LOCUS       sequence-342716-        5726 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 5726)
  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 5726)
  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: pEE074
FEATURES             Location/Qualifiers
     source          1..5726
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             367..384
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(437..455)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      451..498
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      535..990
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(622..641)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     832..853
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1016..1120
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1121..1189,1190..1981)
                     /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(1339..1358)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2152..2740
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2641..2660
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2894..2911
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2926..3065
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2991..3010)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3151..3173
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3167..3358)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4167..5249)
                     /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     5211..5230
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5250..5327)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5533..5552
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5636..5655
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5636..5654
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5655..5679
                     /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             5694..5716
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 gctaacccgt acgaacgtgg tccgaacccg accaactctt cgatcgaagc gctgcgtggt
       61 ccgttccgtg tcgatgaaga acgtgtttcc cgtctgcagg cgcgtggttt tggcggtggc
      121 actatctact atccgactga taacaacacc ttcggcgctg tggctattag cccgggttat
      181 actggcaccc agtccagcat ctcctggctg ggtgaacgtc tggcgtcaca cggttttgtg
      241 gtgatgacca tcgataccaa cactactctg gatcagccgg actctcgcgc gtctcagctg
      301 gacgcagcgc tggattacat ggttgaggat tcttcgtact ccgtacgtaa ccgcatcgat
      361 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
      421 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
      481 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
      541 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
      601 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
      661 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
      721 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
      781 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
      841 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
      901 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
      961 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     1021 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     1081 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     1141 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     1201 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     1261 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     1321 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     1381 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     1441 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     1501 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     1561 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     1621 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac
     1681 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     1741 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     1801 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     1861 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     1921 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     1981 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     2041 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     2101 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     2161 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     2221 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     2281 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     2341 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     2401 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     2461 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     2521 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     2581 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     2641 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     2701 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     2761 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     2821 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     2881 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     2941 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
     3001 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     3061 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     3121 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     3181 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     3241 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     3301 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     3361 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     3421 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     3481 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     3541 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     3601 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     3661 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     3721 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     3781 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg
     3841 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     3901 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     3961 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     4021 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     4081 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     4141 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4201 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     4261 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     4321 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     4381 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     4441 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     4501 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     4561 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     4621 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     4681 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     4741 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     4801 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     4861 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     4921 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     4981 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     5041 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     5101 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     5161 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     5221 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     5281 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     5341 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     5401 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     5461 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     5521 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     5581 gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata
     5641 cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt
     5701 aactttaaga aggagatata catatg
//
