LOCUS       sequence-349077-        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: pEE035
FEATURES             Location/Qualifiers
     source          1..6149
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     921..940
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(960..1037)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1243..1262
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1346..1365
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1346..1364
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1365..1389
                     /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             1404..1426
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2226..2243
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2296..2314)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2310..2357
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2394..2849
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2481..2500)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2691..2712
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2875..2979
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2980..3048,3049..3840)
                     /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(3198..3217)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4011..4599
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4500..4519
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4753..4770
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4785..4924
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4850..4869)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5010..5032
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5026..5217)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6026..6149,1..959))
                     /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 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
       61 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
      121 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
      181 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
      241 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
      301 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
      361 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
      421 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
      481 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
      541 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
      601 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
      661 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
      721 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
      781 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
      841 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
      901 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
      961 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     1021 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     1081 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     1141 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     1201 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     1261 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     1321 gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat
     1381 aacaattccc ctctagaaat aattttgttt aactttaaga aggagatata catatggcgg
     1441 ataacccgta tcagcgcggt ccggatccga ctgaacgttc tgttacggct cgtcgtggcc
     1501 cgtttgcgat cgacgaaatc tcagttaacg gcggtatcgg tgcaggcttt aaccgtggta
     1561 ccatcttcta cccgacggac cgctcccaag gtacgttcgg tgcagttgcc gttatcccgg
     1621 gctttctgtc tccggaaagc ctggttcgtt ggttcggccc gcgtctggcg tcccaaggtt
     1681 tcgtcgttat gaccttaacg acaaacggcc tgacggacac gccagaatct cgttctgaac
     1741 aactgctggc ggctctggat tacctgacta cccgtagcca ggttcgtgac cgaattgatc
     1801 cgtcccgtct ggccgttatg ggccattcta tgggtggtgg cggttcgctg gctgcagctg
     1861 ccaagcgtcc aaccctgcgt gcagccatcc cgctggctcc gtggtccctg accaagaact
     1921 ggagcgacct gaccgtaccg accctgatca tcggtgcaga gaacgataac gtggcgccgg
     1981 tcgccggtca ctctgaacgc ttttacgact ccatgaccaa tgtaccggaa aaagcatact
     2041 tagaaatggc aggtggtaac catgttgacc cgaccgctga atctgatctg gtggccaagt
     2101 tcaccatttc ttggctgaaa cgtttcgttg atgatgatac tcgctatgat cagttcctgt
     2161 gtccagctcc gcgtcctaac cgccagatct cagaataccg cgacacctgc cctcatagcc
     2221 tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg
     2281 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     2341 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc
     2401 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2461 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2521 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2581 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2641 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     2701 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     2761 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     2821 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     2881 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     2941 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     3001 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     3061 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     3121 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     3181 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     3241 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     3301 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     3361 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     3421 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     3481 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg
     3541 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     3601 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     3661 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     3721 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     3781 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     3841 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     3901 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     3961 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     4021 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     4081 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     4141 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     4201 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     4261 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     4321 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     4381 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     4441 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     4501 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     4561 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     4621 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     4681 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     4741 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     4801 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
     4861 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     4921 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     4981 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     5041 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     5101 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     5161 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     5221 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     5281 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     5341 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     5401 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     5461 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     5521 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     5581 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     5641 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc
     5701 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     5761 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     5821 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     5881 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     5941 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     6001 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     6061 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     6121 gggtggtttt tcttttcacc agtgagacg
//
