LOCUS       sequence-349689-        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: pEE069
FEATURES             Location/Qualifiers
     source          1..6149
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1291..1482
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1476..1498)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1584..1723
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1639..1658
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1738..1755)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1909..2497)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1989..2008)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2668..3459,3460..3528))
                     /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     3291..3310
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3529..3633)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3659..4114)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3796..3817)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4008..4027
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4151..4198)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4194..4212
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4265..4282)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             5082..5104
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    5119..5143
                     /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)."
     primer_bind     complement(5143..5162)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5144..5162)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5246..5265)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5471..5548
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5549..6149,1..482)
                     /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     complement(5568..5587)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga
       61 gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg
      121 cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg
      181 ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa gacagctcat
      241 gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg
      301 tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg
      361 tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg
      421 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
      481 gagcgcaacg caattaatgt aagttagctc actcattagg caccgggatc tcgaccgatg
      541 cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc
      601 gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc
      661 tgggtcattt tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt
      721 gcggtattcg gaatcttgca cgccctcgct caagccttcg tcactggtcc cgccaccaaa
      781 cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg ccccacgggt gcgcatgatc
      841 gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat
      901 gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga
      961 gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca
     1021 gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga
     1081 acacctacat ctgtattaac gaagcgctgg cattgaccct gagtgatttt tctctggtcc
     1141 cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca
     1201 tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca tcggtatcat tacccccatg
     1261 aacagaaatc ccccttacac ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac
     1321 atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac
     1381 gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc
     1441 agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     1501 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     1561 gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg
     1621 tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac cattgcggtg
     1681 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc
     1741 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     1801 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     1861 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     1921 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     1981 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     2041 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     2101 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     2161 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     2221 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     2281 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     2341 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     2401 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     2461 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     2521 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     2581 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     2641 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     2701 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     2761 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     2821 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     2881 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     2941 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc
     3001 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     3061 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     3121 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     3181 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     3241 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     3301 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     3361 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     3421 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     3481 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     3541 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     3601 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa
     3661 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     3721 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     3781 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     3841 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     3901 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     3961 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     4021 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     4081 cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgccaatccg gatatagttc
     4141 ctcctttcag caaaaaaccc ctcaagaccc gtttagaggc cccaaggggt tatgctagtt
     4201 attgctcagc ggtggcagca gccaactcag cttcctttcg ggctttgtta gcagccggat
     4261 ctcagtggtg gtggtggtgg tgctcgagga acgggcaagt agaacggtac tcgtcaacat
     4321 cgctcagcag gccggtgcgc ggacccgggc acaggaactg agtgtagcgg gtatcctcgt
     4381 caacgaaacg tttcagccag gccacactgt acataccgat cgttttgttg gtaatgttcg
     4441 gcgcgaaatg ggtggcgtta ttcagttcaa ggtacgcttt gtcagtggag ctcgggatgg
     4501 agttataaaa tggttcgcta tggctggaaa ccggagcaat ggtgtccagg tccgcaccga
     4561 tgatcagggt cggaacggtg atatcacgcc aggacttgtt caggtgccac ggagtgagtg
     4621 gaatcgctgc tttcaggtct gggcgctgac ttgccagacg cagcgtgcca ccgccgccca
     4681 tggaatggcc cattaccgcc agacgagaag cgtcgatacg gttacgaacg gaggaagatg
     4741 catcggtcag catgtaatcc aaagcagcgt tcagctgacg agcacgagag tccggctggt
     4801 caagggtggt gttggtgtcg atcgcgatta ccacaaagcc gtggcttgca atacgttcgc
     4861 ccagccacgc aatggaagac tgagtaccgg tataaccagg ggaaattgca atagcaccat
     4921 acgtgttgtt ttcacgcgga taatagatgg taccgccgcc gaagccatcc gcacccagac
     4981 gagatgcacg ttcttcgctt acagaaaacg ggccgctacg agcctcgagc atgctttccg
     5041 tcgggttcgg accacgctca tacgggttag ccatatgtat atctccttct taaagttaaa
     5101 caaaattatt tctagagggg aattgttatc cgctcacaat tcccctatag tgagtcgtat
     5161 taatttcgcg ggatcgagat ctcgatcctc tacgccggac gcatcgtggc cggcatcacc
     5221 ggcgccacag gtgcggttgc tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg
     5281 gctcgccact tcgggctcat gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg
     5341 gccgggggac tgttgggcgc catctccttg catgcaccat tccttgcggc ggcggtgctc
     5401 aacggcctca acctactact gggctgcttc ctaatgcagg agtcgcataa gggagagcgt
     5461 cgagatcccg gacaccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc
     5521 ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga
     5581 gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc
     5641 tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg
     5701 cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct
     5761 ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg
     5821 tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt
     5881 gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca
     5941 ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc
     6001 tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt
     6061 ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc
     6121 tgtctcggcg cgtctgcgtc tggctggct
//
