LOCUS Exported 5911 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Bacterial expression for structure determination; may not be full ORF. ACCESSION . VERSION . KEYWORDS RHOA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5911) TITLE SGC Oxford REFERENCE 2 (bases 1 to 5911) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5911 /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)." RBS 59..81 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 92..109 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 134..154 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 772..789 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(842..860) /label=T7 Term /note="T7 terminator, reverse primer" terminator 856..903 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 940..1395 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1027..1046) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1237..1258 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(1488..2303) /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 2211..2230 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 2295..2314 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 2425..3013 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2914..2933 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3167..3184 /label=L4440 /note="L4440 vector, forward primer" misc_feature 3199..3341 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(3267..3286) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3427..3449 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(3443..3634) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(4443..5525) /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 5487..5506 /label=LacI-R /note="LacI, reverse primer" promoter complement(5526..5603) /gene="lacI" /label=lacI promoter primer_bind 5809..5828 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" ORIGIN 1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt 61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctggtgt 121 agatctgggt accgagaacc tgtacttcca atccatggct gccatccgga agaaactggt 181 gattgttggt gatggagcct gtggaaagac atgcttgctc atagtcttca gcaaggacca 241 gttcccagag gtgtatgtgc ccacagtgtt tgagaactat gtggcagata tcgaggtgga 301 tggaaagcag gtagaattgg ctttgtggga cacagctggg caggaagatt atgatcgcct 361 gaggcccctc tcctacccag ataccgatgt tatactgatg tgtttttcca tcgacagccc 421 tgatagttta gaaaacatcc cagaaaagtg gaccccagaa gtcaagcatt tctgtcccaa 481 cgtgcccatc atcctggttg ggaataagaa ggatcttcgg aatgatgagc acacaaggcg 541 ggagctagcc aagatgaagc aggagccggt gaaacctgaa gaaggcagag atatggcaaa 601 caggattggc gcttttgggt acatggagtg ttcagcaaag accaaagatg gagtgagaga 661 ggtttttgaa atggctacga gagctgctct gcaagctaga cgtgggtgac agtaaaggtg 721 gatacggatc cgaattcgag ctccgtcgac aagcttgcgg ccgcactcga gcaccaccac 781 caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc 841 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt 901 ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat 961 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 1021 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 1081 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 1141 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt 1201 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 1261 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg 1321 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat 1381 taacgtttac aatttcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 1441 tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca 1501 tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga 1561 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga 1621 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc 1681 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag 1741 aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg 1801 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga 1861 cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc 1921 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc 1981 tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg 2041 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc 2101 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca 2161 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc 2221 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac 2281 gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt 2341 tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc 2401 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt 2461 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac 2521 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt 2581 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 2641 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga 2701 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac 2761 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg 2821 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt 2881 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 2941 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg 3001 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc 3061 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc 3121 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag 3181 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc 3241 acaccgcata tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag 3301 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac 3361 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga 3421 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc 3481 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat 3541 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg 3601 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc 3661 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg 3721 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc 3781 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag 3841 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg 3901 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg 3961 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg 4021 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga 4081 gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga 4141 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata 4201 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga 4261 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg 4321 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca 4381 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc 4441 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc 4501 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt 4561 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg 4621 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata 4681 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc 4741 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc 4801 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg 4861 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta 4921 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg 4981 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg 5041 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca 5101 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc 5161 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg 5221 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc 5281 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc 5341 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc 5401 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg 5461 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt 5521 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag 5581 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat 5641 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc 5701 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc 5761 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc 5821 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc 5881 gtagaggatc gagatctcga tcccgcgaaa t //