LOCUS       sequence-342632-        6209 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 6209)
  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 6209)
  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: pEE032
FEATURES             Location/Qualifiers
     source          1..6209
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             850..867
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(920..938)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      934..981
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1018..1473
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1105..1124)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1315..1336
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1499..1603
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1604..1672,1673..2464)
                     /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(1822..1841)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2635..3223
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3124..3143
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3377..3394
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3409..3548
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3474..3493)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3634..3656
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3650..3841)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4650..5732)
                     /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     5694..5713
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5733..5810)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6016..6035
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6119..6138
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6119..6137
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6138..6162
                     /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             6177..6199
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 gccaacccgc cgggtggtga cccggaccct ggctgccaga ccgactgcaa ctatcagcgc
       61 ggtccggatc cgaccgacgc ttatctggaa gctgcctccg gcccctacac ggtgtctaca
      121 atccgcgtat cctctctggt tccgggtttc ggcggcggta ctatccacta cccgacgaac
      181 gctggtggtg gcaagatggc tggcatcgtt gtgatccctg gttatctctc cttcgaaagc
      241 tccatcgaat ggtggggccc gcgcctggcg tcccacggct tcgttgtaat gactatcgac
      301 accaacacca tctacgacca gccatctcag cgtcgtgacc agatcgaagc agctctgcag
      361 tacctggtca accagtccaa ctctagtagc agcccgattt ctgggatggt tgactcttcc
      421 cgcctcgcgg cagtaggttg gtctatgggc ggtggtggca ccctgcaact ggctgctgac
      481 ggtggtatca aagccgcgat tgccctggct ccgtggaaca gttctatcaa tgattttaac
      541 cgtattcagg taccgaccct gatcttcgct tgtcagctcg atgctatcgc tccagtggcg
      601 ctgcacgcct cgccgttcta caaccgcatc cctaacacca cgccgaaagc gtttttcgaa
      661 atgaccggcg gtgaccactg gtgcgctaac ggcggtaaca tctatagcgc cctgctggga
      721 aaatatggcg tgtcttggat gaaactgcac ctggaccaag atactcgtta tgctccgttc
      781 ctgtgcggcc cgaaccacgc cgctcagacc ctgattagcg aataccgtgg caactgtcct
      841 tacctcgagc accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa
      901 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
      961 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg
     1021 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     1081 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     1141 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     1201 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     1261 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     1321 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     1381 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     1441 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     1501 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     1561 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     1621 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     1681 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     1741 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     1801 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     1861 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     1921 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     1981 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     2041 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     2101 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac
     2161 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     2221 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     2281 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     2341 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     2401 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     2461 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     2521 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     2581 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     2641 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     2701 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     2761 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     2821 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     2881 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     2941 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     3001 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     3061 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     3121 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     3181 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     3241 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     3301 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     3361 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     3421 ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc
     3481 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     3541 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     3601 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     3661 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa
     3721 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
     3781 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
     3841 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
     3901 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
     3961 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
     4021 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga
     4081 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa
     4141 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt
     4201 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc
     4261 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc
     4321 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
     4381 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
     4441 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
     4501 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
     4561 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     4621 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4681 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4741 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     4801 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     4861 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     4921 actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     4981 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     5041 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     5101 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     5161 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     5221 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     5281 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     5341 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     5401 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     5461 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     5521 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     5581 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     5641 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     5701 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     5761 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     5821 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     5881 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
     5941 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
     6001 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
     6061 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta
     6121 atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg
     6181 tttaacttta agaaggagat atacatatg
//
