LOCUS       sequence-349194-        6140 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 6140)
  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 6140)
  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: pEE047
FEATURES             Location/Qualifiers
     source          1..6140
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     951..970
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(990..1067)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1273..1292
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1376..1395
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1376..1394
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1395..1419
                     /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             1434..1456
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2247..2264
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2317..2335)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2331..2378
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2415..2870
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2502..2521)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2712..2733
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2896..3000
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3001..3069,3070..3861)
                     /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(3219..3238)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4032..4620
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4521..4540
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4774..4791
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4806..4945
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4871..4890)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5031..5053
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5047..5238)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6047..6140,1..989))
                     /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 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
       61 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
      121 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
      181 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
      241 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
      301 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
      361 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
      421 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
      481 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
      541 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
      601 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
      661 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
      721 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
      781 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
      841 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
      901 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
      961 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     1021 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     1081 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     1141 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     1201 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     1261 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     1321 gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata
     1381 cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt
     1441 aactttaaga aggagatata catatggaga gtccgtacga acgtggtccg gacccgactt
     1501 ctgcatccgt tctggataat ggaacctttt cactgtcctc cacgtccgtg tcttctcttg
     1561 tgacgggttt cggtggcggc accatttatt atccgacctc caccactcag ggcacgtttg
     1621 gcggcgtagt tttagcaccg ggctacactg cgagcagctc ctcttattct agcgtggccc
     1681 gccgcgtggc atctcacggc tttgtggtct tcgcgattga tactaattcg cgctacgatc
     1741 agccggatag ccgtggtagc cagattctgg cggctgtatc ctacctgaaa aactctgcgt
     1801 cgtccaccgt ggcctcccgc ttggatgaga cccgtatcgc ggttagcggt cattctatgg
     1861 gcgggggcgg caccctggca gccgccaacc aagattcttc catcaaagct gcggtcgcac
     1921 tgcaaccgtg gcacacggat aagacgtggc cgggcatcca aatcccgact atgattatcg
     1981 gcgctgaaaa cgactccgtt gcgccggtcg ccagccactc tattccgttt tatacttcta
     2041 tgaccggcgc tcgcgaaaag gcgtatggtg aaatcaacaa cggtgatcac ttcatcgcta
     2101 acaccgatga cgactggcag ggccgtttgt tcgttacctg gctgaaacgc tatgtcgatg
     2161 atgatacgcg ttactcccag tttctgtgcc cggcgccgtc ctctatctac ttgtctgatt
     2221 atcgcaacac ctgtccggat ctcgagcacc accaccacca ccactgagat ccggctgcta
     2281 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     2341 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     2401 gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     2461 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     2521 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     2581 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     2641 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     2701 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     2761 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     2821 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     2881 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     2941 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     3001 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     3061 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     3121 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     3181 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     3241 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     3301 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     3361 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     3421 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     3481 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     3541 cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     3601 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     3661 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     3721 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     3781 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     3841 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     3901 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     3961 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     4021 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     4081 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     4141 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     4201 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     4261 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     4321 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     4381 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     4441 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     4501 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     4561 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     4621 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     4681 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     4741 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     4801 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
     4861 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     4921 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     4981 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     5041 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     5101 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     5161 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     5221 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     5281 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     5341 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     5401 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     5461 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     5521 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     5581 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     5641 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     5701 ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     5761 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     5821 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     5881 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     5941 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     6001 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     6061 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     6121 cggtttgcgt attgggcgcc
//
