LOCUS       sequence-383074-        6269 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 6269)
  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 6269)
  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: pEE005
FEATURES             Location/Qualifiers
     source          1..6269
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     511..530
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(550..627)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     833..852
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     936..955
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        936..954
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    955..979
                     /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             994..1016
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1936..1953
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2006..2024)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2020..2067
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2104..2559
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2191..2210)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2401..2422
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2585..2689
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2690..2758,2759..3550)
                     /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(2908..2927)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3721..4309
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4210..4229
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4463..4480
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4495..4634
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4560..4579)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4720..4742
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4736..4927)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5736..6269,1..549))
                     /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 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
       61 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
      121 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
      181 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
      241 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
      301 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
      361 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
      421 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
      481 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
      541 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
      601 aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct
      661 gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg
      721 gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca
      781 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
      841 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc
      901 cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt
      961 gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga aggagatata
     1021 catatgtgcc tgtatctgaa tatctggact ccggacctga atggcagcct gccggttatg
     1081 gtgttcattc acggcggcgg taaccagcaa ggttccaccg ctcagatcgc cggtggtgca
     1141 cgtatctacg agggcaagaa cctggcacgt cgtggtcagg tagttgtagt cacccttcaa
     1201 taccgtctgg gtgcactggg ctatctggta cacccgggtc tggaagctga atccacccac
     1261 ggcaaggctg gtaactatgg ggctctggat cagctggcag cactgctctg gattaaagaa
     1321 aacatccgcg cttttggtgg tgacccggag ctggtcacgc tgttcggcga aagcgcgggc
     1381 gcggttaaca tcggcaacct gctggttatg ccggctgcaa aaggtctgtt tcaccgtgct
     1441 atcctgcagt cggggtcccc gcgcctgaaa gcctattctg cagcgcgcaa cgaaggtatc
     1501 gcttttgcac agaagctggg tgctgcaggt actccagagc agcaggtggc ccatctgcgc
     1561 actctgcctg tcgatagcct ggtgaaaggc gattctaacc cgatcagcgg cggcagcatg
     1621 gcgcagggta gctggcagcc ggtactggat ggttactggt tcccacaggc tccgctcgat
     1681 gcgatgcgta gcggtgaaca ccatcgtgta ccgctgatcg ttggttcttc ctcggacgag
     1741 atgagcctgt acgttccgtc tgttgtgact ccgctgatgc tgcagacttt tgttcagacc
     1801 actattccgg cgccttatcg ccagcaggtc ctggcactgt acccgccggg taccaccaac
     1861 gaacaagccc gtgcatctta cgtggccctg gtcggtgacc cgctggagtc cacttgtcgt
     1921 cacgcaagcc tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
     1981 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     2041 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     2101 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     2161 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     2221 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     2281 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     2341 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     2401 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     2461 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     2521 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     2581 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2641 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     2701 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     2761 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     2821 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     2881 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     2941 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     3001 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     3061 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     3121 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     3181 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat
     3241 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     3301 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     3361 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     3421 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     3481 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     3541 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     3601 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     3661 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3721 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     3781 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     3841 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     3901 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     3961 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     4021 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4081 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     4141 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     4201 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     4261 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     4321 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     4381 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     4441 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     4501 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
     4561 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     4621 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     4681 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     4741 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     4801 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     4861 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     4921 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     4981 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     5041 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     5101 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     5161 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     5221 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     5281 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     5341 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     5401 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     5461 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     5521 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     5581 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     5641 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     5701 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5761 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5821 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     5881 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     5941 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     6001 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     6061 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     6121 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     6181 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     6241 gagacagaac ttaatgggcc cgctaacag
//
