LOCUS       sequence-349682-        6152 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 6152)
  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 6152)
  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: pEE051
FEATURES             Location/Qualifiers
     source          1..6152
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             803..820
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(873..891)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      887..934
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      971..1426
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1058..1077)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1268..1289
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1452..1556
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1557..1625,1626..2417)
                     /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(1775..1794)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2588..3176
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3077..3096
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3330..3347
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3362..3501
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3427..3446)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3587..3609
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3603..3794)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4603..5685)
                     /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     5647..5666
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5686..5763)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5969..5988
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6072..6091
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6072..6090
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6091..6115
                     /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             6130..6152
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 tatacatatg gctgctaacc cgtatcagcg tggcccgaac cccactgagg cgagcatcac
       61 tgccgcgcgc ggtccattca atactgcgga aattaccgtt tctcgcctgt ccgtatccgg
      121 tttcggtggt ggcaaaatct actatccaac gaccacctcg gaaggtacct tcggtgctat
      181 cgcaatttct ccgggtttca ccgcatactg gagctctctc gaatggctgg gccaccgtct
      241 ggctagccag ggctttgttg taatcggtat cgaaactaac actactttag accagccgga
      301 ccagcgtggc cagcagctgc tcgctgcgct ggactatctg acccagcgct cagcagttcg
      361 tgatcgtgtt gatgcatctc gtctggcggt agcgggtcat tcgatgggcg gtggtggttc
      421 tctggaagct gcaaaagctc gtacgagtct gaaagcggcg attcctctgg caccctggaa
      481 cctggacaaa acttggccgg aggtgcgcac tccgaccctt attattggtg gtgaactgga
      541 cgccgtcgcg ccggtggcga cccactctat cccgttctac aacagcctga gcaacgctcc
      601 ggagaaagcc tacctcgaac tggataacgc gtctcacttc tttccgaata ttaccaacac
      661 tcagatggcg aaatacatga tcgcatggat gaaacgtttc atcgatgacg atacccgtta
      721 cacccagttc ctgtgcccgc ctccgtctac cggcctgctg agcgactttt cagatgcacg
      781 ttttacatgc ccgatgctcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa
      841 agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag cataacccct
      901 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt
      961 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     1021 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     1081 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     1141 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     1201 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     1261 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     1321 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     1381 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc
     1441 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     1501 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     1561 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     1621 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     1681 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     1741 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     1801 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     1861 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     1921 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     1981 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     2041 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     2101 cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     2161 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     2221 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     2281 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     2341 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     2401 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     2461 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     2521 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     2581 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     2641 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     2701 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     2761 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     2821 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     2881 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     2941 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     3001 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     3061 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     3121 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     3181 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     3241 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     3301 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     3361 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac
     3421 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     3481 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     3541 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     3601 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     3661 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     3721 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     3781 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     3841 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     3901 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     3961 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     4021 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     4081 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     4141 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     4201 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt
     4261 ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca
     4321 gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc
     4381 atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc
     4441 tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc
     4501 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc
     4561 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag
     4621 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4681 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt
     4741 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
     4801 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
     4861 atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc
     4921 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
     4981 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
     5041 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
     5101 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
     5161 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat
     5221 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
     5281 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc
     5341 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
     5401 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
     5461 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
     5521 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
     5581 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga
     5641 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa
     5701 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt
     5761 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta
     5821 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca
     5881 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc
     5941 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
     6001 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga
     6061 tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct
     6121 agaaataatt ttgtttaact ttaagaagga ga
//
