LOCUS Exported 7399 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pKM631 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7399) AUTHORS Kapust RB, Toezser J, Copeland TD, Waugh DS TITLE The P1' specificity of tobacco etch virus protease. JOURNAL Biochem Biophys Res Commun. 2002 Jun 28. 294(5):949-55. PUBMED 12074568 REFERENCE 2 (bases 1 to 7399) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7399 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 3..80 /gene="lacI (mutant)" /label=lacIq promoter /note="In the lacIq allele, a single base change in the promoter boosts expression of the lacI gene about 10-fold." CDS 81..1163 /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 complement(100..119) /label=LacI-R /note="LacI, reverse primer" protein_bind 1176..1197 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 1406..1434 /label=tac promoter /note="strong E. coli promoter; hybrid between the trp and lac UV5 promoters" protein_bind 1442..1458 /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)." primer_bind 1447..1469 /label=M13/pUC Reverse /note="In lacZ gene" CDS 1528..2628 /codon_start=1 /gene="malE (mutated)" /product="maltose binding protein from E. coli" /label=MBP /note="This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol." /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF WYAVRTAVINAASGRQTVDEALKDAQT" regulatory 2485..2494 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" primer_bind 2602..2625 /label=MBP-F /note="Maltose binding protein, forward primer" protein_bind 2638..2662 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" CDS 2665..2685 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 3430..3447 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" protein_bind complement(3453..3477) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" primer_bind complement(3489..3506) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(3489..3505) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3498..3520) /label=M13/pUC Forward /note="In lacZ gene" primer_bind complement(3684..3701) /label=pBAD Reverse /note="For vectors with E. coli araBAD promoter, reverse primer" terminator 3854..3940 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 4032..4059 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" promoter 4079..4170 /gene="bla" /label=AmpR promoter CDS 4171..5031 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERSPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(4389..4408) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin complement(5073..5586) /direction=LEFT /label=M13 ori /note="M13 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5268..5289) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 5480..5499 /label=F1ori-R /note="F1 origin, reverse primer" rep_origin 5697..6285 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6186..6205 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6439..6456 /label=L4440 /note="L4440 vector, forward primer" misc_feature 6471..6611 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(6537..6556) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6697..6719 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(6713..6904) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" ORIGIN 1 ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga 61 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg 121 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa 181 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac 241 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc 301 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg 361 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc 421 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca 481 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga 541 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc 601 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg 661 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag 721 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga 781 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa 841 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg 901 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc 961 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga 1021 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata 1081 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt 1141 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag 1201 gcacaattct catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg 1261 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg 1321 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt 1381 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga 1441 attgtgagcg gataacaatt tcacacagga aacagccagt ccgtttaggt gttttcacga 1501 gcacttcacc aacaaggacc atagattatg aaaatcgaag aaggtaaact ggtaatctgg 1561 attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat 1621 accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt 1681 gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac 1741 gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat 1801 ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt 1861 gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa 1921 gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac 1981 ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag 2041 tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg 2101 ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac 2161 tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg 2221 gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc 2281 aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt 2341 ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga tgaaggtctg 2401 gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag 2461 ttggcgaaag atccacgtat tgccgccacc atggaaaacg cccagaaagg tgaaatcatg 2521 ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc 2581 gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa ttcgatcaca 2641 agtttgtaca aaaaagcagg ctcggagaac ctgtacttcc agggtatgag cgagcaacag 2701 gttcaggaac ttgagaaaaa gtggtacgca ctgcaggtag agcccggaaa agagaacgaa 2761 gctaaggaaa acttactgaa agttctggaa ctggaaggtt taaaggacct cgttgacgag 2821 gtaatagttc ccgcagagga aaaggtagtc ataagggctc agggcaagga aaagtacagg 2881 ctctctctca agggaaacgc gagggacata tccgttttag ggaagaaagg tgttaccaca 2941 ttcaggatag agaacggaga agttaaggtc gttgagagtg ttgaaggaga cacttgcgta 3001 aacgcccctc ccatatcaaa acccggacaa aagatcacgt gtaaggaaaa taaaacggaa 3061 gcaaagatag ttctggataa caaaatattt cccggataca tactcataaa ggctcacatg 3121 aacgacaagc tcctcatggc tattgagaaa actccccacg tcttcagacc tgtaatggtg 3181 ggcggaaagc ccgttcctct gaaagaggaa gaggttcaaa acattctcaa tcaaataaag 3241 aggggtgtta aaccttccaa ggttgagttt gaaaaaggag atcaggtaag ggttatagaa 3301 ggaccgttta tgaactttac gggaactgtt gaggaagttc atcccgagaa gaggaaatta 3361 actgttatga taagtatttt cggaaggatg actccggtag aactggactt tgaccaagtg 3421 gaaaagattc atcaccacca tcatcactaa taacccagct ttcttgtaca aagtggtgat 3481 agcttggcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa 3541 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc 3601 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcagct tggctgtttt 3661 ggcggatgag ataagatttt cagcctgata cagattaaat cagaacgcag aagcggtctg 3721 ataaaacaga atttgcctgg cggcagtagc gcggtggtcc cacctgaccc catgccgaac 3781 tcagaagtga aacgccgtag cgccgatggt agtgtggggt ctccccatgc gagagtaggg 3841 aactgccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat 3901 ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccgggag cggatttgaa 3961 cgttgcgaag caacggcccg gagggtggcg ggcaggacgc ccgccataaa ctgccaggca 4021 tcaaattaag cagaaggcca tcctgacgga tggccttttt gcgtttctac aaactctttt 4081 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa 4141 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt 4201 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa 4261 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac 4321 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ctccaatgat gagcactttt 4381 aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt 4441 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat 4501 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac 4561 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg 4621 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc 4681 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa 4741 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag 4801 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct 4861 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat 4921 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa 4981 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac 5041 caagtttact catatatact ttagattgat ttaccccggt tgataatcag aaaagcccca 5101 aaaacaggaa gattgtataa gcaaatattt aaattgtaaa cgttaatatt ttgttaaaat 5161 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa 5221 tcccttataa atcaaaagaa tagcccgaga tagggttgag tgttgttcca gtttggaaca 5281 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg 5341 gcgatggccc actacgtgaa ccatcaccca aatcaagttt tttggggtcg aggtgccgta 5401 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg 5461 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa 5521 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg 5581 gcgcgtaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 5641 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 5701 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 5761 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 5821 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 5881 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 5941 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 6001 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 6061 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 6121 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 6181 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 6241 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 6301 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 6361 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 6421 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 6481 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca 6541 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg 6601 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 6661 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt 6721 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt 6781 gcagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa 6841 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg 6901 tcacttgatg cctccgtgta agggggaatt tctgttcatg ggggtaatga taccgatgaa 6961 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 7021 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 7081 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 7141 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 7201 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 7261 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 7321 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggccagga 7381 cccaacgctg cccgaaatt //