LOCUS       sequence-342614-        6326 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 6326)
  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 6326)
  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: pEE023
FEATURES             Location/Qualifiers
     source          1..6326
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             967..984
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1037..1055)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1051..1098
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1135..1590
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1222..1241)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1432..1453
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1616..1720
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1721..1789,1790..2581)
                     /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(1939..1958)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2752..3340
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3241..3260
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3494..3511
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3526..3665
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3591..3610)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3751..3773
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3767..3958)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4767..5849)
                     /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     5811..5830
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5850..5927)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6133..6152
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6236..6255
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6236..6254
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6255..6279
                     /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             6294..6316
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 gccacggttc tggtaattcc gaaactcggt ctgacgatga ctgaaggccg tgtcggccgt
       61 tggctgaaac agttaggcga gccggtacag gctggcgaac cagtgcttga agtagagacc
      121 gaaaaactga ccgtagaggt cgaggcccca gctagcggca tcctggcata tattctggcg
      181 gaagaaggcg ttgttctgcc ggtgactgct ccggtggcag tgatcgccga gccgggtgaa
      241 gctgttgacc tggcttctct cctgccggct acttccggtg cggcagcaac cccggttatg
      301 gctgcatcca gcaccatgca agaacaggct cgtgcacagg gtccaacccc gaccggcgaa
      361 atccgcgcaa ccccggctgc tcgtaagctg gcacgtgacc acggcattga cctggcacgt
      421 gttcgcggca ccggtcctgg tggccgtatt acggcggagg atgtagaacg ttacctggcc
      481 agccagggta ccgcttggcc tcgcggtgaa ccggttcgtt tctggtcaga cggtctggct
      541 ctggctggtg aactgttctt acctccgagt accgatacgg cggttcctgg tgttgtgcta
      601 tgcaccggca ttcagggcct gaaagagctc ggtatgccgc tgctggcgca ggcactggca
      661 gatgccgggt acgctgctct gatttttgat tatcgcggct tcggtgcttc cgagggtccg
      721 cgtggccgtc tgctgccaca ggaacgcatt cgtgatgcac gtgcagcgct cactttcctg
      781 gaaactcacc cacttatcga tcgtacccgc ctggccatcc tgggcctgtc gctcggtggc
      841 gcacacgcgc tttccctggc ggcaatcgac gatcgcgttc aggcttgtat cgcaatcgcc
      901 ccgctgacca acggacgccg ctggctgcgt tctctgcgtg ctgaatggca gtggcgtgtt
      961 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
     1021 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     1081 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
     1141 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     1201 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     1261 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     1321 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     1381 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     1441 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     1501 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     1561 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     1621 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     1681 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     1741 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     1801 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     1861 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     1921 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     1981 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     2041 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     2101 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     2161 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     2221 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac
     2281 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     2341 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     2401 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     2461 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     2521 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     2581 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     2641 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     2701 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     2761 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     2821 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     2881 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     2941 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     3001 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     3061 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     3121 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     3181 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     3241 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     3301 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     3361 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     3421 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     3481 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     3541 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
     3601 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     3661 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     3721 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     3781 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     3841 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     3901 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     3961 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     4021 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     4081 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     4141 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     4201 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     4261 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     4321 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     4381 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg
     4441 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     4501 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     4561 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     4621 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     4681 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     4741 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4801 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     4861 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     4921 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     4981 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     5041 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     5101 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     5161 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     5221 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     5281 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     5341 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     5401 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     5461 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     5521 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     5581 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     5641 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     5701 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     5761 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     5821 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     5881 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     5941 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     6001 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     6061 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     6121 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     6181 gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata
     6241 cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt
     6301 aactttaaga aggagatata catatg
//
