LOCUS       sequence-473446-        6233 bp    DNA     circular SYN 15-DEC-2025
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    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1073/pnas.1718804115"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2018-05-8- 8"; volume: "115"; issue: "19"; pages: 
            "E4350-E4357"
COMMENT     Sequence Label: pET21b(+)-Is-PETase-W159H-S238F
FEATURES             Location/Qualifiers
     source          1..6233
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             636..653
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(706..724)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      720..767
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      804..1259
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(891..910)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1101..1122
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1285..1389
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1390..1458,1459..2250)
                     /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(1608..1627)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2421..3009
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2910..2929
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3163..3180
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3195..3334
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3260..3279)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3420..3442
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3436..3627)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4436..5518)
                     /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     5480..5499
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5519..5596)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5802..5821
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5905..5924
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5905..5923
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5924..5948
                     /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             5963..5985
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 ttgcaatcgt ccccgggtac accgcgcgtc aaagcagcat taagtggtgg ggtccgcgct
       61 tagctagcca tggctttgtg gttattacca tcgatacgaa cagcactcta gaccagccca
      121 gcagccgtag ctcgcaacag atggccgcgc ttcgtcaagt tgcgagcttg aacgggacca
      181 gcagtagccc gatttacgga aaggtcgata ctgcccgcat gggtgtgatg ggccactcaa
      241 tggggggcgg cggttcactt attagcgccg cgaacaaccc gagtttaaaa gcagcggcac
      301 cgcaggcgcc atgggactct tcaaccaact tcagcagtgt taccgtgccg acgctgattt
      361 tcgcgtgcga gaatgatagc attgcaccgg tgaacagcag cgcgctgccg atttatgata
      421 gcatgtcccg caacgcaaaa cagtttctgg aaattaacgg cggtagccac ttctgtgcca
      481 actctgggaa cagcaaccag gcactgatcg gaaaaaaagg ggttgcatgg atgaaacgat
      541 tcatggataa tgacacccgt tactcaacct tcgcctgtga gaatcccaac agcacacgcg
      601 tgtcggattt tcgcaccgcg aactgttccc tcgagcacca ccatcaccac cactgagatc
      661 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
      721 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
      781 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
      841 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
      901 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
      961 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     1021 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     1081 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     1141 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     1201 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     1261 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     1321 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     1381 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     1441 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     1501 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     1561 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     1621 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     1681 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     1741 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     1801 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     1861 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     1921 accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     1981 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     2041 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     2101 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     2161 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     2221 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     2281 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     2341 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     2401 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     2461 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     2521 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     2581 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     2641 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     2701 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     2761 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     2821 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     2881 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     2941 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     3001 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     3061 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     3121 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     3181 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     3241 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     3301 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     3361 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     3421 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     3481 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     3541 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     3601 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     3661 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     3721 acatgcccgg ttactggaac gttgtgaggg taaacaactt gcggtatgga tgcggcggga
     3781 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     3841 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     3901 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     3961 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     4021 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca gtagcacgat
     4081 catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt
     4141 ggtggcggga ccagtgacga aggcttgagc gatggcgtgc aagattccga ataccgcaag
     4201 cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag
     4261 cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac
     4321 gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     4381 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     4441 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     4501 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     4561 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     4621 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     4681 agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg
     4741 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     4801 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     4861 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     4921 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     4981 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     5041 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     5101 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     5161 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     5221 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     5281 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     5341 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     5401 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     5461 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     5521 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
     5581 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
     5641 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
     5701 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
     5761 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
     5821 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
     5881 atcgagatct cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata
     5941 acaattcccc tctagaaata attttgttta actttaagaa ggagatatac atatgaactt
     6001 cccccgtgcc tcgcgcctta tgcaggctgc tgtgctgggc ggccttatgg ccgtttccgc
     6061 agcggccacc gcgcagacca atccgtatgc gcgcggcccc aaccctaccg ccgcctcgtt
     6121 ggaagccagc gcgggaccct ttaccgttcg tagctttacc gttagccgtc cgtccggata
     6181 tggtgcaggg accgtctatt acccaaccaa tgcaggcggc accgttggcg cga
//
