LOCUS Exported 5677 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Bacterial expression for structure determination; may not be full ORF. ACCESSION . VERSION . KEYWORDS BAZ2B SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5677) TITLE SGC Oxford REFERENCE 2 (bases 1 to 5677) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5677 /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 538..555 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(608..626) /label=T7 Term /note="T7 terminator, reverse primer" terminator 622..669 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 706..1161 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(793..812) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1003..1024 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(1254..2069) /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 1977..1996 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 2061..2080 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 2191..2779 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2680..2699 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2933..2950 /label=L4440 /note="L4440 vector, forward primer" misc_feature 2965..3107 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(3033..3052) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3193..3215 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(3209..3400) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(4209..5291) /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 5253..5272 /label=LacI-R /note="LacI, reverse primer" promoter complement(5292..5369) /gene="lacI" /label=lacI promoter primer_bind 5575..5594 /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 atccatggat agcaaagatc tggcgctgtg 181 cagcatgatt ctgaccgaaa tggaaaccca tgaagatgcg tggccgtttc tgctgccggt 241 gaacctgaaa ctggttccgg gctataaaaa agtgattaaa aaaccgatgg attttagcac 301 cattcgtgaa aaactgagca gcggtcagta tccgaacctg gaaacctttg cgctggatgt 361 gcgtctggtg tttgataact gcgaaacctt taacgaagat gatagcgata ttggccgtgc 421 cggtcataac atgcgcaaat attttgaaaa aaaatggacc gataccttta aatgacagta 481 aaggtggata cggatccgaa ttcgagctcc gtcgacaagc ttgcggccgc actcgagcac 541 caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct 601 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg 661 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg 721 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 781 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc 841 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc 901 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga 961 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa 1021 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga 1081 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca 1141 aaatattaac gtttacaatt tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta 1201 tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa 1261 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat 1321 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg 1381 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat 1441 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc 1501 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta 1561 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga 1621 gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac 1681 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct 1741 aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga 1801 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg 1861 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct 1921 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg 1981 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag 2041 caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca 2101 gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc 2161 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg 2221 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct 2281 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct 2341 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct 2401 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg 2461 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc 2521 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga 2581 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg 2641 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta 2701 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg 2761 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg 2821 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat 2881 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc 2941 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg 3001 tatttcacac cgcatatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta 3061 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc 3121 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag 3181 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg 3241 cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct 3301 gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa 3361 agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg 3421 gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg 3481 ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat 3541 ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag 3601 atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg 3661 tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc 3721 atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta 3781 tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg 3841 acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct 3901 cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc 3961 cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga 4021 aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca 4081 taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg 4141 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg 4201 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 4261 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc 4321 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc 4381 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc 4441 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca 4501 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca 4561 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg 4621 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga 4681 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac 4741 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct 4801 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc 4861 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta 4921 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg 4981 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat 5041 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca 5101 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc 5161 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc 5221 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt 5281 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg 5341 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc 5401 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa 5461 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc 5521 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat 5581 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg 5641 tccggcgtag aggatcgaga tctcgatccc gcgaaat //