LOCUS       sequence-349197-        6188 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 6188)
  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 6188)
  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: pEE036
FEATURES             Location/Qualifiers
     source          1..6188
                     /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             901..918
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(971..989)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      985..1032
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1069..1524
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1156..1175)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1366..1387
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1550..1654
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1655..1723,1724..2515)
                     /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(1873..1892)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2686..3274
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3175..3194
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3428..3445
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3460..3599
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3525..3544)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3685..3707
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3701..3892)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4701..5783)
                     /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     5745..5764
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5784..5861)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6067..6086
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(6170..6188,1)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6170..6188
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
       61 gatatacata tgcaggcaga taccgatacc actgcagtgg ctcccgcggc ggctaatccg
      121 tatgaacgcg gcccggctcc gactgaagcg tctgtaactg cagctcgcgg tccgtttgct
      181 attgcccagg tgaacgtacc gtctggcagc ggtgctggct tcaacgatgg caccatctac
      241 tatccgactg atacctctca gggtaccttt ggtgcggtcg cggtaatccc gggtttcatc
      301 tcccctcagg ctgtgatcca gtggttcggt ccgcgcttgg catctcaggg cttcgtagtc
      361 ttcactctgg attctaacgg tctggccgat ctgccggatg cgcgcggtcg tcagctgctg
      421 gcggctctgg actacctgac cacccagtct actgtgcgta cccgtattga tccgaatcgc
      481 ctggctgtca tggggcacag catgggtggc ggtggcacgc tgctggcggc ggaaaaccgt
      541 ccaaccctga aagcggccat cccactggcg ccgtgggaac cggatactag ttgggaaggc
      601 gtgaaagtac cgactatgat catcggcggc gaaagcgatg tcgttgctcc ggtttccagt
      661 atggctattc cggactataa ctccctgagc tctgctccag aaaaggctta tctggagttg
      721 cgttctggtg atcacctggc accggcaagc gaatctccta ccgttgcgga atacgcttta
      781 agctggctca agcgctttgt tgatgatgac actcgttatg atcagttcct gtgtccgggt
      841 cctacaccgg atactgatat cagccagtac ctggatacgt gtcctaacgg ttctctcgag
      901 caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg
      961 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     1021 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
     1081 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     1141 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     1201 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     1261 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     1321 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     1381 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     1441 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     1501 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     1561 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     1621 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     1681 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     1741 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     1801 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     1861 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     1921 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     1981 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     2041 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     2101 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     2161 agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg
     2221 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     2281 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     2341 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     2401 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     2461 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     2521 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     2581 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     2641 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     2701 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2761 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     2821 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     2881 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     2941 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3001 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3061 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3121 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3181 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3241 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3301 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3361 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3421 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     3481 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     3541 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     3601 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     3661 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     3721 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     3781 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     3841 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     3901 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     3961 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     4021 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     4081 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     4141 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     4201 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     4261 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     4321 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa
     4381 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     4441 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     4501 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     4561 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     4621 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     4681 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4741 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     4801 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     4861 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     4921 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     4981 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     5041 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     5101 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     5161 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     5221 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     5281 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     5341 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     5401 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     5461 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     5521 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     5581 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     5641 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     5701 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     5761 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     5821 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     5881 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     5941 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     6001 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     6061 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     6121 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc
     6181 actatagg
//
