LOCUS       sequence-342602-        6398 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 6398)
  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 6398)
  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: pEE017
FEATURES             Location/Qualifiers
     source          1..6398
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1039..1056
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1109..1127)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1123..1170
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1207..1662
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1294..1313)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1504..1525
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1688..1792
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1793..1861,1862..2653)
                     /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(2011..2030)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2824..3412
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3313..3332
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3566..3583
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3598..3737
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3663..3682)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3823..3845
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3839..4030)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4839..5921)
                     /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     5883..5902
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5922..5999)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6205..6224
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6308..6327
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6308..6326
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6327..6351
                     /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             6366..6388
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 gttgacatca ccggcaacgg catggcggcc acggccccca ccgacgaacg cattgttgat
       61 aaaccgctgc ctcagccgca aattcgctct ggtaatgtgc gtgcaatgcc ggctgcacgt
      121 aaactggcac aggaacatgg catcgacctg tctaccctga ctggctctgg cccgggtggc
      181 gtgatcgtaa aagaagatgt tgaacgtgcg atcaccgctc gtgccgttcc ggtttctccg
      241 ctgcagcgtg tgaacttcta ttctgcaggc taccgtctcg atggtctgct gtatactccg
      301 cgtcacctgc cagcgggcga acgtcgcccg ggtgtcgtcc tgctggtggg ttacacgtac
      361 cttaaaacca tggtgatgcc agatatcgcc aaagtattaa atgcggcggg ctatgttgca
      421 ctggtttttg actaccgtgg ctttggtgaa tctgaagggc cgcgtggccg cctgatcccg
      481 ctggaacaag tggcagacgc gcgtgcggcg ttgactttcc ttgcggaaca gtccatggta
      541 gatccggacc gtctggctgt gatcggcatc agcctgggtg gtgcgcacgc gatcactacc
      601 gccgctctgg atcagcgcgt gcgcgcagtc gtcgcacttg aaccgcctgg tcacggcgct
      661 cgctggctgc gctcgctgcg tcgtcactgg gaatggcgcc agttcctctc tcgtttagca
      721 gaggatcgtc gtcagcgcgt tctgtctggc ggctctacca tggttgatcc tctggaaatt
      781 gtcctgccgg atccagagtc ccaggccttc ctggatcagg ttgccgcaga attcccgcag
      841 atgaaagtga ccctgccgct ggagtccgcg gaagcgctga tcgaatatgt tagcgaagac
      901 ctggctggac gtattgctcc gcgcccgctg ctgatcatcc acggggacgc agatcagctc
      961 gttccggttg ctgaggccca agctattgcc gaacgtgcag gcagcagtgc gcagctggaa
     1021 atcatcccag gtctcgagca ccaccaccac caccactgag atccggctgc taacaaagcc
     1081 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     1141 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
     1201 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     1261 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     1321 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     1381 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     1441 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     1501 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     1561 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     1621 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     1681 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     1741 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     1801 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     1861 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     1921 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     1981 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     2041 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     2101 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     2161 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     2221 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     2281 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc
     2341 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     2401 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     2461 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     2521 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     2581 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     2641 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     2701 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     2761 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     2821 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     2881 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     2941 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     3001 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     3061 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     3121 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     3181 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     3241 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     3301 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     3361 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     3421 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     3481 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     3541 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     3601 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc
     3661 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
     3721 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     3781 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     3841 agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt
     3901 ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct
     3961 ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct
     4021 gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga
     4081 tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg
     4141 aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc
     4201 agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc
     4261 atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac
     4321 tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc
     4381 agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc
     4441 aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg
     4501 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     4561 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     4621 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     4681 ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg
     4741 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     4801 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4861 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4921 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     4981 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     5041 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     5101 tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc
     5161 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     5221 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     5281 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     5341 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     5401 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     5461 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     5521 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     5581 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     5641 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     5701 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     5761 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     5821 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     5881 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     5941 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     6001 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
     6061 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
     6121 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     6181 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
     6241 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc
     6301 gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa
     6361 ataattttgt ttaactttaa gaaggagata tacatatg
//
