LOCUS Exported 9525 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Level 2 construct. ACCESSION . VERSION . KEYWORDS pUG-320 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9525) 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 9525) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9525 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 55..74 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 158..177 /label=T7 /note="T7 promoter, forward primer" promoter 158..176 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 177..201 /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 255..272 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 3820..3906 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAGYPYDVPDYAGLYPYDVPDY" CDS 3883..3909 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 3922..4008 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAGYPYDVPDYAGLYPYDVPDY" CDS 3985..4011 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" primer_bind complement(4617..4635) /label=T7 Term /note="T7 terminator, reverse primer" terminator 4631..4678 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 4715..5170 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4802..4821) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5012..5033 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(5262..6077) /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 5985..6004 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 6069..6088 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 6199..6787 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6688..6707 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6941..6958 /label=L4440 /note="L4440 vector, forward primer" misc_feature 6973..7112 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(7038..7057) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7198..7220 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(7214..7405) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(8214..9296) /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 9258..9277 /label=LacI-R /note="LacI, reverse primer" promoter complement(9297..9374) /gene="lacI" /label=lacI promoter ORIGIN 1 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat 61 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg 121 tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac tataggggaa 181 ttgtgagcgg ataacaattc ccctctagaa ataattttgc cttaacttta agaaggagat 241 ataccatggg cagccaccac caccaccacc acagcggaat gcagatcttt gttaagactc 301 tcaccggaaa gacaatcacc ctagaggtgg aaagctccga caccatcgac aacgttaagg 361 ccaagatcca ggataaggag ggcattcctc cggatcagca gaggcttatt ttcgccggca 421 agcagctaga ggatggccgt acgttggctg attacaatat ccagaaggaa tccaccctcc 481 acttggtcct caggctccgt ggtggttaag cttgcaatta actttaagaa ggagatatac 541 aatgcttcac aagcgagcta gtgaagctaa cgataagaac gataatacca tcatcggttc 601 cgatttagca tcatctaaga aacgccgtat cgatttcact gaatcttcgt ctgataagtc 661 atcttcgatt ttagcttctg gtagtagcag gggttttcac ggcgatagcg tcgttcagca 721 aatcgacatg gcttttggta attcgaaccg tcaggagatt gatgaagatc tgcacagtcg 781 ccagctcgcc gtctatggtc gtgagactat gaggcgtctc tttgcttcca atgttctcat 841 ctcggggatg cacggtcttg gtgccgagat tgccaagaat cttatacttg ctggtgtgaa 901 gtctgtgacc ttgcatgatg aaagagttgt agagctatgg gacttatcaa gcaactttgt 961 tttctcggag gatgatgttg gcaagaatag agcagatgct tctgttcaga agttacagga 1021 tcttaacaat gctgtggttg tctctagctt gaccaaaagc ttaaacaaag aggatctttc 1081 tggcttccag gttgttgtat tctctgacat aagcatggaa agagcaatcg aatttgatga 1141 ctattgtcac agccatcagc ctcctatcgc ttttgtcaag gctgatgtca ggggtctttt 1201 cggctctgtc ttttgtgatt ttgggcctga atttgcggtt cttgatgttg atggagagga 1261 accccataca ggcatcattg cgtctatctc taatgagaac caggcattta tttcctgtgt 1321 cgacgatgag aggcttgagt ttgaagatgg tgacctagtt gttttctcag aagttgaggg 1381 tatgacagaa ttgaatgatg ggaaaccaag gaagataaaa agtacacggc catattcatt 1441 cacccttgat gaggacacta ctaactatgg aacatatgtg aagggtggta ttgtcactca 1501 agtgaaacag ccaaagttac tgaatttcaa gccattgagg gaagcgctta aggatccagg 1561 ggattttcta tttagtgatt tctctaagtt tgatcggcct ccactccttc acttagcatt 1621 ccaggcactt gatcatttta aagctgaagc tggcagattt cctgttgctg gctcagaaga 1681 ggacgctcag aagcttatat cgattgccac agccatcaat accgggcagg gtgatttaaa 1741 ggtggagaat gttgatcaga agcttctaag acacttttcc tttggagcca aggctgtcct 1801 gaatcctatg gctgcaatgt ttggcggtat tgttggacag gaggttgtta aagcttgctc 1861 tggaaaattt catcctctct tccagttttt ctactttgat tcagtggagt cactcccctc 1921 tgaacctgtg gactcgagtg actttgcacc aagaaacagc cggtatgatg cccaaatatc 1981 tgtatttggg gccaagttcc agaagaaact tgaagatgct aaagttttca cagtagggtc 2041 tggtgctctt ggctgtgagt tcttgaaaaa tttggctctg atgggggttt cttgtggaag 2101 ccaagggaag ctgacagtga cagatgatga tatcattgag aagagtaacc tcagtcgtca 2161 atttctgttc cgtgactgga acattggaca ggctaaatcc acagttgctg cttcggctgc 2221 tgctgttata aatcctcgct tcaacattga ggccctgcaa aaccgtgttg gcgcggagac 2281 tgagaatgta tttgacgatg ccttctggga gaacttaact gttgtcgtca atgcactgga 2341 taatgttaat gcgaggctct atgttgattc gaggtgcttg tatttccaga agcctctcct 2401 tgagtctgga actcttggta caaagtgcaa cacacagtcg gttatcccac atctaactga 2461 aaattacggt gcctcaaggg acccaccaga gaaacaggcc cccatgtgta cagtgcattc 2521 cttcccgcat aacattgatc actgtttaac ttgggctcgc tctgagtttg agggtctgct 2581 ggagaaaact cccgctgaag tgaatgcata tctttctagc ccagttgagt acactaactc 2641 aatgatgagt gctggtgatg ctcaggcgag ggacacattg gagaggattg ttgagtgcct 2701 tgaaaaggag aagtgtgaaa ccttccaaga ctgcttaacc tgggcacgac tgaggtttga 2761 ggattacttt gtgaaccgtg tcaagcaatt gatatacaca tttcctgaag atgctgcgac 2821 tagtaccgga gctccattct ggtctgctcc aaaaagattc cctcgtccac tccagtactc 2881 gtcgtccgac ccaagtctcc ttaactttat cacagcgact gctattttac gagcagagac 2941 atttgggatc ccgatacctg agtggactaa gaacccaaag gaagcagcag aagctgttga 3001 cagagtgata gtcccagact ttgagccaag gcaagatgca aagattgtga ctgatgagaa 3061 agctaccact ttaaccactg cttcggtgga cgacgctgca gtcatcgacg atctcattgc 3121 taagatcgac cagtgtaggc ataacttgtc gccagacttc aggatgaaac ctattcagtt 3181 cgaaaaggat gatgatacaa actatcacat ggatgtgata gcaggactag ccaacatgag 3241 ggcgagaaat tacagcatcc ctgaagtgga caagctgaaa gcaaagttca tagcagggag 3301 aatcatccca gccattgcga cttcaacagc catggctact gggctggtct gcctcgagct 3361 gtacaaggtc cttgatggag gacataaagt ggaagcttac agaaacacat ttgccaacct 3421 ggcacttcca ctattctcca tggctgaacc acttccacca aaggtggtga agcaccgtga 3481 catggcttgg acagtttggg acagatgggt tctcaaggga aacccaactc ttcgtgaggt 3541 gttgcagtgg ctggaggaca aagggcttag cgcatacagc atctcttgtg gaagctgtct 3601 actgttcaac agtatgttca caaggcacaa ggagaggatg gacaagaagg ttgtggatct 3661 cgctcgggat gttgcgaaag tcgagttacc gccttatcgc aaccatcttg atgtagtggt 3721 ggcttgtgag gatgaagatg acaatgacgt cgatattcct ctcgtctcta tctacttcag 3781 gtgagcttac tattaacttt aagaaggaga tataccatgt acccatacga tgttcctgac 3841 tatgcgggct atccctatga cgtcccggac tatgcaggat tgtatccata tgacgttcca 3901 gattacgcca ctagagctgc ttacccatac gatgttcctg actatgcggg ctatccctat 3961 gacgtcccgg actatgcagg attgtatcca tatgacgttc cagattacgc aatggcaacg 4021 agacggatat tgaaagaatt gaaggagttg caaagggacc ctcctgtatc atgcagtgca 4081 ggtccaacag gagaagatat gttccactgg caagctacta taatgggtcc gaatgaaagt 4141 ccttactccg gaggtgtttt ccttgtcaat attcatttcc ctcctgatta tccttttaaa 4201 cctcccaagg ttgtattcag aaccaaagtg tttcacccaa acatcaacag taatggaaac 4261 atatgtttgg acattctcaa agaccaatgg agccctgccc ttaccatttc taaggtgctt 4321 ctctctatat gctctcttct aacagatcca aaccctgacg accctctggt gccagagata 4381 gctcacatat acaaaactga caagaccaaa tatgaagcca tggctcgaag ctggacccag 4441 aagtatgcct tgttttaagc ttttacgctt ttaagccagc cccgacaccc gccaacaccc 4501 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc 4561 gggatgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc 4621 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct 4681 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc 4741 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc 4801 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct 4861 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa 4921 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc 4981 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca 5041 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat 5101 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg 5161 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 5221 tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc 5281 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc 5341 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg 5401 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca 5461 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc 5521 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca 5581 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagtcgaaat 5641 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac 5701 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat 5761 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa 5821 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct 5881 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc 5941 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta 6001 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc 6061 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt 6121 gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga 6181 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac 6241 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt 6301 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc 6361 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat 6421 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag 6481 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc 6541 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag 6601 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac 6661 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg 6721 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct 6781 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc 6841 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga 6901 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga 6961 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg 7021 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc 7081 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg 7141 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgactccg 7201 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt 7261 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca 7321 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa 7381 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg 7441 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac 7501 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc 7561 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac 7621 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact 7681 tccgcgtttc cagactttac gaaacacgga aacccaagac cattcatgtt gttgctcagg 7741 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct 7801 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca 7861 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg 7921 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg 7981 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg 8041 ctgccggcac ctgtcctacg agttgcatga taaagaacac agtcataagt gcggcgacga 8101 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg 8161 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc 8221 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 8281 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgacacgggc 8341 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg 8401 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag 8461 ctgtcctcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac 8521 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg 8581 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag 8641 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca 8701 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg 8761 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcctcatg ggagaaaata 8821 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag 8881 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg 8941 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct 9001 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca 9061 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt 9121 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct 9181 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg 9241 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc 9301 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc 9361 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca 9421 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga 9481 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacc //