LOCUS       sequence-349191-        6140 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 6140)
  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 6140)
  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: pEE045
FEATURES             Location/Qualifiers
     source          1..6140
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        133..237
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(238..306,307..1098)
                     /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(456..475)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1269..1857
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1758..1777
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2011..2028
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2043..2182
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2108..2127)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2268..2290
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2284..2475)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3284..4366)
                     /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     4328..4347
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4367..4444)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4650..4669
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4753..4772
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4753..4771
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4772..4796
                     /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             4811..4833
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5624..5641
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5694..5712)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5708..5755
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      join(5792..6140,1..107)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5879..5898)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6089..6110
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
       61 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
      121 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
      181 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
      241 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
      301 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
      361 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
      421 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
      481 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
      541 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
      601 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
      661 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
      721 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
      781 gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
      841 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
      901 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
      961 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     1021 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     1081 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     1141 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     1201 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     1261 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     1321 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     1381 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     1441 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     1501 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     1561 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     1621 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     1681 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     1741 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     1801 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     1861 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     1921 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     1981 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     2041 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     2101 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     2161 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     2221 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     2281 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     2341 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     2401 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     2461 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     2521 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     2581 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     2641 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     2701 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     2761 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     2821 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     2881 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     2941 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     3001 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     3061 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     3121 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     3181 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     3241 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     3301 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     3361 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     3421 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     3481 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     3541 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
     3601 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     3661 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     3721 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     3781 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     3841 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     3901 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     3961 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     4021 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     4081 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     4141 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     4201 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     4261 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     4321 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     4381 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     4441 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     4501 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
     4561 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
     4621 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
     4681 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg
     4741 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctc
     4801 tagaaataat tttgtttaac tttaagaagg agatatacat atgtccaacc cataccaacg
     4861 tggtccgaac ccgacccgtt ctgccttgac cgccgacggt cctttctcag ttgctaccta
     4921 tactgttagc cgtttatccg tatctggttt cggtggcggc gttatttact atccgactgg
     4981 tacctccctg accttcggcg gcatcgcgat gagcccgggt tacaccgccg atgcttccag
     5041 cctggcgtgg ctgggtcgcc gtctggcttc ccacggcttt gtagttctgg tcattaacac
     5101 caactcacgt ttcgactacc cggactctcg tgcgtctcag ctgtccgccg ctctgaacta
     5161 tctgcgtacg tcatctcctt ctgcagttcg cgctcgtctg gatgctaatc gtctggctgt
     5221 agccggccac agcatgggtg gtggtggtac gctgcgcatc gccgaacaga acccgtctct
     5281 gaaagctgcg gttccgttga ctccgtggca taccgataaa acttttaaca cttccgtgcc
     5341 ggttctcatt gtaggtgccg aagcggatac tgtcgcacca gtctcccagc acgcgatccc
     5401 gttctaccag aacctgccat ccactacccc taaagtgtat gtagaactgg ataacgcatc
     5461 tcactttgcg cctaactcta acaacgcggc tatcagcgtg tacaccatct cgtggatgaa
     5521 actctgggtt gataacgaca ctcgttaccg ccagttcctg tgtaacgtta acgatccagc
     5581 cctgtcagat tttcgtacga acaaccgaca ctgtcaactc gagcaccacc accaccacca
     5641 ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
     5701 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     5761 aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     5821 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     5881 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     5941 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     6001 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     6061 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     6121 aaccctatct cggtctattc
//
