LOCUS       sequence-349634-        6146 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 6146)
  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 6146)
  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: pEE060
FEATURES             Location/Qualifiers
     source          1..6146
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1304..1495
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1489..1511)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1597..1736
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1652..1671
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1751..1768)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1922..2510)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2002..2021)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2681..3472,3473..3541))
                     /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     3304..3323
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3542..3646)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3672..4127)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3809..3830)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4021..4040
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4164..4211)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4207..4225
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4278..4295)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             5092..5114
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    5129..5153
                     /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)."
     primer_bind     complement(5153..5172)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5154..5172)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5256..5275)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5481..5558
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5559..6146,1..495)
                     /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     complement(5578..5597)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg
       61 gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc
      121 atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc
      181 attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc
      241 gaagacagct catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg
      301 gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat
      361 cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc
      421 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
      481 gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcaccggg
      541 atctcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg
      601 catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt
      661 gccggcagcg ctctgggtca ttttcggcga ggaccgcttt cgctggagcg cgacgatgat
      721 cggcctgtcg cttgcggtat tcggaatctt gcacgccctc gctcaagcct tcgtcactgg
      781 tcccgccacc aaacgtttcg gcgagaagca ggccattatc gccggcatgg cggccccacg
      841 ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta ggctggcggg gttgccttac
      901 tggttagcag aatgaatcac cgatacgcga gcgaacgtga agcgactgct gctgcaaaac
      961 gtctgcgacc tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt aaagtctgga
     1021 aacgcggaag tcagcgccct gcaccattat gttccggatc tgcatcgcag gatgctgctg
     1081 gctaccctgt ggaacaccta catctgtatt aacgaagcgc tggcattgac cctgagtgat
     1141 ttttctctgg tcccgccgca tccataccgc cagttgttta ccctcacaac gttccagtaa
     1201 ccgggcatgt tcatcatcag taacccgtat cgtgagcatc ctctctcgtt tcatcggtat
     1261 cattaccccc atgaacagaa atccccctta cacggaggca tcagtgacca aacaggaaaa
     1321 aaccgccctt aacatggccc gctttatcag aagccagaca ttaacgcttc tggagaaact
     1381 caacgagctg gacgcggatg aacaggcaga catctgtgaa tcgcttcacg accacgctga
     1441 tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     1501 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     1561 tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag
     1621 cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg
     1681 caccattgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct
     1741 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     1801 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     1861 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     1921 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     1981 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     2041 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     2101 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     2161 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     2221 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     2281 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     2341 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     2401 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     2461 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     2521 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     2581 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     2641 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     2701 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     2761 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     2821 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     2881 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     2941 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctgcag
     3001 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     3061 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     3121 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     3181 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     3241 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     3301 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     3361 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     3421 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     3481 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     3541 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     3601 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     3661 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     3721 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     3781 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     3841 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     3901 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     3961 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     4021 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     4081 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccaat
     4141 ccggatatag ttcctccttt cagcaaaaaa cccctcaaga cccgtttaga ggccccaagg
     4201 ggttatgcta gttattgctc agcggtggca gcagccaact cagcttcctt tcgggctttg
     4261 ttagcagccg gatctcagtg gtggtggtgg tggtgctcga ggtacgggca ggtagactgg
     4321 tagtcggaaa tagaatcgtc gttctccggc ggcgggcaca ggaactgatc gtaacgagtg
     4381 tcatcgtcca ggaaacgttt cagccatgcg atggagaatt ttgcgatagt ggtgttgtcg
     4441 gtgttcggag caaggtggga agcacctgcc agttccaggt aagctttacc cggatcatct
     4501 ggcagagatt cgtaaaatgg ttttgcgtgc tggcttacag gtgcaacggt gtcgttctgc
     4561 gcaccaatga tcagggtcgg cgtctgaaca ccggagaagt ctttggtggt gtgccacgga
     4621 gtcagtggga ttgccgcttt cagcgccggg ttatccaggg ctgctttcag ggagccgcca
     4681 ccacccatag agtgacccat cacacccaga cgtgccggat cgatgatatc tttcacgtct
     4741 gagtcgttaa ccagatagtc caggcttgcc tgcagctgac gggcacggct atctggctga
     4801 tcggtggtgg taatggtatc aatggtgaag acaacgaaac cctgagatgc cagacgcggg
     4861 ccgtaccaag ccatagtttc ctgtgtgccg gtgaacccgg gagaaataga caccgcggag
     4921 aacagaccat cggtcgtatc cgtggggtag tagatagtgc cgccaccaaa accggacacg
     4981 ctcaggcggc taacggattc ttcgtcgacc tcgaacgggc cgcgcggcgc ggtgatggat
     5041 tcttcggttg ggtccgggcc gtgtacgtca gcgggttcgg ccatatgtat atctccttct
     5101 taaagttaaa caaaattatt tctagagggg aattgttatc cgctcacaat tcccctatag
     5161 tgagtcgtat taatttcgcg ggatcgagat ctcgatcctc tacgccggac gcatcgtggc
     5221 cggcatcacc ggcgccacag gtgcggttgc tggcgcctat atcgccgaca tcaccgatgg
     5281 ggaagatcgg gctcgccact tcgggctcat gagcgcttgt ttcggcgtgg gtatggtggc
     5341 aggccccgtg gccgggggac tgttgggcgc catctccttg catgcaccat tccttgcggc
     5401 ggcggtgctc aacggcctca acctactact gggctgcttc ctaatgcagg agtcgcataa
     5461 gggagagcgt cgagatcccg gacaccatcg aatggcgcaa aacctttcgc ggtatggcat
     5521 gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg
     5581 atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca
     5641 gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca
     5701 ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca
     5761 cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg
     5821 atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta
     5881 aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc
     5941 tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc
     6001 ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc
     6061 gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc
     6121 cattaagttc tgtctcggcg cgtctg
//
