pmirGLO

The pmirGLO Dual-Luciferase miRNA Target Expression Vector(a–d) is designed to quantitatively evaluate microRNA (miRNA) activity by the insertion of miRNA target sites 3? of the firefly luciferase gene (luc2 ). These target sites can be introduced by cloning putative miRNA binding sites alone, or the 3? untranslated region (UTR) of a gene of interest, to study the influence of these sites on transcript stability and activity. Firefly luciferase is the primary reporter gene; reduced firefly luciferase expression indicates the binding of endogenous or introduced miRNAs to the cloned miRNA target sequence. This vector is based on Promega dual-luciferase technology, with firefly luciferase (luc2 ) used as the primary reporter to monitor mRNA regulation and Renilla luciferase (hRluc-neo) acting as a control reporter for normalization and selection. This vector contains the following features:

• Human phosphoglycerate kinase (PGK) promoter provides low translational expression, which is advantageous when reduction of signal is the desired response. The PGK promoter is a nonviral universal promoter, which functions across cell lines (yeast, rat, mouse and human).
• Firefly luciferase reporter gene (luc2 ) inversely reports miRNA activity in mammalian cells.
• Multiple cloning site (MCS) is located 3? of the firefly luciferase reporter gene (luc2 ).
• Humanized Renilla luciferase-neomycin resistance cassette (hRluc-neo) is used as a control reporter for normalization of gene expression and stable cell line selection.
• Ampr gene allows bacterial selection for vector amplification.
• SV40 late poly(A) signal sequence is positioned downstream of luc2 to provide efficient transcription termination and mRNA polyadenylation.
• Synthetic poly(A) signal/transcription stop site.

0001 CATGCAAGCT GATCCGGCTG CTAACAAAGC CCGAAAGGAA GCTGAGTTGG CTGCTGCCAC 
0061 CGCTGAGCAA TAACTAGCAT AACCCCTTGG GGCGGCCGCT TCGAGCAGAC ATGATAAGAT 
0121 ACATTGATGA GTTTGGACAA ACCACAACTA GAATGCAGTG AAAAAAATGC TTTATTTGTG 
0181 AAATTTGTGA TGCTATTGCT TTATTTGTAA CCATTATAAG CTGCAATAAA CAAGTTAACA 
0241 ACAACAATTG CATTCATTTT ATGTTTCAGG TTCAGGGGGA GATGTGGGAG GTTTTTTTAA 
0301 GCAAGTAAAA CCTCTACAAA TGTGGTAAAA TCGAATTTTA ACAAAATATT AACGCTTACA 
0361 ATTTCCTGAT GCGGTATTTT CTCCTTACGC ATCTGTGCGG TATTTCACAC CGCATACGCG 
0421 GATCTGCGCA GCACCATGGC CTGAAATAAC CTCTGAAAGA GGAACTTGGT TAGGTACCTT 
0481 CTGAGGCGGA AAGAACCAGC TGTGGAATGT GTGTCAGTTA GGGTGTGGAA AGTCCCCAGG 
0541 CTCCCCAGCA GGCAGAAGTA TGCAAAGCAT GCATCTCAAT TAGTCAGCAA CCAGGTGTGG 
0601 AAAGTCCCCA GGCTCCCCAG CAGGCAGAAG TATGCAAAGC ATGCATCTCA ATTAGTCAGC 
0661 AACCATAGTC CCGCCCCTAA CTCCGCCCAT CCCGCCCCTA ACTCCGCCCA GTTCCGCCCA 
0721 TTCTCCGCCC CATGGCTGAC TAATTTTTTT TATTTATGCA GAGGCCGAGG CCGCCTCGGC 
0781 CTCTGAGCTA TTCCAGAAGT AGTGAGGAGG CTTTTTTGGA GGCCTAGGCT TTTGCAAAAA 
0841 GCTTGATTCT TCTGACACAA CAGTCTCGAA CCAAAGGCTG GAGCCACCAT GGCTTCCAAG 
0901 GTGTACGACC CCGAGCAACG CAAACGCATG ATCACTGGGC CTCAGTGGTG GGCTCGCTGC 
0961 AAGCAAATGA ACGTGCTGGA CTCCTTCATC AACTACTATG ATTCCGAGAA GCACGCCGAG 
1021 AACGCCGTGA TTTTTCTGCA TGGTAACGCT GCCTCCAGCT ACCTGTGGAG GCACGTCGTG 
1081 CCTCACATCG AGCCCGTGGC TAGATGCATC ATCCCTGATC TGATCGGAAT GGGTAAGTCC 
1141 GGCAAGAGCG GGAATGGCTC ATATCGCCTC CTGGATCACT ACAAGTACCT CACCGCTTGG 
1201 TTCGAGCTGC TGAACCTTCC AAAGAAAATC ATCTTTGTGG GCCACGACTG GGGGGCTTGT 
1261 CTGGCCTTTC ACTACTCCTA CGAGCACCAA GACAAGATCA AGGCCATCGT CCATGCTGAG 
1321 AGTGTCGTGG ACGTGATCGA GTCCTGGGAC GAGTGGCCTG ACATCGAGGA GGATATCGCC 
1381 CTGATCAAGA GCGAAGAGGG CGAGAAAATG GTGCTTGAGA ATAACTTCTT CGTCGAGACC 
1441 ATGCTCCCAA GCAAGATCAT GCGGAAACTG GAGCCTGAGG AGTTCGCTGC CTACCTGGAG 
1501 CCATTCAAGG AGAAGGGCGA GGTTAGACGG CCTACCCTCT CCTGGCCTCG CGAGATCCCT 
1561 CTCGTTAAGG GAGGCAAGCC CGACGTCGTC CAGATTGTCC GCAACTACAA CGCCTACCTT 
1621 CGGGCCAGCG ACGATCTGCC TAAGATGTTC ATCGAGTCCG ACCCTGGGTT CTTTTCCAAC 
1681 GCTATTGTCG AGGGAGCTAA GAAGTTCCCT AACACCGAGT TCGTGAAGGT GAAGGGCCTC 
1741 CACTTCAGCC AGGAGGACGC TCCAGATGAA ATGGGTAAGT ACATCAAGAG CTTCGTGGAG 
1801 CGCGTGCTGA AGAACGAGCA GACCGGTGGT GGGAGCGGAG GTGGCGGATC AGGTGGCGGA 
1861 GGCTCCGGAG GGATTGAACA AGATGGATTG CACGCAGGTT CTCCGGCCGC TTGGGTGGAG 
1921 AGGCTATTCG GCTATGACTG GGCACAACAG ACAATCGGCT GCTCTGATGC CGCCGTGTTC 
1981 CGGCTGTCAG CGCAGGGGCG CCCGGTTCTT TTTGTCAAGA CCGACCTGTC CGGTGCCCTG 
2041 AATGAACTGC AGGACGAGGC AGCGCGGCTA TCGTGGCTGG CCACGACGGG CGTTCCTTGC 
2101 GCAGCTGTGC TCGACGTTGT CACTGAAGCG GGAAGGGACT GGCTGCTATT GGGCGAAGTG 
2161 CCGGGGCAGG ATCTCCTGTC ATCTCACCTT GCTCCTGCCG AGAAAGTATC CATCATGGCT 
2221 GATGCAATGC GGCGGCTGCA TACGCTTGAT CCGGCTACCT GCCCATTCGA CCACCAAGCG 
2281 AAACATCGCA TCGAGCGAGC ACGTACTCGG ATGGAAGCCG GTCTTGTCGA TCAGGATGAT 
2341 CTGGACGAAG AGCATCAGGG GCTCGCGCCA GCCGAACTGT TCGCCAGGCT CAAGGCGCGC 
2401 ATGCCCGACG GCGAGGATCT CGTCGTGACC CATGGCGATG CCTGCTTGCC GAATATCATG 
2461 GTGGAAAATG GCCGCTTTTC TGGATTCATC GACTGTGGCC GGCTGGGTGT GGCGGACCGC 
2521 TATCAGGACA TAGCGTTGGC TACCCGTGAT ATTGCTGAAG AGCTTGGCGG CGAATGGGCT 
2581 GACCGCTTCC TCGTGCTTTA CGGTATCGCC GCTCCCGATT CGCAGCGCAT CGCCTTCTAT 
2641 CGCCTTCTTG ACGAGTTCTT CTGAGCGGGA CTCTGGGGTT CGAAATGACC GACCAAGCGA 
2701 CGCCCAACCT GCCATCACGA TGGCCGCAAT AAAATATCTT TATTTTCATT ACATCTGTGT 
2761 GTTGGTTTTT TGTGTGAATC GATAGCGATA AGGATCCTCT TTGCGCTTGC GTTTTCCCTT 
2821 GTCCAGATAG CCCAGTAGCT GACATTCATC CGGGGTCAGC ACCGTTTCTG CGGACTGGCT 
2881 TTCTACGTAA TGGTTTCTTA GACGTCAGGT GGCACTTTTC GGGGAAATGT GCGCGGAACC 
2941 CCTATTTGTT TATTTTTCTA AATACATTCA AATATGTATC CGCTCATGAG ACAATAACCC 
3001 TGATAAATGC TTCAATAATA TTGAAAAAGG AAGAGTATGA GTATTCAACA TTTCCGTGTC 
3061 GCCCTTATTC CCTTTTTTGC GGCATTTTGC CTTCCTGTTT TTGCTCACCC AGAAACGCTG 
3121 GTGAAAGTAA AAGATGCTGA AGATCAGTTG GGTGCACGAG TGGGTTACAT CGAACTGGAT 
3181 CTCAACAGCG GTAAGATCCT TGAGAGTTTT CGCCCCGAAG AACGTTTTCC AATGATGAGC 
3241 ACTTTCAAAG TTCTGCTATG TGGCGCGGTA TTATCCCGTA TTGACGCCGG GCAAGAGCAA 
3301 CTCGGTCGCC GCATACACTA TTCTCAGAAT GACTTGGTTG AGTACTCACC AGTCACAGAA 
3361 AAGCATCTTA CGGATGGCAT GACAGTAAGA GAATTATGCA GTGCTGCCAT AACCATGAGT 
3421 GATAACACTG CGGCCAACTT ACTTCTGACA ACTATCGGAG GACCGAAGGA GCTAACCGCT 
3481 TTTTTGCACA ACATGGGGGA TCATGTAACT CGCCTTGATC GTTGGGAACC GGAGCTGAAT 
3541 GAAGCCATAC CAAACGACGA GCGTGACACC ACGATGCCTG TAGCAATGGC AACAACGTTG 
3601 CGCAAACTAT TAACTGGCGA ACTACTTACT CTAGCTTCCC GGCAACAATT AATAGACTGG 
3661 ATGGAGGCGG ATAAAGTTGC AGGACCACTT CTGCGCTCGG CCCTTCCGGC TGGCTGGTTT 
3721 ATTGCTGATA AATCTGGAGC CGGTGAGCGT GGGTCTCGCG GTATCATTGC AGCACTGGGG 
3781 CCAGATGGTA AGCCCTCCCG TATCGTAGTT ATCTACACGA CGGGGAGTCA GGCAACTATG 
3841 GATGAACGAA ATAGACAGAT CGCTGAGATA GGTGCCTCAC TGATTAAGCA TTGGTAATTC 
3901 GAAATGACCG ACCAAGCGAC GCCCAACCGG TATCAGCTCA CTCAAAGGCG GTAATACGGT 
3961 TATCCACAGA ATCAGGGGAT AACGCAGGAA AGAACATGTG AGCAAAAGGC CAGCAAAAGG 
4021 CCAGGAACCG TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC CCCCCTGACG 
4081 AGCATCACAA AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA CTATAAAGAT 
4141 ACCAGGCGTT TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC CTGCCGCTTA 
4201 CCGGATACCT GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT AGCTCACGCT 
4261 GTAGGTATCT CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG CACGAACCCC 
4321 CCGTTCAGCC CGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCC AACCCGGTAA 
4381 GACACGACTT ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA GCGAGGTATG 
4441 TAGGCGGTGC TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT AGAAGGACAG 
4501 TATTTGGTAT CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT GGTAGCTCTT 
4561 GATCCGGCAA ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG CAGCAGATTA 
4621 CGCGCAGAAA AAAAGGATTT CAAGAAGATC CTTTGATCTT TTCTACGGGG TCTGACGCTC 
4681 AGTGGAACGA AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA AGGATCTTCA 
4741 CCTAGATCCT TTTATAGTCC GGAAATACAG GAACGCACGC TGGATGGCCC TTCGCTGGGA 
4801 TGGTGAAACC ATGAAAAATG GCAGCTTCAG TGGATTAAGT GGGGGTAATG TGGCCTGTAC 
4861 CCTCTGGTTG CATAGGTATT CATACGGTTA AAATTTATCA GGCGCGATTG CGGCAGTTTT 
4921 TCGGGTGGTT TGTTGCCATT TTTACCTGTC TGCTGCCGTG ATCGCGCTGA ACGCGTTTTA 
4981 GCGGTGCGTA CAATTAAGGG ATTATGGTAA ATCCACTTAC TGTCTGCCCT CGTAGCCATC 
5041 GAGATAAACC GCAGTACTCC GGCCACGATG CGTCCGGCGT AGAGGATCGA GATCTACCGG 
5101 GTAGGGGAGG CGCTTTTCCC AAGGCAGTCT GGAGCATGCG CTTTAGCAGC CCCGCTGGGC 
5161 ACTTGGCGCT ACACAAGTGG CCTCTGGCCT CGCACACATT CCACATCCAC CGGTAGGCGC 
5221 CAACCGGCTC CGTTCTTTGG TGGCCCCTTC GCGCCACCTT CTACTCCTCC CCTAGTCAGG 
5281 AAGTTCCCCC CCGCCCCGCA GCTCGCGTCG TGCAGGACGT GACAAATGGA AGTAGCACGT 
5341 CTCACTAGTC TCGTGCAGAT GGACAGCACC GCTGAGCAAT GGAAGCGGGT AGGCCTTTGG 
5401 GGCAGCGGCC AATAGCAGCT TTGCTCCTTC GCTTTCTGGG CTCAGAGGCT GGGAAGGGGT 
5461 GGGTCCGGGG GCGGGCTCAG GGGCGGGCTC AGGGGCGGGG CGGGCGCCCG AAGGTCCTCC 
5521 GGAGGCCCGG CATTCTGCAC GCTTCAAAAG CGCACGTCTG CCGCGCTGTT CTCCTCTTCC 
5581 TCATCTCCGG GCCTTTCGAC CTGCAGCCCA AGCTTGGCAA TCCGGTACTG TTGGTAAAGC 
5641 CACCATGGAA GATGCCAAAA ACATTAAGAA GGGCCCAGCG CCATTCTACC CACTCGAAGA 
5701 CGGGACCGCC GGCGAGCAGC TGCACAAAGC CATGAAGCGC TACGCCCTGG TGCCCGGCAC 
5761 CATCGCCTTT ACCGACGCAC ATATCGAGGT GGACATTACC TACGCCGAGT ACTTCGAGAT 
5821 GAGCGTTCGG CTGGCAGAAG CTATGAAGCG CTATGGGCTG AATACAAACC ATCGGATCGT 
5881 GGTGTGCAGC GAGAATAGCT TGCAGTTCTT CATGCCCGTG TTGGGTGCCC TGTTCATCGG 
5941 TGTGGCTGTG GCCCCAGCTA ACGACATCTA CAACGAGCGC GAGCTGCTGA ACAGCATGGG 
6001 CATCAGCCAG CCCACCGTCG TATTCGTGAG CAAGAAAGGG CTGCAAAAGA TCCTCAACGT 
6061 GCAAAAGAAG CTACCGATCA TACAAAAGAT CATCATCATG GATAGCAAGA CCGACTACCA 
6121 GGGCTTCCAA AGCATGTACA CCTTCGTGAC TTCCCATTTG CCACCCGGCT TCAACGAGTA 
6181 CGACTTCGTG CCCGAGAGCT TCGACCGGGA CAAAACCATC GCCCTGATCA TGAACAGTAG 
6241 TGGCAGTACC GGATTGCCCA AGGGCGTAGC CCTACCGCAC CGCACCGCTT GTGTCCGATT 
6301 CAGTCATGCC CGCGACCCCA TCTTCGGCAA CCAGATCATC CCCGACACCG CTATCCTCAG 
6361 CGTGGTGCCA TTTCACCACG GCTTCGGCAT GTTCACCACG CTGGGCTACT TGATCTGCGG 
6421 CTTTCGGGTC GTGCTCATGT ACCGCTTCGA GGAGGAGCTA TTCTTGCGCA GCTTGCAAGA 
6481 CTATAAGATT CAATCTGCCC TGCTGGTGCC CACACTATTT AGCTTCTTCG CTAAGAGCAC 
6541 TCTCATCGAC AAGTACGACC TAAGCAACTT GCACGAGATC GCCAGCGGCG GGGCGCCGCT 
6601 CAGCAAGGAG GTAGGTGAGG CCGTGGCCAA ACGCTTCCAC CTACCAGGCA TCCGCCAGGG 
6661 CTACGGCCTG ACAGAAACAA CCAGCGCCAT TCTGATCACC CCCGAAGGGG ACGACAAGCC 
6721 TGGCGCAGTA GGCAAGGTGG TGCCCTTCTT CGAGGCTAAG GTGGTGGACT TGGACACCGG 
6781 TAAGACACTG GGTGTGAACC AGCGCGGCGA GCTGTGCGTC CGTGGCCCCA TGATCATGAG 
6841 CGGCTACGTT AACAACCCCG AGGCTACAAA CGCTCTCATC GACAAGGACG GCTGGCTGCA 
6901 CAGCGGCGAC ATCGCCTACT GGGACGAGGA CGAGCACTTC TTCATCGTGG ACCGGCTGAA 
6961 GAGCCTGATC AAATACAAGG GCTACCAGGT AGCCCCAGCC GAACTGGAGA GCATCCTGCT 
7021 GCAACACCCC AACATCTTCG ACGCCGGGGT CGCCGGCCTG CCCGACGACG ATGCCGGCGA 
7081 GCTGCCCGCC GCAGTCGTCG TGCTGGAACA CGGTAAAACC ATGACCGAGA AGGAGATCGT 
7141 GGACTATGTG GCCAGCCAGG TTACAACCGC CAAGAAGCTG CGCGGTGGTG TTGTGTTCGT 
7201 GGACGAGGTG CCTAAAGGAC TGACCGGCAA GTTGGACGCC CGCAAGATCC GCGAGATTCT 
7261 CATTAAGGCC AAGAAGGGCG GCAAGATCGC CGTGTAATTC TAGTTGTTTA AACGAGCTCG 
7321 CTAGCCTCGA GTCTAGAGTC GACCTGCAGG