LOCUS Exported 5777 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Bacterial expression for structure determination. ACCESSION . VERSION . KEYWORDS 6CKO (DOT1L) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5777) TITLE SGC Toronto Q3-Q4 2017, Q1-Q2 2018 REFERENCE 2 (bases 1 to 5777) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5777 /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 50..73 /codon_start=1 /product="8xHis affinity tag" /label=8xHis /translation="HHHHHHHH" CDS 83..100 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS 101..391 /codon_start=1 /gene="S. cerevisiae SMT3 (truncated)" /product="cleavable ubiquitin-like protein tag" /label=SUMO /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIG" CDS 398..418 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 595..612 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(665..683) /label=T7 Term /note="T7 terminator, reverse primer" terminator 679..726 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 763..1218 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(850..869) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1060..1081 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(1310..2125) /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 2033..2052 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 2117..2136 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 2247..2835 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2736..2755 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2989..3006 /label=L4440 /note="L4440 vector, forward primer" misc_feature 3021..3160 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(3086..3105) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3246..3268 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(3262..3453) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(4262..5344) /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 5306..5325 /label=LacI-R /note="LacI, reverse primer" promoter complement(5345..5422) /gene="lacI" /label=lacI promoter primer_bind 5628..5647 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 5731..5750 /label=T7 /note="T7 promoter, forward primer" promoter 5731..5749 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 5750..5774 /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 atgaaaaaac atcaccatca 61 tcatcatcat cacagcagcg gcctggttcc gcgtggtagt atgtcggact cagaagtcaa 121 tcaagaagct aagccagagg tcaagccaga agtcaagcct gagactcaca tcaatttaaa 181 ggtgtccgat ggatcttcag agatcttctt caagatcaaa aagaccactc ctttaagaag 241 gctgatggaa gcgttcgcta aaagacaggg taaggaaatg gactccttaa gattcttgta 301 cgacggtatt agaatccaag ctgatcagac ccctgaagat ttggacatgg aggataacga 361 tattattgag gctcacagag aacagattgg tggaacagaa aacttgtatt tccagggcac 421 ctacaacgac ctgattcaag cgcagaagga gatctccgcc cataaccagc agctgcggga 481 gcagtcggag cagctggagc aggacaaccg cgcgctccgc ggccagagct tgcagctgct 541 caaggctcgc tgcgaggaga tgcagtgatg acgaagcttg cggccgcact cgagcaccac 601 caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct 661 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt 721 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg 781 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc 841 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc 901 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg 961 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg 1021 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg 1081 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt 1141 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa 1201 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 1261 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact 1321 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt 1381 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa 1441 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc 1501 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg 1561 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct 1621 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga 1681 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc 1741 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata 1801 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac 1861 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca 1921 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg 1981 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag 2041 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag 2101 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca 2161 gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac 2221 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc 2281 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca 2341 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta 2401 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct 2461 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg 2521 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc 2581 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta 2641 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg 2701 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt 2761 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg 2821 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg 2881 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc 2941 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg 3001 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt 3061 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt 3121 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc 3181 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac 3241 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca 3301 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc 3361 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc 3421 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct 3481 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga 3541 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg 3601 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg 3661 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg 3721 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt 3781 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga 3841 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag 3901 cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa 3961 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac 4021 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac 4081 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc 4141 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa 4201 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg 4261 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca 4321 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg 4381 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt 4441 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat 4501 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca 4561 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca 4621 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg 4681 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat 4741 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga 4801 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg 4861 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat 4921 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca 4981 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc 5041 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg 5101 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca 5161 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca 5221 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc 5281 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt 5341 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg 5401 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt 5461 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca 5521 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg 5581 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg 5641 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg 5701 tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag 5761 cggataacaa ttcccct //