LOCUS Exported 10013 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Level 2 construct. ACCESSION . VERSION . KEYWORDS pUG-337 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10013) 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 10013) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10013 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 907..926 /label=LacI-R /note="LacI, reverse primer" promoter complement(946..1023) /gene="lacI" /label=lacI promoter primer_bind 1229..1248 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 1332..1351 /label=T7 /note="T7 promoter, forward primer" promoter 1332..1350 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1351..1375 /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 1429..1446 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 4994..5080 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAGYPYDVPDYAGLYPYDVPDY" CDS 5057..5083 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 5096..5182 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAGYPYDVPDYAGLYPYDVPDY" CDS 5159..5185 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" primer_bind complement(6279..6297) /label=T7 Term /note="T7 terminator, reverse primer" terminator 6293..6340 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 6377..6832 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6464..6483) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6674..6695 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(6924..7739) /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 7647..7666 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 7731..7750 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 7861..8449 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8350..8369 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 8603..8620 /label=L4440 /note="L4440 vector, forward primer" misc_feature 8635..8774 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(8700..8719) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 8860..8882 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(8876..9067) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(9876..945) /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 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag 61 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcctcggt 121 atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc 181 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc 241 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg 301 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag 361 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc 421 gaccagatgc tccacgccca gtcgcgtacc gtcctcatgg gagaaaataa tactgttgat 481 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac 541 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc 601 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac 661 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg 721 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag 781 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc 841 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga 901 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa 961 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt 1021 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta 1081 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca 1141 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc 1201 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg 1261 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga 1321 tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct 1381 agaaataatt ttgccttaac tttaagaagg agatatacca tgggcagcca ccaccaccac 1441 caccacagcg gaatgcagat ctttgttaag actctcaccg gaaagacaat caccctagag 1501 gtggaaagct ccgacaccat cgacaacgtt aaggccaaga tccaggataa ggagggcatt 1561 cctccggatc agcagaggct tattttcgcc ggcaagcagc tagaggatgg ccgtacgttg 1621 gctgattaca atatccagaa ggaatccacc ctccacttgg tcctcaggct ccgtggtggt 1681 taagcttgca attaacttta agaaggagat atacaatgct tcacaagcga gctagtgaag 1741 ctaacgataa gaacgataat accatcatcg gttccgattt agcatcatct aagaaacgcc 1801 gtatcgattt cactgaatct tcgtctgata agtcatcttc gattttagct tctggtagta 1861 gcaggggttt tcacggcgat agcgtcgttc agcaaatcga catggctttt ggtaattcga 1921 accgtcagga gattgatgaa gatctgcaca gtcgccagct cgccgtctat ggtcgtgaga 1981 ctatgaggcg tctctttgct tccaatgttc tcatctcggg gatgcacggt cttggtgccg 2041 agattgccaa gaatcttata cttgctggtg tgaagtctgt gaccttgcat gatgaaagag 2101 ttgtagagct atgggactta tcaagcaact ttgttttctc ggaggatgat gttggcaaga 2161 atagagcaga tgcttctgtt cagaagttac aggatcttaa caatgctgtg gttgtctcta 2221 gcttgaccaa aagcttaaac aaagaggatc tttctggctt ccaggttgtt gtattctctg 2281 acataagcat ggaaagagca atcgaatttg atgactattg tcacagccat cagcctccta 2341 tcgcttttgt caaggctgat gtcaggggtc ttttcggctc tgtcttttgt gattttgggc 2401 ctgaatttgc ggttcttgat gttgatggag aggaacccca tacaggcatc attgcgtcta 2461 tctctaatga gaaccaggca tttatttcct gtgtcgacga tgagaggctt gagtttgaag 2521 atggtgacct agttgttttc tcagaagttg agggtatgac agaattgaat gatgggaaac 2581 caaggaagat aaaaagtaca cggccatatt cattcaccct tgatgaggac actactaact 2641 atggaacata tgtgaagggt ggtattgtca ctcaagtgaa acagccaaag ttactgaatt 2701 tcaagccatt gagggaagcg cttaaggatc caggggattt tctatttagt gatttctcta 2761 agtttgatcg gcctccactc cttcacttag cattccaggc acttgatcat tttaaagctg 2821 aagctggcag atttcctgtt gctggctcag aagaggacgc tcagaagctt atatcgattg 2881 ccacagccat caataccggg cagggtgatt taaaggtgga gaatgttgat cagaagcttc 2941 taagacactt ttcctttgga gccaaggctg tcctgaatcc tatggctgca atgtttggcg 3001 gtattgttgg acaggaggtt gttaaagctt gctctggaaa atttcatcct ctcttccagt 3061 ttttctactt tgattcagtg gagtcactcc cctctgaacc tgtggactcg agtgactttg 3121 caccaagaaa cagccggtat gatgcccaaa tatctgtatt tggggccaag ttccagaaga 3181 aacttgaaga tgctaaagtt ttcacagtag ggtctggtgc tcttggctgt gagttcttga 3241 aaaatttggc tctgatgggg gtttcttgtg gaagccaagg gaagctgaca gtgacagatg 3301 atgatatcat tgagaagagt aacctcagtc gtcaatttct gttccgtgac tggaacattg 3361 gacaggctaa atccacagtt gctgcttcgg ctgctgctgt tataaatcct cgcttcaaca 3421 ttgaggccct gcaaaaccgt gttggcgcgg agactgagaa tgtatttgac gatgccttct 3481 gggagaactt aactgttgtc gtcaatgcac tggataatgt taatgcgagg ctctatgttg 3541 attcgaggtg cttgtatttc cagaagcctc tccttgagtc tggaactctt ggtacaaagt 3601 gcaacacaca gtcggttatc ccacatctaa ctgaaaatta cggtgcctca agggacccac 3661 cagagaaaca ggcccccatg tgtacagtgc attccttccc gcataacatt gatcactgtt 3721 taacttgggc tcgctctgag tttgagggtc tgctggagaa aactcccgct gaagtgaatg 3781 catatctttc tagcccagtt gagtacacta actcaatgat gagtgctggt gatgctcagg 3841 cgagggacac attggagagg attgttgagt gccttgaaaa ggagaagtgt gaaaccttcc 3901 aagactgctt aacctgggca cgactgaggt ttgaggatta ctttgtgaac cgtgtcaagc 3961 aattgatata cacatttcct gaagatgctg cgactagtac cggagctcca ttctggtctg 4021 ctccaaaaag attccctcgt ccactccagt actcgtcgtc cgacccaagt ctccttaact 4081 ttatcacagc gactgctatt ttacgagcag agacatttgg gatcccgata cctgagtgga 4141 ctaagaaccc aaaggaagca gcagaagctg ttgacagagt gatagtccca gactttgagc 4201 caaggcaaga tgcaaagatt gtgactgatg agaaagctac cactttaacc actgcttcgg 4261 tggacgacgc tgcagtcatc gacgatctca ttgctaagat cgaccagtgt aggcataact 4321 tgtcgccaga cttcaggatg aaacctattc agttcgaaaa ggatgatgat acaaactatc 4381 acatggatgt gatagcagga ctagccaaca tgagggcgag aaattacagc atccctgaag 4441 tggacaagct gaaagcaaag ttcatagcag ggagaatcat cccagccatt gcgacttcaa 4501 cagccatggc tactgggctg gtctgcctcg agctgtacaa ggtccttgat ggaggacata 4561 aagtggaagc ttacagaaac acatttgcca acctggcact tccactattc tccatggctg 4621 aaccacttcc accaaaggtg gtgaagcacc gtgacatggc ttggacagtt tgggacagat 4681 gggttctcaa gggaaaccca actcttcgtg aggtgttgca gtggctggag gacaaagggc 4741 ttagcgcata cagcatctct tgtggaagct gtctactgtt caacagtatg ttcacaaggc 4801 acaaggagag gatggacaag aaggttgtgg atctcgctcg ggatgttgcg aaagtcgagt 4861 taccgcctta tcgcaaccat cttgatgtag tggtggcttg tgaggatgaa gatgacaatg 4921 acgtcgatat tcctctcgtc tctatctact tcaggtgagc ttactattaa ctttaagaag 4981 gagatatacc atgtacccat acgatgttcc tgactatgcg ggctatccct atgacgtccc 5041 ggactatgca ggattgtatc catatgacgt tccagattac gccactagag ctgcttaccc 5101 atacgatgtt cctgactatg cgggctatcc ctatgacgtc ccggactatg caggattgta 5161 tccatatgac gttccagatt acgcaatggc caatagtaat cttccccgac gaatcatcaa 5221 ggaaactcaa cgtctgctta gtgaacctgc tcctgggata agtgcgtctc catcagagga 5281 aaatatgaga tatttcaatg ttatgattct tggtcctact caatctcctt atgaaggagg 5341 agttttcaag ttggagctct ttttgcctga agaatatcct atggcagctc ccaaggttag 5401 gtttctcacc aagatttacc atcctaacat cgacaagctt ggaagaatct gccttgacat 5461 actgaaagac aagtggagtc ctgcactaca aatacgaact gtgctcttga gtatccaagc 5521 tcttttaagt gcaccaaacc cagatgatcc tctgtctgag aacatagcta agcattggaa 5581 gagcaatgag gcagaagctg tggaaacagc taaggaatgg actcgtcttt acgcaagtgg 5641 tgcttaagct tttacttaac tttaagaagg agatatacaa tgactcttgg ctcaggatcg 5701 agtgttgtcg ttccgaggaa tttccggttg ctggaggagc ttgaacgtgg agagaaaggt 5761 attggagatg gaactgtgag ctatggaatg gatgatggag atgacattta tatgcgctct 5821 tggactggca ctatcatcgg tcctcacaac gtaactgtac atgagggtcg gatttatcag 5881 ttgaagctct tctgtgacaa agattaccct gagaaacctc cgactgttcg gttccattcg 5941 cgtatcaaca tgacttgtgt caaccatgat accggcgtgg tggattcaaa gaagtttggg 6001 gttcttgcaa actggcaaag acagtacaca atggaggaca tactgactca attgaagaag 6061 gagatggctg catcgcataa ccggaagctt gttcaacctc ccgaaggaac tttcttctaa 6121 gcttcagagc ttttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg 6181 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgggatgag atccggctgc 6241 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata 6301 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc 6361 cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt 6421 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc 6481 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct 6541 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat 6601 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc 6661 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc 6721 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg 6781 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca 6841 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 6901 tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat 6961 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga 7021 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg 7081 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt 7141 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct 7201 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc 7261 aaaccgttat tcattcgtga ttgcgcctga gcgagtcgaa atacgcgatc gctgttaaaa 7321 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca 7381 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc 7441 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga 7501 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg 7561 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag 7621 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca 7681 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata 7741 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc 7801 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 7861 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 7921 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 7981 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 8041 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 8101 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 8161 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 8221 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 8281 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 8341 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 8401 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 8461 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc 8521 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg 8581 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat 8641 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt 8701 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact 8761 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc 8821 tgctcccggc atccgcttac agacaagctg tgaccgactc cgggagctgc atgtgtcaga 8881 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt 8941 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca 9001 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt 9061 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga 9121 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac 9181 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg 9241 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc 9301 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt 9361 acgaaacacg gaaacccaag accattcatg ttgttgctca ggtcgcagac gttttgcagc 9421 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac 9481 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg 9541 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga 9601 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg 9661 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta 9721 cgagttgcat gataaagaac acagtcataa gtgcggcgac gatagtcatg ccccgcgccc 9781 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct 9841 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa 9901 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta 9961 ttgggcgcca gggtggtttt tcttttcacc agtgacacgg gcaacagctg att //