LOCUS       sequence-342640-        6188 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 6188)
  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 6188)
  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: pEE036
FEATURES             Location/Qualifiers
     source          1..6188
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             829..846
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(899..917)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      913..960
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      997..1452
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1084..1103)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1294..1315
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1478..1582
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1583..1651,1652..2443)
                     /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(1801..1820)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2614..3202
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3103..3122
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3356..3373
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3388..3527
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3453..3472)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3613..3635
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3629..3820)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4629..5711)
                     /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     5673..5692
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5712..5789)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5995..6014
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6098..6117
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6098..6116
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6117..6141
                     /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             6156..6178
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 caggcagata ccgataccac tgcagtggct cccgcggcgg ctaatccgta tgaacgcggc
       61 ccggctccga ctgaagcgtc tgtaactgca gctcgcggtc cgtttgctat tgcccaggtg
      121 aacgtaccgt ctggcagcgg tgctggcttc aacgatggca ccatctacta tccgactgat
      181 acctctcagg gtacctttgg tgcggtcgcg gtaatcccgg gtttcatctc ccctcaggct
      241 gtgatccagt ggttcggtcc gcgcttggca tctcagggct tcgtagtctt cactctggat
      301 tctaacggtc tggccgatct gccggatgcg cgcggtcgtc agctgctggc ggctctggac
      361 tacctgacca cccagtctac tgtgcgtacc cgtattgatc cgaatcgcct ggctgtcatg
      421 gggcacagca tgggtggcgg tggcacgctg ctggcggcgg aaaaccgtcc aaccctgaaa
      481 gcggccatcc cactggcgcc gtgggaaccg gatactagtt gggaaggcgt gaaagtaccg
      541 actatgatca tcggcggcga aagcgatgtc gttgctccgg tttccagtat ggctattccg
      601 gactataact ccctgagctc tgctccagaa aaggcttatc tggagttgcg ttctggtgat
      661 cacctggcac cggcaagcga atctcctacc gttgcggaat acgctttaag ctggctcaag
      721 cgctttgttg atgatgacac tcgttatgat cagttcctgt gtccgggtcc tacaccggat
      781 actgatatca gccagtacct ggatacgtgt cctaacggtt ctctcgagca ccaccaccac
      841 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
      901 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
      961 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
     1021 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     1081 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     1141 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     1201 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     1261 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     1321 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     1381 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     1441 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     1501 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     1561 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     1621 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     1681 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     1741 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     1801 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     1861 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     1921 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     1981 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     2041 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     2101 cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac
     2161 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     2221 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     2281 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     2341 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     2401 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     2461 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     2521 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     2581 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     2641 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     2701 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     2761 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     2821 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     2881 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     2941 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     3001 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     3061 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     3121 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     3181 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     3241 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     3301 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     3361 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
     3421 cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     3481 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc
     3541 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     3601 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     3661 cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct
     3721 gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa
     3781 agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg
     3841 gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg
     3901 ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat
     3961 ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag
     4021 atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg
     4081 tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc
     4141 atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta
     4201 tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg
     4261 acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct
     4321 cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc
     4381 cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga
     4441 aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca
     4501 taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     4561 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     4621 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4681 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     4741 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     4801 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     4861 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca
     4921 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     4981 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     5041 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     5101 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     5161 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     5221 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     5281 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     5341 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     5401 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     5461 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     5521 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     5581 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     5641 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     5701 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     5761 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
     5821 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     5881 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     5941 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
     6001 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
     6061 tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac tataggggaa
     6121 ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata
     6181 tacatatg
//
