LOCUS       sequence-349692-        5891 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 5891)
  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 5891)
  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: pEE061
FEATURES             Location/Qualifiers
     source          1..5891
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     937..956
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(976..1053)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1259..1278
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1362..1381
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1362..1380
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1381..1405
                     /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             1420..1442
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1984..2001
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2054..2072)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2068..2115
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2152..2607
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2239..2258)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2449..2470
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2633..2737
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2738..2806,2807..3598)
                     /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(2956..2975)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3769..4357
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4258..4277
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4511..4528
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4543..4682
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4608..4627)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4768..4790
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4784..4975)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5784..5891,1..975))
                     /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"
ORIGIN
        1 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
       61 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
      121 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
      181 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
      241 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
      301 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
      361 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
      421 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
      481 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
      541 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
      601 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
      661 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
      721 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
      781 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
      841 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
      901 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
      961 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     1021 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     1081 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     1141 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     1201 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     1261 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
     1321 tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg
     1381 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
     1441 gatatacata tgccgggttt tctgggttcc tcctccaact acgcgtggat gggtccgcgt
     1501 ctggcttctc aaggtttcat tgtgtttctg atcaacacca atactaggct cgataccccg
     1561 ccgcagcgcg gtgatcagct gctggcagcg ctggattggc tggttgcttc ttctccgtct
     1621 gcggtacgta ctcgtctaga tgcgcgtcgc ttggcggttg cgggccattc tatgggtggc
     1681 ggcggcgcgc tggaagctag cctggatcgc ccgtctctgc aagcaagcct gccgctgcag
     1741 ccgtggcaca ccccggcttc tttcagcggc gtacaggttc cgactatgat cattggtgct
     1801 gaggccgata ctaccgcgcc cgtagcatcg catgctgaac cgttttacga atctctgacc
     1861 tcggcttctg accgtgccta cctggaactc aacggtgcag accaccgcgt gtcaaccacc
     1921 tctagtacca cccaggcgaa attcatgatc gcctggctga aacgtttcgt ggataacctc
     1981 gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag
     2041 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     2101 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
     2161 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     2221 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     2281 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     2341 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     2401 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     2461 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     2521 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     2581 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
     2641 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     2701 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     2761 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     2821 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     2881 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     2941 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     3001 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     3061 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     3121 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     3181 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     3241 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt
     3301 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     3361 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     3421 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     3481 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     3541 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     3601 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     3661 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     3721 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     3781 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     3841 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     3901 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     3961 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4021 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4081 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4141 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4201 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4261 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4321 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4381 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     4441 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     4501 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     4561 ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct
     4621 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     4681 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     4741 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     4801 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     4861 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     4921 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     4981 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     5041 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     5101 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     5161 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     5221 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     5281 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     5341 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     5401 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga
     5461 taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga
     5521 gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa
     5581 agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga
     5641 taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc
     5701 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     5761 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     5821 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     5881 gtggtttttc t
//
