LOCUS       sequence-349240-        5897 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 5897)
  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 5897)
  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: pEE030
FEATURES             Location/Qualifiers
     source          1..5897
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     958..977
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(997..1074)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1280..1299
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1383..1402
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1383..1401
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1402..1426
                     /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             1441..1463
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2011..2028
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2081..2099)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2095..2142
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2179..2634
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2266..2285)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2476..2497
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2660..2764
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2765..2833,2834..3625)
                     /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(2983..3002)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3796..4384
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4285..4304
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4538..4555
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4570..4709
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4635..4654)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4795..4817
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4811..5002)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5811..5897,1..996))
                     /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 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
       61 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
      121 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
      181 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
      241 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
      301 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
      361 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
      421 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
      481 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
      541 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
      601 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
      661 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
      721 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
      781 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
      841 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
      901 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
      961 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     1021 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     1081 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     1141 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     1201 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     1261 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     1321 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa
     1381 attaatacga ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat
     1441 tttgtttaac tttaagaagg agatatacat atgcaaaccg tgaccagcat gctgaaagac
     1501 ctggacgcgg taattactca ggtttcagaa aaatttccgc agattgacaa caagcgcgtc
     1561 tgtctgatcg gtcactctca gggtgcgtac gtatccttcc tgcatgcgac caaagatgaa
     1621 cgtattaaat gcctggtctc ctggatgggt cgtctgtcgg acctgaaaga attttggtct
     1681 aagctgtggt tcgacgagat cgaacgcaaa ggctatatct acgagtggga ttacaaaatc
     1741 accaagaaat atgtgcgtga tagcctgaaa tacaatctgt caaaagctgc atggcgtatc
     1801 aaagtgccga ccctgctgat ttatggtgaa ctggacgata tcgtgccacc ttctgaaggt
     1861 atgaaattct accgcaacat caaatctccg aaaaaaatcg ttattgtaaa ggatctgaac
     1921 cacaccttct ctggtgaaaa agccaaaaaa tccgttatcc gcatcactct gaaatggctg
     1981 tctaaatggc tcaagcgcct ggacctcgag caccaccacc accaccactg agatccggct
     2041 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
     2101 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
     2161 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     2221 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     2281 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     2341 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     2401 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     2461 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     2521 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     2581 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     2641 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     2701 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     2761 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     2821 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     2881 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     2941 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     3001 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     3061 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     3121 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     3181 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     3241 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     3301 gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     3361 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     3421 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     3481 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     3541 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     3601 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     3661 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     3721 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     3781 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     3841 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     3901 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     3961 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     4021 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     4081 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     4141 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     4201 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     4261 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     4321 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     4381 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     4441 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     4501 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     4561 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa
     4621 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     4681 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     4741 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     4801 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     4861 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     4921 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     4981 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
     5041 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
     5101 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga
     5161 gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg
     5221 gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc
     5281 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg
     5341 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct
     5401 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc
     5461 gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg
     5521 cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca
     5581 ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg
     5641 ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag
     5701 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     5761 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     5821 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     5881 gaggcggttt gcgtatt
//
