LOCUS       sequence-349638-        5777 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 5777)
  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 5777)
  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: pEE009
FEATURES             Location/Qualifiers
     source          1..5777
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1..19)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      15..62
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      99..554
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(186..205)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     396..417
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        580..684
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(685..753,754..1545)
                     /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(903..922)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1716..2304
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2205..2224
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2458..2475
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2490..2629
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2555..2574)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2715..2737
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2731..2922)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3731..4813)
                     /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     4775..4794
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4814..4891)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5097..5116
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5200..5219
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5200..5218
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5219..5243
                     /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             5258..5280
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5708..5725
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
       61 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
      121 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
      181 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
      241 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
      301 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
      361 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
      421 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
      481 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
      541 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
      601 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
      661 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
      721 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
      781 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
      841 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
      901 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
      961 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     1021 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     1081 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     1141 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     1201 aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta
     1261 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     1321 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     1381 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     1441 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     1501 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     1561 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     1621 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     1681 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     1741 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     1801 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     1861 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     1921 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     1981 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     2041 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     2101 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     2161 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     2221 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     2281 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     2341 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     2401 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     2461 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     2521 tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     2581 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     2641 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     2701 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     2761 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     2821 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     2881 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     2941 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     3001 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     3061 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     3121 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     3181 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     3241 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     3301 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     3361 cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt
     3421 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
     3481 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc
     3541 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
     3601 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     3661 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     3721 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     3781 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     3841 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     3901 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     3961 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     4021 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     4081 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     4141 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     4201 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     4261 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     4321 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     4381 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     4441 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     4501 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     4561 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     4621 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     4681 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     4741 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     4801 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     4861 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     4921 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     4981 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     5041 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     5101 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     5161 cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg
     5221 aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga
     5281 tatacatatg ctggttcgca tcggtgaaca ggaagacgcg gttgctgcgc tggaattcct
     5341 gctgcagcgc gacgaaatcg acaccgagcg tattgctctg gctggttact ctttcggtgc
     5401 gttcgttggt ctggcagcac tgaatggcaa cgaaaacatt aaagcactgg tgggtgtttc
     5461 accgcctctg actctgttcg aattcagcta cctcaaaaat tgcacgaaac caaaactgct
     5521 gatcatcggt gacatggacc aattcactcc actgaaggtt ttcaaagaat tctacgagaa
     5581 gatcccggaa ccgaaaaaca aacgcatcat tgaaggtgcg gatcacttct attggggtta
     5641 cgagaacgaa gttggtcagg tagttgctga ttttctgaaa aaaaccttca aaaacatccc
     5701 gctcgagcac caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc
     5761 tgagttggct gctgcca
//
