LOCUS Exported 9377 bp ds-DNA circular SYN 18-OCT-2021 DEFINITION Bacterial expression and purification, combined with SAE1 to form the E1 enzyme heterodimer. ACCESSION . VERSION . KEYWORDS SAE2-mGarnet SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9377) AUTHORS Peeples W, Rosen MK TITLE Mechanistic dissection of increased enzymatic rate in a phase-separated compartment. JOURNAL Nat Chem Biol. 2021 Jun;17(6):693-702. doi: 10.1038/s41589-021-00801-x. Epub 2021 May 25. PUBMED 34035521 REFERENCE 2 (bases 1 to 9377) AUTHORS . TITLE Direct Submission JOURNAL Exported Oct 18, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9377 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 734..762 /label=tac promoter /note="strong E. coli promoter; hybrid between the trp and lac UV5 promoters" protein_bind 770..786 /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 775..797 /label=M13/pUC Reverse /note="In lacZ gene" CDS 856..1956 /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="MKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF WYAVRTAVINAASGRQTVDEALKDAQT" regulatory 1813..1822 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" primer_bind 1930..1953 /label=MBP-F /note="Maltose binding protein, forward primer" CDS 2005..2016 /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" CDS 2029..2049 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" primer_bind complement(4773..4790) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4773..4789) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4782..4804) /label=M13/pUC Forward /note="In lacZ gene" primer_bind complement(4968..4985) /label=pBAD Reverse /note="For vectors with E. coli araBAD promoter, reverse primer" terminator 5138..5224 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 5316..5343 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" promoter 5362..5453 /gene="bla" /label=AmpR promoter CDS 5454..6314 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(5672..5691) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin complement(6356..6869) /direction=LEFT /label=M13 ori /note="M13 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6551..6572) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 6763..6782 /label=F1ori-R /note="F1 origin, reverse primer" rep_origin 6980..7568 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7469..7488 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 7722..7739 /label=L4440 /note="L4440 vector, forward primer" primer_bind complement(7845..7864) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 8005..8027 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(8021..8212) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" promoter 8708..8785 /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 8786..491 /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(8805..8824) /label=LacI-R /note="LacI, reverse primer" ORIGIN 1 tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag 61 gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg 121 ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta 181 ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag 241 acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc 301 aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc 361 tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct 421 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa 481 gcgggcagtg agcgcaacgc aattaatgta agttagctca ctcattaggc acaattctca 541 tgtttgacag cttatcatcg actgcacggt gcaccaatgc ttctggcgtc aggcagccat 601 cggaagctgt ggtatggctg tgcaggtcgt aaatcactgc ataattcgtg tcgctcaagg 661 cgcactcccg ttctggataa tgttttttgc gccgacatca taacggttct ggcaaatatt 721 ctgaaatgag ctgttgacaa ttaatcatcg gctcgtataa tgtgtggaat tgtgagcgga 781 taacaatttc acacaggaaa cagccagtcc gtttaggtgt tttcacgagc acttcaccaa 841 caaggaccat agattatgaa aactgaagaa ggtaaactgg taatctggat taacggcgat 901 aaaggctata acggtctcgc tgaagtcggt aagaaattcg agaaagatac cggaattaaa 961 gtcaccgttg agcatccgga taaactggaa gagaaattcc cacaggttgc ggcaactggc 1021 gatggccctg acattatctt ctgggcacac gaccgctttg gtggctacgc tcaatctggc 1081 ctgttggctg aaatcacccc ggacaaagcg ttccaggaca agctgtatcc gtttacctgg 1141 gatgccgtac gttacaacgg caagctgatt gcttacccga tcgctgttga agcgttatcg 1201 ctgatttata acaaagatct gctgccgaac ccgccaaaaa cctgggaaga gatcccggcg 1261 ctggataaag aactgaaagc gaaaggtaag agcgcgctga tgttcaacct gcaagaaccg 1321 tacttcacct ggccgctgat tgctgctgac gggggttatg cgttcaagta tgaaaacggc 1381 aagtacgaca ttaaagacgt gggcgtggat aacgctggcg cgaaagcggg tctgaccttc 1441 ctggttgacc tgattaaaaa caaacacatg aatgcagaca ccgattactc catcgcagaa 1501 gctgccttta ataaaggcga aacagcgatg accatcaacg gcccgtgggc atggtccaac 1561 atcgacacca gcaaagtgaa ttatggtgta acggtactgc cgaccttcaa gggtcaacca 1621 tccaaaccgt tcgttggcgt gctgagcgca ggtattaacg ccgccagtcc gaacaaagag 1681 ctggcaaaag agttcctcga aaactatctg ctgactgatg aaggtctgga agcggttaat 1741 aaagacaaac cgctgggtgc cgtagcgctg aagtcttacg aggaagagtt ggcgaaagat 1801 ccacgtattg ccgccaccat ggaaaacgcc cagaaaggtg aaatcatgcc gaacatcccg 1861 cagatgtccg ctttctggta tgccgtgcgt actgcggtga tcaacgccgc cagcggtcgt 1921 cagactgtcg atgaagccct gaaagacgcg cagactaatt cgagctcgaa caacaacaac 1981 aataacaata acaacaacct cgggatcgag ggaaggattt cagaatttga aaacctgtat 2041 tttcagggcg aattctctgg cggctctggc ggctccggtg gttccatggc actgtcgcgg 2101 gggctgcccc gggagctggc tgaggcggtg gccgggggcc gggtgctggt ggtgggggcg 2161 ggcggcatcg gctgcgagct cctcaagaat ctcgtgctca ccggtttctc ccacatcgac 2221 ctgattgatc tggatactat tgatgtaagc aacctcaaca gacagttttt gtttcaaaag 2281 aaacatgttg gaagatcaaa ggcacaggtt gccaaggaaa gtgtactgca gttttacccg 2341 aaagctaata tcgttgccta ccatgacagc atcatgaacc ctgactataa tgtggaattt 2401 ttccgacagt ttatactggt tatgaatgct ttagataaca gagctgcccg aaaccatgtt 2461 aatagaatgt gcctggcagc tgatgttcct cttattgaaa gtggaacagc tgggtatctt 2521 ggacaagtaa ctactatcaa aaagggtgtg accgagtgtt atgagtgtca tcctaagccg 2581 acccagagaa cctttcctgg ctgtacaatt cgtaacacac cttcagaacc tatacattgc 2641 atcgtttggg caaagtactt gttcaaccag ttgtttgggg aagaagatgc tgatcaagaa 2701 gtatctcctg acagagctga ccctgaagct gcctgggaac caacggaagc cgaagccaga 2761 gctagagcat ctaatgaaga tggtgacatt aaacgtattt ctactaagga atgggctaaa 2821 tcaactggat atgatccagt taaacttttt accaagcttt ttaaagatga catcaggtat 2881 ctgttgacaa tggacaaact atggcggaaa aggaaacctc cagttccgtt ggactgggct 2941 gaagtacaaa gtcaaggaga agaaacgaat gcatcagatc aacagaatga accccagtta 3001 ggcctgaaag accagcaggt tctagatgta aagagctatg cacgtctttt ttcaaagagc 3061 atcgagactt tgagagttca tttagcagaa aagggggatg gagctgagct catatgggat 3121 aaggatgacc catctgcaat ggattttgtc acctctgctg caaacctcag gatgcatatt 3181 ttcagtatga atatgaagag tagatttgat atcaaatcaa tggcagggaa cattattcct 3241 gctattgcta ctactaatgc agtaattgct gggttgatag tattggaagg attgaagatt 3301 ttatcaggaa aaatagacca gtgcagaaca atttttttga ataaacaacc aaacccaaga 3361 aagaagcttc ttgtgccttg tgcactggat cctcccaacc ccaattgtta tgtatgtgcc 3421 agcaagccag aggtgactgt gcggctgaat gtccataaag tgactgttct caccttacaa 3481 gacaagatag tgaaagaaaa atttgctatg gtagcaccag atgtccaaat tgaagatggg 3541 aaaggaacaa tcctaatatc ttccgaagag ggagagacgg aagctaataa tcacaagaag 3601 ttgtcagaat ttggaattag aaatggcagc cggcttcaag cagatgactt cctccaggac 3661 tatactttat tgatcaacat ccttcatagt gaagacctag gaaaggacgt tgaatttgaa 3721 gttgttggtg atgccccgga aaaagtgggg cccaaacaag ctgaagatgc tgccaaaagc 3781 ataaccaatg gcagtgatga tggagctcag ccctccacct ccacagctca agagcaagat 3841 gacgttctca tagttgattc ggatgaagaa gattcttcaa ataatgccga cgtcagtgaa 3901 gaagagagaa gccgcaagag gaaattagat gagaaagaga atctcagtgc aaagaggtca 3961 cgtatagaac agaaggaaga gcttgatgat gtcatagcat tagatctcga cctgagaagc 4021 cgtgctcagg cttcgaactc tgcagttgac ggcaccaaca gccttatcaa ggagaacatg 4081 cgtatgaaag ttgtattaga aggctccgtc aatgggcacc agtttaaatg cacgggcgag 4141 ggcgagggta acccctatat gggaactcag acgatgcgca ttaaagttat tgaaggcgga 4201 ccgttacctt ttgcttttga catccttgca acttcattca tgtatggcag taagactttc 4261 atcaaatatc caaaaggaat ccccgatttc tttaagcagt cattccccga aggcttcacg 4321 tgggagcgtg tgactcgtta cgaggatggt ggggttatca cggtaatgca agacacctca 4381 cttgaggacg gctgcttggt ttatcacgcg caagttcgcg gcgtcaattt tccgagcaac 4441 ggggcagtca tgcaaaagaa aacaaagggg tgggaaccca acactgagat gatgtacccc 4501 gcagatgggg ggttacgcgg ctataatcac atggcattga aggttgatgg cgggggccac 4561 ttatcatgta gcctggtcac tacataccgt agtaagaaaa ccgtcggaaa tatcaaaatg 4621 ccagggattc acgccgttga ccgtcgttta gaacgcttgg aagagtcgga caatgagatg 4681 ttcgtggtcc aacgcgagca tgcagtggct aaatttgccg ggctgggtgg tggataagga 4741 tcctctagag tcgacctgca ggcaagcttg gcactggccg tcgttttaca acgtcgtgac 4801 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc 4861 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat 4921 ggcgaatggc agcttggctg ttttggcgga tgagataaga ttttcagcct gatacagatt 4981 aaatcagaac gcagaagcgg tctgataaaa cagaatttgc ctggcggcag tagcgcggtg 5041 gtcccacctg accccatgcc gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg 5101 gggtctcccc atgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc 5161 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac 5221 aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg 5281 acgcccgcca taaactgcca ggcatcaaat taagcagaag gccatcctga cggatggcct 5341 ttttgcgttt ctacaaactc ttttgtttat ttttctaaat acattcaaat atgtatccgc 5401 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 5461 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 5521 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 5581 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 5641 gtttcccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg 5701 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 5761 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 5821 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac 5881 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 5941 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag 6001 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc 6061 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 6121 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 6181 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 6241 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 6301 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttacccc 6361 ggttgataat cagaaaagcc ccaaaaacag gaagattgta taagcaaata tttaaattgt 6421 aaacgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa 6481 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagcccg agatagggtt 6541 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa 6601 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac ccaaatcaag 6661 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt 6721 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg 6781 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc 6841 cgcgcttaat gcgccgctac agggcgcgta aaaggatcta ggtgaagatc ctttttgata 6901 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 6961 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 7021 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 7081 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 7141 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 7201 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 7261 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 7321 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 7381 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 7441 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 7501 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 7561 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 7621 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 7681 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 7741 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc 7801 gcatgtggtg cactctcagt acaccgcatg tggtgcactc tcagtacaat ctgctctgat 7861 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc 7921 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg 7981 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat 8041 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtgc agcgattcac 8101 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct 8161 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct 8221 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc 8281 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac 8341 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct 8401 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc 8461 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac 8521 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg 8581 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc 8641 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ccaggaccca acgctgcccg 8701 aaattccgac accatcgaat ggtgcaaaac ctttcgcggt atggcatgat agcgcccgga 8761 agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta 8821 tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc 8881 gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt 8941 ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc 9001 cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc 9061 cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca 9121 caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga 9181 tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga 9241 ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga 9301 gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt 9361 ctcggcgcgt ctgcgtc //