LOCUS       sequence-349239-        6149 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 6149)
  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 6149)
  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: pEE058
FEATURES             Location/Qualifiers
     source          1..6149
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     589..608
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(628..705)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     911..930
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1014..1033
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1014..1032
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1033..1057
                     /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             1072..1094
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1894..1911
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1964..1982)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1978..2025
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2062..2517
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2149..2168)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2359..2380
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2543..2647
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2648..2716,2717..3508)
                     /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(2866..2885)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3679..4267
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4168..4187
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4421..4438
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4453..4592
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4518..4537)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4678..4700
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4694..4885)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5694..6149,1..627))
                     /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 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
       61 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
      121 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
      181 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
      241 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
      301 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
      361 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
      421 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
      481 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
      541 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
      601 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
      661 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
      721 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
      781 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
      841 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
      901 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
      961 tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg
     1021 actcactata ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa
     1081 ctttaagaag gagatataca tatggcagac aatccttacc agcgtggtcc ggctccgacc
     1141 aacgcgtcga ttgaggcaac acgtggtccg tacgcggtga gctctacgtc cgtgagctct
     1201 tggctggttt ccggtttcga cggcggtact atttattatc ctactacgac cgctgatggc
     1261 accttcggcg cagtggcgat ctccccgggt tacaccgctt acgaatccag cattgcttgg
     1321 ttcggcgaac gtctggcatc ccaaggcttc gtggttttca ccttcgatac taacaccact
     1381 gtagaccagc cggcccagcg tggtgaccag ctgctggcgg ctctggatta cttaacccaa
     1441 cgttcttccg tgcgttcccg tgtggacgcg agccgtctgg gcgttatggg tcattccatg
     1501 ggcggtggtg gttctttgga agcatctaaa gaccgtccgt ccctgaaggc ggcaatcccg
     1561 atgaccccgt ggaacactga taaaacctgg agcgagatcc gtactccgac cttgattttc
     1621 ggtgctgaaa acgattccgt agctccagtc gcatcccatt ccgaaccgtt ctactccacc
     1681 attccgtcca ccaccaacaa aatgtacatc gaattaaacg gcgcaagcca ctttgctccg
     1741 aactcctcta acacgaccat cgccaaatac agcatcagct ggctgaaacg tttcctggat
     1801 aacgacactc gttatgacca gttcctgtgc cctctgccta cttctgcgct gtacattgaa
     1861 gaatctcgcg gtacctgccc gctgcgtctc gagcaccacc accaccacca ctgagatccg
     1921 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     1981 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     2041 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     2101 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     2161 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     2221 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     2281 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     2341 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     2401 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     2461 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     2521 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     2581 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     2641 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     2701 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     2761 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     2821 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     2881 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     2941 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     3001 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     3061 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     3121 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     3181 cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     3241 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     3301 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     3361 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     3421 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     3481 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     3541 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     3601 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     3661 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     3721 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     3781 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     3841 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     3901 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     3961 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     4021 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     4081 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     4141 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     4201 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     4261 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     4321 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     4381 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     4441 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     4501 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     4561 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     4621 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     4681 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     4741 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     4801 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     4861 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     4921 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     4981 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     5041 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     5101 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     5161 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     5221 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     5281 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     5341 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     5401 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     5461 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     5521 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     5581 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     5641 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     5701 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5761 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     5821 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     5881 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     5941 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac
     6001 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     6061 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     6121 tcgccttccc gttccgctat cggctgaat
//
