LOCUS       sequence-349636-        6149 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 6149)
  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 6149)
  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: pEE078
FEATURES             Location/Qualifiers
     source          1..6149
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     945..964
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(984..1061)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1267..1286
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1370..1389
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1370..1388
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1389..1413
                     /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             1428..1450
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2250..2267
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2320..2338)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2334..2381
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2418..2873
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2505..2524)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2715..2736
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2899..3003
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3004..3072,3073..3864)
                     /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(3222..3241)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4035..4623
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4524..4543
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4777..4794
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4809..4948
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4874..4893)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5034..5056
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5050..5241)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6050..6149,1..983))
                     /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 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
       61 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
      121 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
      181 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
      241 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
      301 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
      361 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
      421 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
      481 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
      541 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
      601 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
      661 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
      721 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
      781 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
      841 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
      901 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
      961 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     1021 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     1081 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     1141 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     1201 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     1261 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     1321 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc
     1381 actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt
     1441 aagaaggaga tatacatatg gcgaacccgt acgaacgcgg cccgaacccg accgaatcta
     1501 tgctggaagc tcgtagcggt ccattctccg ttagcgagga acgtgcttct cgctttggcg
     1561 ctgacggttt cggtggtggc actatctatt atccgcgtga aaacaacacc tacggcgcga
     1621 ttgccatctc tccgggttat accggcactc agagctccat cgcgtggctg ggcgaacgta
     1681 tcgcctccca cggcttcgtt gttatcgcta ttgacaccaa taccaccctg gaccaacctg
     1741 actctcgtgc tcgtcagctg aacgcggcgc tggactatat gcttactgac gcctcctctg
     1801 cagtacgtaa ccgtattgac gcgtcacgcc tggctgtgat gggtcacagc atgggtggcg
     1861 gtgggactct gcgcctggca tcgcagcgcc cggatctgaa agcggctatc ccgttaactc
     1921 cgtggcacct gaacaaatct tggcgcgaca tcacggtacc gaccctgatc atcggtgcag
     1981 agtatgacac catcgcctcc gtgacgctgc attctaaacc gttctacaac tcgattccgt
     2041 ctccgaccga caaagcttat ctcgaactgg acggggcatc tcactttgca ccgaatatca
     2101 ccaataagac tatcggtatg tactctgtgg cctggctgaa acgtttcgtt gatgaggata
     2161 cgcgttacac ccagttcctg tgtccgggcc cgcgtaccgg cctgctgtct gatgtagaag
     2221 agtaccgttc cacttgccca ttcctcgagc accaccacca ccaccactga gatccggctg
     2281 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
     2341 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     2401 ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2461 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     2521 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2581 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     2641 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     2701 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     2761 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     2821 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     2881 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     2941 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     3001 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     3061 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     3121 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     3181 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     3241 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     3301 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     3361 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     3421 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     3481 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     3541 atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     3601 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     3661 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     3721 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     3781 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     3841 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     3901 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     3961 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     4021 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     4081 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     4141 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     4201 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     4261 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     4321 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     4381 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     4441 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     4501 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     4561 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     4621 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     4681 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     4741 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     4801 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat
     4861 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     4921 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     4981 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     5041 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     5101 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     5161 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     5221 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
     5281 aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc
     5341 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
     5401 aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg
     5461 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
     5521 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
     5581 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
     5641 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg
     5701 cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
     5761 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
     5821 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
     5881 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
     5941 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     6001 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     6061 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     6121 aggcggtttg cgtattgggc gccagggtg
//
