LOCUS Exported 9582 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Level 2 construct. ACCESSION . VERSION . KEYWORDS pUG-305 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9582) 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 9582) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9582 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 967..986 /label=LacI-R /note="LacI, reverse primer" promoter complement(1006..1083) /gene="lacI" /label=lacI promoter primer_bind 1289..1308 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 1392..1411 /label=T7 /note="T7 promoter, forward primer" promoter 1392..1410 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1411..1435 /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 1489..1506 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 5054..5140 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAGYPYDVPDYAGLYPYDVPDY" CDS 5117..5143 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 5156..5242 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAGYPYDVPDYAGLYPYDVPDY" CDS 5219..5245 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" primer_bind complement(5908..5926) /label=T7 Term /note="T7 terminator, reverse primer" terminator 5922..5969 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 6006..6461 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6093..6112) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6303..6324 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(6553..7368) /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 7276..7295 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 7360..7379 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 7490..8078 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7979..7998 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 8232..8249 /label=L4440 /note="L4440 vector, forward primer" misc_feature 8264..8403 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(8329..8348) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 8489..8511 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(8505..8696) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(9505..1005) /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 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gacacgggca acagctgatt 61 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag 121 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcctcggt 181 atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc 241 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc 301 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg 361 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag 421 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc 481 gaccagatgc tccacgccca gtcgcgtacc gtcctcatgg gagaaaataa tactgttgat 541 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac 601 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc 661 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac 721 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg 781 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag 841 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc 901 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga 961 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa 1021 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt 1081 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta 1141 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca 1201 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc 1261 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg 1321 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga 1381 tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct 1441 agaaataatt ttgccttaac tttaagaagg agatatacca tgggcagcca ccaccaccac 1501 caccacagcg gaatgcagat ctttgttaag actctcaccg gaaagacaat caccctagag 1561 gtggaaagct ccgacaccat cgacaacgtt aaggccaaga tccaggataa ggagggcatt 1621 cctccggatc agcagaggct tattttcgcc ggcaagcagc tagaggatgg ccgtacgttg 1681 gctgattaca atatccagaa ggaatccacc ctccacttgg tcctcaggct ccgtggtggt 1741 taagcttgca attaacttta agaaggagat atacaatgct tcacaagcga gctagtgaag 1801 ctaacgataa gaacgataat accatcatcg gttccgattt agcatcatct aagaaacgcc 1861 gtatcgattt cactgaatct tcgtctgata agtcatcttc gattttagct tctggtagta 1921 gcaggggttt tcacggcgat agcgtcgttc agcaaatcga catggctttt ggtaattcga 1981 accgtcagga gattgatgaa gatctgcaca gtcgccagct cgccgtctat ggtcgtgaga 2041 ctatgaggcg tctctttgct tccaatgttc tcatctcggg gatgcacggt cttggtgccg 2101 agattgccaa gaatcttata cttgctggtg tgaagtctgt gaccttgcat gatgaaagag 2161 ttgtagagct atgggactta tcaagcaact ttgttttctc ggaggatgat gttggcaaga 2221 atagagcaga tgcttctgtt cagaagttac aggatcttaa caatgctgtg gttgtctcta 2281 gcttgaccaa aagcttaaac aaagaggatc tttctggctt ccaggttgtt gtattctctg 2341 acataagcat ggaaagagca atcgaatttg atgactattg tcacagccat cagcctccta 2401 tcgcttttgt caaggctgat gtcaggggtc ttttcggctc tgtcttttgt gattttgggc 2461 ctgaatttgc ggttcttgat gttgatggag aggaacccca tacaggcatc attgcgtcta 2521 tctctaatga gaaccaggca tttatttcct gtgtcgacga tgagaggctt gagtttgaag 2581 atggtgacct agttgttttc tcagaagttg agggtatgac agaattgaat gatgggaaac 2641 caaggaagat aaaaagtaca cggccatatt cattcaccct tgatgaggac actactaact 2701 atggaacata tgtgaagggt ggtattgtca ctcaagtgaa acagccaaag ttactgaatt 2761 tcaagccatt gagggaagcg cttaaggatc caggggattt tctatttagt gatttctcta 2821 agtttgatcg gcctccactc cttcacttag cattccaggc acttgatcat tttaaagctg 2881 aagctggcag atttcctgtt gctggctcag aagaggacgc tcagaagctt atatcgattg 2941 ccacagccat caataccggg cagggtgatt taaaggtgga gaatgttgat cagaagcttc 3001 taagacactt ttcctttgga gccaaggctg tcctgaatcc tatggctgca atgtttggcg 3061 gtattgttgg acaggaggtt gttaaagctt gctctggaaa atttcatcct ctcttccagt 3121 ttttctactt tgattcagtg gagtcactcc cctctgaacc tgtggactcg agtgactttg 3181 caccaagaaa cagccggtat gatgcccaaa tatctgtatt tggggccaag ttccagaaga 3241 aacttgaaga tgctaaagtt ttcacagtag ggtctggtgc tcttggctgt gagttcttga 3301 aaaatttggc tctgatgggg gtttcttgtg gaagccaagg gaagctgaca gtgacagatg 3361 atgatatcat tgagaagagt aacctcagtc gtcaatttct gttccgtgac tggaacattg 3421 gacaggctaa atccacagtt gctgcttcgg ctgctgctgt tataaatcct cgcttcaaca 3481 ttgaggccct gcaaaaccgt gttggcgcgg agactgagaa tgtatttgac gatgccttct 3541 gggagaactt aactgttgtc gtcaatgcac tggataatgt taatgcgagg ctctatgttg 3601 attcgaggtg cttgtatttc cagaagcctc tccttgagtc tggaactctt ggtacaaagt 3661 gcaacacaca gtcggttatc ccacatctaa ctgaaaatta cggtgcctca agggacccac 3721 cagagaaaca ggcccccatg tgtacagtgc attccttccc gcataacatt gatcactgtt 3781 taacttgggc tcgctctgag tttgagggtc tgctggagaa aactcccgct gaagtgaatg 3841 catatctttc tagcccagtt gagtacacta actcaatgat gagtgctggt gatgctcagg 3901 cgagggacac attggagagg attgttgagt gccttgaaaa ggagaagtgt gaaaccttcc 3961 aagactgctt aacctgggca cgactgaggt ttgaggatta ctttgtgaac cgtgtcaagc 4021 aattgatata cacatttcct gaagatgctg cgactagtac cggagctcca ttctggtctg 4081 ctccaaaaag attccctcgt ccactccagt actcgtcgtc cgacccaagt ctccttaact 4141 ttatcacagc gactgctatt ttacgagcag agacatttgg gatcccgata cctgagtgga 4201 ctaagaaccc aaaggaagca gcagaagctg ttgacagagt gatagtccca gactttgagc 4261 caaggcaaga tgcaaagatt gtgactgatg agaaagctac cactttaacc actgcttcgg 4321 tggacgacgc tgcagtcatc gacgatctca ttgctaagat cgaccagtgt aggcataact 4381 tgtcgccaga cttcaggatg aaacctattc agttcgaaaa ggatgatgat acaaactatc 4441 acatggatgt gatagcagga ctagccaaca tgagggcgag aaattacagc atccctgaag 4501 tggacaagct gaaagcaaag ttcatagcag ggagaatcat cccagccatt gcgacttcaa 4561 cagccatggc tactgggctg gtctgcctcg agctgtacaa ggtccttgat ggaggacata 4621 aagtggaagc ttacagaaac acatttgcca acctggcact tccactattc tccatggctg 4681 aaccacttcc accaaaggtg gtgaagcacc gtgacatggc ttggacagtt tgggacagat 4741 gggttctcaa gggaaaccca actcttcgtg aggtgttgca gtggctggag gacaaagggc 4801 ttagcgcata cagcatctct tgtggaagct gtctactgtt caacagtatg ttcacaaggc 4861 acaaggagag gatggacaag aaggttgtgg atctcgctcg ggatgttgcg aaagtcgagt 4921 taccgcctta tcgcaaccat cttgatgtag tggtggcttg tgaggatgaa gatgacaatg 4981 acgtcgatat tcctctcgtc tctatctact tcaggtgagc ttactattaa ctttaagaag 5041 gagatatacc atgtacccat acgatgttcc tgactatgcg ggctatccct atgacgtccc 5101 ggactatgca ggattgtatc catatgacgt tccagattac gccactagag ctgcttaccc 5161 atacgatgtt cctgactatg cgggctatcc ctatgacgtc ccggactatg caggattgta 5221 tccatatgac gttccagatt acgcaatggc taataaccaa gcaagccttc ttctgcagaa 5281 acagcttaaa gatctgtgta agaaacctgt tgatggtttc tctgctggcc tcgttgatga 5341 gaagaacgtt ttccaatgga gcgtctctat tatgggtcct cctgatactt tgtatgaagg 5401 tggctttttt aatgcaatta tgtcgtttcc ggagaattat ccagtttcac caccaactgt 5461 gacgtttacc tcggagatgt ggcatcctaa tgtttattct gatggaaaag tttgcatttc 5521 tattcttcat cctcctggtg atgatcctca tggatacgag cttgcctctg aacgttggac 5581 ccctgtccat acggtagaaa gcattgtgtt gagtatcata tcgatgctct caggtcccaa 5641 cgatgagtca ccagcgaatg tggaagctgc aaaggaatgg agagacaaca gagcagagtt 5701 taggaagaaa gtgagtcgct gtgtgagaag atcgcaagag atgctatgag cttttacgct 5761 tttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct 5821 cccggcatcc gcttacagac aagctgtgac cgggatgaga tccggctgct aacaaagccc 5881 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg 5941 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga 6001 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt 6061 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct 6121 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg 6181 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag 6241 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa 6301 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga 6361 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa 6421 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga 6481 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc 6541 atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc 6601 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc 6661 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac 6721 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc 6781 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg 6841 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt 6901 cattcgtgat tgcgcctgag cgagtcgaaa tacgcgatcg ctgttaaaag gacaattaca 6961 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc 7021 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag 7081 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc 7141 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc 7201 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc 7261 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga 7321 atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt 7381 attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt 7441 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt 7501 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 7561 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc 7621 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg 7681 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg 7741 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt 7801 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 7861 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg 7921 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg 7981 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat 8041 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 8101 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg 8161 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa 8221 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc 8281 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc 8341 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct 8401 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca 8461 tccgcttaca gacaagctgt gaccgactcc gggagctgca tgtgtcagag gttttcaccg 8521 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat 8581 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat 8641 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat 8701 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg 8761 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt 8821 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag 8881 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag 8941 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg 9001 aaacccaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt 9061 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct 9121 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg 9181 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg 9241 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga 9301 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg 9361 ataaagaaca cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag 9421 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc 9481 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 9541 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gt //