LOCUS Exported 9552 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Level 2 construct. ACCESSION . VERSION . KEYWORDS pUG-312 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9552) AUTHORS Kowarschik K, Hoehenwarter W, Marillonnet S, Trujillo M TITLE UbiGate: a synthetic biology toolbox to analyse ubiquitination. JOURNAL New Phytol. 2018 Mar;217(4):1749-1763. doi: 10.1111/nph.14900. Epub 2017 Nov 30. PUBMED 29194629 REFERENCE 2 (bases 1 to 9552) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9552 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 932..951 /label=LacI-R /note="LacI, reverse primer" promoter complement(971..1048) /gene="lacI" /label=lacI promoter primer_bind 1254..1273 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 1357..1376 /label=T7 /note="T7 promoter, forward primer" promoter 1357..1375 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1376..1400 /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)." CDS 1454..1471 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 5019..5105 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAGYPYDVPDYAGLYPYDVPDY" CDS 5082..5108 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 5121..5207 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAGYPYDVPDYAGLYPYDVPDY" CDS 5184..5210 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" primer_bind complement(5843..5861) /label=T7 Term /note="T7 terminator, reverse primer" terminator 5857..5904 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 5941..6396 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6028..6047) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6238..6259 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(6488..7303) /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 7211..7230 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 7295..7314 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 7425..8013 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7914..7933 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 8167..8184 /label=L4440 /note="L4440 vector, forward primer" misc_feature 8199..8338 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(8264..8283) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 8424..8446 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(8440..8631) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(9440..970) /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" ORIGIN 1 ccagtgacac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca 61 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg 121 ggatataaca tgagctgtcc tcggtatcgt cgtatcccac taccgagata tccgcaccaa 181 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa 241 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg 301 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat 361 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca 421 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtcct 481 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg 541 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa 601 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga 661 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt 721 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa 781 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct 841 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca 901 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta 961 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc 1021 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc 1081 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat 1141 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc 1201 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg 1261 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt 1321 ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat 1381 tgtgagcgga taacaattcc cctctagaaa taattttgcc ttaactttaa gaaggagata 1441 taccatgggc agccaccacc accaccacca cagcggaatg cagatctttg ttaagactct 1501 caccggaaag acaatcaccc tagaggtgga aagctccgac accatcgaca acgttaaggc 1561 caagatccag gataaggagg gcattcctcc ggatcagcag aggcttattt tcgccggcaa 1621 gcagctagag gatggccgta cgttggctga ttacaatatc cagaaggaat ccaccctcca 1681 cttggtcctc aggctccgtg gtggttaagc ttgcaattaa ctttaagaag gagatataca 1741 atgcttcaca agcgagctag tgaagctaac gataagaacg ataataccat catcggttcc 1801 gatttagcat catctaagaa acgccgtatc gatttcactg aatcttcgtc tgataagtca 1861 tcttcgattt tagcttctgg tagtagcagg ggttttcacg gcgatagcgt cgttcagcaa 1921 atcgacatgg cttttggtaa ttcgaaccgt caggagattg atgaagatct gcacagtcgc 1981 cagctcgccg tctatggtcg tgagactatg aggcgtctct ttgcttccaa tgttctcatc 2041 tcggggatgc acggtcttgg tgccgagatt gccaagaatc ttatacttgc tggtgtgaag 2101 tctgtgacct tgcatgatga aagagttgta gagctatggg acttatcaag caactttgtt 2161 ttctcggagg atgatgttgg caagaataga gcagatgctt ctgttcagaa gttacaggat 2221 cttaacaatg ctgtggttgt ctctagcttg accaaaagct taaacaaaga ggatctttct 2281 ggcttccagg ttgttgtatt ctctgacata agcatggaaa gagcaatcga atttgatgac 2341 tattgtcaca gccatcagcc tcctatcgct tttgtcaagg ctgatgtcag gggtcttttc 2401 ggctctgtct tttgtgattt tgggcctgaa tttgcggttc ttgatgttga tggagaggaa 2461 ccccatacag gcatcattgc gtctatctct aatgagaacc aggcatttat ttcctgtgtc 2521 gacgatgaga ggcttgagtt tgaagatggt gacctagttg ttttctcaga agttgagggt 2581 atgacagaat tgaatgatgg gaaaccaagg aagataaaaa gtacacggcc atattcattc 2641 acccttgatg aggacactac taactatgga acatatgtga agggtggtat tgtcactcaa 2701 gtgaaacagc caaagttact gaatttcaag ccattgaggg aagcgcttaa ggatccaggg 2761 gattttctat ttagtgattt ctctaagttt gatcggcctc cactccttca cttagcattc 2821 caggcacttg atcattttaa agctgaagct ggcagatttc ctgttgctgg ctcagaagag 2881 gacgctcaga agcttatatc gattgccaca gccatcaata ccgggcaggg tgatttaaag 2941 gtggagaatg ttgatcagaa gcttctaaga cacttttcct ttggagccaa ggctgtcctg 3001 aatcctatgg ctgcaatgtt tggcggtatt gttggacagg aggttgttaa agcttgctct 3061 ggaaaatttc atcctctctt ccagtttttc tactttgatt cagtggagtc actcccctct 3121 gaacctgtgg actcgagtga ctttgcacca agaaacagcc ggtatgatgc ccaaatatct 3181 gtatttgggg ccaagttcca gaagaaactt gaagatgcta aagttttcac agtagggtct 3241 ggtgctcttg gctgtgagtt cttgaaaaat ttggctctga tgggggtttc ttgtggaagc 3301 caagggaagc tgacagtgac agatgatgat atcattgaga agagtaacct cagtcgtcaa 3361 tttctgttcc gtgactggaa cattggacag gctaaatcca cagttgctgc ttcggctgct 3421 gctgttataa atcctcgctt caacattgag gccctgcaaa accgtgttgg cgcggagact 3481 gagaatgtat ttgacgatgc cttctgggag aacttaactg ttgtcgtcaa tgcactggat 3541 aatgttaatg cgaggctcta tgttgattcg aggtgcttgt atttccagaa gcctctcctt 3601 gagtctggaa ctcttggtac aaagtgcaac acacagtcgg ttatcccaca tctaactgaa 3661 aattacggtg cctcaaggga cccaccagag aaacaggccc ccatgtgtac agtgcattcc 3721 ttcccgcata acattgatca ctgtttaact tgggctcgct ctgagtttga gggtctgctg 3781 gagaaaactc ccgctgaagt gaatgcatat ctttctagcc cagttgagta cactaactca 3841 atgatgagtg ctggtgatgc tcaggcgagg gacacattgg agaggattgt tgagtgcctt 3901 gaaaaggaga agtgtgaaac cttccaagac tgcttaacct gggcacgact gaggtttgag 3961 gattactttg tgaaccgtgt caagcaattg atatacacat ttcctgaaga tgctgcgact 4021 agtaccggag ctccattctg gtctgctcca aaaagattcc ctcgtccact ccagtactcg 4081 tcgtccgacc caagtctcct taactttatc acagcgactg ctattttacg agcagagaca 4141 tttgggatcc cgatacctga gtggactaag aacccaaagg aagcagcaga agctgttgac 4201 agagtgatag tcccagactt tgagccaagg caagatgcaa agattgtgac tgatgagaaa 4261 gctaccactt taaccactgc ttcggtggac gacgctgcag tcatcgacga tctcattgct 4321 aagatcgacc agtgtaggca taacttgtcg ccagacttca ggatgaaacc tattcagttc 4381 gaaaaggatg atgatacaaa ctatcacatg gatgtgatag caggactagc caacatgagg 4441 gcgagaaatt acagcatccc tgaagtggac aagctgaaag caaagttcat agcagggaga 4501 atcatcccag ccattgcgac ttcaacagcc atggctactg ggctggtctg cctcgagctg 4561 tacaaggtcc ttgatggagg acataaagtg gaagcttaca gaaacacatt tgccaacctg 4621 gcacttccac tattctccat ggctgaacca cttccaccaa aggtggtgaa gcaccgtgac 4681 atggcttgga cagtttggga cagatgggtt ctcaagggaa acccaactct tcgtgaggtg 4741 ttgcagtggc tggaggacaa agggcttagc gcatacagca tctcttgtgg aagctgtcta 4801 ctgttcaaca gtatgttcac aaggcacaag gagaggatgg acaagaaggt tgtggatctc 4861 gctcgggatg ttgcgaaagt cgagttaccg ccttatcgca accatcttga tgtagtggtg 4921 gcttgtgagg atgaagatga caatgacgtc gatattcctc tcgtctctat ctacttcagg 4981 tgagcttact attaacttta agaaggagat ataccatgta cccatacgat gttcctgact 5041 atgcgggcta tccctatgac gtcccggact atgcaggatt gtatccatat gacgttccag 5101 attacgccac tagagctgct tacccatacg atgttcctga ctatgcgggc tatccctatg 5161 acgtcccgga ctatgcagga ttgtatccat atgacgttcc agattacgca atgcaggcat 5221 caagagcgcg actgtttaaa gaatacaaag aggtacagcg agagaaagta gcagatcctg 5281 atattcaatt gatctgtgac gataccaaca tattcaaatg gaccgctctt atcaaaggac 5341 cttcggagac tccttacgaa ggcggtgttt ttcagcttgc tttttctgtt cctgaacctt 5401 atcctttgca acctcctcaa gttcgattct tgaccaagat attccatcct aatgttcatt 5461 tcaagacagg agaaatatgt ctcgacattt tgaagaatgc ttggagtcct gcttggacgc 5521 ttcagtctgt gtgtagagct atcatagcat tgatggctca tcctgagccg gacagtcctc 5581 ttaactgcga ctcagggaat cttctaagat ctggtgatgt gagagggttc aattcaatgg 5641 cacaaatgta tacacgcctc gccgctatgc ctaagaaagg atgagctttt acgcttttaa 5701 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg 5761 catccgctta cagacaagct gtgaccggga tgagatccgg ctgctaacaa agcccgaaag 5821 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct 5881 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg 5941 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg 6001 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca 6061 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta 6121 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc 6181 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg 6241 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat 6301 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta 6361 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt 6421 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa 6481 ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat 6541 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc 6601 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa 6661 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag 6721 tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa 6781 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc 6841 gtgattgcgc ctgagcgagt cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag 6901 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat 6961 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc 7021 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca 7081 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt 7141 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt 7201 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta 7261 atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac 7321 tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg 7381 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 7441 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 7501 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 7561 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 7621 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 7681 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 7741 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 7801 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 7861 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 7921 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 7981 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 8041 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct 8101 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc 8161 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt 8221 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg 8281 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc 8341 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc 8401 ttacagacaa gctgtgaccg actccgggag ctgcatgtgt cagaggtttt caccgtcatc 8461 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca 8521 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg 8581 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc 8641 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct 8701 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca 8761 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt 8821 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg 8881 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc 8941 caagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt 9001 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg 9061 ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat 9121 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc 9181 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg 9241 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa 9301 gaacacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac 9361 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact 9421 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 9481 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg 9541 tttttctttt ca //