LOCUS       sequence-349633-        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: pEE076
FEATURES             Location/Qualifiers
     source          1..6149
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     932..951
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(971..1048)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1254..1273
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1357..1376
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1357..1375
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1376..1400
                     /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             1415..1437
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2237..2254
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2307..2325)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2321..2368
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2405..2860
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2492..2511)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2702..2723
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2886..2990
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2991..3059,3060..3851)
                     /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(3209..3228)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4022..4610
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4511..4530
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4764..4781
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4796..4935
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4861..4880)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5021..5043
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5037..5228)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6037..6149,1..970))
                     /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 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
       61 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
      121 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
      181 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
      241 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
      301 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
      361 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
      421 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
      481 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
      541 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
      601 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
      661 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
      721 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
      781 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
      841 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
      901 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
      961 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     1021 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     1081 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     1141 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     1201 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     1261 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     1321 ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat
     1381 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat
     1441 acatatggcc aacccgtatg aacgtgggcc gaatccgacc gatgctctgc tggaagcaag
     1501 cagcggcccg ttcagcgttt cggaagaaaa cgtttctcgt ctgtctgcga gtggcttcgg
     1561 cggcggtact atctactatc cacgtgaaaa caatacctac ggcgcagtcg cgatttctcc
     1621 gggttatact ggcacggaag catcaattgc gtggctgggt gaacgtattg cgagtcatgg
     1681 tttcgttgtt attactatcg acactatcac caccctggac cagccggaca gccgcgcaga
     1741 gcagctgaac gctgctctta accacatgat caaccgtgcg tcctccaccg ttcgcagccg
     1801 catcgactca tcccgtttgg ctgttatggg ccatagcatg ggcggtggcg gcaccctgcg
     1861 tctggcgagc cagcgtccgg atctgaaagc tgcaatcccg ttgactccat ggcatctgaa
     1921 caagaactgg tcctccgtta cggttccgac cctgattatc ggcgcggatc tcgataccat
     1981 cgcaccagtt gccactcatg caaaaccgtt ctacaactca ctgccgagct cgatctctaa
     2041 agcttacctg gaactggatg gcgcgaccca ttttgcgccg aacattccga acaagatcat
     2101 cggcaaatac tccgttgcct ggctgaagcg tttcgttgat aatgacactc gttatacgca
     2161 gttcttgtgc ccgggtcctc gtgacggcct gtttggcgag gtggaagagt atcgttcaac
     2221 ctgtccgttc ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg
     2281 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     2341 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa
     2401 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     2461 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     2521 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     2581 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     2641 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     2701 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     2761 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     2821 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     2881 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     2941 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     3001 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     3061 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     3121 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     3181 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     3241 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     3301 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     3361 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     3421 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     3481 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa
     3541 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     3601 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     3661 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     3721 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     3781 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     3841 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     3901 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     3961 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4021 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4081 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4141 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     4201 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     4261 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     4321 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     4381 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     4441 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     4501 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     4561 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     4621 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     4681 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     4741 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     4801 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
     4861 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     4921 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     4981 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     5041 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     5101 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     5161 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     5221 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     5281 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     5341 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     5401 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     5461 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     5521 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     5581 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     5641 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc
     5701 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
     5761 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
     5821 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
     5881 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
     5941 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     6001 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     6061 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     6121 attgggcgcc agggtggttt ttcttttca
//
