LOCUS       sequence-349241-        6155 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 6155)
  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 6155)
  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: pEE019
FEATURES             Location/Qualifiers
     source          1..6155
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             806..823
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(876..894)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      890..937
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      974..1429
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1061..1080)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1271..1292
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1455..1559
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1560..1628,1629..2420)
                     /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(1778..1797)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2591..3179
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3080..3099
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3333..3350
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3365..3504
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3430..3449)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3590..3612
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3606..3797)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4606..5688)
                     /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     5650..5669
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5689..5766)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5972..5991
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6075..6094
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6075..6093
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6094..6118
                     /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             6133..6155
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 tatacatatg ctgattcgtc cggttacctt ccgcaatatg aaccagcaga ttattggcat
       61 ccttcacact ccggacaaca tccgtctgaa tgaaaaagta ccgggtatcc tgatgttcca
      121 tggcttcact ggtaataaaa ctgaagcgca ccgcctgttt gtgcacgtgg ctcgttctct
      181 gtccgaacat ggtttcatcg tgctgcgttt cgacttccgc ggaagcggtg atagcgatgg
      241 tgaattcgaa gacatgaccc tgccgggtga agttagcgac gcagagcgcg cgctgacctt
      301 tctgttgcgc cagcgtaacg ttgataaaaa ccgtattggt gtaatcggtc tgtccatggg
      361 tggccgtgtt gcggcgattc tggcaagcaa ggaccggcgc gttaaattcg ctgtcctgta
      421 cagcccggcg ctgggtccgc tgcgcgatcg ttctctgtct ttcatgagca aagaaaaaat
      481 tgaacgtctg aactccggtg aggcagtgga attcttcgct gaaggttggt atatcaaaaa
      541 agcattcttt gagaccgtgg actatattgt cccgctggac atcatggatt ccattaaagt
      601 tccggttttg atcgttcatg gcgacaaaga cccgctcatt ccggttggtg aggctatccg
      661 tgcatacgaa aaaatcaaag gtgttaacga gaaaaatgag ctgtacattg tacgtggcgg
      721 tgatcacacc ttctccaaaa aagaacacac cctggaggta atcaagaaaa ctttggactg
      781 gatccgtagc ctgggcattc tcgagcacca ccaccaccac cactgagatc cggctgctaa
      841 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
      901 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
      961 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     1021 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     1081 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     1141 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     1201 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     1261 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     1321 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     1381 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     1441 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     1501 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     1561 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     1621 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     1681 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     1741 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     1801 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     1861 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     1921 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     1981 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     2041 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     2101 ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     2161 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     2221 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     2281 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     2341 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     2401 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     2461 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     2521 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     2581 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     2641 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2701 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     2761 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     2821 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     2881 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     2941 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     3001 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     3061 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     3121 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     3181 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     3241 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     3301 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     3361 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
     3421 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     3481 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     3541 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     3601 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     3661 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     3721 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     3781 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     3841 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     3901 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     3961 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     4021 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     4081 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     4141 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     4201 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     4261 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
     4321 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     4381 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
     4441 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     4501 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     4561 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     4621 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4681 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     4741 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     4801 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     4861 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     4921 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     4981 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     5041 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     5101 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     5161 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     5221 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     5281 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     5341 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     5401 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     5461 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     5521 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     5581 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     5641 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     5701 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     5761 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     5821 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     5881 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     5941 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     6001 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     6061 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     6121 tctagaaata attttgttta actttaagaa ggaga
//
