LOCUS       sequence-349246-        6326 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 6326)
  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 6326)
  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: pEE023
FEATURES             Location/Qualifiers
     source          1..6326
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             53..70
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(123..141)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      137..184
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      221..676
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(308..327)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     518..539
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        702..806
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(807..875,876..1667)
                     /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(1025..1044)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1838..2426
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2327..2346
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2580..2597
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2612..2751
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2677..2696)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2837..2859
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2853..3044)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3853..4935)
                     /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     4897..4916
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4936..5013)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5219..5238
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5322..5341
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5322..5340
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5341..5365
                     /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             5380..5402
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 acgccgctgg ctgcgttctc tgcgtgctga atggcagtgg cgtgttctcg agcaccacca
       61 ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
      121 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
      181 tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca
      241 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
      301 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
      361 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
      421 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
      481 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
      541 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
      601 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
      661 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
      721 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
      781 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
      841 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
      901 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
      961 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     1021 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     1081 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     1141 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     1201 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     1261 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     1321 caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat
     1381 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     1441 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     1501 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     1561 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     1621 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     1681 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     1741 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     1801 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     1861 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     1921 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     1981 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     2041 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     2101 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     2161 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     2221 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     2281 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     2341 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     2401 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     2461 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     2521 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     2581 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     2641 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     2701 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     2761 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     2821 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     2881 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     2941 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     3001 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     3061 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     3121 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     3181 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     3241 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     3301 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     3361 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     3421 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     3481 aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc
     3541 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag
     3601 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg
     3661 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     3721 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     3781 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     3841 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     3901 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     3961 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     4021 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     4081 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
     4141 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     4201 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     4261 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     4321 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     4381 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     4441 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     4501 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     4561 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     4621 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     4681 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     4741 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     4801 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     4861 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     4921 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     4981 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     5041 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     5101 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     5161 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     5221 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
     5281 tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg
     5341 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
     5401 gatatacata tggccacggt tctggtaatt ccgaaactcg gtctgacgat gactgaaggc
     5461 cgtgtcggcc gttggctgaa acagttaggc gagccggtac aggctggcga accagtgctt
     5521 gaagtagaga ccgaaaaact gaccgtagag gtcgaggccc cagctagcgg catcctggca
     5581 tatattctgg cggaagaagg cgttgttctg ccggtgactg ctccggtggc agtgatcgcc
     5641 gagccgggtg aagctgttga cctggcttct ctcctgccgg ctacttccgg tgcggcagca
     5701 accccggtta tggctgcatc cagcaccatg caagaacagg ctcgtgcaca gggtccaacc
     5761 ccgaccggcg aaatccgcgc aaccccggct gctcgtaagc tggcacgtga ccacggcatt
     5821 gacctggcac gtgttcgcgg caccggtcct ggtggccgta ttacggcgga ggatgtagaa
     5881 cgttacctgg ccagccaggg taccgcttgg cctcgcggtg aaccggttcg tttctggtca
     5941 gacggtctgg ctctggctgg tgaactgttc ttacctccga gtaccgatac ggcggttcct
     6001 ggtgttgtgc tatgcaccgg cattcagggc ctgaaagagc tcggtatgcc gctgctggcg
     6061 caggcactgg cagatgccgg gtacgctgct ctgatttttg attatcgcgg cttcggtgct
     6121 tccgagggtc cgcgtggccg tctgctgcca caggaacgca ttcgtgatgc acgtgcagcg
     6181 ctcactttcc tggaaactca cccacttatc gatcgtaccc gcctggccat cctgggcctg
     6241 tcgctcggtg gcgcacacgc gctttccctg gcggcaatcg acgatcgcgt tcaggcttgt
     6301 atcgcaatcg ccccgctgac caacgg
//
