LOCUS Exported 6846 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pET His6 NusA TEV LIC cloning vector (1N) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6846) TITLE LIC N-terminal fusion vectors for E. coli expression REFERENCE 2 (bases 1 to 6846) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6846 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 12..467 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(99..118) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 309..330 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(560..1375) /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind 1283..1302 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 1367..1386 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 1497..2085 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1986..2005 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2239..2256 /label=L4440 /note="L4440 vector, forward primer" misc_feature 2271..2413 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(2339..2358) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 2499..2521 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(2515..2706) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(3515..4597) /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 4559..4578 /label=LacI-R /note="LacI, reverse primer" promoter complement(4598..4675) /gene="lacI" /label=lacI promoter primer_bind 4881..4900 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 4984..5003 /label=T7 /note="T7 promoter, forward primer" promoter 4984..5002 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 5003..5027 /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)." RBS 5042..5064 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 5083..5100 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 5110..6594 /codon_start=1 /gene="nusA" /product="transcription elongation protein (N utilization substance protein A)" /label=NusA /note="highly soluble in E. coli" /translation="MNKEILAVVEAVSNEKALPREKIFEALESALATATKKKYEQEIDV RVQIDRKSGDFDTFRRWLVVDEVTQPTKEITLEAARYEDESLNLGDYVEDQIESVTFDR ITTQTAKQVIVQKVREAERAMVVDQFREHEGEIITGVVKKVNRDNISLDLGNNAEAVIL REDMLPRENFRPGDRVRGVLYSVRPEARGAQLFVTRSKPEMLIELFRIEVPEIGEEVIE IKAAARDPGSRAKIAVKTNDKRIDPVGACVGMRGARVQAVSTELGGERIDIVLWDDNPA QFVINAMAPADVASIVVDEDKHTMDIAVEAGNLAQAIGRNGQNVRLASQLSGWELNVMT VDDLQAKHQAEAHAAIDTFTKYLDIDEDFATVLVEEGFSTLEELAYVPMKELLEIEGLD EPTVEALRERAKNALATIAQAQEESLGDNKPADDLLNLEGVDRDLAFKLAARGVCTLED LAEQGIDDLADIEGLTDEKAGALIMAARNICWFGDEA" CDS 6604..6624 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 6690..6707 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(6760..6778) /label=T7 Term /note="T7 terminator, reverse primer" terminator 6774..6821 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" ORIGIN 1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 421 acaaaaattt aacgcgaatt ttaacaaact agtaacgttt acaatttcag gtggcacttt 481 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 541 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 601 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 661 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 721 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 781 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 841 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 901 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 961 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatgt 1021 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1081 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1141 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1201 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1261 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1321 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1381 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1441 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1501 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1561 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1621 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1681 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1741 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1801 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1861 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1921 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1981 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2041 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2101 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2161 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2221 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2281 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2341 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2401 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2461 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2521 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2581 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2641 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2701 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2761 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2821 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2881 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2941 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3001 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3061 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3121 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3181 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3241 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3301 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3361 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3421 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3481 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3541 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3601 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3661 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3721 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3781 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3841 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3901 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3961 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4021 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4081 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4141 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4201 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4261 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4321 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4381 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4441 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4501 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4561 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4621 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4681 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4741 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4801 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4861 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4921 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4981 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5041 ttttgtttaa ctttaagaag gagatatacc atgggttctt ctcaccatca ccatcaccat 5101 ggttcttcta tgaacaaaga aattttggct gtagttgaag ccgtatccaa tgaaaaggcg 5161 ctacctcgcg agaagatttt cgaagcattg gaaagcgcgc tggcgacagc aacaaagaaa 5221 aaatatgaac aagagatcga cgtccgcgta cagatcgatc gcaaaagcgg tgattttgac 5281 actttccgtc gctggttagt tgttgatgaa gtcacccagc cgaccaagga aatcaccctt 5341 gaagccgcac gttatgaaga tgaaagcctg aacctgggcg attacgttga agatcagatt 5401 gagtctgtta cctttgaccg tatcactacc cagacggcaa aacaggttat cgtgcagaaa 5461 gtgcgtgaag ccgaacgtgc gatggtggtt gatcagttcc gtgaacacga aggtgaaatc 5521 atcaccggcg tggtgaaaaa agtaaaccgc gacaacatct ctctggatct gggcaacaac 5581 gctgaagccg tgatcctgcg cgaagatatg ctgccgcgtg aaaacttccg ccctggcgac 5641 cgcgttcgtg gcgtgctcta ttccgttcgc ccggaagcgc gtggcgcgca actgttcgtc 5701 actcgttcca agccggaaat gctgatcgaa ctgttccgta ttgaagtgcc agaaatcggc 5761 gaagaagtga ttgaaattaa agcagcggct cgcgatccgg gttctcgtgc gaaaatcgcg 5821 gtgaaaacca acgataaacg tatcgatccg gtaggtgctt gcgtaggtat gcgtggcgcg 5881 cgtgttcagg cggtgtctac tgaactgggt ggcgagcgta tcgatatcgt cctgtgggat 5941 gataacccgg cgcagttcgt gattaacgca atggcaccgg cagacgttgc ttctatcgtg 6001 gtggatgaag ataaacacac catggatatc gccgttgaag ccggtaacct ggcgcaggcg 6061 attggccgta acggtcagaa cgtgcgtctg gcttcgcagc tgagcggttg ggaactcaac 6121 gtgatgaccg ttgacgacct gcaggctaag catcaggcgg aagcgcacgc agcgatcgac 6181 accttcacca aatatctcga catcgacgaa gacttcgcga ctgttctggt agaagaaggc 6241 ttctcgacgc tggaagaatt ggcctatgtg ccgatgaaag agctgttgga aatcgaaggc 6301 cttgatgagc cgaccgttga agcactgcgc gagcgtgcta aaaatgcact ggccaccatt 6361 gcacaggccc aggaagaaag cctcggtgat aacaaaccgg ctgacgatct gctgaacctt 6421 gaaggggtag atcgtgattt ggcattcaaa ctggccgccc gtggcgtttg tacgctggaa 6481 gatctcgccg aacagggcat tgatgatctg gctgatatcg aagggttgac cgacgaaaaa 6541 gccggagcac tgattatggc tgcccgtaac atttgctggt tcggtgacga agcggggatc 6601 gaggaaaacc tgtacttcca atccaatatt ggaagtggat aacggatccg aattcgagcg 6661 ccgtcgacaa gcttgcggcc gcactcgagc accaccacca ccaccactga gatccggctg 6721 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat 6781 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat 6841 ccggat //