LOCUS       sequence-342644-        6542 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 6542)
  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 6542)
  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: pEE038
FEATURES             Location/Qualifiers
     source          1..6542
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1183..1200
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1253..1271)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1267..1314
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1351..1806
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1438..1457)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1648..1669
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1832..1936
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1937..2005,2006..2797)
                     /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(2155..2174)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2968..3556
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3457..3476
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3710..3727
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3742..3881
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3807..3826)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3967..3989
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3983..4174)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4983..6065)
                     /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     6027..6046
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6066..6143)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6349..6368
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6452..6471
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6452..6470
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6471..6495
                     /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             6510..6532
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 gcggataacc cgtatcagcg tggcccggat ccgactcgcg attctgtcgc cgcatctcgt
       61 ggcaccttcg ctacggcctc caccaccgta ggctctggca atggttttgg tgctggcttc
      121 atctactacc cgactgacac gtcccagggt acatttggcg ccgtcgcaat cgtgccgggt
      181 tacactgcaa cctgggcagc agaaggcgct tggatgggtc actggctcgc gagcttcggt
      241 tttgtcgtca tcggcatcga taccatcaac cgcaacgact gggacactgc gcgtggtacc
      301 cagctgcttg ccgcgcttga ctacttgact cagcgttcaa ccgttcgtga tcgtgtggat
      361 gcttcccgtc ttgcggttat gggccactcc atgggcggcg gtggtgcaat gtacgccgca
      421 ctgcagcgcc cgagtctgaa agctgctgtg ggtctggcac cgttctcccc gtcacagaac
      481 ttgaacggta tgcgtgtacc gacgatgctg ctggccggac aacacgacac cacgaccacg
      541 ccggcgtcca tcaccagcct gtacaacggc attccggcgg caactgaaaa agcatacctg
      601 gaactgagcg gtgcgggcca cggcttcccg accagcaaca attctgttat gatgcgtaaa
      661 gtaattccgt ggctgaaaat ctttgtagat tcagacgttc gttatacgca gtttctgtgt
      721 ccgctgatgg ataacactgg catccgtagc taccagtcta cctgtcctct gctgcccggt
      781 accccgactc cgccgaaccg ttacgaagcc gagacttcgc cggccgtttg tactggtact
      841 attgctagca accacactgg ttattccggt actggttttt gtgacggtaa caacgctacc
      901 aacgcttacg cccagtttac cgttaacgcg tctgccgctg gttcaatgac cctgcgtgtg
      961 cgtttcgcga acggtaccac caccgctcgc cccgcgagcc tgattgtgaa cggcagcact
     1021 gtccagaccc cgtcctttga aggcactggc gcgtggacca cctgggcaac caaaacactg
     1081 accgtgaccc tgaacgccgg taacaacact atccgtttca acccgaccac cgcgaacggc
     1141 ctgccgaacc ttgattacat cgaaattgcc gctccgctcg agcaccacca ccaccaccac
     1201 tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
     1261 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     1321 ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
     1381 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     1441 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     1501 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     1561 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     1621 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     1681 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     1741 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     1801 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     1861 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     1921 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     1981 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     2041 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     2101 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     2161 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     2221 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     2281 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     2341 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     2401 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     2461 gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga
     2521 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     2581 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     2641 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     2701 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     2761 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     2821 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     2881 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     2941 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     3001 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     3061 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     3121 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     3181 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     3241 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     3301 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     3361 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     3421 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     3481 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     3541 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     3601 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     3661 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     3721 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     3781 ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     3841 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     3901 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     3961 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     4021 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
     4081 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     4141 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     4201 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     4261 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     4321 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     4381 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     4441 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     4501 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     4561 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     4621 gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga
     4681 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
     4741 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
     4801 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     4861 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     4921 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     4981 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     5041 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     5101 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     5161 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     5221 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     5281 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     5341 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     5401 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     5461 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     5521 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     5581 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     5641 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     5701 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     5761 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     5821 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     5881 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     5941 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     6001 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     6061 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     6121 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat
     6181 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc
     6241 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
     6301 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
     6361 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc
     6421 gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga
     6481 gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga gatatacata
     6541 tg
//
