LOCUS       sequence-342658-        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"
     CDS             781..798
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(851..869)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      865..912
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      949..1404
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1036..1055)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1246..1267
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1430..1534
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1535..1603,1604..2395)
                     /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(1753..1772)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2566..3154
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3055..3074
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3308..3325
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3340..3479
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3405..3424)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3565..3587
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3581..3772)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4581..5663)
                     /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     5625..5644
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5664..5741)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5947..5966
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6050..6069
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6050..6068
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6069..6093
                     /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             6108..6130
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 tccaacccat accaacgtgg tccgaacccg acccgttctg ccttgaccgc cgacggtcct
       61 ttctcagttg ctacctatac tgttagccgt ttatccgtat ctggtttcgg tggcggcgtt
      121 atttactatc cgactggtac ctccctgacc ttcggcggca tcgcgatgag cccgggttac
      181 accgccgatg cttccagcct ggcgtggctg ggtcgccgtc tggcttccca cggctttgta
      241 gttctggtca ttaacaccaa ctcacgtttc gactacccgg actctcgtgc gtctcagctg
      301 tccgccgctc tgaactatct gcgtacgtca tctccttctg cagttcgcgc tcgtctggat
      361 gctaatcgtc tggctgtagc cggccacagc atgggtggtg gtggtacgct gcgcatcgcc
      421 gaacagaacc cgtctctgaa agctgcggtt ccgttgactc cgtggcatac cgataaaact
      481 tttaacactt ccgtgccggt tctcattgta ggtgccgaag cggatactgt cgcaccagtc
      541 tcccagcacg cgatcccgtt ctaccagaac ctgccatcca ctacccctaa agtgtatgta
      601 gaactggata acgcatctca ctttgcgcct aactctaaca acgcggctat cagcgtgtac
      661 accatctcgt ggatgaaact ctgggttgat aacgacactc gttaccgcca gttcctgtgt
      721 aacgttaacg atccagccct gtcagatttt cgtacgaaca accgacactg tcaactcgag
      781 caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg
      841 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
      901 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
      961 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     1021 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     1081 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     1141 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     1201 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     1261 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     1321 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     1381 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     1441 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     1501 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     1561 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     1621 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     1681 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     1741 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     1801 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     1861 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     1921 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     1981 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     2041 agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg
     2101 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     2161 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     2221 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     2281 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     2341 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     2401 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     2461 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     2521 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     2581 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2641 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     2701 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     2761 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     2821 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     2881 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     2941 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3001 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3061 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3121 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3181 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3241 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3301 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     3361 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     3421 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     3481 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     3541 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     3601 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     3661 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     3721 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     3781 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     3841 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     3901 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     3961 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     4021 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     4081 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     4141 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     4201 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa
     4261 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     4321 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     4381 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     4441 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     4501 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     4561 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4621 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     4681 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     4741 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     4801 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     4861 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     4921 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     4981 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     5041 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     5101 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     5161 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     5221 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     5281 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     5341 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     5401 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     5461 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     5521 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     5581 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     5641 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     5701 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     5761 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     5821 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     5881 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     5941 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     6001 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc
     6061 actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt
     6121 aagaaggaga tatacatatg
//
