LOCUS       sequence-349200-        6020 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 6020)
  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 6020)
  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: pEE025
FEATURES             Location/Qualifiers
     source          1..6020
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     12..31
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     115..134
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        115..133
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    134..158
                     /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             173..195
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             866..883
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(936..954)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      950..997
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1034..1489
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1121..1140)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1331..1352
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1515..1619
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1620..1688,1689..2480)
                     /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(1838..1857)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2651..3239
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3140..3159
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3393..3410
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3425..3564
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3490..3509)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3650..3672
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3666..3857)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4666..5748)
                     /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     5710..5729
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5749..5826)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
       61 atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac
      121 gactcactat aggggaattg tgagcggata acaattcccc tctagaaata attttgttta
      181 actttaagaa ggagatatac atatggtacg tcgtccgaac aacactacct ttactgcaca
      241 gctgtactat ccggctacgg cgactggtga caacgctccg tacgacggct ctggcgcacc
      301 gtatccggcg gtgtcgtttg gtcacggttt tcttcaaccg cccgaacgct atcgctctat
      361 cctggaacac cttgctagct ggggttacct gaccatcgcc accgaatctg gtcaagagct
      421 gtttccgaac caccgtgcgt acgctgaaga tatgcgttac tgcctgacct acctggaaga
      481 acagaatgcc gatccggcgt cttggttatt tggccaggtg gccactgctc aattcggtat
      541 cagcggtcac tccatgggtg ggggtgcctc catcctggca gctgccgctg atgcacgtat
      601 taaagcggtt gcaaacctgg cagccgcaga aactaacccg agcgcgatcc aagcgagccc
      661 gaacatcacc gttcctcatt ctctcatctc cggttccgcc gacactatca ctccgttgtc
      721 atccaatggt ctgcgtatgt acactgcggg tctgcgtgct gaagcagctg cacgccactc
      781 tcgtcgtctg ggcctgcgcg ttccgaaaac tccgagcatt tttggctgcg atagcggcag
      841 cctgccaccg cgtcacgcgc tcgagcacca ccaccaccac cactgagatc cggctgctaa
      901 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
      961 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     1021 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     1081 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     1141 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     1201 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     1261 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     1321 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     1381 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     1441 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     1501 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     1561 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     1621 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     1681 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     1741 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     1801 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     1861 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     1921 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     1981 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     2041 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     2101 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     2161 ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     2221 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     2281 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     2341 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     2401 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     2461 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     2521 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     2581 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     2641 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     2701 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2761 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     2821 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     2881 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     2941 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     3001 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     3061 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     3121 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     3181 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     3241 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     3301 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     3361 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     3421 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
     3481 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     3541 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     3601 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     3661 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     3721 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     3781 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     3841 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     3901 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     3961 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     4021 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     4081 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     4141 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     4201 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     4261 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     4321 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
     4381 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     4441 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
     4501 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     4561 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     4621 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     4681 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4741 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     4801 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     4861 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     4921 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     4981 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     5041 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     5101 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     5161 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     5221 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     5281 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     5341 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     5401 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     5461 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     5521 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     5581 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     5641 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     5701 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     5761 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     5821 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     5881 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     5941 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     6001 gcccgaagtg gcgagcccga
//
