pIRES2-DsRed2 contains the internal ribosome entry site (IRES; 1, 2) of the encephalomyocarditis virus (ECMV) between the MCS and the Discosoma sp. red fluorescent protein (DsRed2; 3, 4) coding region. This permits both the gene of interest (cloned into the MCS) and the DsRed2 gene to be translated from a single bicistronic mRNA. pIRES2-DsRed2 is designed for the efficient selection (by flow cytometry or other methods) of transiently transfected mammalian cells expressing DsRed2 and the protein of interest. Thisvector can express DsRed2 alone at lower signal intensity. The vector can also be used to obtain stably transfected cell lines by drug and clonal selection.

DsRed2 is a human codon-optimized (5) DsRed variant engineered for faster maturation and lower non-specific aggregation. The MCS in pIRES2-DsRed2 is located between the immediate early promoter of cytomegalovirus (PCMV IE) and the IRES sequence. SV40 polyadenylation signals downstream of the DsRed2 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 (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 pIRES2-DsRed2 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

pIRES2-DsRed2 can be used to quickly identify cells expressing a gene of interest by screening for DsRed2 fluorescence. Genes inserted into the MCS should include the initiating ATG codon. Selection of DsRed2-positive cells is possible 24 hr after transfection by flow cytometry or fluorescence microscopy. However, in some cases, up to 48 hr may be required for detection of red-emitting cells. pIRES2-DsRed2 and its derivatives can be introduced into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (6).

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 GATCCGCCCC TCTCCCTCCC CCCCCCCTAA CGTTACTGGC CGAAGCCGCT TGGAATAAGG 
0721 CCGGTGTGCG TTTGTCTATA TGTTATTTTC CACCATATTG CCGTCTTTTG GCAATGTGAG 
0781 GGCCCGGAAA CCTGGCCCTG TCTTCTTGAC GAGCATTCCT AGGGGTCTTT CCCCTCTCGC 
0841 CAAAGGAATG CAAGGTCTGT TGAATGTCGT GAAGGAAGCA GTTCCTCTGG AAGCTTCTTG 
0901 AAGACAAACA ACGTCTGTAG CGACCCTTTG CAGGCAGCGG AACCCCCCAC CTGGCGACAG 
0961 GTGCCTCTGC GGCCAAAAGC CACGTGTATA AGATACACCT GCAAAGGCGG CACAACCCCA 
1021 GTGCCACGTT GTGAGTTGGA TAGTTGTGGA AAGAGTCAAA TGGCTCTCCT CAAGCGTATT 
1081 CAACAAGGGG CTGAAGGATG CCCAGAAGGT ACCCCATTGT ATGGGATCTG ATCTGGGGCC 
1141 TCGGTGCACA TGCTTTACAT GTGTTTAGTC GAGGTTAAAA AAACGTCTAG GCCCCCCGAA 
1201 CCACGGGGAC GTGGTTTTCC TTTGAAAAAC ACGATGATAA TATGGCCACA ACCATGGCCT 
1261 CCTCCGAGAA CGTCATCACC GAGTTCATGC GCTTCAAGGT GCGCATGGAG GGCACCGTGA 
1321 ACGGCCACGA GTTCGAGATC GAGGGCGAGG GCGAGGGCCG CCCCTACGAG GGCCACAACA 
1381 CCGTGAAGCT GAAGGTGACC AAGGGCGGCC CCCTGCCCTT CGCCTGGGAC ATCCTGTCCC 
1441 CCCAGTTCCA GTACGGCTCC AAGGTGTACG TGAAGCACCC CGCCGACATC CCCGACTACA 
1501 AGAAGCTGTC CTTCCCCGAG GGCTTCAAGT GGGAGCGCGT GATGAACTTC GAGGACGGCG 
1561 GCGTGGCGAC CGTGACCCAG GACTCCTCCC TGCAGGACGG CTGCTTCATC TACAAGGTGA 
1621 AGTTCATCGG CGTGAACTTC CCCTCCGACG GCCCCGTGAT GCAGAAGAAG ACCATGGGCT 
1681 GGGAGGCCTC CACCGAGCGC CTGTACCCCC GCGACGGCGT GCTGAAGGGC GAGACCCACA 
1741 AGGCCCTGAA GCTGAAGGAC GGCGGCCACT ACCTGGTGGA GTTCAAGTCT ATCTACATGG 
1801 CCAAGAAGCC CGTGCAGCTG CCCGGCTACT ACTACGTGGA CGCCAAGCTG GACATCACCT 
1861 CCCACAACGA GGACTACACC ATCGTGGAGC AGTACGAGCG CACCGAGGGC CGCCACCACC 
1921 TGTTCCTGTA GCGGCCGCGA CTCTAGATCA TAATCAGCCA TACCACATTT GTAGAGGTTT 
1981 TACTTGCTTT AAAAAACCTC CCACACCTCC CCCTGAACCT GAAACATAAA ATGAATGCAA 
2041 TTGTTGTTGT TAACTTGTTT ATTGCAGCTT ATAATGGTTA CAAATAAAGC AATAGCATCA 
2101 CAAATTTCAC AAATAAAGCA TTTTTTTCAC TGCATTCTAG TTGTGGTTTG TCCAAACTCA 
2161 TCAATGTATC TTAAGGCGTA AATTGTAAGC GTTAATATTT TGTTAAAATT CGCGTTAAAT 
2221 TTTTGTTAAA TCAGCTCATT TTTTAACCAA TAGGCCGAAA TCGGCAAAAT CCCTTATAAA 
2281 TCAAAAGAAT AGACCGAGAT AGGGTTGAGT GTTGTTCCAG TTTGGAACAA GAGTCCACTA 
2341 TTAAAGAACG TGGACTCCAA CGTCAAAGGG CGAAAAACCG TCTATCAGGG CGATGGCCCA 
2401 CTACGTGAAC CATCACCCTA ATCAAGTTTT TTGGGGTCGA GGTGCCGTAA AGCACTAAAT 
2461 CGGAACCCTA AAGGGAGCCC CCGATTTAGA GCTTGACGGG GAAAGCCGGC GAACGTGGCG 
2521 AGAAAGGAAG GGAAGAAAGC GAAAGGAGCG GGCGCTAGGG CGCTGGCAAG TGTAGCGGTC 
2581 ACGCTGCGCG TAACCACCAC ACCCGCCGCG CTTAATGCGC CGCTACAGGG CGCGTCAGGT 
2641 GGCACTTTTC GGGGAAATGT GCGCGGAACC CCTATTTGTT TATTTTTCTA AATACATTCA 
2701 AATATGTATC CGCTCATGAG ACAATAACCC TGATAAATGC TTCAATAATA TTGAAAAAGG 
2761 AAGAGTCCTG AGGCGGAAAG AACCAGCTGT GGAATGTGTG TCAGTTAGGG TGTGGAAAGT 
2821 CCCCAGGCTC CCCAGCAGGC AGAAGTATGC AAAGCATGCA TCTCAATTAG TCAGCAACCA 
2881 GGTGTGGAAA GTCCCCAGGC TCCCCAGCAG GCAGAAGTAT GCAAAGCATG CATCTCAATT 
2941 AGTCAGCAAC CATAGTCCCG CCCCTAACTC CGCCCATCCC GCCCCTAACT CCGCCCAGTT 
3001 CCGCCCATTC TCCGCCCCAT GGCTGACTAA TTTTTTTTAT TTATGCAGAG GCCGAGGCCG 
3061 CCTCGGCCTC TGAGCTATTC CAGAAGTAGT GAGGAGGCTT TTTTGGAGGC CTAGGCTTTT 
3121 GCAAAGATCG ATCAAGAGAC AGGATGAGGA TCGTTTCGCA TGATTGAACA AGATGGATTG 
3181 CACGCAGGTT CTCCGGCCGC TTGGGTGGAG AGGCTATTCG GCTATGACTG GGCACAACAG 
3241 ACAATCGGCT GCTCTGATGC CGCCGTGTTC CGGCTGTCAG CGCAGGGGCG CCCGGTTCTT 
3301 TTTGTCAAGA CCGACCTGTC CGGTGCCCTG AATGAACTGC AAGACGAGGC AGCGCGGCTA 
3361 TCGTGGCTGG CCACGACGGG CGTTCCTTGC GCAGCTGTGC TCGACGTTGT CACTGAAGCG 
3421 GGAAGGGACT GGCTGCTATT GGGCGAAGTG CCGGGGCAGG ATCTCCTGTC ATCTCACCTT 
3481 GCTCCTGCCG AGAAAGTATC CATCATGGCT GATGCAATGC GGCGGCTGCA TACGCTTGAT 
3541 CCGGCTACCT GCCCATTCGA CCACCAAGCG AAACATCGCA TCGAGCGAGC ACGTACTCGG 
3601 ATGGAAGCCG GTCTTGTCGA TCAGGATGAT CTGGACGAAG AGCATCAGGG GCTCGCGCCA 
3661 GCCGAACTGT TCGCCAGGCT CAAGGCGAGC ATGCCCGACG GCGAGGATCT CGTCGTGACC 
3721 CATGGCGATG CCTGCTTGCC GAATATCATG GTGGAAAATG GCCGCTTTTC TGGATTCATC 
3781 GACTGTGGCC GGCTGGGTGT GGCGGACCGC TATCAGGACA TAGCGTTGGC TACCCGTGAT 
3841 ATTGCTGAAG AGCTTGGCGG CGAATGGGCT GACCGCTTCC TCGTGCTTTA CGGTATCGCC 
3901 GCTCCCGATT CGCAGCGCAT CGCCTTCTAT CGCCTTCTTG ACGAGTTCTT CTGAGCGGGA 
3961 CTCTGGGGTT CGAAATGACC GACCAAGCGA CGCCCAACCT GCCATCACGA GATTTCGATT 
4021 CCACCGCCGC CTTCTATGAA AGGTTGGGCT TCGGAATCGT TTTCCGGGAC GCCGGCTGGA 
4081 TGATCCTCCA GCGCGGGGAT CTCATGCTGG AGTTCTTCGC CCACCCTAGG GGGAGGCTAA 
4141 CTGAAACACG GAAGGAGACA ATACCGGAAG GAACCCGCGC TATGACGGCA ATAAAAAGAC 
4201 AGAATAAAAC GCACGGTGTT GGGTCGTTTG TTCATAAACG CGGGGTTCGG TCCCAGGGCT 
4261 GGCACTCTGT CGATACCCCA CCGAGACCCC ATTGGGGCCA ATACGCCCGC GTTTCTTCCT 
4321 TTTCCCCACC CCACCCCCCA AGTTCGGGTG AAGGCCCAGG GCTCGCAGCC AACGTCGGGG 
4381 CGGCAGGCCC TGCCATAGCC TCAGGTTACT CATATATACT TTAGATTGAT TTAAAACTTC 
4441 ATTTTTAATT TAAAAGGATC TAGGTGAAGA TCCTTTTTGA TAATCTCATG ACCAAAATCC 
4501 CTTAACGTGA GTTTTCGTTC CACTGAGCGT CAGACCCCGT AGAAAAGATC AAAGGATCTT 
4561 CTTGAGATCC TTTTTTTCTG CGCGTAATCT GCTGCTTGCA AACAAAAAAA CCACCGCTAC 
4621 CAGCGGTGGT TTGTTTGCCG GATCAAGAGC TACCAACTCT TTTTCCGAAG GTAACTGGCT 
4681 TCAGCAGAGC GCAGATACCA AATACTGTCC TTCTAGTGTA GCCGTAGTTA GGCCACCACT 
4741 TCAAGAACTC TGTAGCACCG CCTACATACC TCGCTCTGCT AATCCTGTTA CCAGTGGCTG 
4801 CTGCCAGTGG CGATAAGTCG TGTCTTACCG GGTTGGACTC AAGACGATAG TTACCGGATA 
4861 AGGCGCAGCG GTCGGGCTGA ACGGGGGGTT CGTGCACACA GCCCAGCTTG GAGCGAACGA 
4921 CCTACACCGA ACTGAGATAC CTACAGCGTG AGCTATGAGA AAGCGCCACG CTTCCCGAAG 
4981 GGAGAAAGGC GGACAGGTAT CCGGTAAGCG GCAGGGTCGG AACAGGAGAG CGCACGAGGG 
5041 AGCTTCCAGG GGGAAACGCC TGGTATCTTT ATAGTCCTGT CGGGTTTCGC CACCTCTGAC 
5101 TTGAGCGTCG ATTTTTGTGA TGCTCGTCAG GGGGGCGGAG CCTATGGAAA AACGCCAGCA 
5161 ACGCGGCCTT TTTACGGTTC CTGGCCTTTT GCTGGCCTTT TGCTCACATG TTCTTTCCTG 
5221 CGTTATCCCC TGATTCTGTG GATAACCGTA TTACCGCCAT GCAT