LOCUS Exported 6391 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Bacterial expression for structure determination; may not be full ORF. ACCESSION . VERSION . KEYWORDS TPH2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6391) TITLE SGC Oxford REFERENCE 2 (bases 1 to 6391) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6391 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 1..20 /label=T7 /note="T7 promoter, forward primer" promoter 1..19 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 20..44 /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 91..108 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 133..153 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 1251..1268 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(1322..1340) /label=T7 Term /note="T7 terminator, reverse primer" terminator 1336..1383 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 1420..1875 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1507..1526) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1717..1738 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(1968..2783) /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 2691..2710 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 2775..2794 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 2905..3493 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3394..3413 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3647..3664 /label=L4440 /note="L4440 vector, forward primer" misc_feature 3679..3821 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(3747..3766) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3907..3929 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(3923..4114) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(4923..6005) /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 5967..5986 /label=LacI-R /note="LacI, reverse primer" promoter complement(6006..6083) /gene="lacI" /label=lacI promoter primer_bind 6289..6308 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" ORIGIN 1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt 61 tgtttaactt tagaaggaga tatacatatg caccatcatc atcatcattc ttctggtgta 121 gatctgggta ccgagaacct gtacttccaa tccatgctag aggatgtgcc ctggttccct 181 cggaagatct ctgagttaga caaatgctct cacagagttc tcatgtatgg ttctgagctt 241 gatgctgacc acccaggatt taaggacaat gtctatcgac agagaagaaa gtattttgtg 301 gatgtggcca tgggttataa atatggtcag cccattccca gggtggagta tactgaagaa 361 gaaactaaaa cttggggtgt tgtattccgg gagctctcca aactctatcc cactcatgct 421 tgccgagagt atttgaaaaa cttccctctg ctgactaaat actgtggcta cagagaggac 481 aatgtgcctc aactcgaaga tgtctccatg tttctgaaag aaaggtctgg cttcacggtg 541 aggccggtgg ctggatacct gagcccacga gactttctgg caggactggc ctacagagtg 601 ttccactgta cccagtacat ccggcatggc tcagatcccc tctacacccc ggaaccagac 661 acatgccatg aactcttggg acatgttcca ctacttgcgg atcctaagtt tgctcagttt 721 tcacaagaaa taggtctggc gtctctggga gcatcagatg aagatgttca gaaactagcc 781 acgtgctatt tcttcacaat cgagtttggc ctttgcaagc aagaagggca actgcgggct 841 tatggagcag gactcctttc ctccattgga gaattaaagc acgccctttc tgacaaggca 901 tgtgtgaaag cctttgaccc aaagacaact tgcttacagg aatgccttat caccaccttc 961 caggaagcct actttgtttc agaaagtttt gaagaagcca aagaaaagat gagggacttt 1021 gcaaagtcaa ttacccgtcc cttctcagta tacttcaatc cctacacaca gagtattgaa 1081 attctgaaag acaccagaag tattgaaaat gtggtgcagg accttcgcag cgacttgaat 1141 acagtgtgtg atgctttaaa caaaatgaac caatatctgg ggatttgaca gtaaaggtgg 1201 atacggatcc gaattcgagc tccgtcgaca agcttgcggc cgcactcgag caccaccacc 1261 accaccactg agatcccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc 1321 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt 1381 ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat 1441 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 1501 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 1561 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 1621 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt 1681 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 1741 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg 1801 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat 1861 taacgtttac aatttcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 1921 tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca 1981 tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga 2041 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga 2101 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc 2161 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag 2221 aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg 2281 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga 2341 cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc 2401 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc 2461 tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg 2521 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc 2581 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca 2641 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc 2701 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac 2761 gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt 2821 tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc 2881 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt 2941 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac 3001 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt 3061 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 3121 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga 3181 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac 3241 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg 3301 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt 3361 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 3421 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg 3481 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc 3541 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc 3601 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag 3661 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc 3721 acaccgcata tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag 3781 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac 3841 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga 3901 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc 3961 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat 4021 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg 4081 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc 4141 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg 4201 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc 4261 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag 4321 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg 4381 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg 4441 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg 4501 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga 4561 gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga 4621 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata 4681 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga 4741 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg 4801 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca 4861 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc 4921 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc 4981 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt 5041 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg 5101 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata 5161 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc 5221 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc 5281 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg 5341 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta 5401 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg 5461 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg 5521 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca 5581 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc 5641 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg 5701 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc 5761 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc 5821 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc 5881 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg 5941 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt 6001 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag 6061 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat 6121 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc 6181 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc 6241 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc 6301 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc 6361 gtagaggatc gagatctcga tcccgcgaaa t //