LOCUS       sequence-349201-        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: pEE043
FEATURES             Location/Qualifiers
     source          1..6212
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(117..908,909..977))
                     /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     740..759
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(978..1082)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1108..1563)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1245..1266)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1457..1476
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(1600..1647)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     1643..1661
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(1714..1731)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             2594..2616
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2631..2655
                     /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)."
     primer_bind     complement(2655..2674)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2656..2674)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2758..2777)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2983..3060
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3061..4143
                     /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     complement(3080..3099)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4952..5143
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5137..5159)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5245..5384
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5300..5319
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5399..5416)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5570..6158)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5650..5669)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
       61 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
      121 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
      181 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
      241 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
      301 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
      361 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
      421 gttgccattg ctgcaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
      481 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
      541 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
      601 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
      661 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
      721 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
      781 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
      841 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
      901 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
      961 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     1021 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     1081 cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg ttaaaattcg
     1141 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     1201 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     1261 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     1321 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     1381 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     1441 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     1501 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     1561 cgtcccattc gccaatccgg atatagttcc tcctttcagc aaaaaacccc tcaagacccg
     1621 tttagaggcc ccaaggggtt atgctagtta ttgctcagcg gtggcagcag ccaactcagc
     1681 ttcctttcgg gctttgttag cagccggatc tcagtggtgg tggtggtggt gctcgagctg
     1741 cggcaggttt tcggtacgcc aggtagccac atcggaggat ggacgtttga tgtaggaaac
     1801 ccaacgggtg tcgccatcca gaaacgtttt gtggaacgcc agtgcgatac ccgcaacgga
     1861 acgattcgct gcggttgggg aaccccagtc gaagtgccca gcgctggcaa cttcgtagat
     1921 aactttcgga ataccgcgcg ggatagcgtt gtaaacaccg atagactggc ccaggccacc
     1981 cagcatggtc aggtccagcg cgccgttgaa caggagggtc ggaacacggg tgttaccacc
     2041 tttgctgtcc agatcgatag cggtcatatt gtgacctgcg aggctcatag ccgttttgag
     2101 accgttgtac tccgcagagt tgatccaggt agctccgcca cccatactcc agcccacggc
     2161 acccagacgg gaagtgtcca gcttaccacg gagcggagag cccgaacgcg tgttttcctg
     2221 tttcagaacg tccagtacgc gtttctgctg gctcgcacgc tgatcaacgg tgtccactgt
     2281 agtggacgtg tccatggtta ccagaacgat accgtgagat gcaaaaaatg ggccccatgc
     2341 gcgaaacata gcctgagtac cggtgtacgg cggagtgaaa accagaccgc tgtacggtgg
     2401 ttcggcgtta gacgggtagt atacggttgc gccgcccggg gtctggaaac gagacagggt
     2461 gtagctccgg gaaccatacg ggccgttacc agaatagcta cccggcgcct cgcctcggca
     2521 gatagctgta gacgggcaat cacccatcgg atcatcggtc ggcggggtcg gcggaacctg
     2581 ggacatatgt atatctcctt cttaaagtta aacaaaatta tttctagagg ggaattgtta
     2641 tccgctcaca attcccctat agtgagtcgt attaatttcg cgggatcgag atctcgatcc
     2701 tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct
     2761 atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt
     2821 gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct
     2881 tgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct
     2941 tcctaatgca ggagtcgcat aagggagagc gtcgagatcc cggacaccat cgaatggcgc
     3001 aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat
     3061 gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt
     3121 tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg
     3181 gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag
     3241 tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc
     3301 gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa
     3361 cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt
     3421 gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc
     3481 actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt
     3541 ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag
     3601 caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc
     3661 tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg
     3721 agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact
     3781 gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc
     3841 gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca
     3901 tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc
     3961 gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc
     4021 gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc
     4081 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     4141 tgagcgcaac gcaattaatg taagttagct cactcattag gcaccgggat ctcgaccgat
     4201 gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt
     4261 cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct
     4321 ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct
     4381 tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa
     4441 acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccccacggg tgcgcatgat
     4501 cgtgctcctg tcgttgagga cccggctagg ctggcggggt tgccttactg gttagcagaa
     4561 tgaatcaccg atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg
     4621 agcaacaaca tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc
     4681 agcgccctgc accattatgt tccggatctg catcgcagga tgctgctggc taccctgtgg
     4741 aacacctaca tctgtattaa cgaagcgctg gcattgaccc tgagtgattt ttctctggtc
     4801 ccgccgcatc cataccgcca gttgtttacc ctcacaacgt tccagtaacc gggcatgttc
     4861 atcatcagta acccgtatcg tgagcatcct ctctcgtttc atcggtatca ttacccccat
     4921 gaacagaaat cccccttaca cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa
     4981 catggcccgc tttatcagaa gccagacatt aacgcttctg gagaaactca acgagctgga
     5041 cgcggatgaa caggcagaca tctgtgaatc gcttcacgac cacgctgatg agctttaccg
     5101 cagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     5161 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     5221 agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt
     5281 gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccattgcggt
     5341 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct
     5401 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     5461 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     5521 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     5581 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     5641 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     5701 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     5761 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     5821 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     5881 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     5941 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     6001 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     6061 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     6121 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     6181 cggggtctga cgctcagtgg aacgaaaact ca
//
