LOCUS       sequence-349678-        6149 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 6149)
  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 6149)
  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: pEE062
FEATURES             Location/Qualifiers
     source          1..6149
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             15..37
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             837..854
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(907..925)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      921..968
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1005..1460
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1092..1111)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1302..1323
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1486..1590
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1591..1659,1660..2451)
                     /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(1809..1828)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2622..3210
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3111..3130
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3364..3381
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3396..3535
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3461..3480)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3621..3643
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3637..3828)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4637..5719)
                     /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     5681..5700
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5720..5797)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6003..6022
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6106..6125
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6106..6124
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6125..6149
                     /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)."
ORIGIN
        1 cctctagaaa taattttgtt taactttaag aaggagatat acatatggcg aacccgtatg
       61 aacggggtcc gaaccctacg gacgctctgc tggaagcacg tagcggtccg tttagtgttt
      121 ccgaggagaa cgtttctcgc ctttctgctt ccggttttgg cggcggtacc atctactacc
      181 cgcgtgaaaa caacacgtat ggtgctgttg ctatcagccc gggttatact ggtactgaag
      241 cttccattgc ttggctgggt aaacgtatcg ctagccacgg ttttgtagtc atcaccatcg
      301 ataccatcac taccctcgat cagccagata gccgtgcgga acagctgaac gcggcactga
      361 accacatgat caaccgtgcg tcgtcgaccg ttcgttctcg tattgactct tcccgcctgg
      421 cggtaatggg ccactctatg ggtggtggtg gctcgcttcg cttagcctct cagcggccgg
      481 atctcaaggc agctattccg ctgaccccgt ggcacttaaa caaaaactgg tctagcgttc
      541 gtgtaccgac cctgatcatc ggcgcggacc tggatactat tgcgccggtt ctgacccacg
      601 cgcgcccgtt ctacaattcg ctgccgacct ccatctctaa agcatacttg gaactggacg
      661 gtgcgacgca cttcgcgccg aacattccga acaagattat cggcaaatac tccgtggctt
      721 ggctgaaacg tttcgtagac aacgatactc gttacacaca gttcctgtgt ccgggtccgc
      781 gtgatggtct gtttggtgaa gttgaagaat accgctccac ctgcccgttt ctcgagcacc
      841 accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg
      901 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
      961 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     1021 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     1081 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     1141 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     1201 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     1261 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     1321 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     1381 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     1441 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     1501 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     1561 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     1621 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     1681 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     1741 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     1801 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     1861 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     1921 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     1981 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     2041 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     2101 ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa
     2161 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     2221 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     2281 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     2341 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     2401 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     2461 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     2521 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     2581 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     2641 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     2701 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     2761 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     2821 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     2881 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     2941 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     3001 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     3061 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     3121 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     3181 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     3241 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     3301 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     3361 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
     3421 catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg
     3481 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     3541 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     3601 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     3661 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
     3721 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct
     3781 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt
     3841 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac
     3901 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact
     3961 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt
     4021 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa
     4081 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa
     4141 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg
     4201 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt
     4261 cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc
     4321 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
     4381 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
     4441 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
     4501 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
     4561 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     4621 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4681 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     4741 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     4801 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     4861 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat
     4921 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     4981 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     5041 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     5101 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     5161 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     5221 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     5281 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     5341 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     5401 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     5461 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     5521 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     5581 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     5641 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     5701 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     5761 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     5821 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     5881 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     5941 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     6001 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
     6061 acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta
     6121 taggggaatt gtgagcggat aacaattcc
//
