LOCUS       pET21b_FAST-PETase-His  6155 bp ds-DNA     circular     14-OCT-2024
DEFINITION  .
KEYWORDS    "accession:addgene_112202_334619"
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
COMMENT     Imported from database: AddGene Entry: pET21b(+)-Is-PETase URL:
            https://www.addgene.org/browse/sequence/334619/ GENERAL Depositing
            Labs: Gregg Beckham, Christopher Johnson Publication: Austin et al
            Proc Natl Acad Sci U S A. 2018 May 8;115(19):E4350-E4357. doi:
            10.1073/pnas.1718804115. Epub 2018 Apr 17. Purpose: pET-21b(+)
            based plasmid for expression of PETase from Ideonella sakaiensis
            201-F6 (Genbank GAP38373.1), codon optimized for expression in E.
            coli K12 BACKBONE Backbone manufacturer: Novagen/Millipore Sigma
            Backbone size w/o insert (bp): 5367 Modifications to backbone: His
            tag: CACCACCATCACCACCAC Total vector size (bp): 6237 Vector
            backbone: pET-21b(+) Vector type: Bacterial Expression CLONING
            INFORMATION 3′ sequencing primer: N/A 5′ sequencing primer: N/A
            Cloning method: Gibson Cloning GROWTH IN BACTERIA Bacterial
            Resistance(s): Ampicillin Copy number: Unknown Growth Strain(s):
            DH5alpha Growth Temperature: 37°C GENE/INSERT GenBank ID:
            Gene/Insert name: PETase gene from Ideonella sakaiensis 201-F6,
            codon optimized for expression in E. coli K12 Promoter: N/A
            Species: Ideonella sakaiensis Tag / Fusion Protein:  TERMS AND
            LICENSES Academic/Nonprofit Terms:  Industry Terms:
FEATURES             Location/Qualifiers
     rep_origin      12..467
                     /label="f1 ori"
                     /note="direction: RIGHT note: f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis"
     primer_bind     complement(99..118)
                     /label="F1ori-R"
                     /note="note: F1 origin, reverse primer"
     primer_bind     309..330
                     /label="F1ori-F"
                     /note="note: F1 origin, forward primer"
     promoter        493..597
                     /label="AmpR promoter"
                     /note="gene: bla"
     CDS             598..1458
                     /label="AmpR"
                     /note="gene: bla product: beta-lactamase note: confers resistance to ampicillin, carbenicillin, andrelated antibiotics translation: MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW"
     CDS             598..1458
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW*"
     primer_bind     complement(816..835)
                     /label="Amp-R"
                     /note="note: Ampicillin resistance gene, reverse primer"
     rep_origin      1629..2217
                     /label="ori"
                     /note="direction: RIGHT note: high-copy-number ColE1/pMB1/pBR322/pUC origin of replication"
     primer_bind     2118..2137
                     /label="pBR322ori-F"
                     /note="note: pBR322 origin, forward primer"
     primer_bind     2371..2388
                     /label="L4440"
                     /note="note: L4440 vector, forward primer"
     misc_feature    2403..2542
                     /label="bom"
                     /note="note: basis of mobility region from pBR322"
     primer_bind     complement(2468..2487)
                     /label="pRS-marker"
                     /note="note: pRS vectors, use to sequence yeast selectable marker"
     primer_bind     2628..2650
                     /label="pGEX 3'"
                     /note="note: pGEX vectors, reverse primer"
     CDS             complement(2644..2835)
                     /label="rop"
                     /note="gene: rop product: Rop protein, which maintains plasmids at low copynumber translation: MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHADELYRSCLARFGDDGENL"
     CDS             complement(2644..2835)
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHADELYRSCLARFGDDGENL*"
     CDS             complement(3644..4726)
                     /label="lacI"
                     /note="gene: lacI product: lac repressor 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: MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ"
     CDS             complement(3644..4726)
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ*"
     primer_bind     4688..4707
                     /label="LacI-R"
                     /note="note: LacI, reverse primer"
     promoter        complement(4727..4804)
                     /label="lacI promoter"
                     /note="gene: lacI"
     primer          4772..4791
                     /label="pCDF Seq for (JB89)"
                     /note="sequence: accgcgaaaggttttgcgcc"
     primer          5007..5029
                     /label="pSFB seq for (JB 121)"
                     /note="sequence: atcggtgatgtcggcgatatagg"
     primer_bind     5010..5029
                     /label="pBRrevBam"
                     /note="note: pBR322 vectors, tet region, downstream of BamHI, reverse primer"
     primer          5106..5132
                     /label="pET Seq For (JB 24)"
                     /note="sequence: gcgaaattaatacgactcactataggg"
     promoter        5113..5131
                     /label="T7 promoter"
                     /note="note: promoter for bacteriophage T7 RNA polymerase"
     primer_bind     5113..5132
                     /label="T7"
                     /note="note: T7 promoter, forward primer"
     protein_bind    5132..5156
                     /label="lac operator"
                     /note="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
                     /label="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)"
     misc_feature    5201..5998
                     /label="FAST PETase"
     CDS             5201..5998
                     /label="Translation 5201-5998"
                     /translation="MQTNPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPESRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWHSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSQNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTAVSDFRTANCSLE"
     CDS             5201..6019
                     /label="PETase_FAST_noSS"
                     /translation="MQTNPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPESRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWHSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSQNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTAVSDFRTANCSLEHHHHHH*"
     CDS             5999..6016
                     /label="6xHis"
                     /note="product: 6xHis affinity tag translation: HHHHHH"
     misc_feature    5999..6019
                     /label="PETase-his"
     CDS             5999..6019
                     /label="Translation 5999-6019"
                     /translation="HHHHHH*"
     primer_bind     complement(6069..6087)
                     /label="T7 Term"
                     /note="note: T7 terminator, reverse primer"
     primer          complement(6069..6087)
                     /label="pET Seq Rev (JB 23)"
                     /note="sequence: gctagttattgctcagcgg"
     terminator      6083..6130
                     /label="T7 terminator"
                     /note="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 aacaactggc 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 caacgacagg agcacgatca tgcgcacccg
     3301 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     3361 agtgacgaag gcttgagcga gggcgtgcaa 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 ATGCAGACTA ATCCATACGC
     5221 CCGCGGACCT AACCCTACGG CGGCGAGTTT GGAGGCTTCT GCTGGTCCGT TCACAGTTCG
     5281 TTCTTTTACG GTGTCGCGTC CAAGCGGGTA CGGCGCTGGA ACCGTCTACT ATCCCACTAA
     5341 CGCTGGAGGC ACAGTAGGAG CTATCGCCAT TGTCCCCGGC TACACTGCTC GCCAAAGCAG
     5401 TATCAAATGG TGGGGTCCGC GCTTGGCCTC ACATGGGTTT GTTGTGATTA CCATCGACAC
     5461 TAATTCCACA TTGGACCAGC CAgaaTCACG TTCTTCACAA CAAATGGCTG CCTTACGCCA
     5521 AGTGGCGTCG CTTAATGGGA CTTCTAGTTC ACCAATCTAC GGTAAGGTAG ATACTGCTCG
     5581 CATGGGAGTT ATGGGATGGA GCATGGGCGG TGGGGGGAGC CTTATTTCCG CAGCGAATAA
     5641 CCCATCCCTG AAGGCTGCCG CACCTCAGGC CCCGTGGcat TCGTCAACTA ATTTCAGCTC
     5701 GGTCACCGTC CCTACTCTTA TTTTCGCTTG TGAAAACGAC AGTATCGCAC CTGTTAATTC
     5761 ATCGGCACTG CCAATCTATG ATTCCATGTC CcagAACGCT AAGCAATTCC TGGAGATTaa
     5821 aGGGGGAAGT CACTCGTGCG CTAATAGTGG GAACAGTAAT CAAGCTCTTA TCGGGAAGAA
     5881 GGGTGTTGCA TGGATGAAAC GCTTTATGGA CAACGACACA CGTTATAGTA CCTTTGCGTG
     5941 CGAAAATCCT AATTCTACGg cgGTGAGTGA CTTCCGCACG GCAAATTGCA GCCTCGAGca
     6001 ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc
     6061 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     6121 gggttttttg ctgaaaggag gaactatatc cggat
//