LOCUS       sequence-342628-        5897 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 5897)
  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 5897)
  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: pEE030
FEATURES             Location/Qualifiers
     source          1..5897
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             538..555
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(608..626)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      622..669
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      706..1161
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(793..812)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1003..1024
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1187..1291
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1292..1360,1361..2152)
                     /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(1510..1529)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2323..2911
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2812..2831
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3065..3082
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3097..3236
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3162..3181)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3322..3344
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3338..3529)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4338..5420)
                     /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     5382..5401
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5421..5498)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5704..5723
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5807..5826
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5807..5825
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5826..5850
                     /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             5865..5887
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 caaaccgtga ccagcatgct gaaagacctg gacgcggtaa ttactcaggt ttcagaaaaa
       61 tttccgcaga ttgacaacaa gcgcgtctgt ctgatcggtc actctcaggg tgcgtacgta
      121 tccttcctgc atgcgaccaa agatgaacgt attaaatgcc tggtctcctg gatgggtcgt
      181 ctgtcggacc tgaaagaatt ttggtctaag ctgtggttcg acgagatcga acgcaaaggc
      241 tatatctacg agtgggatta caaaatcacc aagaaatatg tgcgtgatag cctgaaatac
      301 aatctgtcaa aagctgcatg gcgtatcaaa gtgccgaccc tgctgattta tggtgaactg
      361 gacgatatcg tgccaccttc tgaaggtatg aaattctacc gcaacatcaa atctccgaaa
      421 aaaatcgtta ttgtaaagga tctgaaccac accttctctg gtgaaaaagc caaaaaatcc
      481 gttatccgca tcactctgaa atggctgtct aaatggctca agcgcctgga cctcgagcac
      541 caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct
      601 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
      661 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
      721 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
      781 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
      841 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
      901 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
      961 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     1021 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     1081 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     1141 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
     1201 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     1261 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     1321 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     1381 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     1441 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     1501 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     1561 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     1621 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     1681 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     1741 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     1801 cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa
     1861 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     1921 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     1981 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     2041 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     2101 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     2161 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     2221 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     2281 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     2341 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     2401 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     2461 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     2521 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     2581 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     2641 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     2701 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     2761 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     2821 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     2881 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     2941 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     3001 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     3061 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     3121 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc
     3181 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     3241 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     3301 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     3361 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     3421 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     3481 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     3541 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     3601 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     3661 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     3721 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     3781 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     3841 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     3901 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     3961 tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg
     4021 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
     4081 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt
     4141 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
     4201 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
     4261 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     4321 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4381 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     4441 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     4501 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     4561 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
     4621 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     4681 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     4741 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     4801 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     4861 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     4921 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     4981 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     5041 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     5101 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     5161 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     5221 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     5281 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     5341 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     5401 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     5461 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     5521 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     5581 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
     5641 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
     5701 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
     5761 cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact
     5821 ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag
     5881 aaggagatat acatatg
//
