LOCUS Exported 6293 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Bacterial expression for structure determination. ACCESSION . VERSION . KEYWORDS 6DUB (LOC149281) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6293) TITLE SGC Toronto Q3-Q4 2017, Q1-Q2 2018 REFERENCE 2 (bases 1 to 6293) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6293 /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 1111..1128 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(1181..1199) /label=T7 Term /note="T7 terminator, reverse primer" terminator 1195..1242 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 1279..1734 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1366..1385) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1576..1597 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(1826..2641) /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 2549..2568 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 2633..2652 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 2763..3351 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3252..3271 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3505..3522 /label=L4440 /note="L4440 vector, forward primer" misc_feature 3537..3676 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(3602..3621) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3762..3784 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(3778..3969) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(4778..5860) /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 5822..5841 /label=LacI-R /note="LacI, reverse primer" promoter complement(5861..5938) /gene="lacI" /label=lacI promoter primer_bind 6144..6163 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 6247..6266 /label=T7 /note="T7 promoter, forward primer" promoter 6247..6265 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 6266..6290 /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 gtcccaggtt attaacggtg aaatgcagtt ctacgcgcgg gccaagcttt tttatcaaga 481 ggtcccagca actgaagaag ggatgatggg aaatttcata gagctgtcga gccctgacat 541 tcaggcttct caaaaatttc tgcgcaaatt cgtgggcggt cccgggagag cgggaacaga 601 ttgtgcctta gactgcggca gtggtatcgg ccgagtatca aagcatgttt tgcttccggt 661 ctttaactcc gtagaactcg tggatatgat ggagtcgttt ttattggaag cacagaatta 721 ccttcaagtt aaaggtgata aagtcgaaag ctatcactgt tactctctcc aggagttcac 781 cccacctttt cggcgttatg acgtgatatg gattcagtgg gtaagtgggc atttaacgga 841 taaggacctg ttggctttcc tttcacgctg cagagatgga ctgaaagaaa acggcatcat 901 tattctgaaa gacaatgtgg cgcgagaggg ttgtatcctc gatttatccg attcgagcgt 961 tactcgtgac atggatatac tgcgctcttt gatccggaag agtggccttg tcgttctggg 1021 tcaagaaaaa caggacgggt ttccggaaca atgcattccc gtatggatgt tcgccctgca 1081 ctgatgacga agcttgcggc cgcactcgag caccaccacc accaccactg agatccggct 1141 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca 1201 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata 1261 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg 1321 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct 1381 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc 1441 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg 1501 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt 1561 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg 1621 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc 1681 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg 1741 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa 1801 tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca 1861 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag 1921 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc 1981 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa 2041 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt 2101 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa 2161 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa 2221 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa 2281 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga 2341 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa 2401 gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa 2461 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat 2521 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag 2581 catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca 2641 taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc 2701 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct 2761 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 2821 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc 2881 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac 2941 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct 3001 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat 3061 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg 3121 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa 3181 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg 3241 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga 3301 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc 3361 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct 3421 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct 3481 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg 3541 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca 3601 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga 3661 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg 3721 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca 3781 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg 3841 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc 3901 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg 3961 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat 4021 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga 4081 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca 4141 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca 4201 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact 4261 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca 4321 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca 4381 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc 4441 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac 4501 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt 4561 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc 4621 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc 4681 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc 4741 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg 4801 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 4861 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct 4921 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc 4981 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt 5041 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca 5101 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat 5161 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg 5221 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg 5281 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca 5341 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg 5401 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa 5461 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa 5521 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca 5581 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt 5641 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt 5701 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg 5761 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac 5821 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac 5881 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg 5941 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg 6001 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt 6061 cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag 6121 tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct 6181 gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg 6241 cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cct //