pIRES2-DsRed-Express

pIRES2-DsRed-Express contains the internal ribosome entry site (IRES; 1, 2) of the encephalomyocarditis virus (ECMV) between the MCS and a mutant of the Discosoma sp. red fluorescent protein DsRed-Express coding region (3–5). This permits both the gene of interest (cloned into the MCS) and the DsRed-Express gene to be translated from a single bicistronic mRNA. pIRES2-DsRed-Express is designed for the efficient selection by flow cytometry of transiently transfected mammalian cells expressing DsRed-Express and the protein of interest. This vector can also be used to express DsRed-Express alone or to obtain stably transfected cell lines without timeconsuming drug and clonal selection.

DsRed-Express is a human codon-optimized (4) DsRed variant of Discosoma sp. red fluorescent protein (6). Its coding sequence contains nine amino acid substitutions which improve solubility of the protein and reduce the time from transfection to detection of red fluorescence (exitation and emission maxima = 557 nm and 579 nm, respectively). In addition, these substitutions reduce the level of residual green emission (5). Although DsRed-Express most likely forms the same tetrameric structure as wild-type DsRed, DsRed-Express displays a reduced tendency to aggregate (3, 5).

The MCS in pIRES2-DsRed-Express is between the immediate early promoter of cytomegalovirus (P CMV IE ) and the IRES sequence. SV40 polyadenylation signals downstream of the DsRed-Express gene direct proper processing of the 3' end of the bicistronic mRNA. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen. A neomycin-resistance cassette (Neo r ), 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 pIRES2-DsRed-Express backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

0001 ATGCATTAGT TATTAATAGT AATCAATTAC GGGGTCATTA GTTCATAGCC CATATATGGA 
0061 GTTCCGCGTT ACATAACTTA CGGTAAATGG CCCGCCTGGC TGACCGCCCA ACGACCCCCG 
0121 CCCATTGACG TCAATAATGA CGTATGTTCC CATAGTAACG CCAATAGGGA CTTTCCATTG 
0181 ACGTCAATGG GTGGAGTATT TACGGTAAAC TGCCCACTTG GCAGTACATC AAGTGTATCA 
0241 TATGCCAAGT ACGCCCCCTA TTGACGTCAA TGACGGTAAA TGGCCCGCCT GGCATTATGC 
0301 CCAGTACATG ACCTTATGGG ACTTTCCTAC TTGGCAGTAC ATCTACGTAT TAGTCATCGC 
0361 TATTACCATG GTGATGCGGT TTTGGCAGTA CATCAATGGG CGTGGATAGC GGTTTGACTC 
0421 ACGGGGATTT CCAAGTCTCC ACCCCATTGA CGTCAATGGG AGTTTGTTTT GGCACCAAAA 
0481 TCAACGGGAC TTTCCAAAAT GTCGTAACAA CTCCGCCCCA TTGACGCAAA TGGGCGGTAG 
0541 GCGTGTACGG TGGGAGGTCT ATATAAGCAG AGCTGGTTTA GTGAACCGTC AGATCCGCTA 
0601 GCGATTACGC CAAGCTCGAA ATTAACCCTC ACTAAAGGGA ACAAAAGCTG GAGCTCCACC 
0661 GCGGTGGCGG CCGCCATGGA GCAGAAACTC ATCTCTGAAG AGGATCTGGC CCGGGCGGAT 
0721 CCCCCGGGCT GCAGGAATTC GATATCAAGC TTATCGATAC CGTCGACCTC GAGGGGGGGC 
0781 CCGGTACCTT AATTAATTAA GGTACCAGGT AAGTGTACCC AATTCGCCCT ATAGTGAGTC 
0841 GTATTACAAT TCACTCGATC GCCCTTCCCA ACAGTTGCGC AGCCTGAATG GCGAATGGAG 
0901 ATCCAATTTT TAAGTGTATA ATGTGTTAAA CTACTGATTC TAATTGTTTG TGTATTTTAG 
0961 ATTCACAGTC CCAAGGCTCA TTTCAGGCCC CTCAGTCCTC ACAGTCTGTT CATGATCATA 
1021 ATCAGCCATA CCACATTTGT AGAGGTTTTA CTTGCTTTAA AAAACCTCCC ACACCTCCCC 
1081 CTGAACCTGA AACATAAAAT GAATGCAATT GTTGTTGTTA ACTTGTTTAT TGCAGCTTAT 
1141 AATGGTTACA AATAAAGCAA TAGCATCACA AATTTCACAA ATAAAGCATT TTTTTCACTG 
1201 CATTCTAGTT GTGGTTTGTC CAAACTCATC AATGTATCTT AACGCGTAAA TTGTAAGCGT 
1261 TAATATTTTG TTAAAATTCG CGTTAAATTT TTGTTAAATC AGCTCATTTT TTAACCAATA 
1321 GGCCGAAATC GGCAAAATCC CTTATAAATC AAAAGAATAG ACCGAGATAG GGTTGAGTGT 
1381 TGTTCCAGTT TGGAACAAGA GTCCACTATT AAAGAACGTG GACTCCAACG TCAAAGGGCG 
1441 AAAAACCGTC TATCAGGGCG ATGGCCCACT ACGTGAACCA TCACCCTAAT CAAGTTTTTT 
1501 GGGGTCGAGG TGCCGTAAAG CACTAAATCG GAACCCTAAA GGGAGCCCCC GATTTAGAGC 
1561 TTGACGGGGA AAGCCGGCGA ACGTGGCGAG AAAGGAAGGG AAGAAAGCGA AAGGAGCGGG 
1621 CGCTAGGGCG CTGGCAAGTG TAGCGGTCAC GCTGCGCGTA ACCACCACAC CCGCCGCGCT 
1681 TAATGCGCCG CTACAGGGCG CGTCAGGTGG CACTTTTCGG GGAAATGTGC GCGGAACCCC 
1741 TATTTGTTTA TTTTTCTAAA TACATTCAAA TATGTATCCG CTCATGAGAC AATAACCCTG 
1801 ATAAATGCTT CAATAATATT GAAAAAGGAA GAATCCTGAG GCGGAAAGAA CCAGCTGTGG 
1861 AATGTGTGTC AGTTAGGGTG TGGAAAGTCC CCAGGCTCCC CAGCAGGCAG AAGTATGCAA 
1921 AGCATGCATC TCAATTAGTC AGCAACCAGG TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC 
1981 AGAAGTATGC AAAGCATGCA TCTCAATTAG TCAGCAACCA TAGTCCCGCC CCTAACTCCG 
2041 CCCATCCCGC CCCTAACTCC GCCCAGTTCC GCCCATTCTC CGCCCCATGG CTGACTAATT 
2101 TTTTTTATTT ATGCAGAGGC CGAGGCCGCC TCGGCCTCTG AGCTATTCCA GAAGTAGTGA 
2161 GGAGGCTTTT TTGGAGGCCT AGGCTTTTGC AAAGATCGAT CAAGAGACAG GATGAGGATC 
2221 GTTTCGCATG ATTGAACAAG ATGGATTGCA CGCAGGTTCT CCGGCCGCTT GGGTGGAGAG 
2281 GCTATTCGGC TATGACTGGG CACAACAGAC AATCGGCTGC TCTGATGCCG CCGTGTTCCG 
2341 GCTGTCAGCG CAGGGGCGCC CGGTTCTTTT TGTCAAGACC GACCTGTCCG GTGCCCTGAA 
2401 TGAACTGCAA GACGAGGCAG CGCGGCTATC GTGGCTGGCC ACGACGGGCG TTCCTTGCGC 
2461 AGCTGTGCTC GACGTTGTCA CTGAAGCGGG AAGGGACTGG CTGCTATTGG GCGAAGTGCC 
2521 GGGGCAGGAT CTCCTGTCAT CTCACCTTGC TCCTGCCGAG AAAGTATCCA TCATGGCTGA 
2581 TGCAATGCGG CGGCTGCATA CGCTTGATCC GGCTACCTGC CCATTCGACC ACCAAGCGAA 
2641 ACATCGCATC GAGCGAGCAC GTACTCGGAT GGAAGCCGGT CTTGTCGATC AGGATGATCT 
2701 GGACGAAGAA CATCAGGGGC TCGCGCCAGC CGAACTGTTC GCCAGGCTCA AGGCGAGCAT 
2761 GCCCGACGGC GAGGATCTCG TCGTGACCCA TGGCGATGCC TGCTTGCCGA ATATCATGGT 
2821 GGAAAATGGC CGCTTTTCTG GATTCATCGA CTGTGGCCGG CTGGGTGTGG CGGACCGCTA 
2881 TCAGGACATA GCGTTGGCTA CCCGTGATAT TGCTGAAGAA CTTGGCGGCG AATGGGCTGA 
2941 CCGCTTCCTC GTGCTTTACG GTATCGCCGC TCCCGATTCG CAGCGCATCG CCTTCTATCG 
3001 CCTTCTTGAC GAGTTCTTCT GAGCGGGACT CTGGGGTTCG AAATGACCGA CCAAGCGACG 
3061 CCCAACCTGC CATCACGAGA TTTCGATTCC ACCGCCGCCT TCTATGAAAG GTTGGGCTTC 
3121 GGAATCGTTT TCCGGGACGC CGGCTGGATG ATCCTCCAGC GCGGGGATCT CATGCTGGAG 
3181 TTCTTCGCCC ACCCTAGGGG GAGGCTAACT GAAACACGGA AGGAGACAAT ACCGGAAGGA 
3241 ACCCGCGCTA TGACGGCAAT AAAAAGACAG AATAAAACGC ACGGTGTTGG GTCGTTTGTT 
3301 CATAAACGCG GGGTTCGGTC CCAGGGCTGG CACTCTGTCG ATACCCCACC GAGACCCCAT 
3361 TGGGGCCAAT ACGCCCGCGT TTCTTCCTTT TCCCCACCCC ACCCCCCAAG TTCGGGTGAA 
3421 GGCCCAGGGC TCGCAGCCAA CGTCGGGGCG GCAGGCCCTG CCATAGCCTC AGGTTACTCA 
3481 TATATACTTT AGATTGATTT AAAACTTCAT TTTTAATTTA AAAGGATCTA GGTGAAGATC 
3541 CTTTTTGATA ATCTCATGAC CAAAATCCCT TAACGTGAGT TTTCGTTCCA CTGAGCGTCA 
3601 GACCCCGTAG AAAAGATCAA AGGATCTTCT TGAGATCCTT TTTTTCTGCG CGTAATCTGC 
3661 TGCTTGCAAA CAAAAAAACC ACCGCTACCA GCGGTGGTTT GTTTGCCGGA TCAAGAGCTA 
3721 CCAACTCTTT TTCCGAAGGT AACTGGCTTC AGCAGAGCGC AGATACCAAA TACTGTCCTT 
3781 CTAGTGTAGC CGTAGTTAGG CCACCACTTC AAGAACTCTG TAGCACCGCC TACATACCTC 
3841 GCTCTGCTAA TCCTGTTACC AGTGGCTGCT GCCAGTGGCG ATAAGTCGTG TCTTACCGGG 
3901 TTGGACTCAA GACGATAGTT ACCGGATAAG GCGCAGCGGT CGGGCTGAAC GGGGGGTTCG 
3961 TGCACACAGC CCAGCTTGGA GCGAACGACC TACACCGAAC TGAGATACCT ACAGCGTGAG 
4021 CTATGAGAAA GCGCCACGCT TCCCGAAGGG AGAAAGGCGG ACAGGTATCC GGTAAGCGGC 
4081 AGGGTCGGAA CAGGAGAGCG CACGAGGGAG CTTCCAGGGG GAAACGCCTG GTATCTTTAT 
4141 AGTCCTGTCG GGTTTCGCCA CCTCTGACTT GAGCGTCGAT TTTTGTGATG CTCGTCAGGG 
4201 GGGCGGAGCC TATGGAAAAA CGCCAGCAAC GCGGCCTTTT TACGGTTCCT GGCCTTTTGC 
4261 TGGCCTTTTG CTCACATGTT CTTTCCTGCG TTATCCCCTG ATTCTGTGGA TAACCGTATT 
4321 ACCGCC