LOCUS       sequence-349671-        6212 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 6212)
  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 6212)
  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: pEE041
FEATURES             Location/Qualifiers
     source          1..6212
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             15..37
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             900..917
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(970..988)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      984..1031
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1068..1523
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1155..1174)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1365..1386
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1549..1653
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1654..1722,1723..2514)
                     /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(1872..1891)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2685..3273
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3174..3193
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3427..3444
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3459..3598
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3524..3543)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3684..3706
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3700..3891)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4700..5782)
                     /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     5744..5763
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5783..5860)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6066..6085
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6169..6188
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6169..6187
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6188..6212
                     /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)."
ORIGIN
        1 cctctagaaa taattttgtt taactttaag aaggagatat acatatgtcc caagtcccgc
       61 caacggatcc tcaggacgcg ccgttgggcg aatgccctgc taccgccttg tgtcgttcag
      121 aagcgccggg ttcttacagc ggcaacggtc cgtacggtta tcgcagctat tccctgtctc
      181 gtctgcaaac cccgggcggc gcaaccgttt attatccggc aaacgcggag ccaccgtact
      241 cgggtctcgt tttcacgccg ccgtacaccg gcgtgcaatt catgtacgcc gcgtggggtc
      301 cgttttttgc gtcccacggc atcgtactgg tgactatgga taccactact accctggaca
      361 ctgttgatca acgcgcacgt caacagaaaa ctgtactgga tgttctgaaa ggcgaaaaca
      421 atcgtgcagc atcgccgctg cgcggtaaac tggatacctc acgtattggt gctgttggct
      481 ggtccatggg tggaggcgcg acctggatca atgcagctga atatgcaggt ctgaaaaccg
      541 cgatgtcttt ggctggccat aacctgtccg ctatcgatcc gaatgcgcgt ggctacaaca
      601 ctcgcgtgcc gaccttactg ttcaacggtg cactggacgc gacctacctg ggcggtctgg
      661 gtcagagcga tggggtgtat aatgcaatcc cggcgggcat ccctaaggta ttctacgaag
      721 ttgccagcgc ggggcatttc gattggggtt cccctaccgc cgctaaccgt gatgtagcgg
      781 gtattgcact ggcgttccac aaagcattcc tggacggcga cacccgctgg gtcgattaca
      841 tccgccgccc ttctcgtgac gttgcaactt ggcgcaccgc atacctgcca gacctcgagc
      901 accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
      961 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     1021 ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag
     1081 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     1141 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     1201 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     1261 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     1321 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     1381 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     1441 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     1501 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     1561 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     1621 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     1681 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     1741 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     1801 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     1861 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     1921 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     1981 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     2041 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     2101 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     2161 gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc
     2221 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     2281 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     2341 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     2401 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     2461 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     2521 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     2581 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     2641 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     2701 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     2761 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     2821 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     2881 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     2941 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3001 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3061 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     3121 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     3181 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     3241 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     3301 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     3361 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     3421 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     3481 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg
     3541 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     3601 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     3661 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     3721 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca
     3781 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     3841 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     3901 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     3961 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     4021 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     4081 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     4141 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
     4201 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
     4261 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
     4321 ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat
     4381 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc
     4441 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg
     4501 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa
     4561 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac
     4621 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     4681 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     4741 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     4801 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     4861 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     4921 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     4981 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     5041 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     5101 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     5161 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     5221 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     5281 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     5341 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     5401 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     5461 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     5521 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     5581 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     5641 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     5701 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     5761 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     5821 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     5881 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     5941 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     6001 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     6061 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
     6121 gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca
     6181 ctatagggga attgtgagcg gataacaatt cc
//
