pMR101, 4095 bp pMR101 is a plasmid vector designed for the high-level expression of Rop in Escherichia coli. The low-copy p15A origin of this plasmid is not regulated by Rop (which regulates ColE1 plasmids). The plasmid confers kanamycin resistance and can be co-maintained with ColE1 plasmids with compatible resistance markers (Amp, Cam, Tet). Despite the low plasmid copy number, high-level expression of a variety of proteins has been observed from the T7(lac) promoter in BL21(DE3). We recommend chloramphenicol amplification when preparing large quantities of the vector. Single-stranded DNA can be prepared for Kunkel mutagenesis by means of the M13 origin. This sequence was constructed from other GenBank and commercial plasmid files. It is so far consistent with all of the digests we have attempted, but it is not extensively confirmed by sequencing. We typically clone the gene to be expressed from the T7(lac) promoter between NcoI (101) and BamHI (137), with the ATG in the NcoI site as the initiating Met. Features (approximate positions) 1- 101 T7(lac) promoter 101 NcoI site (initiator Met overlaps) 137 BamHI site (typical cloning site) 303 BstEII (alternate cloning site) 852-1665 Kan resistance 2000-2700 p15A origin 3300-3800 M13 origin --Thomas J. Magliery, Ph.D., March 15, 2003 Reference: Munson M, Predki PF, Regan L. 1994. ColE1-compatible vectors for high-level expression of cloned DNAs from the T7 promoter. Gene 144: 59. A scanned PDF version of this paper is available at the Regan Lab Web Site: http://www.csb.yale.edu/people/regan/publications.html 1 TCGATCCCGC GAAATTAATA CGACTCACTA TAGGGGAATT GTGAGCGGAT 51 AACAATTCCC CTCTAGAAAT AATTTTGTTT AACTTTAAGA AGGAGATATA 101 CCATGGCTAG CATGACTGGT GGACAGCAAA TGGGTCGGAT CCGGCTGCTA 151 ACAAAGCCCG AAAGGAAGCT GAGTTGGCTG CTGCCACCGC TGAGCAATAA 201 CTAGCATAAC CCCTTGGGGC CTCTAAACGG GTCTTGAGGG GTTTTTTGCT 251 GAAAGGAGGA ACTATATCCG GATATCCACA GGACGGGTGT GGTCGCCATG 301 ATGGTGACCA CCATTTGCCC CATATCACCG TCGGTACTGA TCCCGTCGTC 351 AATAAACCGA ACCGCTACAC CCTGAGCATC AAACTCTTTT ATCAGTTGGA 401 TCATGTCGGC GGTGTCGCGG CCAAGACGGT CGAGCTTCTT CACCAGAATG 451 ACATCACCTT CCTCCACCTT CATCCTCAGC AAATCCAGCC CTTCCCGATC 501 TGTTGAACTG CCGGATGCCT TGTCGGTAAA GATGCGGTTA GCTTTTACCC 551 CTGCATCTTT GAGCGCTGAG GTCTGCCTCG TGAAGAAGGT GTTGCTGACT 601 CATACCAGGC CTGAATCGCC CCATCATCCA GCCAGAAAGT GAGGGAGCCA 651 CGGTTGATGA GAGCTTTGTT GTAGGTGGAC CAGTTGGTGA TTTTGAACTT 701 TTGCTTTGCC ACGGAACGGT CTGCGTTGTC GGGAAGATGC GTGATCTGAT 751 CCTTCAACTC AGCAAAAGTT CGATTTATTC AACAAAGCCG CCGTCCCGTC 801 AAGTCAGCGT AATGCTCTGC CAGTGTTACA ACCAATTAAC CAATTCTGAT 851 TAGAAAAACT CATCGAGCAT CAAATGAAAC TGCAATTTAT TCATATCAGG 901 ATTATCAATA CCATATTTTT GAAAAAGCCG TTTCTGTAAT GAAGGAGAAA 951 ACTCACCGAG GCAGTTCCAT AGGATGGCAA GATCCTGGTA TCGGTCTGCG 1001 ATTCCGACTC GTCCAACATC AATACAACCT ATTAATTTCC CCTCGTCAAA 1051 AATAAGGTTA TCAAGTGAGA AATCACCATG AGTGACGACT GAATCCGGTG 1101 AGAATGGCAA AAGCTTATGC ATTTCTTTCC AGACTTGTTC AACAGGCCAG 1151 CCATTACGCT CGTCATCAAA ATCACTCGCA TCAACCAAAC CGTTATTCAT 1201 TCGTGATTGC GCCTGAGCGA GACGAAATAC GCGATCGCTG TTAAAAGGAC 1251 AATTACAAAC AGGAATCGAA TGCAACCGGC GCAGGAACAC TGCCAGCGCA 1301 TCAACAATAT TTTCACCTGA ATCAGGATAT TCTTCTAATA CCTGGAATGC 1351 TGTTTTCCCG GGGATCGCAG TGGTGAGTAA CCATGCATCA TCAGGAGTAC 1401 GGATAAAATG CTTGATGGTC GGAAGAGGCA TAAATTCCGT CAGCCAGTTT 1451 AGTCTGACCA TCTCATCTGT AACATCATTG GCAACGCTAC CTTTGCCATG 1501 TTTCAGAAAC AACTCTGGCG CATCGGGCTT CCCATACAAT CGATAGATTG 1551 TCGCACCTGA TTGCCCGACA TTATCGCGAG CCCATTTATA CCCATATAAA 1601 TCAGCATCCA TGTTGGAATT TAATCGCGGC CTCGAGCAAG ACGTTTCCCG 1651 TTGAATATGG CTCATAACAC CCCTTGTATT ACTGTTTATG TAAGCAGACA 1701 GTTTTATTGT TCATGATGAT ATATTTTTAT CTTGTGCAAT GTAACATCAG 1751 AGATTTTGAG ACACAACGTG GCTTTGTTGA ATAAATCGAA CTTTTGCTGA 1801 GTTGAAGGAT CAGATCACGC ATCTTCCCGA CAACGCAGAC CGTTCCGTGG 1851 CAAAGCAAAA GTTCAAAATC ACCAACTGGT CCACCTACAA CAAAGCTCTC 1901 ATCAACCGTG GCTCCCTCAC TTTCTGGCTG GATGATGGGG CGATTCAGGC 1951 CTGGTATGAG TCAGCAACAC CTTCTTCACG AGGCAGACCT CAGCGCTAGC 2001 GGAGTGTATA CTGGCTTACT ATGTTGGCAC TGATGAGGGT GTCAGTGAAG 2051 TGCTTCATGT GGCAGGAGAA AAAAGGCTGC ACCGGTGCGT CAGCAGAATA 2101 TGTGATACAG GATATATTCC GCTTCCTCGC TCACTGACTC GCTACGCTCG 2151 GTCGTTCGAC TGCGGCGAGC GGAAATGGCT TACGAACGGG GCGGAGATTT 2201 CCTGGAAGAT GCCAGGAAGA TACTTAACAG GGAAGTGAGA GGGCCGCGGC 2251 AAAGCCGTTT TTCCATAGGC TCCGCCCCCC TGACAAGCAT CACGAAATCT 2301 GACGCTCAAA TCAGTGGTGG CGAAACCCGA CAGGACTATA AAGATACCAG 2351 GCGTTTCCCC TGGCGGCTCC CTCGTGCGCT CTCCTGTTCC TGCCTTTCGG 2401 TTTACCGGTG TCATTCCGCT GTTATGGCCG CGTTTGTCTC ATTCCACGCC 2451 TGACACTCAG TTCCGGGTAG GCAGTTCGCT CCAAGCTGGA CTGTATGCAC 2501 GAACCCCCCG TTCAGTCCGA CCGCTGCGCC TTATCCGGTA ACTATCGTCT 2551 TGAGTCCAAC CCGGAAAGAC ATGCAAAAGC ACCACTGGCA GCAGCCACTG 2601 GTAATTGATT TAGAGGAGTT AGTCTTGAAG TCATGCGCCG GTTAAGGCTA 2651 AACTGAAAGG ACAAGTTTTG GTGACTGCGC TCCTCCAAGC CAGTTACCTC 2701 GGTTCAAAGA GTTGGTAGCT CAGAGAACCT TCGAAAAACC GCCCTGCAAG 2751 GCGGTTTTTT CGTTTTCAGA GCAAGAGATT ACGCGCAGAC CAAAACGATC 2801 TCAAGAAGAT CATCTTATTA AGGGGTCTGA CGCTCAGTGG AACGAAAACT 2851 CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGAT CTTCACCTAG 2901 ATCCTTTTAA ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA 2951 GTAAACTTGG TCTGACAGTT ACCAATGCTT AATCAGTGAG GCACCTATCT 3001 CAGCGATCTG TCTATTTCGT TCATCCATAG TTGCCTGACT CCCCGTCGTG 3051 TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA GTGCTGCAAT 3101 GATACCGCGA GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC 3151 AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC TTTATCCGCC 3201 TCCATCCAGT CTATTAATTG TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC 3251 AGTTAATAGT TTGCGCAACG TTGTTGCCAT TGCTGCAGGG TTGAAGGCTC 3301 TCAAGGGCAT CGGTCGACAC CCCGGTTGAT AATCAGAAAA GCCCCAAAAA 3351 CAGGAAGATT GTATAAGCAA ATATTTAAAT TGTAAACGTT AATATTTTGT 3401 TAAAATTCGC GTTAAATTTT TGTTAAATCA GCTCATTTTT TAACCAATAG 3451 GCCGAAATCG GCAAAATCCC TTATAAATCA AAAGAATAGA CCGAGATAGG 3501 GTTGAGTGTT GTTCCAGTTT GGAACAAGAG TCCACTATTA AAGAACGTGG 3551 ACTCCAACGT CAAAGGGCGA AAAACCGTCT ATCAGGGCGA TGGCCCACTA 3601 CGTGAACCAT CACCCTAATC AAGTTTTTTG GGGTCGAGGT GCCGTAAAGC 3651 ACTAAATCGG AACCCTAAAG GGAGCCCCCG ATTTAGAGCT TGACGGGGAA 3701 AGCCGGCGAA CGTGGCGAGA AAGGAAGGGA AGAAAGCGAA AGGAGCGGGC 3751 GCTAGGGCGC TGGCAAGTGT AGCGGTCACG CTGCGCGTAA CCACCACACC 3801 CGCCGCGCTT AATGCGCCGC TACAGGGCGC GTCCTCAATT AGGAAGCAGC 3851 CCAGTAGTAG GTTGAGGCCG TTGAGCACCG CCGCCGCAAG GAATGGTGCA 3901 TGCAAGGAGA TGGCGCCCAA CAGTCCCCCG GCCACGGGGC CTGCCACCAT 3951 ACCCACGCCG AAACAAGCGC TCATGAGCCC GAAGTGGCGA GCCCGATCTT 4001 CCCCATCGGT GATGTCGGCG ATATAGGCGC CAGCAACCGC ACCTGTGGCG 4051 CCGGTGATGC CGGCCACGAT GCGTCCGGCG TAGAGGATCG AGATC