LOCUS       sequence-349204-        6263 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 6263)
  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 6263)
  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: pEE020
FEATURES             Location/Qualifiers
     source          1..6263
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(25..1107)
                     /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     1069..1088
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1108..1185)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1391..1410
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1494..1513
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1494..1512
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1513..1537
                     /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             1552..1574
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2488..2505
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2558..2576)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2572..2619
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2656..3111
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2743..2762)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2953..2974
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3137..3241
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3242..3310,3311..4102)
                     /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(3460..3479)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4273..4861
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4762..4781
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5015..5032
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5047..5186
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5112..5131)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5272..5294
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5288..5479)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
       61 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
      121 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
      181 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
      241 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
      301 cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
      361 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
      421 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
      481 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
      541 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
      601 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
      661 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
      721 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
      781 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
      841 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
      901 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
      961 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     1021 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     1081 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     1141 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     1201 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     1261 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     1321 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     1381 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     1441 tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg
     1501 actcactata ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa
     1561 ctttaagaag gagatataca tatgctgact atcacagcga tcatctacct cctggccacc
     1621 attatcgcag ccatcctcct tgttgtatat attatttcct cctccgcgtc taaaaaactg
     1681 gccacgccgc cgcgaaaaac cggttcctgg tcaccgcgcg atctgggttt cgagtacgag
     1741 aaagtggaag tgaaaacctc cgatggactg actctgcgtg gctggctgat tccgcgcggc
     1801 tccgaaaaaa ccgtcatcgt gatccacggt tacacttctt gcaagtggga tgaatggtat
     1861 atgaagccgg ttatcaacat cctggcacgt cacgatttca acgtcgttgc atttgatatg
     1921 cgtgcgcacg gcgaatctga tggcgaaaag actaccctgg gctaccgtga ggtagatgac
     1981 atcggcgcaa ttattaatta cctgaaagaa cgtggcctgg caagtcgcct gggcatcatt
     2041 ggctactcga tgggcggtgc tatcactctg atgtccctgg cgcgctacga ggaactgaaa
     2101 gcgggtgtgg ccgactcgcc gtacatcgac attcgtgcgt cgggtaaacg ttggattaat
     2161 cgcgttggcg ctcctctgcg ctacatcctc ctggcatcgt acccgctgat catgcgtctt
     2221 actgcctccc gtactggcgc atctccggaa aaactggtta tgtaccaata tgccaagtcg
     2281 atcaccaaac cactgcttat cattggtggt cagcaggacg acctggtggc catcgacgaa
     2341 gtacgtaaat tctatgaaga agttaaaaaa gtgaactcca atgtcgaact gtgggaaacc
     2401 acctccaaac acgtttctgc catccaagat tacccgcgtg aatacgaaga gcgtatcgtc
     2461 ggcttcttca accgctggct gctcgagcac caccaccacc accactgaga tccggctgct
     2521 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     2581 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     2641 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2701 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2761 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     2821 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     2881 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     2941 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     3001 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     3061 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     3121 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3181 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     3241 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     3301 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     3361 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     3421 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     3481 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     3541 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     3601 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     3661 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     3721 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     3781 gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     3841 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     3901 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     3961 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     4021 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     4081 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     4141 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     4201 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     4261 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     4321 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     4381 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     4441 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     4501 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     4561 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     4621 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     4681 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     4741 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     4801 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     4861 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     4921 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     4981 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     5041 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     5101 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     5161 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     5221 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     5281 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     5341 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     5401 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     5461 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     5521 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     5581 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     5641 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     5701 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     5761 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     5821 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     5881 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     5941 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     6001 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     6061 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     6121 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     6181 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     6241 atcccggtgc ctaatgagtg agc
//
