LOCUS       sequence-349627-        6143 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 6143)
  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 6143)
  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: pEE049
FEATURES             Location/Qualifiers
     source          1..6143
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             221..238
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(291..309)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      305..352
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      389..844
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(476..495)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     686..707
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        870..974
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(975..1043,1044..1835)
                     /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(1193..1212)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2006..2594
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2495..2514
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2748..2765
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2780..2919
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2845..2864)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3005..3027
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3021..3212)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4021..5103)
                     /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     5065..5084
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5104..5181)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5387..5406
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5490..5509
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5490..5508
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5509..5533
                     /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             5548..5570
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 cccgtctacc gtgaaaaaag cactggcggt tctgaatggc gcatctcacc tgacgttcac
       61 ctcctatgac gaacgtgcgg cgcgctacgg tgttgcgttc gcgaaacgct tcgctgacgg
      121 tgacacccgt tatactccat tcctgtgtgg cgctgaacac acagcttacg caacttcatc
      181 tcgtttcacg gaatacagta gcaattgccc gtatctcgag caccaccacc accaccactg
      241 agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
      301 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
      361 aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
      421 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
      481 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
      541 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
      601 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
      661 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
      721 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
      781 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
      841 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
      901 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
      961 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     1021 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     1081 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     1141 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     1201 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     1261 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     1321 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     1381 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     1441 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     1501 gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac
     1561 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     1621 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     1681 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     1741 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     1801 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     1861 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     1921 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     1981 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2041 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2101 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     2161 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     2221 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     2281 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     2341 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     2401 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     2461 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     2521 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     2581 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     2641 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     2701 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     2761 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     2821 cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     2881 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
     2941 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     3001 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     3061 ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc
     3121 gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc
     3181 atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg
     3241 ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat
     3301 gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg
     3361 cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt
     3421 gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc
     3481 gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt
     3541 gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat
     3601 tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc
     3661 acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa
     3721 cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc
     3781 gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc
     3841 cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg
     3901 gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     3961 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     4021 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     4081 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     4141 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     4201 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     4261 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag
     4321 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     4381 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     4441 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     4501 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     4561 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
     4621 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     4681 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     4741 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     4801 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     4861 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
     4921 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     4981 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     5041 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     5101 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     5161 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta
     5221 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
     5281 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     5341 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
     5401 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
     5461 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
     5521 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tatacatatg
     5581 gtggaaatcg gcccagcacc gacctccacc tcgctgaaca gtgacggctc cttcgctgtg
     5641 tcatctgcgt ctgtgagctc gtctgcatgc ggctccggtt gcgctggtgg tactgtatac
     5701 tacccgaaca ctgctggcag ctacggtgtg atcgcggttt gtccgggctt caccaatacc
     5761 tccagtgcca tctcctggtt tgcacgtcgc atggctactc acggtttcgt cactatcgca
     5821 atgaacacca actcccgtta tgactttcca gcaagtcgcg ctacccagct gcgtgcggtt
     5881 ctgaattacc tggtgaacag ctctagcagc acgattcgtt cccgtatccg ctccgcagat
     5941 cgtggtgtgt ctggttattc catgggcggt ggcggtaccc tgttggctag ccgtgacgac
     6001 agcaccttga agaccggtgt cccgatggcg ccgtataatt caggtactat cagcggcgtg
     6061 aacgtaccac agatgattat cggcggttcc aacgatagta tcgcgccggt ttcctctatg
     6121 gctcgtccat tctacaacaa cat
//
