA CTCCTCCCTG 
1021 CAGGACGGCT GCTTCATCTA CAAGGTGAAG TTCATCGGCG TGAACTTCCC CTCCGACGGC 
1081 CCCGTAATGC AGAAGAAGAC CATGGGCTGG GAGGCCTCCA CCGAGCGCCT GTACCCCCGC 
1141 GACGGCGTGC TGAAGGGCGA GATCCACAAG GCCCTGAAGC TGAAGGACGG CGGCCACTAC 
1201 CTGGTGGAGT TCAAGTCCAT CTACATGGCC AAGAAGCCCG TGCAGCTGCC CGGCTACTAC 
1261 TACGTGGACT CCAAGCTGGA CATCACCTCC CACAACGAGG ACTACACCAT CGTGGAGCAG 
1321 TACGAGCGCA CCGAGGGCCG CCACCACCTG TTCCTGTAGC GGCCGCGACT CTAGATCATA 
1381 ATCAGCCATA CCACATTTGT AGAGGTTTTA CTTGCTTTAA AAAACCTCCC ACACCTCCCC 
1441 CTGAACCTGA AACATAAAAT GAATGCAATT GTTGTTGTTA ACTTGTTTAT TGCAGCTTAT 
1501 AATGGTTACA AATAAAGCAA TAGCATCACA AATTTCACAA ATAAAGCATT TTTTTCACTG 
1561 CATTCTAGTT GTGGTTTGTC CAAACTCATC AATGTATCTT AAGGCGTAAA TTGTAAGCGT 
1621 TAATATTTTG TTAAAATTCG CGTTAAATTT TTGTTAAATC AGCTCATTTT TTAACCAATA 
1681 GGCCGAAATC GGCAAAATCC CTTATAAATC AAAAGAATAG ACCGAGATAG GGTTGAGTGT 
1741 TGTTCCAGTT TGGAACAAGA GTCCACTATT AAAGAACGTG GACTCCAACG TCAAAGGGCG 
1801 AAAAACCGTC TATCAGGGCG ATGGCCCACT ACGTGAACCA TCACCCTAAT CAAGTTTTTT 
1861 GGGGTCGAGG TGCCGTAAAG CACTAAATCG GAACCCTAAA GGGAGCCCCC GATTTAGAGC 
1921 TTGACGGGGA AAGCCGGCGA ACGTGGCGAG AAAGGAAGGG AAGAAAGCGA AAGGAGCGGG 
1981 CGCTAGGGCG CTGGCAAGTG TAGCGGTCAC GCTGCGCGTA ACCACCACAC CCGCCGCGCT 
2041 TAATGCGCCG CTACAGGGCG CGTCAGGTGG CACTTTTCGG GGAAATGTGC GCGGAACCCC 
2101 TATTTGTTTA TTTTTCTAAA TACATTCAAA TATGTATCCG CTCATGAGAC AATAACCCTG 
2161 ATAAATGCTT CAATAATATT GAAAAAGGAA GAGTCCTGAG GCGGAAAGAA CCAGCTGTGG 
2221 AATGTGTGTC AGTTAGGGTG TGGAAAGTCC CCAGGCTCCC CAGCAGGCAG AAGTATGCAA 
2281 AGCATGCATC TCAATTAGTC AGCAACCAGG TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC 
2341 AGAAGTATGC AAAGCATGCA TCTCAATTAG TCAGCAACCA TAGTCCCGCC CCTAACTCCG 
2401 CCCATCCCGC CCCTAACTCC GCCCAGTTCC GCCCATTCTC CGCCCCATGG CTGACTAATT 
2461 TTTTTTATTT ATGCAGAGGC CGAGGCCGCC TCGGCCTCTG AGCTATTCCA GAAGTAGTGA 
2521 GGAGGCTTTT TTGGAGGCCT AGGCTTTTGC AAAGATCGAT CAAGAGACAG GATGAGGATC 
2581 GTTTCGCATG ATTGAACAAG ATGGATTGCA CGCAGGTTCT CCGGCCGCTT GGGTGGAGAG 
2641 GCTATTCGGC TATGACTGGG CACAACAGAC AATCGGCTGC TCTGATGCCG CCGTGTTCCG 
2701 GCTGTCAGCG CAGGGGCGCC CGGTTCTTTT TGTCAAGACC GACCTGTCCG GTGCCCTGAA 
2761 TGAACTGCAA GACGAGGCAG CGCGGCTATC GTGGCTGGCC ACGACGGGCG TTCCTTGCGC 
2821 AGCTGTGCTC GACGTTGTCA CTGAAGCGGG AAGGGACTGG CTGCTATTGG GCGAAGTGCC 
2881 GGGGCAGGAT CTCCTGTCAT CTCACCTTGC TCCTGCCGAG AAAGTATCCA TCATGGCTGA 
2941 TGCAATGCGG CGGCTGCATA CGCTTGATCC GGCTACCTGC CCATTCGACC ACCAAGCGAA 
3001 ACATCGCATC GAGCGAGCAC GTACTCGGAT GGAAGCCGGT CTTGTCGATC AGGATGATCT 
3061 GGACGAAGAG CATCAGGGGC TCGCGCCAGC CGAACTGTTC GCCAGGCTCA AGGCGAGCAT 
3121 GCCCGACGGC GAGGATCTCG TCGTGACCCA TGGCGATGCC TGCTTGCCGA ATATCATGGT 
3181 GGAAAATGGC CGCTTTTCTG GATTCATCGA CTGTGGCCGG CTGGGTGTGG CGGACCGCTA 
3241 TCAGGACATA GCGTTGGCTA CCCGTGATAT TGCTGAAGAG CTTGGCGGCG AATGGGCTGA 
3301 CCGCTTCCTC GTGCTTTACG GTATCGCCGC TCCCGATTCG CAGCGCATCG CCTTCTATCG 
3361 CCTTCTTGAC GAGTTCTTCT GAGCGGGACT CTGGGGTTCG AAATGACCGA CCAAGCGACG 
3421 CCCAACCTGC CATCACGAGA TTTCGATTCC ACCGCCGCCT TCTATGAAAG GTTGGGCTTC 
3481 GGAATCGTTT TCCGGGACGC CGGCTGGATG ATCCTCCAGC GCGGGGATCT CATGCTGGAG 
3541 TTCTTCGCCC ACCCTAGGGG GAGGCTAACT GAAACACGGA AGGAGACAAT ACCGGAAGGA 
3601 ACCCGCGCTA TGACGGCAAT AAAAAGACAG AATAAAACGC ACGGTGTTGG GTCGTTTGTT 
3661 CATAAACGCG GGGTTCGGTC CCAGGGCTGG CACTCTGTCG ATACCCCACC GAGACCCCAT 
3721 TGGGGCCAAT ACGCCCGCGT TTCTTCCTTT TCCCCACCCC ACCCCCCAAG TTCGGGTGAA 
3781 GGCCCAGGGC TCGCAGCCAA CGTCGGGGCG GCAGGCCCTG CCATAGCCTC AGGTTACTCA 
3841 TATATACTTT AGATTGATTT AAAACTTCAT TTTTAATTTA AAAGGATCTA GGTGAAGATC 
3901 CTTTTTGATA ATCTCATGAC CAAAATCCCT TAACGTGAGT TTTCGTTCCA CTGAGCGTCA 
3961 GACCCCGTAG AAAAGATCAA AGGATCTTCT TGAGATCCTT TTTTTCTGCG CGTAATCTGC 
4021 TGCTTGCAAA CAAAAAAACC ACCGCTACCA GCGGTGGTTT GTTTGCCGGA TCAAGAGCTA 
4081 CCAACTCTTT TTCCGAAGGT AACTGGCTTC AGCAGAGCGC AGATACCAAA TACTGTCCTT 
4141 CTAGTGTAGC CGTAGTTAGG CCACCACTTC AAGAACTCTG TAGCACCGCC TACATACCTC 
4201 GCTCTGCTAA TCCTGTTACC AGTGGCTGCT GCCAGTGGCG ATAAGTCGTG TCTTACCGGG 
4261 TTGGACTCAA GACGATAGTT ACCGGATAAG GCGCAGCGGT CGGGCTGAAC GGGGGGTTCG 
4321 TGCACACAGC CCAGCTTGGA GCGAACGACC TACACCGAAC TGAGATACCT ACAGCGTGAG 
4381 CTATGAGAAA GCGCCACGCT TCCCGAAGGG AGAAAGGCGG ACAGGTATCC GGTAAGCGGC 
4441 AGGGTCGGAA CAGGAGAGCG CACGAGGGAG CTTCCAGGGG GAAACGCCTG GTATCTTTAT 
4501 AGTCCTGTCG GGTTTCGCCA CCTCTGACTT GAGCGTCGAT TTTTGTGATG CTCGTCAGGG 
4561 GGGCGGAGCC TATGGAAAAA CGCCAGCAAC GCGGCCTTTT TACGGTTCCT GGCCTTTTGC 
4621 TGGCCTTTTG CTCACATGTT CTTTCCTGCG TTATCCCCTG ATTCTGTGGA TAACCGTATT 
4681 ACCGCCATGC AT