LOCUS Exported 10013 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Level 2 construct. ACCESSION . VERSION . KEYWORDS pUG-334 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 584..603 /label=LacI-R /note="LacI, reverse primer" promoter complement(623..700) /gene="lacI" /label=lacI promoter primer_bind 906..925 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 1009..1028 /label=T7 /note="T7 promoter, forward primer" promoter 1009..1027 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1028..1052 /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 1106..1123 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 4671..4757 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAGYPYDVPDYAGLYPYDVPDY" CDS 4734..4760 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 4773..4859 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAGYPYDVPDYAGLYPYDVPDY" CDS 4836..4862 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" primer_bind complement(5956..5974) /label=T7 Term /note="T7 terminator, reverse primer" terminator 5970..6017 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 6054..6509 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6141..6160) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6351..6372 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(6601..7416) /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 7324..7343 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 7408..7427 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 7538..8126 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8027..8046 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 8280..8297 /label=L4440 /note="L4440 vector, forward primer" misc_feature 8312..8451 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(8377..8396) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 8537..8559 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(8553..8744) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(9553..622) /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 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg 61 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc 121 gcgtaccgtc ctcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa 181 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca 241 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt 301 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat 361 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg 421 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa 481 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc 541 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat 601 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc 661 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc 721 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc 781 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct 841 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc 901 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg 961 gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca 1021 ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg ccttaacttt 1081 aagaaggaga tataccatgg gcagccacca ccaccaccac cacagcggaa tgcagatctt 1141 tgttaagact ctcaccggaa agacaatcac cctagaggtg gaaagctccg acaccatcga 1201 caacgttaag gccaagatcc aggataagga gggcattcct ccggatcagc agaggcttat 1261 tttcgccggc aagcagctag aggatggccg tacgttggct gattacaata tccagaagga 1321 atccaccctc cacttggtcc tcaggctccg tggtggttaa gcttgcaatt aactttaaga 1381 aggagatata caatgcttca caagcgagct agtgaagcta acgataagaa cgataatacc 1441 atcatcggtt ccgatttagc atcatctaag aaacgccgta tcgatttcac tgaatcttcg 1501 tctgataagt catcttcgat tttagcttct ggtagtagca ggggttttca cggcgatagc 1561 gtcgttcagc aaatcgacat ggcttttggt aattcgaacc gtcaggagat tgatgaagat 1621 ctgcacagtc gccagctcgc cgtctatggt cgtgagacta tgaggcgtct ctttgcttcc 1681 aatgttctca tctcggggat gcacggtctt ggtgccgaga ttgccaagaa tcttatactt 1741 gctggtgtga agtctgtgac cttgcatgat gaaagagttg tagagctatg ggacttatca 1801 agcaactttg ttttctcgga ggatgatgtt ggcaagaata gagcagatgc ttctgttcag 1861 aagttacagg atcttaacaa tgctgtggtt gtctctagct tgaccaaaag cttaaacaaa 1921 gaggatcttt ctggcttcca ggttgttgta ttctctgaca taagcatgga aagagcaatc 1981 gaatttgatg actattgtca cagccatcag cctcctatcg cttttgtcaa ggctgatgtc 2041 aggggtcttt tcggctctgt cttttgtgat tttgggcctg aatttgcggt tcttgatgtt 2101 gatggagagg aaccccatac aggcatcatt gcgtctatct ctaatgagaa ccaggcattt 2161 atttcctgtg tcgacgatga gaggcttgag tttgaagatg gtgacctagt tgttttctca 2221 gaagttgagg gtatgacaga attgaatgat gggaaaccaa ggaagataaa aagtacacgg 2281 ccatattcat tcacccttga tgaggacact actaactatg gaacatatgt gaagggtggt 2341 attgtcactc aagtgaaaca gccaaagtta ctgaatttca agccattgag ggaagcgctt 2401 aaggatccag gggattttct atttagtgat ttctctaagt ttgatcggcc tccactcctt 2461 cacttagcat tccaggcact tgatcatttt aaagctgaag ctggcagatt tcctgttgct 2521 ggctcagaag aggacgctca gaagcttata tcgattgcca cagccatcaa taccgggcag 2581 ggtgatttaa aggtggagaa tgttgatcag aagcttctaa gacacttttc ctttggagcc 2641 aaggctgtcc tgaatcctat ggctgcaatg tttggcggta ttgttggaca ggaggttgtt 2701 aaagcttgct ctggaaaatt tcatcctctc ttccagtttt tctactttga ttcagtggag 2761 tcactcccct ctgaacctgt ggactcgagt gactttgcac caagaaacag ccggtatgat 2821 gcccaaatat ctgtatttgg ggccaagttc cagaagaaac ttgaagatgc taaagttttc 2881 acagtagggt ctggtgctct tggctgtgag ttcttgaaaa atttggctct gatgggggtt 2941 tcttgtggaa gccaagggaa gctgacagtg acagatgatg atatcattga gaagagtaac 3001 ctcagtcgtc aatttctgtt ccgtgactgg aacattggac aggctaaatc cacagttgct 3061 gcttcggctg ctgctgttat aaatcctcgc ttcaacattg aggccctgca aaaccgtgtt 3121 ggcgcggaga ctgagaatgt atttgacgat gccttctggg agaacttaac tgttgtcgtc 3181 aatgcactgg ataatgttaa tgcgaggctc tatgttgatt cgaggtgctt gtatttccag 3241 aagcctctcc ttgagtctgg aactcttggt acaaagtgca acacacagtc ggttatccca 3301 catctaactg aaaattacgg tgcctcaagg gacccaccag agaaacaggc ccccatgtgt 3361 acagtgcatt ccttcccgca taacattgat cactgtttaa cttgggctcg ctctgagttt 3421 gagggtctgc tggagaaaac tcccgctgaa gtgaatgcat atctttctag cccagttgag 3481 tacactaact caatgatgag tgctggtgat gctcaggcga gggacacatt ggagaggatt 3541 gttgagtgcc ttgaaaagga gaagtgtgaa accttccaag actgcttaac ctgggcacga 3601 ctgaggtttg aggattactt tgtgaaccgt gtcaagcaat tgatatacac atttcctgaa 3661 gatgctgcga ctagtaccgg agctccattc tggtctgctc caaaaagatt ccctcgtcca 3721 ctccagtact cgtcgtccga cccaagtctc cttaacttta tcacagcgac tgctatttta 3781 cgagcagaga catttgggat cccgatacct gagtggacta agaacccaaa ggaagcagca 3841 gaagctgttg acagagtgat agtcccagac tttgagccaa ggcaagatgc aaagattgtg 3901 actgatgaga aagctaccac tttaaccact gcttcggtgg acgacgctgc agtcatcgac 3961 gatctcattg ctaagatcga ccagtgtagg cataacttgt cgccagactt caggatgaaa 4021 cctattcagt tcgaaaagga tgatgataca aactatcaca tggatgtgat agcaggacta 4081 gccaacatga gggcgagaaa ttacagcatc cctgaagtgg acaagctgaa agcaaagttc 4141 atagcaggga gaatcatccc agccattgcg acttcaacag ccatggctac tgggctggtc 4201 tgcctcgagc tgtacaaggt ccttgatgga ggacataaag tggaagctta cagaaacaca 4261 tttgccaacc tggcacttcc actattctcc atggctgaac cacttccacc aaaggtggtg 4321 aagcaccgtg acatggcttg gacagtttgg gacagatggg ttctcaaggg aaacccaact 4381 cttcgtgagg tgttgcagtg gctggaggac aaagggctta gcgcatacag catctcttgt 4441 ggaagctgtc tactgttcaa cagtatgttc acaaggcaca aggagaggat ggacaagaag 4501 gttgtggatc tcgctcggga tgttgcgaaa gtcgagttac cgccttatcg caaccatctt 4561 gatgtagtgg tggcttgtga ggatgaagat gacaatgacg tcgatattcc tctcgtctct 4621 atctacttca ggtgagctta ctattaactt taagaaggag atataccatg tacccatacg 4681 atgttcctga ctatgcgggc tatccctatg acgtcccgga ctatgcagga ttgtatccat 4741 atgacgttcc agattacgcc actagagctg cttacccata cgatgttcct gactatgcgg 4801 gctatcccta tgacgtcccg gactatgcag gattgtatcc atatgacgtt ccagattacg 4861 caatggccaa cagtaatttg ccgcgaagaa tcatcaagga aactcaacgt ctgctcagtg 4921 aaccggctcc cggtataagt gcctctccat ctgaggataa tatgcggtat ttcaacgtta 4981 tgattcttgg tcctacacaa tcaccttatg aaggaggagt tttcaagttg gagctctttt 5041 tgcctgaaga ataccctatg gcagctccca aggttaggtt tctcacaaag atataccatc 5101 ctaacattga caagcttgga agaatctgtc ttgatattct caaggacaaa tggagccctg 5161 cactacaaat acgaacagtg ctcttaagta ttcaagctct tctgagtgca ccaaacccag 5221 atgatccgct ttctgagaac attgctaagc actggaagag taatgaggca gaagctgtgg 5281 atacagctaa ggaatggacc cgtctttatg caagcggcgc atgagctttt acttaacttt 5341 aagaaggaga tatacaatga ctcttggctc aggaggatct agcgttgttg ttccgaggaa 5401 ttttcggttg ttggaggagc ttgaacgtgg ggagaaaggt attggggatg gaactgtgag 5461 ctatggaatg gatgatggag atgacatcta tatgcgttct tggactggca ccatcatcgg 5521 tcctcacaac actgttcatg aaggtagaat ctatcagttg aagctctttt gtgacaaaga 5581 ttacccggag aaacctccaa ctgtccgatt ccattcacgt gtcaacatgg cttgtgtcaa 5641 ccacgaaaca ggagtggtgg atcctaagaa atttgggcta cttgcaaact ggcaaagaga 5701 gtacacaatg gaggatatac tggtacagtt gaagaaggaa atgtctacgt cgcataaccg 5761 taagcttgtt caacctccgg aaggtacttg cttctaggct tcagagcttt taagccagcc 5821 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc 5881 ttacagacaa gctgtgaccg ggatgagatc cggctgctaa caaagcccga aaggaagctg 5941 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg 6001 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc 6061 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact 6121 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc 6181 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt 6241 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc 6301 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt 6361 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat 6421 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa 6481 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga 6541 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc 6601 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat 6661 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca 6721 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc 6781 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac 6841 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca 6901 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg 6961 cgcctgagcg agtcgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga 7021 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata 7081 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc 7141 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt 7201 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa 7261 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac 7321 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg 7381 cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat 7441 gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact 7501 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 7561 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 7621 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 7681 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 7741 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 7801 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 7861 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 7921 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 7981 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 8041 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 8101 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 8161 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata 8221 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca 8281 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc 8341 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata 8401 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac 8461 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga 8521 caagctgtga ccgactccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa 8581 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct 8641 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg 8701 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa 8761 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata 8821 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg 8881 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat 8941 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata 9001 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa acccaagacc 9061 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg 9121 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc 9181 aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc 9241 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag 9301 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg 9361 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaacaca 9421 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg 9481 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta 9541 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 9601 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct 9661 tttcaccagt gacacgggca acagctgatt gcccttcacc gcctggccct gagagagttg 9721 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa 9781 cggcgggata taacatgagc tgtcctcggt atcgtcgtat cccactaccg agatatccgc 9841 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt 9901 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa 9961 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gat //