LOCUS Exported 8447 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pDEST-HisMBP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8447) AUTHORS Nallamsetty S, Austin BP, Penrose KJ, Waugh DS TITLE Gateway vectors for the production of combinatorially-tagged His6-MBP fusion proteins in the cytoplasm and periplasm of Escherichia coli. JOURNAL Protein Sci. 2005 Dec . 14(12):2964-71. PUBMED 16322578 REFERENCE 2 (bases 1 to 8447) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8447 /organism="synthetic DNA construct" /mol_type="other DNA" CDS complement(129..434) /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind complement(231..250) /label=ccdB-fwd /note="ccdB gene, forward primer" CDS complement(776..1435) /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind 1350..1369 /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" promoter complement(1436..1538) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" protein_bind complement(1712..1836) /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(1849..1872) /label=MBP-F /note="Maltose binding protein, forward primer" regulatory 1980..1989 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS complement(2938..2955) /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(3023..3045) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3034..3050 /label=lac operator /bound_moiety="lac repressor encoded by 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)." promoter complement(3058..3086) /label=tac promoter /note="strong E. coli promoter; hybrid between the trp and lac UV5 promoters" CDS complement(3329..4411) /codon_start=1 /gene="lacI" /product="lac repressor" /label=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" primer_bind 4373..4392 /label=LacI-R /note="LacI, reverse primer" promoter complement(4412..4489) /gene="lacI (mutant)" /label=lacIq promoter /note="In the lacIq allele, a single base change in the promoter boosts expression of the lacI gene about 10-fold." CDS 4985..5176 /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" primer_bind complement(5170..5192) /label=pGEX 3' /note="pGEX vectors, reverse primer" misc_feature 5278..5418 /label=bom /note="basis of mobility region from pBR322" primer_bind 5333..5352 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind complement(5433..5450) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5604..6192) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5684..5703) /label=pBR322ori-F /note="pBR322 origin, forward primer" rep_origin 6303..6816 /direction=RIGHT /label=M13 ori /note="M13 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6390..6409) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6600..6621 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(6858..7718) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 7481..7500 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7719..7810) /gene="bla" /label=AmpR promoter terminator 7829..7856 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" terminator 7948..8034 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" primer_bind 8187..8204 /label=pBAD Reverse /note="For vectors with E. coli araBAD promoter, reverse primer" primer_bind 8368..8390 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 8382..8399 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 8383..8399 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" protein_bind 8411..88 /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" ORIGIN 1 atgatataaa tatcaatata ttaaattaga ttttgcataa aaaacagact acataatact 61 gtaaaacaca acatatccag tcactatggt cgacctgcag actggctgtg tataagggag 121 cctgacattt atattcccca gaacatcagg ttaatggcgt ttttgatgtc attttcgcgg 181 tggctgagat cagccacttc ttccccgata acggagaccg gcacactggc catatcggtg 241 gtcatcatgc gccagctttc atccccgata tgcaccaccg ggtaaagttc acgggagact 301 ttatctgaca gcagacgtgc actggccagg gggatcacca tccgtcgccc gggcgtgtca 361 ataatatcac tctgtacatc cacaaacaga cgataacggc tctctctttt ataggtgtaa 421 accttaaact gcatttcacc agcccctgtt ctcgtcagca aaagagccgt tcatttcaat 481 aaaccgggcg acctcagcca tcccttcctg attttccgct ttccagcgtt cggcacgcag 541 acgacgggct tcattctgca tggttgtgct taccagaccg gagatattga catcatatat 601 gccttgagca actgatagct gtcgctgtca actgtcactg taatacgctg cttcatagca 661 tacctctttt tgacatactt cgggtataca tatcagtata tattcttata ccgcaaaaat 721 cagcgcgcaa atacgcatac tgttatctgg cttttagtaa gccggatcca cgcgtttacg 781 ccccgccctg ccactcatcg cagtactgtt gtaattcatt aagcattctg ccgacatgga 841 agccatcaca aacggcatga tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt 901 gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt 961 ttaaatcaaa actggtgaaa ctcacccagg gattggctga gacgaaaaac atattctcaa 1021 taaacccttt agggaaatag gccaggtttt caccgtaaca cgccacatct tgcgaatata 1081 tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa aacgtttcag 1141 tttgctcatg gaaaacggtg taacaagggt gaacactatc ccatatcacc agctcaccgt 1201 ctttcattgc catacggaat tccggatgag cattcatcag gcgggcaaga atgtgaataa 1261 aggccggata aaacttgtgc ttatttttct ttacggtctt taaaaaggcc gtaatatcca 1321 gctgaacggt ctggttatag gtacattgag caactgactg aaatgcctca aaatgttctt 1381 tacgatgcca ttgggatata tcaacggtgg tatatccagt gatttttttc tccattttag 1441 cttccttagc tcctgaaaat ctcgataact caaaaaatac gcccggtagt gatcttattt 1501 cattatggtg aaagttggaa cctcttgcgt gccgatcaac gtctcatttt cgccaaaagt 1561 tggcccaggg cttcccggta tcaacaggga caccaggatt tatttattct gcgaagtgat 1621 cttccgtcac aggtatttat tcggcgcaaa gtgcgtcggg tgatgctgcc aacttagcgg 1681 ccgctaagtt ggcagcatca cccgcggccg ccatagtgac tggatatgtt gtgttttaca 1741 gtattatgta gtctgttttt tatgcaaaat ctaatttaat atattgatat ttatatcatt 1801 ttacgtttct cgttcagctt ttttgtacaa acttgtgatc gaattagtct gcgcgtcttt 1861 cagggcttca tcgacagtct gacgaccgct ggcggcgttg atcaccgcag tacgcacggc 1921 ataccagaaa gcggacatct gcgggatgtt cggcatgatt tcacctttct gggcgttttc 1981 catggtggcg gcaatacgtg gatctttcgc caactcttcc tcgtaagact tcagcgctac 2041 ggcacccagc ggtttgtctt tattaaccgc ttccagacct tcatcagtca gcagatagtt 2101 ttcgaggaac tcttttgcca gctctttgtt cggactggcg gcgttaatac ctgcgctcag 2161 cacgccaacg aacggtttgg atggttgacc cttgaaggtc ggcagtaccg ttacaccata 2221 attcactttg ctggtgtcga tgttggacca tgcccacggg ccgttgatgg tcatcgctgt 2281 ttcgccttta ttaaaggcag cttctgcgat ggagtaatcg gtgtctgcat tcatgtgttt 2341 gtttttaatc aggtcaacca ggaaggtcag acccgctttc gcgccagcgt tatccacgcc 2401 cacgtcttta atgtcgtact tgccgttttc atacttgaac gcataacccc cgtcagcagc 2461 aatcagcggc caggtgaagt acggttcttg caggttgaac atcagcgcgc tcttaccttt 2521 cgctttcagt tctttatcca gcgccgggat ctcttcccag gtttttggcg ggttcggcag 2581 cagatctttg ttataaatca gcgataacgc ttcaacagcg atcgggtaag caatcagctt 2641 gccgttgtaa cgtacggcat cccaggtaaa cggatacagc ttgtcctgga acgctttgtc 2701 cggggtgatt tcagccaaca ggccagattg agcgtagcca ccaaagcggt cgtgtgccca 2761 gaagataatg tcagggccat cgccagttgc cgcaacctgt gggaatttct cttccagttt 2821 atccggatgc tcaacggtga ctttaattcc ggtatctttc tcgaatttct taccgacttc 2881 agcgagaccg ttatagcctt tatcgccgtt aatccagatt accagtttac cttcttcgtg 2941 atggtgatgg tgatggattt tcataatcta tggtccttgt tggtgaagtg ctcgtgaaaa 3001 cacctaaacg gactggctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacat 3061 tatacgagcc gatgattaat tgtcaacagc tcatttcaga atatttgcca gaaccgttat 3121 gatgtcggcg caaaaaacat tatccagaac gggagtgcgc cttgagcgac acgaattatg 3181 cagtgattta cgacctgcac agccatacca cagcttccga tggctgcctg acgccagaag 3241 cattggtgca ccgtgcagtc gatgataagc tgtcaaacat gagaattgtg cctaatgagt 3301 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc 3361 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg 3421 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct 3481 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct 3541 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca 3601 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca 3661 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt 3721 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct 3781 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag 3841 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca 3901 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag 3961 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct 4021 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca 4081 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac 4141 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca 4201 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc 4261 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag 4321 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact 4381 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg 4441 ggcgctatca tgccataccg cgaaaggttt tgcaccattc gatggtgtcg gaatttcggg 4501 cagcgttggg tcctggccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct 4561 aggctggcgg ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg 4621 aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt 4681 ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat 4741 ctgcatcgca ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg 4801 ctggcattga ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt 4861 accctcacaa cgttccagta accgggcatg ttcatcatca gtaacccgta tcgtgagcat 4921 cctctctcgt ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc 4981 atcagtgacc aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac 5041 attaacgctt ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga 5101 atcgctgcac gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga 5161 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga 5221 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc 5281 agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca 5341 gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg 5401 agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc 5461 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa 5521 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 5581 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 5641 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 5701 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 5761 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc 5821 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 5881 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 5941 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 6001 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc 6061 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 6121 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 6181 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 6241 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt 6301 ttacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 6361 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 6421 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 6481 tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg 6541 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 6601 tggactcttg ttccaaactg gaacaacact caaccctatc tcgggctatt cttttgattt 6661 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 6721 taacgcgaat tttaacaaaa tattaacgtt tacaatttaa atatttgctt atacaatctt 6781 cctgtttttg gggcttttct gattatcaac cggggtaaat caatctaaag tatatatgag 6841 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 6901 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 6961 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 7021 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 7081 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 7141 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg 7201 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc 7261 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 7321 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 7381 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt 7441 atgcggcgac cgagttgctc ttgcccggcg tcaacacggg ataataccgc gccacatagc 7501 agaactttaa aagtgctcat cattgggaaa cgttcttcgg ggcgaaaact ctcaaggatc 7561 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 7621 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 7681 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 7741 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 7801 aataaacaaa agagtttgta gaaacgcaaa aaggccatcc gtcaggatgg ccttctgctt 7861 aatttgatgc ctggcagttt atggcgggcg tcctgcccgc caccctccgg gccgttgctt 7921 cgcaacgttc aaatccgctc ccggcggatt tgtcctactc aggagagcgt tcaccgacaa 7981 acaacagata aaacgaaagg cccagtcttt cgactgagcc tttcgtttta tttgatgcct 8041 ggcagttccc tactctcgca tggggagacc ccacactacc atcggcgcta cggcgtttca 8101 cttctgagtt cggcatgggg tcaggtggga ccaccgcgct actgccgcca ggcaaattct 8161 gttttatcag accgcttctg cgttctgatt taatctgtat caggctgaaa atcttatctc 8221 atccgccaaa acagccaagc tgccattcgc cattcaggct gcgcaactgt tgggaagggc 8281 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc 8341 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg 8401 ccaagctatc accactttgt acaagaaagc tgaacgagaa acgtaaa //