LOCUS Exported 9532 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Level 2 construct. ACCESSION . VERSION . KEYWORDS pUG-303 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9532) 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 9532) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9532 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 12..467 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(99..118) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 309..330 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(560..1375) /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 1283..1302 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 1367..1386 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 1497..2085 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1986..2005 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2239..2256 /label=L4440 /note="L4440 vector, forward primer" misc_feature 2271..2413 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(2339..2358) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 2499..2521 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(2515..2706) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(3515..4597) /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 4559..4578 /label=LacI-R /note="LacI, reverse primer" promoter complement(4598..4675) /gene="lacI" /label=lacI promoter primer_bind 4881..4900 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 4984..5003 /label=T7 /note="T7 promoter, forward primer" promoter 4984..5002 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 5003..5027 /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 5081..5098 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 8646..8732 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAGYPYDVPDYAGLYPYDVPDY" CDS 8709..8735 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 8748..8834 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAGYPYDVPDYAGLYPYDVPDY" CDS 8811..8837 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" primer_bind complement(9446..9464) /label=T7 Term /note="T7 terminator, reverse primer" terminator 9460..9507 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" ORIGIN 1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 421 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 481 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 541 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 601 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 661 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 721 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 781 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 841 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 901 cgttattcat tcgtgattgc gcctgagcga gtcgaaatac gcgatcgctg ttaaaaggac 961 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1021 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1081 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1141 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1201 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1261 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1321 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1381 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1441 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1501 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1561 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1621 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1681 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1741 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1801 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1861 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1921 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1981 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2041 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2101 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2161 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2221 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2281 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2341 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2401 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2461 gctcccggca tccgcttaca gacaagctgt gaccgactcc gggagctgca tgtgtcagag 2521 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2581 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2641 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2701 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2761 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2821 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2881 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2941 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3001 cgaaacacgg aaacccaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3061 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3121 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3181 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3241 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3301 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3361 gagttgcatg ataaagaaca cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3421 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3481 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3541 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3601 tgggcgccag ggtggttttt cttttcacca gtgacacggg caacagctga ttgcccttca 3661 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3721 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcctcg gtatcgtcgt 3781 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3841 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3901 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3961 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4021 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4081 gctccacgcc cagtcgcgta ccgtcctcat gggagaaaat aatactgttg atgggtgtct 4141 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4201 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4261 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4321 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4381 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4441 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4501 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4561 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4621 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4681 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4741 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4801 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4861 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4921 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4981 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5041 ttttgcctta actttaagaa ggagatatac catgggcagc caccaccacc accaccacag 5101 cggaatgcag atctttgtta agactctcac cggaaagaca atcaccctgg aggtggaaag 5161 ctccgacacc atcgacaacg ttaaggccaa gatccaggat aaggagggca ttcctccgga 5221 tcagcagagg cttattttcg ccggcaagca gctagaggat ggccgtacgt tggctgatta 5281 caatatccag aaggaatcca ccctccactt ggtcctcagg ctccgtggtg gttaagcttg 5341 caattaactt taagaaggag atatacaatg cttcacaagc gagctagtga agctaacgat 5401 aagaacgata ataccatcat cggttccgat ttagcatcat ctaagaaacg ccgtatcgat 5461 ttcactgaat cttcgtctga taagtcatct tcgattttag cttctggtag tagcaggggt 5521 tttcacggcg atagcgtcgt tcagcaaatc gacatggctt ttggtaattc gaaccgtcag 5581 gagattgatg aagatctgca cagtcgccag ctcgccgtct atggtcgtga gactatgagg 5641 cgtctctttg cttccaatgt tctcatctcg gggatgcacg gtcttggtgc cgagattgcc 5701 aagaatctta tacttgctgg tgtgaagtct gtgaccttgc atgatgaaag agttgtagag 5761 ctatgggact tatcaagcaa ctttgttttc tcggaggatg atgttggcaa gaatagagca 5821 gatgcttctg ttcagaagtt acaggatctt aacaatgctg tggttgtctc tagcttgacc 5881 aaaagcttaa acaaagagga tctttctggc ttccaggttg ttgtattctc tgacataagc 5941 atggaaagag caatcgaatt tgatgactat tgtcacagcc atcagcctcc tatcgctttt 6001 gtcaaggctg atgtcagggg tcttttcggc tctgtctttt gtgattttgg gcctgaattt 6061 gcggttcttg atgttgatgg agaggaaccc catacaggca tcattgcgtc tatctctaat 6121 gagaaccagg catttatttc ctgtgtcgac gatgagaggc ttgagtttga agatggtgac 6181 ctagttgttt tctcagaagt tgagggtatg acagaattga atgatgggaa accaaggaag 6241 ataaaaagta cacggccata ttcattcacc cttgatgagg acactactaa ctatggaaca 6301 tatgtgaagg gtggtattgt cactcaagtg aaacagccaa agttactgaa tttcaagcca 6361 ttgagggaag cgcttaagga tccaggggat tttctattta gtgatttctc taagtttgat 6421 cggcctccac tccttcactt agcattccag gcacttgatc attttaaagc tgaagctggc 6481 agatttcctg ttgctggctc agaagaggac gctcagaagc ttatatcgat tgccacagcc 6541 atcaataccg ggcagggtga tttaaaggtg gagaatgttg atcagaagct tctaagacac 6601 ttttcctttg gagccaaggc tgtcctgaat cctatggctg caatgtttgg cggtattgtt 6661 ggacaggagg ttgttaaagc ttgctctgga aaatttcatc ctctcttcca gtttttctac 6721 tttgattcag tggagtcact cccctctgaa cctgtggact cgagtgactt tgcaccaaga 6781 aacagccggt atgatgccca aatatctgta tttggggcca agttccagaa gaaacttgaa 6841 gatgctaaag ttttcacagt agggtctggt gctcttggct gtgagttctt gaaaaatttg 6901 gctctgatgg gggtttcttg tggaagccaa gggaagctga cagtgacaga tgatgatatc 6961 attgagaaga gtaacctcag tcgtcaattt ctgttccgtg actggaacat tggacaggct 7021 aaatccacag ttgctgcttc ggctgctgct gttataaatc ctcgcttcaa cattgaggcc 7081 ctgcaaaacc gtgttggcgc ggagactgag aatgtatttg acgatgcctt ctgggagaac 7141 ttaactgttg tcgtcaatgc actggataat gttaatgcga ggctctatgt tgattcgagg 7201 tgcttgtatt tccagaagcc tctccttgag tctggaactc ttggtacaaa gtgcaacaca 7261 cagtcggtta tcccacatct aactgaaaat tacggtgcct caagggaccc accagagaaa 7321 caggccccca tgtgtacagt gcattccttc ccgcataaca ttgatcactg tttaacttgg 7381 gctcgctctg agtttgaggg tctgctggag aaaactcccg ctgaagtgaa tgcatatctt 7441 tctagcccag ttgagtacac taactcaatg atgagtgctg gtgatgctca ggcgagggac 7501 acattggaga ggattgttga gtgccttgaa aaggagaagt gtgaaacctt ccaagactgc 7561 ttaacctggg cacgactgag gtttgaggat tactttgtga accgtgtcaa gcaattgata 7621 tacacatttc ctgaagatgc tgcgactagt accggagctc cattctggtc tgctccaaaa 7681 agattccctc gtccactcca gtactcgtcg tccgacccaa gtctccttaa ctttatcaca 7741 gcgactgcta ttttacgagc agagacattt gggatcccga tacctgagtg gactaagaac 7801 ccaaaggaag cagcagaagc tgttgacaga gtgatagtcc cagactttga gccaaggcaa 7861 gatgcaaaga ttgtgactga tgagaaagct accactttaa ccactgcttc ggtggacgac 7921 gctgcagtca tcgacgatct cattgctaag atcgaccagt gtaggcataa cttgtcgcca 7981 gacttcagga tgaaacctat tcagttcgaa aaggatgatg atacaaacta tcacatggat 8041 gtgatagcag gactagccaa catgagggcg agaaattaca gcatccctga agtggacaag 8101 ctgaaagcaa agttcatagc agggagaatc atcccagcca ttgcgacttc aacagccatg 8161 gctactgggc tggtctgcct cgagctgtac aaggtccttg atggaggaca taaagtggaa 8221 gcttacagaa acacatttgc caacctggca cttccactat tctccatggc tgaaccactt 8281 ccaccaaagg tggtgaagca ccgtgacatg gcttggacag tttgggacag atgggttctc 8341 aagggaaacc caactcttcg tgaggtgttg cagtggctgg aggacaaagg gcttagcgca 8401 tacagcatct cttgtggaag ctgtctactg ttcaacagta tgttcacaag gcacaaggag 8461 aggatggaca agaaggttgt ggatctcgct cgggatgttg cgaaagtcga gttaccgcct 8521 tatcgcaacc atcttgatgt agtggtggct tgtgaggatg aagatgacaa tgacgtcgat 8581 attcctctcg tctctatcta cttcaggtga gcttactatt aactttaaga aggagatata 8641 ccatgtaccc atacgatgtt cctgactatg cgggctatcc ctatgacgtc ccggactatg 8701 caggattgta tccatatgac gttccagatt acgccactag agctgcttac ccatacgatg 8761 ttcctgacta tgcgggctat ccctatgacg tcccggacta tgcaggattg tatccatatg 8821 acgttccaga ttacgcaatg gcttctaaga ggatttcaag ggaactgagg gatatgcaga 8881 gacatcctcc agcaaactgt agcgcaggtc ctgtggctga ggaggacata ttccattggc 8941 aagcaactat catgggaccg catgacagtc cgtattccgg aggcgtattt acggtttcta 9001 ttgatttctc ttcggattat cctttcaagc caccaaaggt gaatttcaag actaaggtgt 9061 accacccaaa catcgacagc aaaggaagca tttgccttga catattgaaa gagcaatgga 9121 gtcctgctcc taccacatcc aaggttttgt tgtcgatttg ctcgctgctg actgacccga 9181 acccaaatga tcctcttgtg ccggagatag ctcatctcta caaggtagat aaatcaaaat 9241 acgagtcaac tgcacaaaaa tggacacaga agtacgcgat gggatgagct tttacgcttt 9301 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc 9361 cggcatccgc ttacagacaa gctgtgaccg ggatgagatc cggctgctaa caaagcccga 9421 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc 9481 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg at //