LOCUS       sequence-349238-        5957 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 5957)
  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 5957)
  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: pEE011
FEATURES             Location/Qualifiers
     source          1..5957
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1..23
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             631..648
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(701..719)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      715..762
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      799..1254
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(886..905)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1096..1117
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1280..1384
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1385..1453,1454..2245)
                     /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(1603..1622)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2416..3004
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2905..2924
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3158..3175
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3190..3329
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3255..3274)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3415..3437
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3431..3622)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4431..5513)
                     /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     5475..5494
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5514..5591)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5797..5816
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5900..5919
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5900..5918
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5919..5943
                     /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)."
ORIGIN
        1 tttgtttaac tttaagaagg agatatacat atggttccgc tgatcctgaa cgtgcatggt
       61 ggtccggcag gcgttttcca gcagaccttt accggtggtc gcagcatcta tccgatcgct
      121 accttcgcag cgcgcggtta cgctgttctg cgtccgaacc cgcgcggttc ctctggttat
      181 ggtgtggaat tccgccgtgc caacctcaaa gattggggtg gcatggacta ccaggacctc
      241 atggctggtg ttgacaaagt aatcgaaatg ggtgtggcgg actcctcccg tctgggtgtt
      301 atgggttggt cctatggtgg ttttatgacg tcttggatcg taactcagac aaaccgtttc
      361 aaagcggctt cagcaggcgc tccggtcacc aacctgacgt cattcactac taccgccgac
      421 atcccggcgt tcatcccgga ctatttcggc ggccagttct gggactctcc ggaagtgtac
      481 cgtgcacact ccccaatttc gttcgtgaaa tctgtcacca ctcctactat gatccagcac
      541 ggtaccgcag acatgcgtgt tccgatcagc cagggtttcg aattctacaa cgctctcaaa
      601 gctcgtggca tcccgacccg catgctcgag caccaccacc accaccactg agatccggct
      661 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
      721 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
      781 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
      841 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
      901 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
      961 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     1021 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     1081 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     1141 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     1201 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     1261 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     1321 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     1381 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     1441 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     1501 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     1561 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     1621 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     1681 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     1741 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     1801 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     1861 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     1921 gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     1981 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     2041 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     2101 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     2161 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     2221 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     2281 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     2341 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     2401 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     2461 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     2521 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     2581 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     2641 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     2701 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     2761 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     2821 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     2881 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     2941 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     3001 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     3061 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     3121 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     3181 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa
     3241 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     3301 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     3361 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     3421 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     3481 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     3541 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     3601 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
     3661 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
     3721 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga
     3781 gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg
     3841 gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc
     3901 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg
     3961 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct
     4021 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc
     4081 gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg
     4141 cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca
     4201 ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg
     4261 ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag
     4321 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     4381 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     4441 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4501 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
     4561 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
     4621 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
     4681 tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg
     4741 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
     4801 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
     4861 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
     4921 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
     4981 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
     5041 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
     5101 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
     5161 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
     5221 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
     5281 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
     5341 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
     5401 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
     5461 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     5521 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     5581 tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc
     5641 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat
     5701 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct
     5761 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc
     5821 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga
     5881 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
     5941 tcccctctag aaataat
//
