LOCUS       sequence-349690-        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: pEE073
FEATURES             Location/Qualifiers
     source          1..5726
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     970..989
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1009..1086)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1292..1311
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1395..1414
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1395..1413
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1414..1438
                     /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             1453..1475
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1852..1869
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1922..1940)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1936..1983
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2020..2475
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2107..2126)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2317..2338
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2501..2605
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2606..2674,2675..3466)
                     /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(2824..2843)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3637..4225
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4126..4145
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4379..4396
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4411..4550
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4476..4495)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4636..4658
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4652..4843)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5652..5726,1..1008))
                     /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 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
       61 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
      121 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
      181 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
      241 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
      301 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
      361 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
      421 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
      481 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
      541 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
      601 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
      661 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
      721 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
      781 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
      841 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
      901 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
      961 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     1021 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     1081 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     1141 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     1201 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     1261 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     1321 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     1381 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     1441 tctagaaata attttgttta actttaagaa ggagatatac atatggcgaa tccgtacgaa
     1501 cgtggtccta acccaaccaa ctcaagcatc gaggctctgc gcgggccata cagcgtgtca
     1561 gaggactcgg tttcgagctt ggtgagcggt ttcgggggcg gcaccatcta ctacccgacc
     1621 ggtaccaatg aaacttttgg cgcggtggca atcagcccgg gttacacggg tacgcagtct
     1681 tctatttctt ggctgggccc tcgtctggcg tcccagggtt tcgttgttat gaccattgat
     1741 actaacacta ccctggatca gccggactct cgcgcctctc agctggatgc agcactggac
     1801 tatatggtga accgttcttc atctaccgtg cgcaatcgta tcgacctcga gcaccaccac
     1861 caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
     1921 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     1981 ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat
     2041 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     2101 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     2161 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     2221 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     2281 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     2341 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     2401 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     2461 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     2521 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     2581 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     2641 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     2701 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     2761 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     2821 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     2881 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     2941 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     3001 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     3061 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     3121 aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt
     3181 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     3241 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     3301 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     3361 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     3421 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     3481 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     3541 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     3601 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     3661 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     3721 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     3781 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     3841 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     3901 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     3961 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     4021 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     4081 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     4141 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     4201 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     4261 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     4321 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     4381 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     4441 gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag
     4501 ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc
     4561 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     4621 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     4681 gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg
     4741 cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga
     4801 taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag
     4861 ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac
     4921 gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg
     4981 tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata
     5041 cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa
     5101 tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca
     5161 ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc
     5221 gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca
     5281 acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct
     5341 tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga
     5401 ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc
     5461 cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag
     5521 tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga
     5581 aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa
     5641 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5701 gaatcggcca acgcgcgggg agaggc
//
