LOCUS Exported 6362 bp ds-DNA circular SYN 11-2-2014 DEFINITION Bacterial vector encoding a C-terminal 10xHis-TVMV-MBP cassette, for high-throughput purification of recombinant proteins. ACCESSION . VERSION . KEYWORDS pMCSG27 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6362) AUTHORS Eschenfeldt WH, Maltseva N, Stols L, Donnelly MI, Gu M, Nocek B, Tan K, Kim Y, Joachimiak A. TITLE Cleavable C-terminal His-tag vectors for structure determination. JOURNAL J. Struct. Funct. Genomics 2010;11:31-9. PUBMED 20213425 REFERENCE 2 (bases 1 to 6362) AUTHORS Midwest Center for Structural Genomics TITLE Direct Submission COMMENT For ligation-independent cloning (LIC), linearize with SmaI and treat with T4 DNA polymerase plus dATP. FEATURES Location/Qualifiers source 1..6362 /organism="synthetic DNA construct" /lab_host="Escherichia coli" /mol_type="other DNA" terminator 26..73 /note="T7 terminator" /note="transcription terminator for bacteriophage T7 RNA polymerase" CDS complement(140..157) /codon_start=1 /product="6xHis affinity tag" /note="6xHis" /translation="HHHHHH" CDS complement(213..1310) /codon_start=1 /gene="malE (mutated)" /product="maltose binding protein from E. coli" /note="MBP" /note="This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol." /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW YAVRTAVINAASGRQTVDEALKDAQT" CDS complement(1311..1331) /codon_start=1 /product="tobacco vein mottling virus (TVMV) NIa protease recognition and cleavage site" /note="TVMV site" /translation="ETVRFQS" CDS complement(1332..1361) /codon_start=1 /product="10xHis affinity tag" /note="10xHis" /translation="HHHHHHHHHH" RBS 1373..1378 /note="ribosome binding site" protein_bind 1409..1433 /bound_moiety="lac repressor encoded by lacI" /note="lac operator" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(1434..1452) /note="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" promoter 1765..1842 /gene="lacI" /note="lacI promoter" /note=" " CDS 1843..2925 /codon_start=1 /gene="lacI" /product="lac repressor" /note="lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" CDS 3734..3925 /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /note="rop" /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" rep_origin complement(4355..4943) /direction=LEFT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(5114..5974) /codon_start=1 /gene="bla" /product="beta-lactamase" /note="AmpR" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(5975..6078) /gene="bla" /note="AmpR promoter" ORIGIN 1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa 61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt 121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt 181 cgacggagct cgaattcgga tccttacgaa ttagtctgcg cgtctttcag ggcttcatcg 241 acagtctgac gaccgctggc ggcgttgatc accgcagtac gcacggcata ccagaaagcg 301 gacatctgcg ggatgttcgg catgatttca cctttctggg cgttttccat ggtggcggca 361 atacgtggat ctttcgccaa ctcttcctcg taagacttca gcgctacggc acccagcggt 421 ttgtctttat taaccgcttc cagaccttca tcagtcagca gatagttttc gaggaactct 481 tttgccagct ctttgttcgg actggcggcg ttaatacctg cgctcagcac gccaacgaac 541 ggtttggatg gttgaccctt gaaggtcggc agtaccgtta caccataatt cactttgctg 601 gtgtcgatgt tggaccatgc ccacgggccg ttgatggtca tcgctgtttc gcctttatta 661 aaggcagctt ctgcgatgga gtaatcggtg tctgcattca tgtgtttgtt tttaatcagg 721 tcaaccagga aggtcagacc cgctttcgcg ccagcgttat ccacgcccac gtctttaatg 781 tcgtacttgc cgttttcata cttgaacgca taacccccgt cagcagcaat cagcggccag 841 gtgaagtacg gttcttgcag gttgaacatc agcgcgctct tacctttcgc tttcagttct 901 ttatccagcg ccgggatctc ttcccaggtt tttggcgggt tcggcagcag atctttgtta 961 taaatcagcg ataacgcttc aacagcgatc gggtaagcaa tcagcttgcc gttgtaacgt 1021 acggcatccc aggtaaacgg atacagcttg tcctggaacg ctttgtccgg ggtgatttca 1081 gccaacaggc cagattgagc gtagccacca aagcggtcgt gtgcccagaa gataatgtca 1141 gggccatcgc cagttgccgc aacctgtggg aatttctctt ccagtttatc cggatgctca 1201 acggtgactt taattccggt atctttctcg aatttcttac cgacttcagc gagaccgtta 1261 tagcctttat cgccgttaat ccagattacc agtttacctt cttcgatttt agactggaaa 1321 cgcacggttt cgtgatggtg gtgatgatga tgatggtggt gcccgggaga gactccttct 1381 taaagttaaa caaaattatt tctagagggg aattgttatc cgctcacaat tcccctatag 1441 tgagtcgtat taatttcgcg ggatcgagat cgatctcgat cctctacgcc ggacgcatcg 1501 tggccggcat caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg 1561 atggggaaga tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg 1621 tggcaggccc cgtggccggg ggactgttgg gcgccatctc cttgcatgca ccattccttg 1681 cggcggcggt gctcaacggc ctcaacctac tactgggctg cttcctaatg caggagtcgc 1741 ataagggaga gcgtcgagat cccggacacc atcgaatggc gcaaaacctt tcgcggtatg 1801 gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc agtaacgtta 1861 tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag 1921 gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc ggagctgaat 1981 tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt 2041 gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc 2101 gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc 2161 tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat 2221 ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta 2281 tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt 2341 acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg 2401 ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact 2461 cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt 2521 caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac 2581 gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg 2641 gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat cccgccgtta 2701 accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa 2761 ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga 2821 aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta 2881 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa 2941 tgtaagttag ctcactcatt aggcaccggg atctcgaccg atgcccttga gagccttcaa 3001 cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac ttatgactgt 3061 cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca ttttcggcga 3121 ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat tcggaatctt 3181 gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca 3241 ggccattatc gccggcatgg cggccccacg ggtgcgcatg atcgtgctcc tgtcgttgag 3301 gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga 3361 gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt 3421 cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct gcaccattat 3481 gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta catctgtatt 3541 aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca tccataccgc 3601 cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag taacccgtat 3661 cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta 3721 cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag 3781 aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga 3841 catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc tcgcgcgttt 3901 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct 3961 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg 4021 tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat 4081 gcggcatcag agcagattgt actgagagtg caccatatat gcggtgtgaa ataccgcaca 4141 gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc 4201 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 4261 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 4321 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 4381 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 4441 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 4501 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 4561 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 4621 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 4681 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 4741 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag 4801 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 4861 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 4921 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 4981 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 5041 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 5101 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 5161 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 5221 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt 5281 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 5341 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 5401 atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc tcgtcgtttg 5461 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 5521 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 5581 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 5641 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 5701 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 5761 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 5821 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 5881 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg 5941 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa attattgaag 6001 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa 6061 acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat 6121 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga 6181 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa 6241 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc 6301 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc 6361 ca //