LOCUS Exported 10012 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Level 2 construct. ACCESSION . VERSION . KEYWORDS pUG-338 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10012) 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 10012) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10012 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 564..583 /label=LacI-R /note="LacI, reverse primer" promoter complement(603..680) /gene="lacI" /label=lacI promoter primer_bind 886..905 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 989..1008 /label=T7 /note="T7 promoter, forward primer" promoter 989..1007 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1008..1032 /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 1086..1103 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 4651..4737 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAGYPYDVPDYAGLYPYDVPDY" CDS 4714..4740 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 4753..4839 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAGYPYDVPDYAGLYPYDVPDY" CDS 4816..4842 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" primer_bind complement(5935..5953) /label=T7 Term /note="T7 terminator, reverse primer" terminator 5949..5996 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 6033..6488 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6120..6139) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6330..6351 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(6580..7395) /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 7303..7322 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 7387..7406 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 7517..8105 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8006..8025 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 8259..8276 /label=L4440 /note="L4440 vector, forward primer" misc_feature 8291..8430 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(8356..8375) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 8516..8538 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(8532..8723) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(9532..602) /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 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt 61 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ctcatgggag 121 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta 181 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc 241 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt 301 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc 361 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac 421 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc 481 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg 541 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc 601 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt 661 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag 721 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg 781 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa 841 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat 901 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta 961 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg 1021 gataacaatt cccctctaga aataattttg ccttaacttt aagaaggaga tataccatgg 1081 gcagccacca ccaccaccac cacagcggaa tgcagatctt tgttaagact ctcaccggaa 1141 agacaatcac cctagaggtg gaaagctccg acaccatcga caacgttaag gccaagatcc 1201 aggataagga gggcattcct ccggatcagc agaggcttat tttcgccggc aagcagctag 1261 aggatggccg tacgttggct gattacaata tccagaagga atccaccctc cacttggtcc 1321 tcaggctccg tggtggttaa gcttgcaatt aactttaaga aggagatata caatgcttca 1381 caagcgagct agtgaagcta acgataagaa cgataatacc atcatcggtt ccgatttagc 1441 atcatctaag aaacgccgta tcgatttcac tgaatcttcg tctgataagt catcttcgat 1501 tttagcttct ggtagtagca ggggttttca cggcgatagc gtcgttcagc aaatcgacat 1561 ggcttttggt aattcgaacc gtcaggagat tgatgaagat ctgcacagtc gccagctcgc 1621 cgtctatggt cgtgagacta tgaggcgtct ctttgcttcc aatgttctca tctcggggat 1681 gcacggtctt ggtgccgaga ttgccaagaa tcttatactt gctggtgtga agtctgtgac 1741 cttgcatgat gaaagagttg tagagctatg ggacttatca agcaactttg ttttctcgga 1801 ggatgatgtt ggcaagaata gagcagatgc ttctgttcag aagttacagg atcttaacaa 1861 tgctgtggtt gtctctagct tgaccaaaag cttaaacaaa gaggatcttt ctggcttcca 1921 ggttgttgta ttctctgaca taagcatgga aagagcaatc gaatttgatg actattgtca 1981 cagccatcag cctcctatcg cttttgtcaa ggctgatgtc aggggtcttt tcggctctgt 2041 cttttgtgat tttgggcctg aatttgcggt tcttgatgtt gatggagagg aaccccatac 2101 aggcatcatt gcgtctatct ctaatgagaa ccaggcattt atttcctgtg tcgacgatga 2161 gaggcttgag tttgaagatg gtgacctagt tgttttctca gaagttgagg gtatgacaga 2221 attgaatgat gggaaaccaa ggaagataaa aagtacacgg ccatattcat tcacccttga 2281 tgaggacact actaactatg gaacatatgt gaagggtggt attgtcactc aagtgaaaca 2341 gccaaagtta ctgaatttca agccattgag ggaagcgctt aaggatccag gggattttct 2401 atttagtgat ttctctaagt ttgatcggcc tccactcctt cacttagcat tccaggcact 2461 tgatcatttt aaagctgaag ctggcagatt tcctgttgct ggctcagaag aggacgctca 2521 gaagcttata tcgattgcca cagccatcaa taccgggcag ggtgatttaa aggtggagaa 2581 tgttgatcag aagcttctaa gacacttttc ctttggagcc aaggctgtcc tgaatcctat 2641 ggctgcaatg tttggcggta ttgttggaca ggaggttgtt aaagcttgct ctggaaaatt 2701 tcatcctctc ttccagtttt tctactttga ttcagtggag tcactcccct ctgaacctgt 2761 ggactcgagt gactttgcac caagaaacag ccggtatgat gcccaaatat ctgtatttgg 2821 ggccaagttc cagaagaaac ttgaagatgc taaagttttc acagtagggt ctggtgctct 2881 tggctgtgag ttcttgaaaa atttggctct gatgggggtt tcttgtggaa gccaagggaa 2941 gctgacagtg acagatgatg atatcattga gaagagtaac ctcagtcgtc aatttctgtt 3001 ccgtgactgg aacattggac aggctaaatc cacagttgct gcttcggctg ctgctgttat 3061 aaatcctcgc ttcaacattg aggccctgca aaaccgtgtt ggcgcggaga ctgagaatgt 3121 atttgacgat gccttctggg agaacttaac tgttgtcgtc aatgcactgg ataatgttaa 3181 tgcgaggctc tatgttgatt cgaggtgctt gtatttccag aagcctctcc ttgagtctgg 3241 aactcttggt acaaagtgca acacacagtc ggttatccca catctaactg aaaattacgg 3301 tgcctcaagg gacccaccag agaaacaggc ccccatgtgt acagtgcatt ccttcccgca 3361 taacattgat cactgtttaa cttgggctcg ctctgagttt gagggtctgc tggagaaaac 3421 tcccgctgaa gtgaatgcat atctttctag cccagttgag tacactaact caatgatgag 3481 tgctggtgat gctcaggcga gggacacatt ggagaggatt gttgagtgcc ttgaaaagga 3541 gaagtgtgaa accttccaag actgcttaac ctgggcacga ctgaggtttg aggattactt 3601 tgtgaaccgt gtcaagcaat tgatatacac atttcctgaa gatgctgcga ctagtaccgg 3661 agctccattc tggtctgctc caaaaagatt ccctcgtcca ctccagtact cgtcgtccga 3721 cccaagtctc cttaacttta tcacagcgac tgctatttta cgagcagaga catttgggat 3781 cccgatacct gagtggacta agaacccaaa ggaagcagca gaagctgttg acagagtgat 3841 agtcccagac tttgagccaa ggcaagatgc aaagattgtg actgatgaga aagctaccac 3901 tttaaccact gcttcggtgg acgacgctgc agtcatcgac gatctcattg ctaagatcga 3961 ccagtgtagg cataacttgt cgccagactt caggatgaaa cctattcagt tcgaaaagga 4021 tgatgataca aactatcaca tggatgtgat agcaggacta gccaacatga gggcgagaaa 4081 ttacagcatc cctgaagtgg acaagctgaa agcaaagttc atagcaggga gaatcatccc 4141 agccattgcg acttcaacag ccatggctac tgggctggtc tgcctcgagc tgtacaaggt 4201 ccttgatgga ggacataaag tggaagctta cagaaacaca tttgccaacc tggcacttcc 4261 actattctcc atggctgaac cacttccacc aaaggtggtg aagcaccgtg acatggcttg 4321 gacagtttgg gacagatggg ttctcaaggg aaacccaact cttcgtgagg tgttgcagtg 4381 gctggaggac aaagggctta gcgcatacag catctcttgt ggaagctgtc tactgttcaa 4441 cagtatgttc acaaggcaca aggagaggat ggacaagaag gttgtggatc tcgctcggga 4501 tgttgcgaaa gtcgagttac cgccttatcg caaccatctt gatgtagtgg tggcttgtga 4561 ggatgaagat gacaatgacg tcgatattcc tctcgtctct atctacttca ggtgagctta 4621 ctattaactt taagaaggag atataccatg tacccatacg atgttcctga ctatgcgggc 4681 tatccctatg acgtcccgga ctatgcagga ttgtatccat atgacgttcc agattacgcc 4741 actagagctg cttacccata cgatgttcct gactatgcgg gctatcccta tgacgtcccg 4801 gactatgcag gattgtatcc atatgacgtt ccagattacg caatggccaa tagtaatctt 4861 ccccgacgaa tcatcaagga aactcaacgt ctgcttagtg aacctgctcc tgggataagt 4921 gcgtctccat cagaggaaaa tatgagatat ttcaatgtta tgattcttgg tcctactcaa 4981 tctccttatg aaggaggagt tttcaagttg gagctctttt tgcctgaaga atatcctatg 5041 gcagctccca aggttaggtt tctcaccaag atttaccatc ctaacatcga caagcttgga 5101 agaatctgcc ttgacatact gaaagacaag tggagtcctg cactacaaat acgaactgtg 5161 ctcttgagta tccaagctct tttaagtgca ccaaacccag atgatcctct gtctgagaac 5221 atagctaagc attggaagag caatgaggca gaagctgtgg aaacagctaa ggaatggact 5281 cgtctttacg caagtggtgc ttaagctttt acttaacttt aagaaggaga tatacaatga 5341 ctcttggctc aggaggatct agcgttgttg ttccgaggaa ttttcggttg ttggaggagc 5401 ttgaacgtgg ggagaaaggt attggggatg gaactgtgag ctatggaatg gatgatggag 5461 atgacatcta tatgcgttct tggactggca ccatcatcgg tcctcacaac actgttcatg 5521 aaggtagaat ctatcagttg aagctctttt gtgacaaaga ttacccggag aaacctccaa 5581 ctgtccgatt ccattcacgt gtcaacatgg cttgtgtcaa ccacgaaaca ggagtggtgg 5641 atcctaagaa atttgggcta cttgcaaact ggcaaagaga gtacacaatg gaggatatac 5701 tggtacagtt gaagaaggaa atgtctacgt cgcataaccg taagcttgtt caacctccgg 5761 aaggtacttg cttctaggct tcagagcttt taagccagcc ccgacacccg ccaacacccg 5821 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgacgg 5881 gatgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg 5941 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga 6001 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc 6061 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc 6121 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct 6181 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa 6241 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc 6301 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact 6361 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg 6421 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct 6481 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 6541 taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat 6601 caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg 6661 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta 6721 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa 6781 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa 6841 aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa 6901 atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gtcgaaatac 6961 gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac 7021 tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc 7081 tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg 7141 cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt 7201 aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt 7261 cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata 7321 cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg 7381 ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt 7441 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 7501 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 7561 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 7621 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt 7681 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 7741 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 7801 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 7861 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 7921 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 7981 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 8041 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 8101 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 8161 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 8221 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 8281 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca 8341 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg 8401 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg 8461 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgactccggg 8521 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa 8581 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc 8641 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg 8701 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg 8761 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat 8821 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag 8881 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag 8941 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc 9001 cgcgtttcca gactttacga aacacggaaa cccaagacca ttcatgttgt tgctcaggtc 9061 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc 9121 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg 9181 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg 9241 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac 9301 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct 9361 gccggcacct gtcctacgag ttgcatgata aagaacacag tcataagtgc ggcgacgata 9421 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt 9481 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc 9541 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 9601 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg acacgggcaa 9661 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt 9721 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct 9781 gtcctcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc 9841 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg 9901 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc 9961 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gc //