LOCUS       sequence-349198-        5726 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 5726)
  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 5726)
  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: pEE074
FEATURES             Location/Qualifiers
     source          1..5726
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     582..601
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(621..698)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     904..923
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1007..1026
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1007..1025
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1026..1050
                     /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             1065..1087
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1464..1481
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1534..1552)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1548..1595
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1632..2087
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1719..1738)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1929..1950
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2113..2217
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2218..2286,2287..3078)
                     /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(2436..2455)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3249..3837
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3738..3757
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3991..4008
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4023..4162
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4088..4107)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4248..4270
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4264..4455)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5264..5726,1..620))
                     /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 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
       61 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
      121 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
      181 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
      241 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
      301 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
      361 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
      421 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
      481 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
      541 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
      601 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
      661 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
      721 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
      781 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
      841 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
      901 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
      961 cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact
     1021 ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag
     1081 aaggagatat acatatggct aacccgtacg aacgtggtcc gaacccgacc aactcttcga
     1141 tcgaagcgct gcgtggtccg ttccgtgtcg atgaagaacg tgtttcccgt ctgcaggcgc
     1201 gtggttttgg cggtggcact atctactatc cgactgataa caacaccttc ggcgctgtgg
     1261 ctattagccc gggttatact ggcacccagt ccagcatctc ctggctgggt gaacgtctgg
     1321 cgtcacacgg ttttgtggtg atgaccatcg ataccaacac tactctggat cagccggact
     1381 ctcgcgcgtc tcagctggac gcagcgctgg attacatggt tgaggattct tcgtactccg
     1441 tacgtaaccg catcgatctc gagcaccacc accaccacca ctgagatccg gctgctaaca
     1501 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     1561 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     1621 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     1681 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     1741 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     1801 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     1861 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     1921 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     1981 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     2041 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     2101 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     2161 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     2221 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     2281 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     2341 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     2401 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     2461 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     2521 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     2581 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     2641 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     2701 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     2761 gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     2821 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     2881 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     2941 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     3001 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     3061 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     3121 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     3181 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3241 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     3301 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     3361 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     3421 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     3481 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     3541 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     3601 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     3661 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     3721 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     3781 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     3841 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     3901 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     3961 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     4021 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     4081 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     4141 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     4201 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4261 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     4321 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     4381 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     4441 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     4501 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     4561 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     4621 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     4681 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     4741 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     4801 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     4861 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     4921 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     4981 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     5041 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     5101 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     5161 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     5221 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     5281 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5341 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     5401 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     5461 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     5521 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
     5581 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     5641 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     5701 gttccgctat cggctgaatt tgattg
//
