LOCUS       sequence-342606-        6155 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 6155)
  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 6155)
  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: pEE019
FEATURES             Location/Qualifiers
     source          1..6155
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             796..813
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(866..884)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      880..927
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      964..1419
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1051..1070)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1261..1282
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1445..1549
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1550..1618,1619..2410)
                     /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(1768..1787)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2581..3169
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3070..3089
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3323..3340
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3355..3494
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3420..3439)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3580..3602
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3596..3787)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4596..5678)
                     /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     5640..5659
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5679..5756)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5962..5981
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6065..6084
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6065..6083
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6084..6108
                     /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             6123..6145
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 ctgattcgtc cggttacctt ccgcaatatg aaccagcaga ttattggcat ccttcacact
       61 ccggacaaca tccgtctgaa tgaaaaagta ccgggtatcc tgatgttcca tggcttcact
      121 ggtaataaaa ctgaagcgca ccgcctgttt gtgcacgtgg ctcgttctct gtccgaacat
      181 ggtttcatcg tgctgcgttt cgacttccgc ggaagcggtg atagcgatgg tgaattcgaa
      241 gacatgaccc tgccgggtga agttagcgac gcagagcgcg cgctgacctt tctgttgcgc
      301 cagcgtaacg ttgataaaaa ccgtattggt gtaatcggtc tgtccatggg tggccgtgtt
      361 gcggcgattc tggcaagcaa ggaccggcgc gttaaattcg ctgtcctgta cagcccggcg
      421 ctgggtccgc tgcgcgatcg ttctctgtct ttcatgagca aagaaaaaat tgaacgtctg
      481 aactccggtg aggcagtgga attcttcgct gaaggttggt atatcaaaaa agcattcttt
      541 gagaccgtgg actatattgt cccgctggac atcatggatt ccattaaagt tccggttttg
      601 atcgttcatg gcgacaaaga cccgctcatt ccggttggtg aggctatccg tgcatacgaa
      661 aaaatcaaag gtgttaacga gaaaaatgag ctgtacattg tacgtggcgg tgatcacacc
      721 ttctccaaaa aagaacacac cctggaggta atcaagaaaa ctttggactg gatccgtagc
      781 ctgggcattc tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
      841 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
      901 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
      961 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     1021 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     1081 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     1141 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     1201 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     1261 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     1321 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     1381 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     1441 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     1501 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     1561 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     1621 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     1681 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     1741 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     1801 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     1861 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     1921 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     1981 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     2041 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat
     2101 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     2161 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     2221 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     2281 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     2341 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     2401 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     2461 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     2521 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     2581 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     2641 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     2701 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     2761 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     2821 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     2881 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     2941 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     3001 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     3061 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3121 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3181 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     3241 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     3301 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     3361 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
     3421 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     3481 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     3541 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     3601 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     3661 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     3721 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     3781 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     3841 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     3901 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     3961 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     4021 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     4081 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     4141 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     4201 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     4261 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     4321 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     4381 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     4441 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     4501 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     4561 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4621 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4681 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     4741 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     4801 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     4861 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     4921 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     4981 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     5041 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     5101 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     5161 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     5221 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     5281 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     5341 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     5401 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     5461 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     5521 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     5581 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     5641 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     5701 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     5761 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     5821 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     5881 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     5941 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     6001 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     6061 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     6121 attttgttta actttaagaa ggagatatac atatg
//
