LOCUS       sequence-349691-        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: pEE068
FEATURES             Location/Qualifiers
     source          1..6149
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             800..817
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(870..888)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      884..931
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      968..1423
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1055..1074)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1265..1286
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1449..1553
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1554..1622,1623..2414)
                     /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(1772..1791)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2585..3173
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3074..3093
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3327..3344
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3359..3498
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3424..3443)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3584..3606
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3600..3791)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4600..5682)
                     /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     5644..5663
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5683..5760)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5966..5985
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6069..6088
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6069..6087
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6088..6112
                     /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             6127..6149
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 tatacatatg gctaacccat acgaacgtgg tccgaaccca actgacgcac tgctggaagc
       61 tagctccggc ccgttttccg tgtccgaaga aaacgtttct cgtctgagcg cttccggctt
      121 cggcggtggc accatctact atccgcgtga gaacaacact tatggcgccg tagccatctc
      181 cccgggctac accggtactg aagcttcaat cgcatggctg ggtggtcgta tcgccagcca
      241 cggtttcgtt gttatcacca tcgatactat caccacactg gatcagccgg attctcgcgc
      301 tgaacagctg aacgcggccc tgaaccacat gattaatcgc gcctcctcca ctgttcgttc
      361 ccgcattgat tcctcccgcc tggcggtgat gggccatagt atgggtggtg gcggaacccc
      421 gcgtctggcc tctcagcgcc cggatctgaa agcggcgatt cctctgaccc cgtggcacct
      481 gaacaaaaac cgttcctctg tgactgttcc gaccctgatc atcggcgcgg acctggatac
      541 catcgctccg gtagccactc acgctaaacc gttctacaat agcctgccgt ctagcatctc
      601 caaagcttac ctagagctgg atggtgcgac ccactttgcg ccgaacattc cgaataaaat
      661 catcggtaaa tatagcgttg catggctgaa acgtttcgtt gataacgata cccgctacac
      721 ccagtttctg tgtccgggcc cacgtgacgg tctgttcggc gaagtcgaag aatattgctc
      781 tacctgcccg ttcctcgagc accaccacca ccaccactga gatccggctg ctaacaaagc
      841 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
      901 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc
      961 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     1021 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     1081 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     1141 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     1201 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     1261 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     1321 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     1381 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     1441 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     1501 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     1561 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     1621 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     1681 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     1741 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     1801 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     1861 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     1921 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     1981 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     2041 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag
     2101 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     2161 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     2221 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     2281 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     2341 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     2401 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     2461 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     2521 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     2581 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     2641 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     2701 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     2761 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     2821 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     2881 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     2941 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     3001 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     3061 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     3121 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     3181 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     3241 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     3301 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     3361 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct
     3421 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     3481 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     3541 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     3601 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
     3661 tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
     3721 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
     3781 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
     3841 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
     3901 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
     3961 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
     4021 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
     4081 ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg
     4141 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg
     4201 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg
     4261 gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg
     4321 acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc
     4381 gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt
     4441 cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc
     4501 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     4561 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     4621 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     4681 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     4741 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     4801 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     4861 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
     4921 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     4981 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     5041 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     5101 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     5161 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     5221 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     5281 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     5341 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     5401 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     5461 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     5521 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     5581 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     5641 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     5701 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     5761 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
     5821 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
     5881 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
     5941 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
     6001 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc
     6061 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
     6121 aataattttg tttaacttta agaaggaga
//
