LOCUS       sequence-349193-        6059 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 6059)
  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 6059)
  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: pEE028
FEATURES             Location/Qualifiers
     source          1..6059
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..25
                     /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             40..62
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             772..789
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(842..860)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      856..903
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      940..1395
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1027..1046)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1237..1258
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1421..1525
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1526..1594,1595..2386)
                     /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(1744..1763)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2557..3145
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3046..3065
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3299..3316
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3331..3470
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3396..3415)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3556..3578
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3572..3763)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4572..5654)
                     /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     5616..5635
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5655..5732)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5938..5957
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(6041..6059,1)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6041..6059
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
       61 gatatacata tgcaagtaat tcagcagacc gttacactgc aaaaaaccca actgcgcctg
      121 accaaggaag gcttcgttac caattatcgt ttcccggtgg atttctacta ccctgattct
      181 ccggaatctt tcccggtaat tctgatctct catggttttg gctcggtccg cgaaaacttc
      241 cgcactctgg cacagcatct ggcctctcac ggcttcctgg tagccgttcc gcagcacatc
      301 ggctcggatc tgcagtaccg tcaagagctg atcaaaggta ctttatcctc cgcactgtcc
      361 ccagttgaat ttttggcgcg tccgaccgac ctgtctacca tcattgacta tctgcaggcg
      421 actcagaaca ccggctcctg gcagaagcgt gcaaatctgc agcagatcgg cgttatcggt
      481 gatagtctgg gcggtaccac tgctctgacg attggtggtg caccgctgga tattccgcgt
      541 ctgcagacta aatgtacctc ggacaacgtt attgtgaacg ttgccctgat cctgcaatgc
      601 caggcctcgt tcctgccgcc gagcgaatac aacctggctg attcccgtgt caaagccgtt
      661 attgccacgc acccgctgat ctcaggcatt ttttctccgg actctctggc gaaaattcag
      721 atcccagtga tgattaccgc gggcaacttt gacatcatca ccccgctcga gcaccaccac
      781 caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
      841 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
      901 ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat
      961 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     1021 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     1081 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     1141 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     1201 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     1261 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     1321 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     1381 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     1441 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     1501 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     1561 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     1621 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     1681 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     1741 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     1801 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     1861 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     1921 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     1981 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     2041 aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt
     2101 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     2161 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     2221 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     2281 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     2341 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     2401 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     2461 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     2521 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     2581 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     2641 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     2701 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     2761 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     2821 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     2881 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     2941 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3001 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3061 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3121 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3181 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     3241 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     3301 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     3361 gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag
     3421 ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc
     3481 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     3541 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     3601 gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg
     3661 cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga
     3721 taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag
     3781 ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac
     3841 gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg
     3901 tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata
     3961 cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa
     4021 tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca
     4081 ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc
     4141 gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca
     4201 acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct
     4261 tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga
     4321 ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc
     4381 cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag
     4441 tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga
     4501 aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa
     4561 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4621 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt
     4681 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc
     4741 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac
     4801 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca
     4861 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg
     4921 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa
     4981 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg
     5041 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
     5101 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
     5161 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
     5221 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag
     5281 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
     5341 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
     5401 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
     5461 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
     5521 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
     5581 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
     5641 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
     5701 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga
     5761 ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag
     5821 gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat
     5881 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt
     5941 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat
     6001 gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact cactatagg
//
