LOCUS       sequence-349672-        6173 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 6173)
  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 6173)
  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: pEE042
FEATURES             Location/Qualifiers
     source          1..6173
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             824..841
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(894..912)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      908..955
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      992..1447
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1079..1098)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1289..1310
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1473..1577
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1578..1646,1647..2438)
                     /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(1796..1815)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2609..3197
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3098..3117
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3351..3368
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3383..3522
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3448..3467)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3608..3630
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3624..3815)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4624..5706)
                     /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     5668..5687
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5707..5784)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5990..6009
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6093..6112
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6093..6111
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6112..6136
                     /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             6151..6173
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 tatacatatg gcggactgcc ctgccggtgc gatctgccgc tatgacgaac agccgggcgg
       61 ctatactggt gacggtccat accgcgtggg tgattactcc atcagcactt tccaagcagc
      121 gggcggtgcg accgtttact atccgaccaa cgcaactccg cctttcgctg ctctggtttt
      181 ctgcccgccg tataccggcg ttcagtacat gtaccgcgac tggggtccat tcttcgcgtc
      241 gcacggcatc gttatggtta ccatggactc cgaaaccacg ctggacaccg ttgatcaacg
      301 tgctgaccag cagcgtgaag ttttggactt tctgaagcgt gaaaatacta actcccgctc
      361 ccctctgtat gggaaattgg ctactgatcg ttttggtgtt acgggttggt ctatgggtgg
      421 cggcgctacc tggatcaact ctgccgatta cagcggtctt aaaactgcga tgtccctggc
      481 cggccataac ctgactgccc tcgatccgga cagccgtggc tattccactc gcatcccgac
      541 tctgatcatg aacggtgcac tggatactac ctacctgggt ggcctgggtc agagcgacgg
      601 cgtctataac gcgatcccgt atggtgtccc gaaagtgttc tacgaagttt cctctgcagg
      661 ccacttcgca tgggggtctc cgacgtctgc gtcggacgat gtggcaaaag tagcgctggc
      721 ttttcagaag actttcctgg aaggtgacac tcgctgggcc gaatacatcc gtcgtccgtt
      781 ctggggcgct tctgagtggg agaccgcaaa tctgccgctc gagcaccacc accaccacca
      841 ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
      901 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
      961 aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     1021 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     1081 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     1141 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     1201 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     1261 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     1321 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     1381 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     1441 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     1501 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     1561 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     1621 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     1681 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     1741 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     1801 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     1861 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     1921 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     1981 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     2041 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     2101 agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     2161 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     2221 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     2281 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     2341 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     2401 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     2461 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     2521 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     2581 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     2641 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     2701 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     2761 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     2821 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     2881 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     2941 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     3001 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     3061 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     3121 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     3181 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     3241 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     3301 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     3361 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     3421 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     3481 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     3541 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     3601 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     3661 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     3721 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     3781 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     3841 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     3901 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     3961 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     4021 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     4081 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     4141 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     4201 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     4261 agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg
     4321 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat
     4381 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg
     4441 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt
     4501 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     4561 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     4621 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4681 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     4741 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     4801 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     4861 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg
     4921 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     4981 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     5041 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     5101 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     5161 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     5221 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     5281 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     5341 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     5401 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     5461 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     5521 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     5581 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     5641 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
     5701 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
     5761 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
     5821 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
     5881 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
     5941 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
     6001 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
     6061 cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
     6121 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg aga
//
