LOCUS       sequence-349679-        6149 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 6149)
  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 6149)
  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: pEE064
FEATURES             Location/Qualifiers
     source          1..6149
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     956..975
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(995..1072)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1278..1297
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1381..1400
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1381..1399
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1400..1424
                     /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             1439..1461
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2261..2278
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2331..2349)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2345..2392
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2429..2884
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2516..2535)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2726..2747
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2910..3014
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3015..3083,3084..3875)
                     /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(3233..3252)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4046..4634
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4535..4554
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4788..4805
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4820..4959
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4885..4904)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5045..5067
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5061..5252)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6061..6149,1..994))
                     /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"
ORIGIN
        1 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
       61 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
      121 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
      181 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
      241 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
      301 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
      361 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
      421 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
      481 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
      541 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
      601 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
      661 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
      721 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
      781 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
      841 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
      901 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
      961 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     1021 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     1081 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     1141 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     1201 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     1261 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     1321 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
     1381 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
     1441 tgtttaactt taagaaggag atatacatat ggccaacccg tacgaacgcg gtccaaaccc
     1501 gaccgacgcg cttcttgagg cccgtagcgg tccattcagc gtaagcgaag aaaacgtgtc
     1561 ccgcctgagc gcctctggtt ttggtggtgg caccatctac tatccgcgcg aaaacaacac
     1621 atacggtgcg gtcgctatct ccccaggtta taccggtacc gaagcatcca tcgcatggct
     1681 tggtgaacgc attgcaagcc atggctttgt cgtcatcacg attgatacga tcaccactct
     1741 ggaccagccg gattcccgcg cggaacagct gaacgcggct ctcaatcaca tgatcaaccg
     1801 tgcgtcctct accgtacgtt cgcgtatcga cagctcgcgc ctggctgtta tgggccatag
     1861 catgggtggc ggcggttcgc ttcgtctggc ttcgcagcgt ccggacttga aggccgcaat
     1921 cccactgacc ccgtggcacc tgaataaaaa ttggagctcc gttcgtgtgc cgaccctgat
     1981 catcggtgcg gatctggaca ccatcgcgcc ggttctgact cacgcgcgcc cattctacaa
     2041 ctctctgccg acctctatct ccaaagcata ccttgaactg gacggcgcga cccacttcgc
     2101 tccgaacatt cctaacaaaa tcatcggcaa gtatagcgta gcctggctga aacgcttcgt
     2161 ggacaacgat acccgctaca cccagttcct gtgcccgggt ccgcgcgacg gcctgttcgg
     2221 cgaagtagaa gaatatcgct ctacctgccc tttcctcgag caccaccacc accaccactg
     2281 agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     2341 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     2401 aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     2461 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     2521 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     2581 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     2641 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     2701 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     2761 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     2821 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     2881 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     2941 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     3001 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     3061 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     3121 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     3181 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     3241 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     3301 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     3361 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     3421 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     3481 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     3541 gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac
     3601 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     3661 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     3721 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     3781 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     3841 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     3901 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     3961 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     4021 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     4081 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     4141 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     4201 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     4261 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     4321 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     4381 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     4441 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     4501 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     4561 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     4621 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     4681 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     4741 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     4801 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     4861 cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     4921 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
     4981 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     5041 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     5101 ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc
     5161 gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc
     5221 atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg
     5281 ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat
     5341 gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg
     5401 cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt
     5461 gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc
     5521 gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt
     5581 gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat
     5641 tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc
     5701 acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa
     5761 cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc
     5821 gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc
     5881 cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg
     5941 gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     6001 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     6061 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     6121 cgcgcgggga gaggcggttt gcgtattgg
//
