LOCUS Exported 6233 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION pET-21b(+) based plasmid for expression of PETase from Ideonella sakaiensis 201-F6 (Genbank GAP38373.1) with W159H and S238F mutations, codon optimized for expression in E. coli K12. ACCESSION . VERSION . KEYWORDS pET21b(+)-Is-PETase-W159H-S238F SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6233) AUTHORS Austin HP, Allen MD, Donohoe BS, Rorrer NA, Kearns FL, Silveira RL, Pollard BC, Dominick G, Duman R, El Omari K, Mykhaylyk V, Wagner A, Michener WE, Amore A, Skaf MS, Crowley MF, Thorne AW, Johnson CW, Woodcock HL, McGeehan JE, Beckham GT TITLE Characterization and engineering of a plastic-degrading aromatic polyesterase. JOURNAL Proc Natl Acad Sci U S A. 2018 May 8;115(19):E4350-E4357. doi: 10.1073/pnas.1718804115. Epub 2018 Apr 17. PUBMED 29666242 REFERENCE 2 (bases 1 to 6233) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6233 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 12..467 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(99..118) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 309..330 /label=F1ori-F /note="F1 origin, forward primer" promoter 493..597 /gene="bla" /label=AmpR promoter CDS 598..1458 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(816..835) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1629..2217 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2118..2137 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2371..2388 /label=L4440 /note="L4440 vector, forward primer" misc_feature 2403..2542 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(2468..2487) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 2628..2650 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(2644..2835) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(3644..4726) /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 4688..4707 /label=LacI-R /note="LacI, reverse primer" promoter complement(4727..4804) /gene="lacI" /label=lacI promoter primer_bind 5010..5029 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 5113..5132 /label=T7 /note="T7 promoter, forward primer" promoter 5113..5131 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 5132..5156 /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 5171..5193 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 6077..6094 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(6147..6165) /label=T7 Term /note="T7 terminator, reverse primer" terminator 6161..6208 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" ORIGIN 1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 421 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt 481 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat 541 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg 601 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt 661 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga 721 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa 781 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt 841 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt 901 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc 961 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga 1021 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat 1081 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct 1141 gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc 1201 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg 1261 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc 1321 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg 1381 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca 1441 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta 1501 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc 1561 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa 1621 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca 1681 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta 1741 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc 1801 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca 1861 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta 1921 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag 1981 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt 2041 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc 2101 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac 2161 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac 2221 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc 2281 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat 2341 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag 2401 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca 2461 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct 2521 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg 2581 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat 2641 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc 2701 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag 2761 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt 2821 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat 2881 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt 2941 actggaacgt tgtgagggta aacaacttgc ggtatggatg cggcgggacc agagaaaaat 3001 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca 3061 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc 3121 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt 3181 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt 3241 aaggcaaccc cgccagccta gccgggtcct caacgacagt agcacgatca tgcgcacccg 3301 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc 3361 agtgacgaag gcttgagcga tggcgtgcaa gattccgaat accgcaagcg acaggccgat 3421 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac 3481 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc 3541 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc 3601 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca 3661 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg 3721 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat 3781 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca 3841 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg 3901 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg 3961 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc 4021 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc 4081 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca 4141 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg 4201 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga 4261 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca 4321 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg 4381 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca 4441 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg 4501 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca 4561 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt 4621 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag 4681 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga 4741 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg 4801 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt 4861 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc 4921 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc 4981 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc 5041 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg 5101 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctc 5161 tagaaataat tttgtttaac tttaagaagg agatatacat atgaacttcc cccgtgcctc 5221 gcgccttatg caggctgctg tgctgggcgg ccttatggcc gtttccgcag cggccaccgc 5281 gcagaccaat ccgtatgcgc gcggccccaa ccctaccgcc gcctcgttgg aagccagcgc 5341 gggacccttt accgttcgta gctttaccgt tagccgtccg tccggatatg gtgcagggac 5401 cgtctattac ccaaccaatg caggcggcac cgttggcgcg attgcaatcg tccccgggta 5461 caccgcgcgt caaagcagca ttaagtggtg gggtccgcgc ttagctagcc atggctttgt 5521 ggttattacc atcgatacga acagcactct agaccagccc agcagccgta gctcgcaaca 5581 gatggccgcg cttcgtcaag ttgcgagctt gaacgggacc agcagtagcc cgatttacgg 5641 aaaggtcgat actgcccgca tgggtgtgat gggccactca atggggggcg gcggttcact 5701 tattagcgcc gcgaacaacc cgagtttaaa agcagcggca ccgcaggcgc catgggactc 5761 ttcaaccaac ttcagcagtg ttaccgtgcc gacgctgatt ttcgcgtgcg agaatgatag 5821 cattgcaccg gtgaacagca gcgcgctgcc gatttatgat agcatgtccc gcaacgcaaa 5881 acagtttctg gaaattaacg gcggtagcca cttctgtgcc aactctggga acagcaacca 5941 ggcactgatc ggaaaaaaag gggttgcatg gatgaaacga ttcatggata atgacacccg 6001 ttactcaacc ttcgcctgtg agaatcccaa cagcacacgc gtgtcggatt ttcgcaccgc 6061 gaactgttcc ctcgagcacc accatcacca ccactgagat ccggctgcta acaaagcccg 6121 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc 6181 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gat //