LOCUS       sequence-349242-        6398 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 6398)
  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 6398)
  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: pEE017
FEATURES             Location/Qualifiers
     source          1..6398
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     549..568
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(588..665)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     871..890
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     974..993
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        974..992
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    993..1017
                     /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             1032..1054
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2103..2120
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2173..2191)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2187..2234
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2271..2726
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2358..2377)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2568..2589
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2752..2856
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2857..2925,2926..3717)
                     /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(3075..3094)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3888..4476
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4377..4396
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4630..4647
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4662..4801
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4727..4746)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4887..4909
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4903..5094)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5903..6398,1..587))
                     /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 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
       61 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
      121 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
      181 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
      241 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
      301 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
      361 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
      421 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
      481 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
      541 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
      601 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
      661 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
      721 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
      781 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
      841 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
      901 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc
      961 gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa caattcccct
     1021 ctagaaataa ttttgtttaa ctttaagaag gagatataca tatggttgac atcaccggca
     1081 acggcatggc ggccacggcc cccaccgacg aacgcattgt tgataaaccg ctgcctcagc
     1141 cgcaaattcg ctctggtaat gtgcgtgcaa tgccggctgc acgtaaactg gcacaggaac
     1201 atggcatcga cctgtctacc ctgactggct ctggcccggg tggcgtgatc gtaaaagaag
     1261 atgttgaacg tgcgatcacc gctcgtgccg ttccggtttc tccgctgcag cgtgtgaact
     1321 tctattctgc aggctaccgt ctcgatggtc tgctgtatac tccgcgtcac ctgccagcgg
     1381 gcgaacgtcg cccgggtgtc gtcctgctgg tgggttacac gtaccttaaa accatggtga
     1441 tgccagatat cgccaaagta ttaaatgcgg cgggctatgt tgcactggtt tttgactacc
     1501 gtggctttgg tgaatctgaa gggccgcgtg gccgcctgat cccgctggaa caagtggcag
     1561 acgcgcgtgc ggcgttgact ttccttgcgg aacagtccat ggtagatccg gaccgtctgg
     1621 ctgtgatcgg catcagcctg ggtggtgcgc acgcgatcac taccgccgct ctggatcagc
     1681 gcgtgcgcgc agtcgtcgca cttgaaccgc ctggtcacgg cgctcgctgg ctgcgctcgc
     1741 tgcgtcgtca ctgggaatgg cgccagttcc tctctcgttt agcagaggat cgtcgtcagc
     1801 gcgttctgtc tggcggctct accatggttg atcctctgga aattgtcctg ccggatccag
     1861 agtcccaggc cttcctggat caggttgccg cagaattccc gcagatgaaa gtgaccctgc
     1921 cgctggagtc cgcggaagcg ctgatcgaat atgttagcga agacctggct ggacgtattg
     1981 ctccgcgccc gctgctgatc atccacgggg acgcagatca gctcgttccg gttgctgagg
     2041 cccaagctat tgccgaacgt gcaggcagca gtgcgcagct ggaaatcatc ccaggtctcg
     2101 agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt
     2161 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     2221 tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg
     2281 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     2341 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     2401 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     2461 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     2521 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     2581 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     2641 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     2701 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     2761 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     2821 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     2881 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     2941 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     3001 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     3061 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     3121 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     3181 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     3241 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     3301 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     3361 gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg
     3421 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     3481 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     3541 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     3601 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     3661 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     3721 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     3781 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     3841 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     3901 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     3961 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4021 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4081 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4141 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4201 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4261 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4321 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4381 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4441 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4501 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     4561 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     4621 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     4681 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     4741 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     4801 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     4861 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     4921 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     4981 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     5041 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     5101 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     5161 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     5221 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     5281 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     5341 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     5401 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     5461 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     5521 ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat
     5581 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag
     5641 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa
     5701 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
     5761 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
     5821 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     5881 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     5941 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     6001 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     6061 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     6121 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     6181 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     6241 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     6301 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     6361 gattgcgagt gagatattta tgccagccag ccagacgc
//
