pIRES2-ZsGreen1 contains the internal ribosome entry site (IRES; 1, 2) of the encephalomyocarditis virus (ECMV) between an MCS and the Zoanthus sp. green fluorescent protein (ZsGreen; 3, 4) coding region. This design permits both the gene of interest (cloned into the MCS) and the ZsGreen1 gene to be translated from a single bicistronic mRNA. pIRES2-ZsGreen1 is designed for the efficient selection (by flow cytometry or other methods) of transiently transfected mammalian cells expressing ZsGreen1 and the protein of interest. This vector can be used to obtain stably transfected cell lines without time-consuming drug and clonal selection.

ZsGreen1 is a human codon-optimized (5) ZsGreen variant that encodes the brightest commercially available green fluorescent protein. The MCS in pIRES2-ZsGreen1 is located between the immediate early promoter of cytomegalovirus (PCMV IE) and the IRES sequence. SV40 polyadenylation signals downstream of the ZsGreen1 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-ZsGreen1 backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

載體應用

pIRES2-ZsGreen1 can be used to quickly identify cells expressing a gene of interest by screening for ZsGreen1 fluorescence. Genes inserted into the MCS should include an initiating ATG codon. Selection of ZsGreen1-positive cells is possible 24–36 hours after transfection by flow cytometry or fluorescence microscopy. pIRES2-ZsGreen1 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 TCGGTACACA TGCTTTACAT GTGTTTAGTC GAGGTTAAAA AAACGTCTAG GCCCCCCGAA 
1201 CCACGGGGAC GTGGTTTTCC TTTGAAAAAC ACGATGATAA TATGGCCACA ACCATGGCCC 
1261 AGTCCAAGCA CGGCCTGACC AAGGAGATGA CCATGAAGTA CCGCATGGAG GGCTGCGTGG 
1321 ACGGCCACAA GTTCGTGATC ACCGGCGAGG GCATCGGCTA CCCCTTCAAG GGCAAGCAGG 
1381 CCATCAACCT GTGCGTGGTG GAGGGCGGCC CCTTGCCCTT CGCCGAGGAC ATCTTGTCCG 
1441 CCGCCTTCAT GTACGGCAAC CGCGTGTTCA CCGAGTACCC CCAGGACATC GTCGACTACT 
1501 TCAAGAACTC CTGCCCCGCC GGCTACACCT GGGACCGCTC CTTCCTGTTC GAGGACGGCG 
1561 CCGTGTGCAT CTGCAACGCC GACATCACCG TGAGCGTGGA GGAGAACTGC ATGTACCACG 
1621 AGTCCAAGTT CTACGGCGTG AACTTCCCCG CCGACGGCCC CGTGATGAAG AAGATGACCG 
1681 ACAACTGGGA GCCCTCCTGC GAGAAGATCA TCCCCGTGCC CAAGCAGGGC ATCTTGAAGG 
1741 GCGACGTGAG CATGTACCTG CTGCTGAAGG ACGGTGGCCG CTTGCGCTGC CAGTTCGACA 
1801 CCGTGTACAA GGCCAAGTCC GTGCCCCGCA AGATGCCCGA CTGGCACTTC ATCCAGCACA 
1861 AGCTGACCCG CGAGGACCGC AGCGACGCCA AGAACCAGAA GTGGCACCTG ACCGAGCACG 
1921 CCATCGCCTC CGGCTCCGCC TTGCCCTGAG CGGCCGCGAC TCTAGATCAT AATCAGCCAT 
1981 ACCACATTTG TAGAGGTTTT ACTTGCTTTA AAAAACCTCC CACACCTCCC CCTGAACCTG 
2041 AAACATAAAA TGAATGCAAT TGTTGTTGTT AACTTGTTTA TTGCAGCTTA TAATGGTTAC 
2101 AAATAAAGCA ATAGCATCAC AAATTTCACA AATAAAGCAT TTTTTTCACT GCATTCTAGT 
2161 TGTGGTTTGT CCAAACTCAT CAATGTATCT TAAGGCGTAA ATTGTAAGCG TTAATATTTT 
2221 GTTAAAATTC GCGTTAAATT TTTGTTAAAT CAGCTCATTT TTTAACCAAT AGGCCGAAAT 
2281 CGGCAAAATC CCTTATAAAT CAAAAGAATA GACCGAGATA GGGTTGAGTG TTGTTCCAGT 
2341 TTGGAACAAG AGTCCACTAT TAAAGAACGT GGACTCCAAC GTCAAAGGGC GAAAAACCGT 
2401 CTATCAGGGC GATGGCCCAC TACGTGAACC ATCACCCTAA TCAAGTTTTT TGGGGTCGAG 
2461 GTGCCGTAAA GCACTAAATC GGAACCCTAA AGGGAGCCCC CGATTTAGAG CTTGACGGGG 
2521 AAAGCCGGCG AACGTGGCGA GAAAGGAAGG GAAGAAAGCG AAAGGAGCGG GCGCTAGGGC 
2581 GCTGGCAAGT GTAGCGGTCA CGCTGCGCGT AACCACCACA CCCGCCGCGC TTAATGCGCC 
2641 GCTACAGGGC GCGTCAGGTG GCACTTTTCG GGGAAATGTG CGCGGAACCC CTATTTGTTT 
2701 ATTTTTCTAA ATACATTCAA ATATGTATCC GCTCATGAGA CAATAACCCT GATAAATGCT 
2761 TCAATAATAT TGAAAAAGGA AGAGTCCTGA GGCGGAAAGA ACCAGCTGTG GAATGTGTGT 
2821 CAGTTAGGGT GTGGAAAGTC CCCAGGCTCC CCAGCAGGCA GAAGTATGCA AAGCATGCAT 
2881 CTCAATTAGT CAGCAACCAG GTGTGGAAAG TCCCCAGGCT CCCCAGCAGG CAGAAGTATG 
2941 CAAAGCATGC ATCTCAATTA GTCAGCAACC ATAGTCCCGC CCCTAACTCC GCCCATCCCG 
3001 CCCCTAACTC CGCCCAGTTC CGCCCATTCT CCGCCCCATG GCTGACTAAT TTTTTTTATT 
3061 TATGCAGAGG CCGAGGCCGC CTCGGCCTCT GAGCTATTCC AGAAGTAGTG AGGAGGCTTT 
3121 TTTGGAGGCC TAGGCTTTTG CAAAGATCGA TCAAGAGACA GGATGAGGAT CGTTTCGCAT 
3181 GATTGAACAA GATGGATTGC ACGCAGGTTC TCCGGCCGCT TGGGTGGAGA GGCTATTCGG 
3241 CTATGACTGG GCACAACAGA CAATCGGCTG CTCTGATGCC GCCGTGTTCC GGCTGTCAGC 
3301 GCAGGGGCGC CCGGTTCTTT TTGTCAAGAC CGACCTGTCC GGTGCCCTGA ATGAACTGCA 
3361 AGACGAGGCA GCGCGGCTAT CGTGGCTGGC CACGACGGGC GTTCCTTGCG CAGCTGTGCT 
3421 CGACGTTGTC ACTGAAGCGG GAAGGGACTG GCTGCTATTG GGCGAAGTGC CGGGGCAGGA 
3481 TCTCCTGTCA TCTCACCTTG CTCCTGCCGA GAAAGTATCC ATCATGGCTG ATGCAATGCG 
3541 GCGGCTGCAT ACGCTTGATC CGGCTACCTG CCCATTCGAC CACCAAGCGA AACATCGCAT 
3601 CGAGCGAGCA CGTACTCGGA TGGAAGCCGG TCTTGTCGAT CAGGATGATC TGGACGAAGA 
3661 GCATCAGGGG CTCGCGCCAG CCGAACTGTT CGCCAGGCTC AAGGCGAGCA TGCCCGACGG 
3721 CGAGGATCTC GTCGTGACCC ATGGCGATGC CTGCTTGCCG AATATCATGG TGGAAAATGG 
3781 CCGCTTTTCT GGATTCATCG ACTGTGGCCG GCTGGGTGTG GCGGACCGCT ATCAGGACAT 
3841 AGCGTTGGCT ACCCGTGATA TTGCTGAAGA GCTTGGCGGC GAATGGGCTG ACCGCTTCCT 
3901 CGTGCTTTAC GGTATCGCCG CTCCCGATTC GCAGCGCATC GCCTTCTATC GCCTTCTTGA 
3961 CGAGTTCTTC TGAGCGGGAC TCTGGGGTTC GAAATGACCG ACCAAGCGAC GCCCAACCTG 
4021 CCATCACGAG ATTTCGATTC CACCGCCGCC TTCTATGAAA GGTTGGGCTT CGGAATCGTT 
4081 TTCCGGGACG CCGGCTGGAT GATCCTCCAG CGCGGGGATC TCATGCTGGA GTTCTTCGCC 
4141 CACCCTAGGG GGAGGCTAAC TGAAACACGG AAGGAGACAA TACCGGAAGG AACCCGCGCT 
4201 ATGACGGCAA TAAAAAGACA GAATAAAACG CACGGTGTTG GGTCGTTTGT TCATAAACGC 
4261 GGGGTTCGGT CCCAGGGCTG GCACTCTGTC GATACCCCAC CGAGACCCCA TTGGGGCCAA 
4321 TACGCCCGCG TTTCTTCCTT TTCCCCACCC CACCCCCCAA GTTCGGGTGA AGGCCCAGGG 
4381 CTCGCAGCCA ACGTCGGGGC GGCAGGCCCT GCCATAGCCT CAGGTTACTC ATATATACTT 
4441 TAGATTGATT TAAAACTTCA TTTTTAATTT AAAAGGATCT AGGTGAAGAT CCTTTTTGAT 
4501 AATCTCATGA CCAAAATCCC TTAACGTGAG TTTTCGTTCC ACTGAGCGTC AGACCCCGTA 
4561 GAAAAGATCA AAGGATCTTC TTGAGATCCT TTTTTTCTGC GCGTAATCTG CTGCTTGCAA 
4621 ACAAAAAAAC CACCGCTACC AGCGGTGGTT TGTTTGCCGG ATCAAGAGCT ACCAACTCTT 
4681 TTTCCGAAGG TAACTGGCTT CAGCAGAGCG CAGATACCAA ATACTGTTCT TCTAGTGTAG 
4741 CCGTAGTTAG GCCACCACTT CAAGAACTCT GTAGCACCGC CTACATACCT CGCTCTGCTA 
4801 ATCCTGTTAC CAGTGGCTGC TGCCAGTGGC GATAAGTCGT GTCTTACCGG GTTGGACTCA 
4861 AGACGATAGT TACCGGATAA GGCGCAGCGG TCGGGCTGAA CGGGGGGTTC GTGCACACAG 
4921 CCCAGCTTGG AGCGAACGAC CTACACCGAA CTGAGATACC TACAGCGTGA GCTATGAGAA 
4981 AGCGCCACGC TTCCCGAAGG GAGAAAGGCG GACAGGTATC CGGTAAGCGG CAGGGTCGGA 
5041 ACAGGAGAGC GCACGAGGGA GCTTCCAGGG GGAAACGCCT GGTATCTTTA TAGTCCTGTC 
5101 GGGTTTCGCC ACCTCTGACT TGAGCGTCGA TTTTTGTGAT GCTCGTCAGG GGGGCGGAGC 
5161 CTATGGAAAA ACGCCAGCAA CGCGGCCTTT TTACGGTTCC TGGCCTTTTG CTGGCCTTTT 
5221 GCTCACATGT TCTTTCCTGC GTTATCCCCT GATTCTGTGG ATAACCGTAT TACCGCCATG 
5281 CAT