LOCUS       sequence-349628-        6143 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes codon optimized putative thermophilic PET hydrolase enzyme 
            for bacterial expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6143)
  AUTHORS   Erickson E, Gado JE, Avilan L, Bratti F, Brizendine RK, Cox PA, Gill
            R, Graham R, Kim DJ, Konig G, Michener WE, Poudel S, Ramirez KJ, 
            Shakespeare TJ, Zahn M, Boyd ES, Payne CM, DuBois JL, Pickford AR, 
            Beckham GT, McGeehan JE
  TITLE     Sourcing thermotolerant poly(ethylene terephthalate) hydrolase 
            scaffolds from natural diversity.
  JOURNAL   Nat Commun. 2022 Dec 21;13(1):7850. doi: 10.1038/s41467-022-35237-x.
  PUBMED    36543766
REFERENCE   2  (bases 1 to 6143)
  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"; journalName: "Nat 
            Commun."; date: "2022-12-21"; volume: "13(1):7850. doi"; pages: " 
            10.1038/s41467-022-35237-x"
COMMENT     Sequence Label: pEE053
FEATURES             Location/Qualifiers
     source          1..6143
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             15..37
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             831..848
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(901..919)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      915..962
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      999..1454
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1086..1105)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1296..1317
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1480..1584
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1585..1653,1654..2445)
                     /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(1803..1822)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2616..3204
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3105..3124
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3358..3375
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3390..3529
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3455..3474)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3615..3637
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3631..3822)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4631..5713)
                     /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     5675..5694
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5714..5791)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5997..6016
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6100..6119
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6100..6118
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6119..6143
                     /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)."
ORIGIN
        1 cctctagaaa taattttgtt taactttaag aaggagatat acatatggct tccccgtacg
       61 aacgcgggcc ggccccaacc tctgcgattt tggaagcaag ccgtggccct ttcgcgactt
      121 cgtccatcaa cgtttcttct ctgtcagtta ccggcttcgg tggtggtgta atttattacc
      181 cgaccagcac cgctgaaggc accttcggcg ccgtcgcgat tagcccgggt tatactgctt
      241 cttggagtag cctgtcttgg ctgggtccac gtatcgcatc tcatggcttc gtggttatcg
      301 gtatcgagac caacacgcgc ttggatcagc ctgcttcccg cggccgtcag ctgctggctg
      361 ctctggacta cctgaccgaa cgttcctccg tacgtggccg catcgattcg tctcgtctgg
      421 ctgtagcggg tcactcgatg ggtggtggcg gttccttgga ggctgcagca gcccgcccta
      481 gtctgcaagc agccgtaccg ctggcgccgt ggaatcttga taaaacctgg tctgacgtcc
      541 gtgttccgac tctgatcatc ggtggtgaaa ccgattccgt agcaccggtt gcaacccatt
      601 ctattccttt ttacaactcg atcccggcat ctagcgaaaa agcttacctt gagcttgatg
      661 gcgcatccca ctttttcccg cagaccacta acaccccgac cgcaaagcag atggtagcgt
      721 ggctgaaacg tttcgtagat gacgacactc gttatgaaca gttcctgtgc ccgggtcctt
      781 ccggctccgc gatccaggag taccgtaaca cctgtccatc tgcactcgag caccaccacc
      841 accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
      901 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
      961 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
     1021 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     1081 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     1141 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     1201 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     1261 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     1321 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     1381 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     1441 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     1501 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     1561 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     1621 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     1681 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     1741 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     1801 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     1861 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     1921 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     1981 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     2041 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     2101 aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta
     2161 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     2221 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     2281 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     2341 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     2401 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     2461 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     2521 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     2581 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     2641 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     2701 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     2761 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     2821 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     2881 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     2941 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     3001 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     3061 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     3121 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     3181 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     3241 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     3301 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     3361 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     3421 tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     3481 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     3541 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     3601 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     3661 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     3721 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     3781 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     3841 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     3901 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     3961 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     4021 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     4081 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     4141 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     4201 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     4261 cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt
     4321 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
     4381 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc
     4441 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
     4501 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     4561 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     4621 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4681 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     4741 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     4801 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     4861 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     4921 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     4981 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     5041 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     5101 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     5161 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     5221 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     5281 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     5341 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     5401 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     5461 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     5521 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     5581 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     5641 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     5701 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     5761 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     5821 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     5881 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     5941 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     6001 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     6061 cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg
     6121 aattgtgagc ggataacaat tcc
//
