LOCUS       sequence-342598-        6008 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 6008)
  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 6008)
  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: pEE015
FEATURES             Location/Qualifiers
     source          1..6008
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             649..666
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(719..737)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      733..780
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      817..1272
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(904..923)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1114..1135
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1298..1402
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1403..1471,1472..2263)
                     /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(1621..1640)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2434..3022
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2923..2942
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3176..3193
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3208..3347
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3273..3292)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3433..3455
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3449..3640)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4449..5531)
                     /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     5493..5512
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5532..5609)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5815..5834
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5918..5937
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5918..5936
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5937..5961
                     /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             5976..5998
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 ggctttaccg gcaacaaagc cgaggctggt cgtctgtaca ccgacatggc tcgtgtcctg
       61 tgtgcggctg gctacgctgc gctgcgcttc gacttccgtt gtcatggcga ctccccgctg
      121 ccgtttgaag aattccgcat ttcgtacgcg gtagaagacg cacgtaacgc ggcttctttc
      181 ctgaagattc aaccgtccgt agacggttct cgtttcgccg taatcggcct gtcgatgggt
      241 ggtggtgtag ccgtgagtct ggcagcgggt cgtgatgatg tagctgccct cgtgctcctg
      301 tctccggctc tggattggcc ggaactggcg gcgcgcattc cgcagccgaa ggtggaaggc
      361 ggctacgtgt acatgggtcc aaaccgcatg aaggtggagt gtgttacgga aaccatgaaa
      421 ttcactgtca tggacctggc agaacgtgtt aaagctccca ctctgatcat tcatgccgct
      481 gatgatatgg ttgttccgat tagccaatcc aaacgcttct atgaaaaact gaaagtagag
      541 aaaaagttta tggagatcga acgtagcggt catgttttcg atgattacaa cgtacgccgc
      601 cgtgtcgaag cggaagttct tgattggatc aaaaaacacc tgctcgagca ccaccaccac
      661 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
      721 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
      781 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
      841 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
      901 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
      961 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     1021 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     1081 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     1141 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     1201 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     1261 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     1321 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     1381 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     1441 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     1501 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     1561 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     1621 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     1681 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     1741 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     1801 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     1861 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     1921 cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac
     1981 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     2041 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     2101 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     2161 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     2221 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     2281 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     2341 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     2401 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     2461 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     2521 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     2581 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     2641 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     2701 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     2761 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     2821 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     2881 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     2941 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     3001 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     3061 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     3121 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     3181 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
     3241 cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     3301 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc
     3361 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     3421 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     3481 cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct
     3541 gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa
     3601 agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg
     3661 gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg
     3721 ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat
     3781 ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag
     3841 atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg
     3901 tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc
     3961 atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta
     4021 tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg
     4081 acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct
     4141 cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc
     4201 cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga
     4261 aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca
     4321 taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     4381 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     4441 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4501 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     4561 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     4621 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     4681 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca
     4741 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     4801 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     4861 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     4921 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     4981 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     5041 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     5101 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     5161 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     5221 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     5281 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     5341 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     5401 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     5461 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     5521 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     5581 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
     5641 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     5701 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     5761 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
     5821 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
     5881 tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac tataggggaa
     5941 ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata
     6001 tacatatg
//
