LOCUS       sequence-349211-        6008 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 6008)
  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 6008)
  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: pEE015
FEATURES             Location/Qualifiers
     source          1..6008
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..25
                     /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             40..62
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             721..738
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(791..809)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      805..852
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      889..1344
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(976..995)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1186..1207
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1370..1474
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1475..1543,1544..2335)
                     /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(1693..1712)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2506..3094
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2995..3014
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3248..3265
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3280..3419
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3345..3364)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3505..3527
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3521..3712)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4521..5603)
                     /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     5565..5584
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5604..5681)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5887..5906
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(5990..6008,1)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5990..6008
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
       61 gatatacata tgggctttac cggcaacaaa gccgaggctg gtcgtctgta caccgacatg
      121 gctcgtgtcc tgtgtgcggc tggctacgct gcgctgcgct tcgacttccg ttgtcatggc
      181 gactccccgc tgccgtttga agaattccgc atttcgtacg cggtagaaga cgcacgtaac
      241 gcggcttctt tcctgaagat tcaaccgtcc gtagacggtt ctcgtttcgc cgtaatcggc
      301 ctgtcgatgg gtggtggtgt agccgtgagt ctggcagcgg gtcgtgatga tgtagctgcc
      361 ctcgtgctcc tgtctccggc tctggattgg ccggaactgg cggcgcgcat tccgcagccg
      421 aaggtggaag gcggctacgt gtacatgggt ccaaaccgca tgaaggtgga gtgtgttacg
      481 gaaaccatga aattcactgt catggacctg gcagaacgtg ttaaagctcc cactctgatc
      541 attcatgccg ctgatgatat ggttgttccg attagccaat ccaaacgctt ctatgaaaaa
      601 ctgaaagtag agaaaaagtt tatggagatc gaacgtagcg gtcatgtttt cgatgattac
      661 aacgtacgcc gccgtgtcga agcggaagtt cttgattgga tcaaaaaaca cctgctcgag
      721 caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg
      781 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
      841 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
      901 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
      961 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     1021 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     1081 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     1141 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     1201 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     1261 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     1321 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     1381 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     1441 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     1501 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     1561 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     1621 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     1681 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     1741 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     1801 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     1861 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     1921 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     1981 agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg
     2041 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     2101 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     2161 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     2221 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     2281 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     2341 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     2401 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     2461 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     2521 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2581 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     2641 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     2701 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     2761 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     2821 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     2881 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     2941 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3001 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3061 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3121 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3181 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3241 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     3301 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     3361 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     3421 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     3481 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     3541 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     3601 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     3661 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     3721 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     3781 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     3841 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     3901 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     3961 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     4021 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     4081 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     4141 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa
     4201 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     4261 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     4321 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     4381 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     4441 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     4501 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4561 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     4621 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     4681 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     4741 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     4801 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     4861 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     4921 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     4981 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     5041 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     5101 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     5161 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     5221 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     5281 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     5341 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     5401 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     5461 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     5521 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     5581 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     5641 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     5701 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     5761 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     5821 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     5881 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     5941 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc
     6001 actatagg
//
