LOCUS Exported 5672 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Bacterial expression for structure determination. ACCESSION . VERSION . KEYWORDS 6ASB (FBXL19) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5672) TITLE SGC Toronto Q3-Q4 2017, Q1-Q2 2018 REFERENCE 2 (bases 1 to 5672) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5672 /organism="synthetic DNA construct" /mol_type="other DNA" RBS 12..34 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 44..61 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 74..94 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 490..507 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(560..578) /label=T7 Term /note="T7 terminator, reverse primer" terminator 574..621 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 658..1113 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(745..764) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 955..976 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(1205..2020) /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 1928..1947 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 2012..2031 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 2142..2730 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2631..2650 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2884..2901 /label=L4440 /note="L4440 vector, forward primer" misc_feature 2916..3055 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(2981..3000) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3141..3163 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(3157..3348) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(4157..5239) /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 5201..5220 /label=LacI-R /note="LacI, reverse primer" promoter complement(5240..5317) /gene="lacI" /label=lacI promoter primer_bind 5523..5542 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 5626..5645 /label=T7 /note="T7 promoter, forward primer" promoter 5626..5644 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 5645..5669 /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)." ORIGIN 1 ctagaaataa ttttgtttaa ctttaagaag gagatatacc atgcatcatc atcatcatca 61 cagcagcggc agagaaaact tgtatttcca gggcgcccgt cgccgtcgca cccgttgccg 121 tcgctgtcgt gcatgcgttc gcacggaatg tggcgattgc catttttgtc gcgacatgaa 181 aaaatttggc ggtccgggtc gtatgaaaca gagctgtctg ctgcgtcaat gcaccgcacc 241 ggtgctgccg cacacggccg tttgcctgct gtgtggcgaa gccggtaaag aagataccgt 301 cgaaggcgaa gaagaaaaat ttggtctgtc tctgatggaa tgcacgattt gtaacgaaat 361 cgtccatccg ggttgtctga aaatgggcaa agcagaaggt gtgattaacg ctgaaatccc 421 gaattgctgg gaatgtccgc gttgcaccca ggaaggtcgt tgatgacgaa gcttgcggcc 481 gcactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa 541 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa 601 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg 661 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta 721 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt 781 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg 841 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat 901 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac 961 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag 1021 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg 1081 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg 1141 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta 1201 attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat 1261 caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt 1321 tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac 1381 aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga 1441 cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag 1501 gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg 1561 attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa 1621 tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag 1681 gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg 1741 catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc 1801 agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca 1861 gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc 1921 cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc 1981 gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt 2041 ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc 2101 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 2161 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 2221 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 2281 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 2341 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 2401 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 2461 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 2521 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 2581 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 2641 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 2701 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 2761 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg 2821 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag 2881 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg 2941 catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg 3001 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg 3061 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta 3121 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc 3181 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat 3241 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct 3301 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt 3361 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac 3421 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact 3481 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt 3541 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa 3601 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa 3661 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg 3721 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt 3781 cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc 3841 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg 3901 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc 3961 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa 4021 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg 4081 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac 4141 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 4201 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt 4261 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga 4321 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg 4381 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat 4441 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat 4501 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt 4561 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc 4621 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc 4681 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg 4741 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa 4801 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg 4861 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac 4921 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg 4981 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg 5041 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt 5101 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg 5161 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt 5221 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg 5281 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta 5341 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc 5401 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc 5461 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca 5521 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc 5581 acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta 5641 taggggaatt gtgagcggat aacaattccc ct //