LOCUS       sequence-349630-        6089 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 6089)
  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 6089)
  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: pEE014
FEATURES             Location/Qualifiers
     source          1..6089
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             588..605
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(658..676)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      672..719
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      756..1211
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(843..862)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1053..1074
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1237..1341
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1342..1410,1411..2202)
                     /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(1560..1579)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2373..2961
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2862..2881
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3115..3132
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3147..3286
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3212..3231)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3372..3394
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3388..3579)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4388..5470)
                     /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     5432..5451
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5471..5548)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5754..5773
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5857..5876
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5857..5875
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5876..5900
                     /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             5915..5937
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 ctgccgggta tgcagcgctg cgctttgact ttcgttgcca tggggattcc ccgctgccat
       61 tcgaggaatt ccgcatctca ctggcggttg aagatgcgcg taatgccgct ggctttctga
      121 aaaatgttcc tgaagttgat ggcaccaaat tcggcgtggt tggtctgtct atgggaggtg
      181 gtgttgctgt ttcgctcgcc gcgggccgtg aggatgtagg tgctctggta ctgctgtctc
      241 cggcccttga ttggccggag ctgttccagc gcgcacgtgg cttcttccgc gcggaagaag
      301 gttacgtgta ctggggtccg aaccgtatgc gtgatgtata cgcaatggag accatgaact
      361 tcagcgtgat gggcctggca gaagaaatta aagcgccgac tctgatcatt cactcggtgg
      421 atgatgtggt agtgccgatc agtcaggcta aacgtttcta cgaaaaactg aaagttgaaa
      481 aaaaatttat cgaaatcgaa cagggcggcc acgtgtttga agattacaac gttcgtcgtc
      541 gtatcgaacg tgaagttctg gactgggtga agcgccattt actcgagcac caccaccacc
      601 accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg
      661 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
      721 tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag
      781 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
      841 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
      901 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
      961 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     1021 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     1081 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     1141 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     1201 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     1261 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     1321 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     1381 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     1441 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     1501 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     1561 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     1621 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     1681 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     1741 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     1801 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     1861 gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact
     1921 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     1981 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     2041 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     2101 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     2161 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     2221 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     2281 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     2341 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     2401 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     2461 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     2521 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     2581 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     2641 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     2701 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     2761 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     2821 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     2881 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     2941 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     3001 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     3061 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     3121 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     3181 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     3241 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     3301 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     3361 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     3421 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     3481 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     3541 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     3601 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     3661 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
     3721 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
     3781 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     3841 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
     3901 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
     3961 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
     4021 caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc
     4081 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
     4141 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
     4201 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat
     4261 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     4321 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     4381 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4441 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     4501 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     4561 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     4621 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     4681 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     4741 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     4801 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     4861 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     4921 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     4981 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     5041 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     5101 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     5161 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     5221 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     5281 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     5341 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     5401 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     5461 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     5521 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     5581 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     5641 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     5701 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     5761 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     5821 ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat
     5881 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat
     5941 acatatggtt ccatccgcgg gtgtaggcct gtctggcgtt cttcacctgc cggcaggcgt
     6001 aagccgcccg gtgctgtttc tgcacggttt caccggtaac aaaaccgaat ccggccgcct
     6061 ttatactgac atggctcgtg ttctgtgtt
//
