LOCUS       sequence-349632-        6215 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 6215)
  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 6215)
  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: pEE031
FEATURES             Location/Qualifiers
     source          1..6215
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..25
                     /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             40..62
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             928..945
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(998..1016)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1012..1059
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1096..1551
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1183..1202)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1393..1414
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1577..1681
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1682..1750,1751..2542)
                     /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(1900..1919)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2713..3301
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3202..3221
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3455..3472
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3487..3626
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3552..3571)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3712..3734
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3728..3919)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4728..5810)
                     /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     5772..5791
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5811..5888)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6094..6113
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(6197..6215,1)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6197..6215
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
       61 gatatacata tgctgaaaat catcgaagat tttgcgagtc tggacaccgg tgttaaagtg
      121 ttctatcgtt gtatcctgcc ggaatctttc aaagaactgg cgatcgtgtc tcatggtttc
      181 acctcgcact ctggtttcta catccatatt ggcaaggaat tggcgtccta tggctatggt
      241 gtttgcatcc acgatcagcg cggtcacggt cgtaccgcgc agaacctgga acgtggctac
      301 gtagattctt ttaacgattt cctggtggac ctggaaactt tcactatgca tgttcagcgc
      361 gttttcggtg gtgaacgcac cgtcctaatc ggacacagca tgggcggtct gattgttctg
      421 ctgtatgcgg gcaaatacgg tcgcgttggc gacgccgtag ttgcagtggc tccggccgtt
      481 ctgattccgg aaacccgccg ttttagcact ctgatctttg ccaccattgc ctccatcctg
      541 tttccgcgta agcgtattga actcccgttt accgaacagc agattgagga aggtatgaaa
      601 cgtatggacc gtgagctcct ggaagcaatg ggtaaagatg aactggtact gcgtgacacc
      661 accattaaac tgctggttga aatctggaaa gcgtcgcgtg aattttggcg ctacgtggaa
      721 cgtatccaga tcccgaccct gctgatccac ggcgaaaaag acaatatcat cccgattgaa
      781 gctagccgcc gtacctattc tcgcctgaaa actctgaaaa aagagctgat cgtttacccg
      841 gaatgcggcc attctccgct gcatgaaatc ggttggcgtg aacgtatcaa aaacatggta
      901 gaatggatcc gcaacaacat cctcgagcac caccaccacc accactgaga tccggctgct
      961 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     1021 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     1081 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     1141 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     1201 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     1261 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     1321 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     1381 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     1441 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     1501 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     1561 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     1621 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     1681 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     1741 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     1801 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     1861 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     1921 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     1981 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     2041 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     2101 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     2161 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     2221 gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     2281 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     2341 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     2401 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     2461 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     2521 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     2581 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     2641 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     2701 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     2761 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     2821 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     2881 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     2941 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3001 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3061 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3121 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3181 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3241 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3301 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     3361 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     3421 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     3481 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     3541 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     3601 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     3661 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     3721 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     3781 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     3841 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     3901 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     3961 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     4021 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     4081 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     4141 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     4201 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     4261 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     4321 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     4381 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     4441 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     4501 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     4561 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     4621 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     4681 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     4741 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     4801 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
     4861 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
     4921 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
     4981 tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta
     5041 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
     5101 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
     5161 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
     5221 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
     5281 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
     5341 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
     5401 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
     5461 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
     5521 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
     5581 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
     5641 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
     5701 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
     5761 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc
     5821 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg
     5881 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag
     5941 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc
     6001 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat
     6061 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc
     6121 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat
     6181 ctcgatcccg cgaaattaat acgactcact atagg
//
