LOCUS       sequence-349244-        6209 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 6209)
  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 6209)
  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: pEE032
FEATURES             Location/Qualifiers
     source          1..6209
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     946..965
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(985..1062)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1268..1287
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1371..1390
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1371..1389
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1390..1414
                     /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             1429..1451
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2311..2328
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2381..2399)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2395..2442
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2479..2934
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2566..2585)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2776..2797
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2960..3064
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3065..3133,3134..3925)
                     /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(3283..3302)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4096..4684
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4585..4604
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4838..4855
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4870..5009
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4935..4954)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5095..5117
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5111..5302)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6111..6209,1..984))
                     /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 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
       61 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
      121 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
      181 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
      241 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
      301 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
      361 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
      421 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
      481 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
      541 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
      601 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
      661 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
      721 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
      781 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
      841 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
      901 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
      961 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     1021 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
     1081 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
     1141 ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     1201 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     1261 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     1321 cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact
     1381 cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt
     1441 taagaaggag atatacatat ggccaacccg ccgggtggtg acccggaccc tggctgccag
     1501 accgactgca actatcagcg cggtccggat ccgaccgacg cttatctgga agctgcctcc
     1561 ggcccctaca cggtgtctac aatccgcgta tcctctctgg ttccgggttt cggcggcggt
     1621 actatccact acccgacgaa cgctggtggt ggcaagatgg ctggcatcgt tgtgatccct
     1681 ggttatctct ccttcgaaag ctccatcgaa tggtggggcc cgcgcctggc gtcccacggc
     1741 ttcgttgtaa tgactatcga caccaacacc atctacgacc agccatctca gcgtcgtgac
     1801 cagatcgaag cagctctgca gtacctggtc aaccagtcca actctagtag cagcccgatt
     1861 tctgggatgg ttgactcttc ccgcctcgcg gcagtaggtt ggtctatggg cggtggtggc
     1921 accctgcaac tggctgctga cggtggtatc aaagccgcga ttgccctggc tccgtggaac
     1981 agttctatca atgattttaa ccgtattcag gtaccgaccc tgatcttcgc ttgtcagctc
     2041 gatgctatcg ctccagtggc gctgcacgcc tcgccgttct acaaccgcat ccctaacacc
     2101 acgccgaaag cgtttttcga aatgaccggc ggtgaccact ggtgcgctaa cggcggtaac
     2161 atctatagcg ccctgctggg aaaatatggc gtgtcttgga tgaaactgca cctggaccaa
     2221 gatactcgtt atgctccgtt cctgtgcggc ccgaaccacg ccgctcagac cctgattagc
     2281 gaataccgtg gcaactgtcc ttacctcgag caccaccacc accaccactg agatccggct
     2341 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
     2401 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
     2461 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     2521 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     2581 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     2641 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     2701 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     2761 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     2821 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     2881 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     2941 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     3001 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     3061 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     3121 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     3181 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     3241 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     3301 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     3361 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     3421 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     3481 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     3541 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     3601 gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     3661 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     3721 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     3781 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     3841 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     3901 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     3961 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     4021 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     4081 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     4141 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     4201 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     4261 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     4321 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     4381 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     4441 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     4501 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     4561 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     4621 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     4681 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     4741 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     4801 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     4861 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa
     4921 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     4981 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     5041 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     5101 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     5161 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     5221 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     5281 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
     5341 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
     5401 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga
     5461 gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg
     5521 gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc
     5581 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg
     5641 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct
     5701 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc
     5761 gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg
     5821 cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca
     5881 ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg
     5941 ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag
     6001 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     6061 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     6121 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     6181 gaggcggttt gcgtattggg cgccagggt
//
