LOCUS       sequence-342686-        6146 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 6146)
  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 6146)
  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: pEE059
FEATURES             Location/Qualifiers
     source          1..6146
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             787..804
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(857..875)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      871..918
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      955..1410
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1042..1061)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1252..1273
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1436..1540
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1541..1609,1610..2401)
                     /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(1759..1778)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2572..3160
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3061..3080
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3314..3331
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3346..3485
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3411..3430)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3571..3593
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3587..3778)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4587..5669)
                     /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     5631..5650
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5670..5747)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5953..5972
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6056..6075
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6056..6074
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6075..6099
                     /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             6114..6136
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 gttgaagcca ccgatgttca cggcccggac ccgacggaag aaacgattac cgctccgcgt
       61 ggccctttcg acgttgagca ggaatcagtt tcccgcttcg aagttgaagg cttcggtggc
      121 ggtaccatct attacccgac cgacaccacc gacggtctgt ttagcgctgt gtccatctcc
      181 ccgggttata ctggcaccca ggaaagcatg gcctggtatg gcccacgcct cgcatcacac
      241 ggttttgtgg tttttaccat tgatacgatc accaccacgg atcagccgga ttcccgtgcc
      301 cgccagctgc aggcatcgct ggaccatctc gtagacgatt cctccgtacg tgaccgcgtt
      361 gacccggcac gtctgggtgt tatgggccac tccatgggcg gcggtggttc actgaaagcg
      421 gctctggata acccagcgct gcaggcagcg atcccactga cgccgtggca tactactaag
      481 gatttctccg gcgtacgcac tccaaccctg atcatcggtg ctcaaaacga tacggtggca
      541 ccggttagcc aacatgcaga accgttctac gaaagtttac cggacgaccc gggcaaagcg
      601 tatctggaac tggcgggcgc gggtcacctg gcgccgaaca ccccggacac taccattgcg
      661 aagtattccc tggcgtggct gaaacgcttc ctggacgatg acactcgtta cgaccaattt
      721 ctgtgcccgc caccgcagga cgacccggag atcgcagaac atcgttctac ttgcccgtac
      781 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
      841 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
      901 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
      961 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     1021 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     1081 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     1141 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     1201 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     1261 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     1321 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     1381 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     1441 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     1501 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     1561 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     1621 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     1681 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     1741 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     1801 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     1861 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     1921 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     1981 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     2041 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac
     2101 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     2161 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     2221 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     2281 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     2341 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     2401 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     2461 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     2521 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     2581 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     2641 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     2701 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     2761 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     2821 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     2881 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     2941 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     3001 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     3061 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     3121 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     3181 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     3241 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     3301 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     3361 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
     3421 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     3481 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     3541 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     3601 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     3661 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     3721 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     3781 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     3841 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     3901 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     3961 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     4021 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     4081 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     4141 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     4201 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg
     4261 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     4321 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     4381 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     4441 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     4501 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     4561 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4621 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     4681 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     4741 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     4801 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     4861 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     4921 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     4981 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     5041 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     5101 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     5161 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     5221 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     5281 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     5341 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     5401 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     5461 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     5521 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     5581 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     5641 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     5701 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     5761 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     5821 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     5881 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     5941 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     6001 gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata
     6061 cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt
     6121 aactttaaga aggagatata catatg
//
