LOCUS       sequence-342608-        6263 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 6263)
  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 6263)
  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: pEE020
FEATURES             Location/Qualifiers
     source          1..6263
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             904..921
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(974..992)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      988..1035
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1072..1527
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1159..1178)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1369..1390
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1553..1657
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1658..1726,1727..2518)
                     /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(1876..1895)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2689..3277
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3178..3197
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3431..3448
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3463..3602
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3528..3547)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3688..3710
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3704..3895)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4704..5786)
                     /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     5748..5767
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5787..5864)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6070..6089
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6173..6192
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6173..6191
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6192..6216
                     /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             6231..6253
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 ctgactatca cagcgatcat ctacctcctg gccaccatta tcgcagccat cctccttgtt
       61 gtatatatta tttcctcctc cgcgtctaaa aaactggcca cgccgccgcg aaaaaccggt
      121 tcctggtcac cgcgcgatct gggtttcgag tacgagaaag tggaagtgaa aacctccgat
      181 ggactgactc tgcgtggctg gctgattccg cgcggctccg aaaaaaccgt catcgtgatc
      241 cacggttaca cttcttgcaa gtgggatgaa tggtatatga agccggttat caacatcctg
      301 gcacgtcacg atttcaacgt cgttgcattt gatatgcgtg cgcacggcga atctgatggc
      361 gaaaagacta ccctgggcta ccgtgaggta gatgacatcg gcgcaattat taattacctg
      421 aaagaacgtg gcctggcaag tcgcctgggc atcattggct actcgatggg cggtgctatc
      481 actctgatgt ccctggcgcg ctacgaggaa ctgaaagcgg gtgtggccga ctcgccgtac
      541 atcgacattc gtgcgtcggg taaacgttgg attaatcgcg ttggcgctcc tctgcgctac
      601 atcctcctgg catcgtaccc gctgatcatg cgtcttactg cctcccgtac tggcgcatct
      661 ccggaaaaac tggttatgta ccaatatgcc aagtcgatca ccaaaccact gcttatcatt
      721 ggtggtcagc aggacgacct ggtggccatc gacgaagtac gtaaattcta tgaagaagtt
      781 aaaaaagtga actccaatgt cgaactgtgg gaaaccacct ccaaacacgt ttctgccatc
      841 caagattacc cgcgtgaata cgaagagcgt atcgtcggct tcttcaaccg ctggctgctc
      901 gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag
      961 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     1021 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
     1081 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     1141 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     1201 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     1261 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     1321 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     1381 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     1441 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     1501 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
     1561 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     1621 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     1681 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     1741 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     1801 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     1861 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     1921 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     1981 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     2041 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     2101 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     2161 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt
     2221 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     2281 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     2341 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     2401 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     2461 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     2521 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     2581 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     2641 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     2701 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     2761 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     2821 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     2881 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     2941 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     3001 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     3061 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     3121 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     3181 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     3241 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     3301 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     3361 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     3421 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     3481 ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct
     3541 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     3601 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     3661 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     3721 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     3781 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     3841 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     3901 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     3961 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     4021 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     4081 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     4141 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     4201 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     4261 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     4321 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga
     4381 taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga
     4441 gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa
     4501 agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga
     4561 taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc
     4621 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     4681 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4741 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     4801 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     4861 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     4921 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     4981 gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     5041 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     5101 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     5161 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     5221 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     5281 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     5341 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     5401 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     5461 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     5521 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     5581 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     5641 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     5701 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     5761 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     5821 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     5881 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     5941 cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg
     6001 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc
     6061 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
     6121 gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga
     6181 ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac
     6241 tttaagaagg agatatacat atg
//
