LOCUS       sequence-342624-        6059 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 6059)
  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 6059)
  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: pEE028
FEATURES             Location/Qualifiers
     source          1..6059
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             700..717
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(770..788)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      784..831
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      868..1323
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(955..974)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1165..1186
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1349..1453
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1454..1522,1523..2314)
                     /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(1672..1691)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2485..3073
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2974..2993
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3227..3244
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3259..3398
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3324..3343)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3484..3506
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3500..3691)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4500..5582)
                     /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     5544..5563
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5583..5660)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5866..5885
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5969..5988
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5969..5987
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5988..6012
                     /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             6027..6049
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 caagtaattc agcagaccgt tacactgcaa aaaacccaac tgcgcctgac caaggaaggc
       61 ttcgttacca attatcgttt cccggtggat ttctactacc ctgattctcc ggaatctttc
      121 ccggtaattc tgatctctca tggttttggc tcggtccgcg aaaacttccg cactctggca
      181 cagcatctgg cctctcacgg cttcctggta gccgttccgc agcacatcgg ctcggatctg
      241 cagtaccgtc aagagctgat caaaggtact ttatcctccg cactgtcccc agttgaattt
      301 ttggcgcgtc cgaccgacct gtctaccatc attgactatc tgcaggcgac tcagaacacc
      361 ggctcctggc agaagcgtgc aaatctgcag cagatcggcg ttatcggtga tagtctgggc
      421 ggtaccactg ctctgacgat tggtggtgca ccgctggata ttccgcgtct gcagactaaa
      481 tgtacctcgg acaacgttat tgtgaacgtt gccctgatcc tgcaatgcca ggcctcgttc
      541 ctgccgccga gcgaatacaa cctggctgat tcccgtgtca aagccgttat tgccacgcac
      601 ccgctgatct caggcatttt ttctccggac tctctggcga aaattcagat cccagtgatg
      661 attaccgcgg gcaactttga catcatcacc ccgctcgagc accaccacca ccaccactga
      721 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
      781 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
      841 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
      901 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
      961 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     1021 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     1081 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     1141 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     1201 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     1261 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     1321 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     1381 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     1441 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     1501 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     1561 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     1621 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     1681 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     1741 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     1801 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     1861 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     1921 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     1981 tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     2041 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     2101 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     2161 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     2221 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     2281 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     2341 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     2401 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     2461 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     2521 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     2581 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     2641 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     2701 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     2761 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     2821 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     2881 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     2941 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     3001 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     3061 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     3121 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     3181 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     3241 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     3301 acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     3361 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     3421 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     3481 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     3541 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     3601 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     3661 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     3721 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     3781 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
     3841 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
     3901 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     3961 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
     4021 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
     4081 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
     4141 cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac
     4201 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
     4261 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
     4321 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
     4381 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     4441 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     4501 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     4561 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     4621 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     4681 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     4741 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc
     4801 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     4861 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     4921 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
     4981 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     5041 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
     5101 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     5161 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     5221 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
     5281 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     5341 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     5401 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     5461 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     5521 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     5581 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     5641 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
     5701 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     5761 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     5821 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     5881 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
     5941 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
     6001 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacatatg
//
