LOCUS       sequence-349676-        6503 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 6503)
  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 6503)
  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: pEE010
FEATURES             Location/Qualifiers
     source          1..6503
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..25
                     /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             40..62
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1216..1233
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1286..1304)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1300..1347
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1384..1839
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1471..1490)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1681..1702
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1865..1969
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1970..2038,2039..2830)
                     /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(2188..2207)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3001..3589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3490..3509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3743..3760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3775..3914
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3840..3859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4000..4022
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4016..4207)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5016..6098)
                     /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     6060..6079
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6099..6176)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6382..6401
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(6485..6503,1)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6485..6503
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
       61 gatatacata tggttgatat cactggcaac ggtatggctg ctaccgcgcc gaccgacgaa
      121 cgtattgtag acaaacctct gcctcagccg cagattcgtt ctggtaacgt tcgtgcaatg
      181 ccggcggctc gcaagctggc gcaggagcac ggtattgacc tgtccactct gaccggtagc
      241 ggtccaggtg gtgttatcgt taaagaggac gtcgaacgtg caatcaccgc tcgtgctgtt
      301 cctgtatctc cgctgcagcg tgtcaacttt tattctgccg gttatcgtct ggacggtctg
      361 ctgtacaccc cgcgtcacct gccagctggt gaacgtagac cgggtgtcgt tctcctggtg
      421 ggttacactt acctcaaaac tatggtaatg ccggacatcg cgaaagttct gaacgctgcg
      481 ggttacgttg ccctggtttt cgactaccgc ggcttcggcg aatccgaggg cccgcgcggt
      541 cgtctaatcc cgttagaaca agtagctgat gcacgtgcag cgctgacctt tctggcggaa
      601 cagtcaatgg ttgatccgga tcgtctcgcg gtaattggca tttctctggg tggtgcacat
      661 gcaattacca ctgctgcact ggatcagcgt gtccgcgcgg tcgtggctct ggaaccgcca
      721 ggccatggtg cgcgttggct gcgtagcctg cgtcgtcact gggaatggcg tcagttcctg
      781 tctcgtctgg ctgaagatcg tcgtcagcgc gtgctaagcg gtggcagcac catggttgac
      841 ccgctggaga tcgttctgcc agacccggag tctcaggctt tcctggacca agttgccgca
      901 gaatttccgc agatgaaagt gacgctgccg ctggaatctg ccgaagcact gatcgaatat
      961 gtgtccgaag acctcgccgg ccgtatcgct ccgcgtccac tgctgatcat tcactctgac
     1021 gccgaccagc tggttccggt tgcggaagct caggcgatcg cagagcgcgc gggctcttct
     1081 gcacagctgg agatcattcc aggcatgtcc catttcaatt gggtaatgcc aggcagcccg
     1141 ggcttcactc gtgttactga ttctatcgtt aaattcctgc gtaacaccct gccggtatct
     1201 gcggacaatc tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
     1261 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     1321 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     1381 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     1441 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     1501 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     1561 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     1621 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     1681 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     1741 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     1801 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     1861 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     1921 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     1981 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     2041 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     2101 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     2161 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     2221 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     2281 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     2341 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     2401 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     2461 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat
     2521 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     2581 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     2641 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     2701 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     2761 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     2821 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     2881 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     2941 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3001 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     3061 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     3121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     3181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     3241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     3301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     3361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     3421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     3481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     3661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     3721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     3781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
     3841 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     3901 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     3961 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     4021 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     4081 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     4141 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     4201 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     4261 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     4321 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     4381 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     4441 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     4501 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     4561 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     4621 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     4681 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     4741 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     4801 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     4861 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     4921 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     4981 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5041 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5101 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     5161 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     5221 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     5281 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     5341 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     5401 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     5461 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     5521 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     5581 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     5641 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     5701 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     5761 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     5821 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     5881 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     5941 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     6001 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     6061 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     6121 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     6181 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     6241 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     6301 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     6361 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     6421 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     6481 aaattaatac gactcactat agg
//
