LOCUS Exported 6389 bp ds-DNA circular SYN 11-2-2014 DEFINITION Bacterial vector encoding a C-terminal TEV-10xHis-TVMV-MBP cassette, for high-throughput purification of recombinant proteins. ACCESSION . VERSION . KEYWORDS pMCSG29 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6389) 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 6389) 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..6389 /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" CDS complement(1368..1388) /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /note="TEV site" /translation="ENLYFQS" RBS 1400..1405 /note="ribosome binding site" protein_bind 1436..1460 /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(1461..1479) /note="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" promoter 1792..1869 /gene="lacI" /note="lacI promoter" /note=" " CDS 1870..2952 /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 3761..3952 /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /note="rop" /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" rep_origin complement(4382..4970) /direction=LEFT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(5141..6001) /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(6002..6105) /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 gcccggcgga ttggaagtac 1381 aggttctccc cgggagagac tccttcttaa agttaaacaa aattatttct agaggggaat 1441 tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcgcggga tcgagatcga 1501 tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg 1561 ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac ttcgggctca 1621 tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga ctgttgggcg 1681 ccatctcctt gcatgcacca ttccttgcgg cggcggtgct caacggcctc aacctactac 1741 tgggctgctt cctaatgcag gagtcgcata agggagagcg tcgagatccc ggacaccatc 1801 gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg 1861 gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat 1921 cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa 1981 gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg 2041 ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg 2101 caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg 2161 atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa 2221 cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa 2281 gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac 2341 agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg 2401 ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt 2461 ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa 2521 ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc 2581 gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt 2641 accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa 2701 gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg 2761 caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag 2821 ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc 2881 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa 2941 agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg caccgggatc 3001 tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat 3061 gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc 3121 ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg 3181 cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg tcactggtcc 3241 cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg ccccacgggt 3301 gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg 3361 ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc 3421 tgcgacctga gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac 3481 gcggaagtca gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct 3541 accctgtgga acacctacat ctgtattaac gaagcgctgg cattgaccct gagtgatttt 3601 tctctggtcc cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg 3661 ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca tcggtatcat 3721 tacccccatg aacagaaatc ccccttacac ggaggcatca gtgaccaaac aggaaaaaac 3781 cgcccttaac atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa 3841 cgagctggac gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga 3901 gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca 3961 gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca 4021 gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga 4081 tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac 4141 catatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct 4201 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 4261 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 4321 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 4381 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 4441 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 4501 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 4561 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 4621 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 4681 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 4741 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 4801 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta 4861 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 4921 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 4981 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 5041 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 5101 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 5161 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 5221 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 5281 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 5341 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 5401 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctgcag 5461 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 5521 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 5581 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 5641 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 5701 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 5761 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 5821 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 5881 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 5941 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 6001 tactcttcct ttttcaaatt attgaagcat ttatcagggt tattgtctca tgagcggata 6061 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 6121 agtgccacct aaattgtaag cgttaatgtg aaccatcacc ctaatcaagt tttttggggt 6181 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac 6241 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta 6301 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg 6361 cgccgctaca gggcgcgtcc cattcgcca //