LOCUS       sequence-349673-        5951 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 5951)
  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 5951)
  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: pEE013
FEATURES             Location/Qualifiers
     source          1..5951
                     /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             639..656
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(709..727)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      723..770
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      807..1262
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(894..913)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1104..1125
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1288..1392
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1393..1461,1462..2253)
                     /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(1611..1630)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2424..3012
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2913..2932
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3166..3183
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3198..3337
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3263..3282)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3423..3445
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3439..3630)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4439..5521)
                     /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     5483..5502
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5522..5599)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5805..5824
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5908..5927
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5908..5926
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5927..5951
                     /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 acatatgcgc gtactgtgct
       61 cggctggtta tgcggtactg cgcttcgact atcgttgcca cggcgactct ccgctgccgt
      121 tcgaagaatt ccgtattagc atggcggtag aagacgcgga aaacgcagta aaatacgtaa
      181 aatcccttga acgtgtggac ggctcatcct ttgccgtcat tggtctgtct atgggcggtg
      241 gcgttgcagt caaattggct gcgggtcgtg acgacgtggc ggcactggta ctgctttctc
      301 cggccctgga ttggccggaa ctgaccggcc gtgttccgtt taaagttgaa gaaggttacg
      361 tttacatggg tccgttccgt atgcgtgcag aaaacgcgat ggaaaacgcc cgtttcactg
      421 ttatggatat tgcagaacaa gtgaaagctc cgacgctgat cgttcatgct accgatgacg
      481 aagttgtacc tatctctcag gctaagcgtt tctatgagaa acttcgtgta gaaaaacgtt
      541 tcctggaagt taagtccggt catgttttca acgattacca cgttcgtcgt aacctggaag
      601 gtgagatcct gtcgtgggtt aaaagccacc tgctcgagca ccaccaccac caccactgag
      661 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
      721 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
      781 gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
      841 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
      901 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
      961 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     1021 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     1081 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     1141 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     1201 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     1261 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     1321 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     1381 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     1441 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     1501 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     1561 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     1621 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     1681 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     1741 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     1801 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     1861 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     1921 gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     1981 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     2041 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     2101 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     2161 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     2221 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     2281 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     2341 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     2401 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     2461 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     2521 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     2581 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     2641 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     2701 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     2761 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     2821 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     2881 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     2941 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     3001 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     3061 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     3121 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     3181 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     3241 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     3301 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     3361 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     3421 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     3481 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     3541 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     3601 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     3661 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     3721 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     3781 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     3841 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     3901 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     3961 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     4021 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     4081 gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     4141 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     4201 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     4261 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     4321 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     4381 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     4441 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     4501 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     4561 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     4621 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     4681 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc
     4741 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     4801 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     4861 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     4921 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     4981 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     5041 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     5101 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     5161 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     5221 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     5281 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     5341 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     5401 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     5461 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     5521 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     5581 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     5641 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     5701 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     5761 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     5821 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     5881 aggatcgaga tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg
     5941 ataacaattc c
//
