pGEMEX-1

The pGEMEX?Vectors(a,b) are T7 expression vectors that can be used for cloning, as templates for in vitro transcription and for production of single-stranded DNA (ssDNA). Expression from the pGEMEX?Vectors is based on the T7 expression system (a) developed by Studier (1), which uses a convenient vector/host combination for high-level expression of cloned genes in vivo. Sequences cloned into the pGEMEX? Vectors are expressed as T7 gene 10 fusion proteins in JM109(DE3)(a) or BL21(DE3)pLysS(c) host strains containing an inducible gene for T7 RNA polymerase. JM109(DE3) is a specially constructed host strain that contains an IPTGinducible gene for T7 RNA polymerase; JM109(DE3) and JM109 are supplied with the pGEMEX?Vectors. The pGEMEX?-1 and pGEMEX?-2 Vectors differ by only two base pairs resulting in a shift in the reading frame (Figure 1). The bacteriophage T7 gene 10 leader peptide (260 amino acids) is expressed very efficiently in this host and can accumulate to greater than 50% of total cell protein in three hours or less. Typical levels of fusion protein production are on the order of 10mg per liter of induced culture.

0001 GAATACTCAA GCTTATGCAT GCGGCCGCAT CTAGAGGGCC CGGATCCCTC GAGGTCGACG 
0061 AATTCGAGCT CGGCCGACTT GGCCTTCCCT TTAGTGAGGG TTAATAAACT TGGCCAATTC 
0121 GATCCGGTAC CAGCACCACC AGCGGTGAGG TGCGGAACTT CTACAACCTC AAAGCCCATA 
0181 ACGTTGCGGA TAGAACCCTT CTCAGGGTCA ATCAGAGCAG CGTAGTTTGC TGCGTTCGGC 
0241 ATCAGTGCTG CCAGAATCGC AGAGTAGCTA TCTGGGTCAC AGTAGAACAC ACGGTCAGCA 
0301 GCCGGAACAT AGTTCTTGGT CAGAGCCGCA CGAGCCTTAG TCAGAGCCGC AATAATCTCC 
0361 TTACCCAGCG CAACTTGGTC GGTAAGTGCG GCCTTGTTCT GAGTGGTCTC AATTACGGTA 
0421 GCAGTACCTA AGCCCTCGAT GTTCTCATTA TATTTGCTTT CCACGTTACA CAGACCGGCA 
0481 ATCTCAGCCA GAACCGCACC ATCCGCAGCC ATCGCCAGAG ATTCACCCAA CTGAGAGGTA 
0541 TACTCAGAGC GAACGTCGTA GTGGTTCATC GCGTCCTCAA TATCATAAAT CAGAACGTCA 
0601 GCCGTCAGGA GACCGTCAAT GGTGATTACC TTCTCGGTGT GTTTGATGTC CTTACGTTTA 
0661 TCGTCGAGGT TCTCGCCCGG AGCCAGATAC GCTGCCTGAG TGCGACCCAG AACAGGGAAC 
0721 TGAGCGGATT TACCGCTGGA GATGGAACGT ACCATGTGGC GAGAAGTGGT CACGGAGGTA 
0781 CGAGCGAACG CAGTCAGGAC TTCACCGCCA AATACCTTCA AGAACAACGC CAGTTTATCT 
0841 CCAGCAGCAA CTACACCTTT ACCTTGGTTA GTACCCATTT GCTGTCCACC AGTCATGCTA 
0901 GCCATATGTA TATCTCCTTC TTAAAGTTAA ACAAAATTAT TTCTAGAGGG AAACCGTTGT 
0961 GGTCTCCCTA TAGTGAGTCG TATTAATTTC GCGGGATCGA GATCTCCCGG GCTGCATTAA 
1021 TGAATCGGCC AACGCGCGGG GAGAGGCGGT TTGCGTATTG GGCGCTCTTC CGCTTCCTCG 
1081 CTCACTGACT CGCTGCGCTC GGTCGTTCGG CTGCGGCGAG CGGTATCAGC TCACTCAAAG 
1141 GCGGTAATAC GGTTATCCAC AGAATCAGGG GATAACGCAG GAAAGAACAT GTGAGCAAAA 
1201 GGCCAGCAAA AGGCCAGGAA CCGTAAAAAG GCCGCGTTGC TGGCGTTTTT CGATAGGCTC 
1261 CGCCCCCCTG ACGAGCATCA CAAAAATCGA CGCTCAAGTC AGAGGTGGCG AAACCCGACA 
1321 GGACTATAAA GATACCAGGC GTTTCCCCCT GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG 
1381 ACCCTGCCGC TTACCGGATA CCTGTCCGCC TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT 
1441 CATAGCTCAC GCTGTAGGTA TCTCAGTTCG GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT 
1501 GTGCACGAAC CCCCCGTTCA GCCCGACCGC TGCGCCTTAT CCGGTAACTA TCGTCTTGAG 
1561 TCCAACCCGG TAAGACACGA CTTATCGCCA CTGGCAGCAG CCACTGGTAA CAGGATTAGC 
1621 AGAGCGAGGT ATGTAGGCGG TGCTACAGAG TTCTTGAAGT GGTGGCCTAA CTACGGCTAC 
1681 ACTAGAAGGA CAGTATTTGG TATCTGCGCT CTGCTGAAGC CAGTTACCTT CGGAAAAAGA 
1741 GTTGGTAGCT CTTGATCCGG CAAACAAACC ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC 
1801 AAGCAGCAGA TTACGCGCAG AAAAAAAGGA TCTCAAGAAG ATCCTTTGAT CTTTTCTACG 
1861 GGGTCTGACG CTCAGTGGAA CGAAAACTCA CGTTAAGGGA TTTTGGTCAT GAGATTATCA 
1921 AAAAGGATCT TCACCTAGAT CCTTTTAAAT TAAAAATGAA GTTTTAAATC AATCTAAAGT 
1981 ATATATGAGT AAACTTGGTC TGACAGTTAC CAATGCTTAA TCAGTGAGGC ACCTATCTCA 
2041 GCGATCTGTC TATTTCGTTC ATCCATAGTT GCCTGACTCC CCGTCGTGTA GATAACTACG 
2101 ATACGGGAGG GCTTACCATC TGGCCCCAGT GCTGCAATGA TACCGCGAGA CCCACGCTCA 
2161 CCGGCTCCAG ATTTATCAGC AATAAACCAG CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT 
2221 CCTGCAACTT TATCCGCCTC CATCCAGTCT ATTAATTGTT GCCGGGAAGC TAGAGTAAGT 
2281 AGTTCGCCAG TTAATAGTTT GCGCAACGTT GTTGGCATTG CTACAGGCAT CGTGGTGTCA 
2341 CGCTCGTCGT TTGGTATGGC TTCATTCAGC TCCGGTTCCC AACGATCAAG GCGAGTTACA 
2401 TGATCCCCCA TGTTGTGCAA AAAAGCGGTT AGCTCCTTCG GTCCTCCGAT CGTTGTCAGA 
2461 AGTAAGTTGG CCGCAGTGTT ATCACTCATG GTTATGGCAG CACTGCATAA TTCTCTTACT 
2521 GTCATGCCAT CCGTAAGATG CTTTTCTGTG ACTGGTGAGT ACTCAACCAA GTCATTCTGA 
2581 GAATACCGCG CCCGGCGACC GAGTTGCTCT TGCCCGGCGT CAATACGGGA TAATAGTGTA 
2641 TGACATAGCA GAACTTTAAA AGTGCTCATC ATTGGAAAAC GTTCTTCGGG GCGAAAACTC 
2701 TCAAGGATCT TACCGCTGTT GAGATCCAGT TCGATGTAAC CCACTCGTGC ACCCAACTGA 
2761 TCTTCAGCAT CTTTTACTTT CACCAGCGTT TCTGGGTGAG CAAAAACAGG AAGGCAAAAT 
2821 GCCGCAAAAA AGGGAATAAG GGCGACACGG AAATGTTGAA TACTCATACT CTTCCTTTTT 
2881 CAATATTATT GAAGCATTTA TCAGGGTTAT TGTCTCATGA GCGGATACAT ATTTGAATGT 
2941 ATTTAGAAAA ATAAACAAAT AGGGGTTCCG CGCACATTTC CCCGAAAAGT GCCACCTGAC 
3001 GTCTAAGAAA CCATTATTAT CATGACATTA ACCTATAAAA ATAGGCGTAT CACGAGGCCC 
3061 TTTCGTCTCG CGCGTTTCGG TGATGACGGT GAAAACCTCT GACACATGCA GCTCCCGGAG 
3121 ACGGTCACAG CTTGTCTGTA AGCGGATGCC GGGAGCAGAC AAGCCCGTCA GGGCGCGTCA 
3181 GCGGGTGTTG GCGGGTGTCG GGGCTGGCTT AACTATGCGG CATCAGAGCA GATTGTACTG 
3241 AGAGTGCACC ATATGCGGTG TGAAATACCG CACAGATGCG TAAGGAGAAA ATACCGCATC 
3301 AGGCGACGCG CCCTGTAGCG GCGCATTAAG CGCGGCGGGT GTGGTGGTTA CGCGCAGCGT 
3361 GACCGCTACA CTTGCCAGCG CCCTAGCGCC CGCTCCTTTC GCTTTCTTCC CTTCCTTTCT 
3421 CGCCACGTTC GCCGGCTTTC CCCGTCAAGC TCTAAATCGG GGGCTCCCTT TAGGGTTCCG 
3481 ATTTAGAGCT TTACGGCACC TCGACCGCAA AAAACTTGAT TTGGGTGATG GTTCACGTAG 
3541 TGGGCCATCG CCCTGATAGA CGGTTTTTCG CCCTTTGACG TTGGAGTCCA CGTTCTTTAA 
3601 TAGTGGACTC TTGTTCCAAA CTGGAACAAC ACTCAACCCT ATCTCGGTCT ATTCTTTTGA 
3661 TTTATAAGGG ATTTTGCCGA TTTCGGCCTA TTGGTTAAAA AATGAGCTGA TTTAACAAAT 
3721 ATTTAACGCG AATTTTAACA AAATATTAAC GTTTACAATT TCCATTCGCC ATTCAGGCTG 
3781 CGCAACTGTT GGGAAGGGCG ATCGGTGCGG GCCTCTTCGC TATTACGCCA GGTTATCCGG 
3841 ATATAGTTCC TCCTTTCAGC AAAAAACCCC TCAAGACCCG TTTAGAGGCC CCAAGGGGTT 
3901 ATGCTAGTTA TTGCTCAGCG GTGGCAGCAG CCAACTCAGC TTCCTTTCGG GCTTTGTTAG 
3961 CAGCCGGATC AAGCTGGGAT TTAGGTGACA CTATA