LOCUS       sequence-342620-        6380 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 6380)
  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 6380)
  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: pEE026
FEATURES             Location/Qualifiers
     source          1..6380
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1021..1038
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1091..1109)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1105..1152
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1189..1644
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1276..1295)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1486..1507
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1670..1774
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1775..1843,1844..2635)
                     /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(1993..2012)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2806..3394
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3295..3314
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3548..3565
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3580..3719
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3645..3664)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3805..3827
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3821..4012)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4821..5903)
                     /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     5865..5884
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5904..5981)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6187..6206
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6290..6309
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6290..6308
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6309..6333
                     /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             6348..6370
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atctggtatc cggctgttcg tgtccgtggc caaccgcagc gtaccacgta ccagtacggc
       61 ccactgattg gggaaggacg tgcatatcgc gacgcgccgg cagatctgcg cggcgcccca
      121 tacccgttac tgatcttctc tcacggtctg ggtggtgcgc gtatccagtc cgtgttctac
      181 gcagagcacc tggcaagcca tggtttcgtc gttatggcgg ccgatcacac gggttcgact
      241 tttgctgacc tgctgcgtgg ccgtgcggat agcatcctgg aatcgttcgc ccgccgcccg
      301 ctggaaatcc tgcgtcagat tgaatacgcg gcagcgttaa acgcagacga cgatacgctg
      361 cgtggcgcga tcgatgcaga gaccgtcggc gtaacaggcc actcttttgg cggttacact
      421 gccctcgcgg ccgcaggtgc tcaactgaat atcaatgcga tccgtgaagg ttgtgaaagc
      481 ggcaaactgc cggaacagca gtgtctgttt gtacgctccg aagaaatcat ctggcgtgca
      541 cgtggcctgt ctgccgcccc tgaaggcctg tatccgccga cgactgaccc gcgcatcaaa
      601 gctgttgtgg cactggcgcc gtcgtctgca ccgaccttcg gtgaagcagg tgccgctgct
      661 cttcgtgtac cgctgatgat cattgtaggt tctaaagacc aagccacccc gccggagcgc
      721 gattcctatc cgatctacca gagcgtgtcc tctgctcaga aagcgctggt agtcttcgaa
      781 aacgctggtc attatatctt cgtggaacag tgcgtcccgg ctttgattgc actgggtcgt
      841 ttcgaacagt gctctgacct agtatgggac atgcagcgtg ctcacgatct gattaatcac
      901 ttcgcgactg ccttctttct gcacgccctg aaaggtgatc cggcggccaa agcggcgctg
      961 gacccgactg cggttcagtt catcggcatc acttaccgcc gtgacggcgc ttggctcgag
     1021 caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg
     1081 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     1141 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
     1201 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     1261 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     1321 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     1381 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     1441 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     1501 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     1561 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     1621 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     1681 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     1741 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     1801 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     1861 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     1921 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     1981 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     2041 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     2101 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     2161 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     2221 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     2281 agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg
     2341 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     2401 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     2461 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     2521 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     2581 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     2641 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     2701 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     2761 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     2821 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2881 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     2941 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3001 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3061 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3121 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3181 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3241 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3301 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3361 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3421 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3481 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3541 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     3601 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     3661 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     3721 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     3781 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     3841 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     3901 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     3961 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     4021 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     4081 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     4141 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     4201 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     4261 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     4321 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     4381 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     4441 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa
     4501 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     4561 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     4621 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     4681 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     4741 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     4801 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4861 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     4921 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     4981 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     5041 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     5101 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     5161 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     5221 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     5281 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     5341 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     5401 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     5461 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     5521 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     5581 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     5641 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     5701 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     5761 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     5821 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     5881 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     5941 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     6001 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     6061 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     6121 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     6181 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     6241 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc
     6301 actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt
     6361 aagaaggaga tatacatatg
//
