LOCUS       sequence-349225-        5996 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 5996)
  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 5996)
  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: pEE024
FEATURES             Location/Qualifiers
     source          1..5996
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             647..664
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(717..735)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      731..778
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      815..1270
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(902..921)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1112..1133
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1296..1400
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1401..1469,1470..2261)
                     /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(1619..1638)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2432..3020
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2921..2940
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3174..3191
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3206..3345
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3271..3290)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3431..3453
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3447..3638)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4447..5529)
                     /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     5491..5510
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5530..5607)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5813..5832
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5916..5935
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5916..5934
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5935..5959
                     /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             5974..5996
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 tatacatatg caaccgtacc cggtcggtac tcgtaccatt acttaccagg atccggtacg
       61 taacaaccgc aacatccaga cgtacctgta ctatccggcg accgcagccg gtgctaacca
      121 gcctgttgct ggtggtcagt ttccggtcgt agtggtgggg cacggtttca ctatgaatta
      181 cgcgccgtat gcgttttggg gtaacgcgct ggctgagtct ggttatatcg tagctatccc
      241 gaacacggaa accggctttt ctccgtccca tagcgccttc gctgctgata tggctttcct
      301 ggtggcgaaa ctgtacaccg aaaacaccaa ctcctcctcc cctttttatc agcatgttca
      361 gtacaattct tgcattattg gtcactctat gggtggtgga tgcacttacc tggcggccca
      421 aaacaacgca gacgtgagcg ctacggttac cttcgcagcc gcagaaacca acccgtctgc
      481 taccgcggct gcagcaaacg ttaactgtcc gtctctggtt ttctctggtt ccgccgactg
      541 catcaccccg ccggctcagc accaggtacc gatgtataac gctctgccgg actgtaaagc
      601 gtacggcggt tcttcccgcg ttgacctgca agcatgcaaa ctcgagcacc accaccacca
      661 ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
      721 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
      781 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
      841 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
      901 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
      961 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     1021 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     1081 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     1141 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     1201 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     1261 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     1321 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     1381 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     1441 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     1501 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     1561 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     1621 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     1681 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     1741 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     1801 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     1861 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     1921 acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg
     1981 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     2041 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     2101 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     2161 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     2221 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     2281 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     2341 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     2401 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     2461 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     2521 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     2581 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     2641 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     2701 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     2761 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     2821 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     2881 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     2941 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     3001 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     3061 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     3121 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     3181 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     3241 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     3301 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     3361 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     3421 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     3481 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     3541 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     3601 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     3661 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     3721 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     3781 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     3841 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     3901 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     3961 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     4021 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     4081 aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg
     4141 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     4201 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa
     4261 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     4321 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     4381 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     4441 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4501 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     4561 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     4621 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     4681 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     4741 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     4801 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     4861 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     4921 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     4981 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     5041 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     5101 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     5161 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     5221 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     5281 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     5341 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     5401 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     5461 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     5521 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     5581 aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct
     5641 gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg
     5701 gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca
     5761 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
     5821 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc
     5881 cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt
     5941 gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga aggaga
//
