LOCUS       sequence-349640-        6464 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 6464)
  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 6464)
  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: pEE008
FEATURES             Location/Qualifiers
     source          1..6464
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     956..975
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(995..1072)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1278..1297
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1381..1400
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1381..1399
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1400..1424
                     /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             1439..1461
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2576..2593
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2646..2664)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2660..2707
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2744..3199
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2831..2850)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3041..3062
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3225..3329
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3330..3398,3399..4190)
                     /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(3548..3567)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4361..4949
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4850..4869
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5103..5120
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5135..5274
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5200..5219)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5360..5382
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5376..5567)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6376..6464,1..994))
                     /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 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
       61 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
      121 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
      181 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
      241 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
      301 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
      361 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
      421 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
      481 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
      541 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
      601 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
      661 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
      721 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
      781 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
      841 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
      901 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
      961 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     1021 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     1081 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     1141 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     1201 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     1261 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     1321 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
     1381 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
     1441 tgtttaactt taagaaggag atatacatat ggttttcctt ggctggcagg gctcaccggt
     1501 gcagctgccg gcacacgcgg gcgaacaagc accgagcccg gttgagccac tcaacctgcc
     1561 tgatccggca cgtccgggag cttacccggt agcgctgtta acttacggct ctggccaaga
     1621 taaactgcgt caggagtacg cgcaaggtgc agctctgctg accccgagcg tggacgcgtc
     1681 tctgctgctg gaaggctggt cctctctccg taccgcttat tggggttttt cccctgccga
     1741 actgccgctg aatggtcgtg tgtggtatcc gcaggcggaa gggcgttttc cgctggttat
     1801 cgcagtacac ggcaaccacc ctatggagga aacctcagag agtggctatg actacctggg
     1861 cgaactgctg gcttcccgcg gttttatctt cgtggctgtt gacgaaaact tccttaatat
     1921 ctccgcatgg ggtgatgtgc tgtttttcaa ccgcctggaa ggtgaaagcg atgcgcgtgg
     1981 atggctggtt ctggaacatc ttcgtctgtg gcaaagctgg aacgaacaac caggcaaccc
     2041 gttctatcag cgcgtagatc tgaaccagat cgcgctgctg ggccactccc gtggtggaga
     2101 agcaattgtt atcgccgcag cattcaaccg cctgtcccac tacccggaca atgcggccct
     2161 gagtttcgat tacggcttca agattcgcag cctgatcgcg ctggctccag ccgacggcca
     2221 gtaccagcca ggcggtctgc ctaccccgct gcaggatgtt aactacctgc tgctgcatgg
     2281 ttcccacgac atggacgtgc tgactatgat gggcgcggcg ccgttcgaac gtctgacctt
     2341 cagcggccag gatgacttct tcaagtctgc agtttacatc tacggcgcta accacggtca
     2401 gttcaactct gtgtggggca ataaggatat cgcagaaccg atcccacgtc tgtacaatct
     2461 gcgtcagctg ctgccgcaga ccgaacagca acgtatcgcg caggtactga ttagcgcatt
     2521 cctggaagat actctgcgtg gtgaacgtgc ctatcgtccg ctgtttcaac tcgagcacca
     2581 ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     2641 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     2701 ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc
     2761 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2821 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2881 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     2941 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     3001 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     3061 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     3121 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     3181 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     3241 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     3301 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     3361 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     3421 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     3481 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     3541 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     3601 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     3661 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     3721 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     3781 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     3841 taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac
     3901 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     3961 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     4021 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     4081 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     4141 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     4201 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     4261 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     4321 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     4381 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     4441 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     4501 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     4561 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4621 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4681 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     4741 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     4801 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     4861 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     4921 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     4981 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     5041 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     5101 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     5161 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     5221 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     5281 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     5341 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     5401 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     5461 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     5521 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     5581 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     5641 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     5701 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     5761 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     5821 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     5881 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     5941 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     6001 ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc
     6061 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
     6121 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc
     6181 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag
     6241 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
     6301 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     6361 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     6421 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgg
//
