LOCUS       sequence-349684-        6152 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 6152)
  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 6152)
  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: pEE063
FEATURES             Location/Qualifiers
     source          1..6152
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             15..37
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             840..857
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(910..928)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      924..971
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1008..1463
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1095..1114)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1305..1326
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1489..1593
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1594..1662,1663..2454)
                     /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(1812..1831)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2625..3213
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3114..3133
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3367..3384
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3399..3538
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3464..3483)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3624..3646
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3640..3831)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4640..5722)
                     /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     5684..5703
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5723..5800)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6006..6025
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6109..6128
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6109..6127
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6128..6152
                     /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)."
ORIGIN
        1 cctctagaaa taattttgtt taactttaag aaggagatat acatatggct gcaaacccgt
       61 atgaacgcgg tccgaatccg accgacgcac tgttagaagc gcgatctggt ccattctccg
      121 tatcagagga aaatgtgtcc cgtctgtccg cgtcgggctt cggcggtggc accatttact
      181 acccgcgtga aagtaacacc tatggcgctg tagctatctc cccgggctat actggtaccg
      241 aagcgtctat tgcatggctg ggtgaacgta tcgcatccca tggttttgta gttattacta
      301 ttgacaccat tactacgctg gatcaaccag actcacgtgc tgagcagctg aacgcagcgc
      361 tcaatcacat gattaaccgc gcatcgagca ccgtgcgttc tcgcatcgat agctctcgtc
      421 tggcggtgat gggtcactcc atgggtggcg gtggcacgct gcgtctggca agccagcgtc
      481 cggatctcaa agcagcgatt ccgctgactc catggcattt gaacaaaaac tggagctctg
      541 tgaccgtgcc gaccctgatc atcggcgccg atctggacac catcgcaccg gtggccactc
      601 atgccaaacc attctataac tccctgccgt catctatctc caaggcttac ctggaactgg
      661 acggtgcgac ccacttcgct ccaaacatcc cgaacaagat tatcggtaaa tattcagtag
      721 catggctgaa atggttcgtt gataacgata cccgttacac tcagttcctg tgtccgggtc
      781 cgcgcgacgg tctgttcggc gaagtggaag agtaccgttc gacctgtccg tttctcgagc
      841 accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
      901 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
      961 ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag
     1021 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     1081 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     1141 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     1201 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     1261 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     1321 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     1381 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     1441 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     1501 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     1561 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     1621 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     1681 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     1741 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     1801 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     1861 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     1921 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     1981 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     2041 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     2101 gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc
     2161 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     2221 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     2281 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     2341 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     2401 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     2461 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     2521 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     2581 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     2641 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     2701 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     2761 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     2821 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     2881 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     2941 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3001 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     3061 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     3121 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     3181 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     3241 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     3301 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     3361 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     3421 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg
     3481 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     3541 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     3601 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     3661 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca
     3721 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     3781 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     3841 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     3901 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     3961 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     4021 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     4081 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
     4141 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
     4201 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
     4261 ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat
     4321 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc
     4381 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg
     4441 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa
     4501 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac
     4561 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     4621 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     4681 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     4741 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     4801 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     4861 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     4921 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     4981 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     5041 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     5101 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     5161 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     5221 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     5281 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     5341 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     5401 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     5461 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     5521 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     5581 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     5641 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     5701 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     5761 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     5821 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     5881 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     5941 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     6001 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
     6061 gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca
     6121 ctatagggga attgtgagcg gataacaatt cc
//
