LOCUS       sequence-349683-        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: pEE065
FEATURES             Location/Qualifiers
     source          1..6149
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             881..898
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(951..969)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      965..1012
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1049..1504
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1136..1155)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1346..1367
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1530..1634
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1635..1703,1704..2495)
                     /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(1853..1872)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2666..3254
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3155..3174
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3408..3425
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3440..3579
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3505..3524)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3665..3687
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3681..3872)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4681..5763)
                     /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     5725..5744
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5764..5841)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6047..6066
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat ggcgaaccct tatgaacgtg gcccgaaccc
      121 gaccgatgcg ctgctggaag ctcgatctgg tccattctct gtgtctgaag aaaacgtgtc
      181 ccgcctgggc gcgagcggtt ttggcggtgg gactatctat tatccgcgcg aaaacaacac
      241 ctacggagct gttgcgatct ctccgggtta tacgggcacc caggcatccg tggcgtggct
      301 tggcaaacgt atcgcaagtc acgggttcgt agtcatcacc atcgacacca tcaccacgct
      361 ggaccagccg gattcgcgtg cgcgtcagct taacgcggca ctggactata tgatcaatga
      421 cgcgtcctcc gcagttcgca gccgcatcga cagctcccgc ctggcggtca tgggccatag
      481 catgggcggt ggtggctctc tgcgtctggc gtctcagcgt ccggacctga aagcggccat
      541 cccgctgacc ccgtggcatc tcaacaaaaa ctggtcttct gtgcgtgtgc cgactctgat
      601 catcggggct gatctggata ccatcgcgcc agtactgacc cacgcccgtc cattctacaa
      661 cagcctcccg acgagcatca gcaaggctta cctggaactg gacggtgcta cccatttcgc
      721 accgaacatc ccgaacaaaa ttatcggtaa gtactctgtt gcttggttga aacgctttgt
      781 tgacaacgat acccgttaca cccagttcct gtgcccgggc cctcgtgacg gcctctttgg
      841 tgaagttgaa gaatatcgtt ctacctgtcc attcctcgag caccaccacc accaccactg
      901 agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
      961 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     1021 aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     1081 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     1141 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     1201 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     1261 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     1321 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     1381 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     1441 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     1501 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     1561 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     1621 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     1681 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     1741 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     1801 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     1861 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     1921 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     1981 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     2041 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     2101 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     2161 gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac
     2221 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     2281 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     2341 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     2401 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     2461 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     2521 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     2581 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     2641 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2701 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2761 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     2821 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     2881 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     2941 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     3001 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     3061 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     3121 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     3181 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3241 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3301 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     3361 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     3421 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     3481 cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     3541 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
     3601 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     3661 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     3721 ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc
     3781 gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc
     3841 atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg
     3901 ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat
     3961 gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg
     4021 cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt
     4081 gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc
     4141 gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt
     4201 gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat
     4261 tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc
     4321 acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa
     4381 cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc
     4441 gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc
     4501 cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg
     4561 gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     4621 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     4681 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     4741 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     4801 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     4861 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     4921 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag
     4981 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     5041 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     5101 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     5161 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     5221 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
     5281 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     5341 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     5401 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     5461 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     5521 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
     5581 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     5641 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     5701 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     5761 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     5821 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta
     5881 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
     5941 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     6001 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
     6061 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
     6121 agaggatcga gatctcgatc ccgcgaaat
//
