LOCUS       sequence-349072-        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: pEE075
FEATURES             Location/Qualifiers
     source          1..6152
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     562..581
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(601..678)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     884..903
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     987..1006
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        987..1005
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1006..1030
                     /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             1045..1067
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1870..1887
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1940..1958)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1954..2001
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2038..2493
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2125..2144)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2335..2356
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2519..2623
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2624..2692,2693..3484)
                     /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(2842..2861)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3655..4243
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4144..4163
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4397..4414
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4429..4568
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4494..4513)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4654..4676
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4670..4861)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5670..6152,1..600))
                     /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"
ORIGIN
        1 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
       61 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
      121 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
      181 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
      241 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
      301 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
      361 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
      421 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
      481 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
      541 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
      601 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
      661 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
      721 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
      781 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
      841 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
      901 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
      961 ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
     1021 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat acatatggcg
     1081 aacccttacg aacgcggtcc gaacccgacc gatgccctgc tcgaagctcg ctcgggcccg
     1141 ttctctgtct ccgaagaaaa cgtgagccgt ctgtcggctt ccggctttgg cggtggcaca
     1201 atttactatc ctcgcgagaa caacacctac ggtgctgttg cgatctctcc gggctatact
     1261 ggtacagagg cttccatcgc ctggctgggc gagcgcatcg cttctcacgg tttcgttgtc
     1321 attaccatcg atactattac caccctggac cagccggact cgcgtgctga acagcttaat
     1381 gcagcgctta accatatgat caatcgtgct tcgtcaaccg ttcgcagccg tatcgattct
     1441 tctcgtctgg cggtgatggg tcattctatg ggtggcggtg gttcgctccg tctggccagc
     1501 cagcgcccgg atctgaaagc ggcaatcccg ctgactccgt ggcatctgaa caaaaactgg
     1561 tcttcggtta ccgtgccgac cctgattatc ggtgcagacc tggacacgat tgcaccggtt
     1621 gcgactcacg caaaaccgtt ctacaactcc ctgccgtctt ctatttctaa ggcatacctt
     1681 gaactggacg gtgcaaccca tttcgctccg aacattccga acaaaatcat cggtaaatac
     1741 agcgtggcct ggctgaaacg ttttgttgac aacgacaccc gttacacaca gttcctgtgc
     1801 ccgggtccgc gtgacggtct tttcggcgag gtggaagaat atcgtagcac ctgtccattc
     1861 tacctcgagc accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa
     1921 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     1981 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg
     2041 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     2101 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     2161 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     2221 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     2281 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     2341 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     2401 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     2461 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     2521 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     2581 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     2641 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     2701 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     2761 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     2821 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     2881 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     2941 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     3001 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     3061 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     3121 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac
     3181 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     3241 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     3301 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     3361 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     3421 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     3481 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     3541 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     3601 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3661 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     3721 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     3781 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     3841 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     3901 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     3961 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     4021 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     4081 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     4141 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     4201 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     4261 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     4321 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     4381 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     4441 ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc
     4501 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     4561 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     4621 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     4681 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa
     4741 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
     4801 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
     4861 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
     4921 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
     4981 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
     5041 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga
     5101 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa
     5161 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt
     5221 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc
     5281 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc
     5341 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
     5401 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
     5461 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
     5521 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
     5581 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     5641 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     5701 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     5761 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     5821 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     5881 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     5941 actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     6001 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     6061 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     6121 tgaatttgat tgcgagtgag atatttatgc ca
//
