LOCUS Exported 5597 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Bacterial Expression. ACCESSION . VERSION . KEYWORDS 6WAJ SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5597) TITLE SGC Toronto 2019-Q1 to 2020-Q2 REFERENCE 2 (bases 1 to 5597) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5597 /organism="synthetic DNA construct" /mol_type="other DNA" CDS complement(279..299) /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS complement(312..329) /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" RBS 339..361 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" protein_bind 376..400 /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)." primer_bind complement(400..419) /label=T7 /note="T7 promoter, forward primer" promoter complement(401..419) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(503..522) /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" promoter 728..805 /gene="lacI" /label=lacI promoter CDS 806..1888 /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 complement(825..844) /label=LacI-R /note="LacI, reverse primer" CDS 2697..2888 /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" primer_bind complement(2882..2904) /label=pGEX 3' /note="pGEX vectors, reverse primer" misc_feature 2990..3129 /label=bom /note="basis of mobility region from pBR322" primer_bind 3045..3064 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind complement(3144..3161) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3315..3903) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3395..3414) /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind complement(4014..4033) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 4025..4840 /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 complement(4098..4117) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin complement(4932..5387) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5069..5090) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 5281..5300 /label=F1ori-R /note="F1 origin, reverse primer" terminator 5424..5471 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" primer_bind 5467..5485 /label=T7 Term /note="T7 terminator, reverse primer" CDS complement(5538..5555) /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" ORIGIN 1 tggtagatga tgtctaggac cttctctgtc tccactgcct gggactccaa cgtcttcccc 61 agtgaggaga cgatctcagc atcgtggaca aagaaagcca gtggcagggg atcctcctgg 121 gccagtagcg cgttgcacac gagctgcagc ctgtccgggg tgatgtccac gggcacgtcg 181 aacggggaac ccagcagctg cccgccctca tcctggaact gcactagcaa ccgctgcaca 241 tcgcgcgcca ccgcctcgtc cgccactgct gccgccatgc cctggaaata caagttttct 301 ctgccgctgc tgtgatgatg atgatgatgc atggtatatc tccttcttaa agttaaacaa 361 aattatttct agaggggaat tgttatccgc tcacaattcc cctatagtga gtcgtattaa 421 tttcgcggga tcgagatctc gatcctctac gccggacgca tcgtggccgg catcaccggc 481 gccacaggtg cggttgctgg cgcctatatc gccgacatca ccgatgggga agatcgggct 541 cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc 601 gggggactgt tgggcgccat ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac 661 ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgtcga 721 gatcccggac accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga 781 agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta 841 tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc 901 gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt 961 ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc 1021 cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc 1081 cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca 1141 caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga 1201 tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga 1261 ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga 1321 gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt 1381 ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc 1441 gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat 1501 gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg 1561 cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg 1621 atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga 1681 ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc 1741 ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc 1801 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca 1861 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc 1921 attaggcacc gggatctcga ccgatgccct tgagagcctt caacccagtc agctccttcc 1981 ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac 2041 tcgtaggaca ggtgccggca gcgctctggg tcattttcgg cgaggaccgc tttcgctgga 2101 gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag 2161 ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt atcgccggca 2221 tggcggcccc acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc 2281 ggggttgcct tactggttag cagaatgaat caccgatacg cgagcgaacg tgaagcgact 2341 gctgctgcaa aacgtctgcg acctgagcaa caacatgaat ggtcttcggt ttccgtgttt 2401 cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg atctgcatcg 2461 caggatgctg ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctggcatt 2521 gaccctgagt gatttttctc tggtcccgcc gcatccatac cgccagttgt ttaccctcac 2581 aacgttccag taaccgggca tgttcatcat cagtaacccg tatcgtgagc atcctctctc 2641 gtttcatcgg tatcattacc cccatgaaca gaaatccccc ttacacggag gcatcagtga 2701 ccaaacagga aaaaaccgcc cttaacatgg cccgctttat cagaagccag acattaacgc 2761 ttctggagaa actcaacgag ctggacgcgg atgaacaggc agacatctgt gaatcgcttc 2821 acgaccacgc tgatgagctt taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa 2881 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga 2941 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga 3001 cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat 3061 tgtactgaga gtgcaccatt gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac 3121 cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 3181 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 3241 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 3301 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 3361 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 3421 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 3481 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 3541 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 3601 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 3661 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 3721 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg 3781 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 3841 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 3901 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 3961 taagggattt tggtcatgaa caataaaact gtctgcttac ataaacagta atacaagggg 4021 tgttatgagc catattcaac gggaaacgtc ttgctctagg ccgcgattaa attccaacat 4081 ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac 4141 aatctatcga ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg 4201 tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat 4261 gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac 4321 tgcgatcccc gggaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa 4381 tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg 4441 tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg 4501 tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg 4561 gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt 4621 ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg 4681 agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt 4741 ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa 4801 taaattgcag tttcatttga tgctcgatga gtttttctaa gaattaattc atgagcggat 4861 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 4921 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta 4981 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga 5041 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa 5101 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga 5161 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc 5221 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga 5281 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg 5341 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccaat 5401 ccggatatag ttcctccttt cagcaaaaaa cccctcaaga cccgtttaga ggccccaagg 5461 ggttatgcta gttattgctc agcggtggca gcagccaact cagcttcctt tcgggctttg 5521 ttagcagccg gatctcagtg gtggtggtgg tggtgctcga gtgcggccgc aagcttcgtc 5581 atcagatagc ctgtggc //