LOCUS       sequence-342592-        6089 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 6089)
  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 6089)
  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: pEE012
FEATURES             Location/Qualifiers
     source          1..6089
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             730..747
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(800..818)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      814..861
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      898..1353
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(985..1004)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1195..1216
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1379..1483
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1484..1552,1553..2344)
                     /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(1702..1721)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2515..3103
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3004..3023
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3257..3274
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3289..3428
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3354..3373)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3514..3536
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3530..3721)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4530..5612)
                     /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     5574..5593
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5613..5690)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5896..5915
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5999..6018
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5999..6017
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6018..6042
                     /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             6057..6079
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 gtgccaagcg cgggtgtagg tctttctggc gtcctccatc tgccggctgg cgtttcccgc
       61 ccggtgctgt tcctgcatgg tttcacgggc aacaagacgg aaagcggtcg tttgtacacc
      121 gacatggcgc gcgttctgtg ttctgcgggc tacgcagcct tgcgtttcga ttttcgtggt
      181 cacggtgata gccctctgcc attcgaggaa tttcgtatca gtctggcagt tgaagacgcc
      241 cgtaacgcgg ccggtttcct gaaaaacgta ccggaagtgg acggaactcg ctttggtgta
      301 gtgggcttgt ctatgggtgg cggcgtggca gtgagcctgg cggctggtcg cgaagacgtt
      361 ggtgcgctcg tgctgctttc tccggctctg gattggcctg aactcttcca gcgtgcgcgt
      421 ggcttctttc gtgcggaaga gggctacgtg tactggggcc cgcaccgtat gcgcgatgtt
      481 tacgctatgg aaaccatgaa cttctctgta atgggcctgg ccgaagaaat ccaagcgccg
      541 actctgatca tccactctgt tgatgacatg gttgttccga ttagtcaagc caaacgcttc
      601 tatgaaaaac tgaaagtaga aaaaaagttt atcgagatcg aacacggtgg tcacgttttt
      661 gatgactaca acgtgcgtcg ccgtatcgag caggaggttc tcgactgggt gaaacgccac
      721 ctgctcgagc accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa
      781 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
      841 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg
      901 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
      961 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     1021 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     1081 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     1141 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     1201 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     1261 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     1321 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     1381 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     1441 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     1501 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     1561 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     1621 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     1681 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     1741 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     1801 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     1861 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     1921 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     1981 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac
     2041 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     2101 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     2161 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     2221 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     2281 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     2341 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     2401 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     2461 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     2521 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     2581 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     2641 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     2701 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     2761 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     2821 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     2881 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     2941 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     3001 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     3061 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     3121 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     3181 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     3241 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     3301 ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc
     3361 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     3421 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     3481 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     3541 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa
     3601 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
     3661 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
     3721 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
     3781 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
     3841 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
     3901 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga
     3961 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa
     4021 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt
     4081 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc
     4141 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc
     4201 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
     4261 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
     4321 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
     4381 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
     4441 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     4501 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4561 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4621 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     4681 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     4741 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     4801 actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     4861 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     4921 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     4981 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     5041 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     5101 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     5161 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     5221 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     5281 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     5341 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     5401 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     5461 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     5521 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     5581 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     5641 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     5701 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     5761 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
     5821 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
     5881 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
     5941 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta
     6001 atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg
     6061 tttaacttta agaaggagat atacatatg
//
