LOCUS       sequence-342684-        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: pEE058
FEATURES             Location/Qualifiers
     source          1..6149
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             790..807
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(860..878)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      874..921
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      958..1413
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1045..1064)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1255..1276
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1439..1543
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1544..1612,1613..2404)
                     /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(1762..1781)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2575..3163
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3064..3083
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3317..3334
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3349..3488
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3414..3433)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3574..3596
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3590..3781)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4590..5672)
                     /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     5634..5653
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5673..5750)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5956..5975
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6059..6078
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6059..6077
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6078..6102
                     /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             6117..6139
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 gcagacaatc cttaccagcg tggtccggct ccgaccaacg cgtcgattga ggcaacacgt
       61 ggtccgtacg cggtgagctc tacgtccgtg agctcttggc tggtttccgg tttcgacggc
      121 ggtactattt attatcctac tacgaccgct gatggcacct tcggcgcagt ggcgatctcc
      181 ccgggttaca ccgcttacga atccagcatt gcttggttcg gcgaacgtct ggcatcccaa
      241 ggcttcgtgg ttttcacctt cgatactaac accactgtag accagccggc ccagcgtggt
      301 gaccagctgc tggcggctct ggattactta acccaacgtt cttccgtgcg ttcccgtgtg
      361 gacgcgagcc gtctgggcgt tatgggtcat tccatgggcg gtggtggttc tttggaagca
      421 tctaaagacc gtccgtccct gaaggcggca atcccgatga ccccgtggaa cactgataaa
      481 acctggagcg agatccgtac tccgaccttg attttcggtg ctgaaaacga ttccgtagct
      541 ccagtcgcat cccattccga accgttctac tccaccattc cgtccaccac caacaaaatg
      601 tacatcgaat taaacggcgc aagccacttt gctccgaact cctctaacac gaccatcgcc
      661 aaatacagca tcagctggct gaaacgtttc ctggataacg acactcgtta tgaccagttc
      721 ctgtgccctc tgcctacttc tgcgctgtac attgaagaat ctcgcggtac ctgcccgctg
      781 cgtctcgagc accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa
      841 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
      901 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg
      961 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     1021 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     1081 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     1141 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     1201 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     1261 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     1321 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     1381 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     1441 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     1501 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     1561 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     1621 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     1681 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     1741 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     1801 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     1861 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     1921 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     1981 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     2041 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac
     2101 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     2161 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     2221 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     2281 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     2341 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     2401 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     2461 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     2521 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     2581 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     2641 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     2701 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     2761 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     2821 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     2881 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     2941 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     3001 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     3061 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     3121 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     3181 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     3241 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     3301 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     3361 ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc
     3421 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     3481 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     3541 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     3601 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa
     3661 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
     3721 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
     3781 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
     3841 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
     3901 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
     3961 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga
     4021 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa
     4081 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt
     4141 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc
     4201 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc
     4261 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
     4321 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
     4381 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
     4441 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
     4501 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     4561 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4621 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4681 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     4741 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     4801 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     4861 actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     4921 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     4981 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     5041 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     5101 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     5161 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     5221 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     5281 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     5341 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     5401 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     5461 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     5521 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     5581 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     5641 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     5701 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     5761 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     5821 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
     5881 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
     5941 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
     6001 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta
     6061 atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg
     6121 tttaacttta agaaggagat atacatatg
//
