LOCUS       sequence-349631-        6170 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 6170)
  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 6170)
  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: pEE040
FEATURES             Location/Qualifiers
     source          1..6170
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             821..838
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(891..909)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      905..952
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      989..1444
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1076..1095)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1286..1307
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1470..1574
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1575..1643,1644..2435)
                     /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(1793..1812)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2606..3194
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3095..3114
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3348..3365
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3380..3519
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3445..3464)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3605..3627
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3621..3812)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4621..5703)
                     /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     5665..5684
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5704..5781)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5987..6006
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6090..6109
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6090..6108
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6109..6133
                     /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             6148..6170
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 tatacatatg ggtgattgtc cagcaactgc tatctgtcgc agcgaaagcc cgggcgcgta
       61 ctccggtaac ggcccctatg gttctcgctc ctacaccctg agccgcttcc agacgccggg
      121 tggtgctacc gtgtactatc cggcgaacgc agaaccgccg tacgctggta tggtctttac
      181 cccgccgtat accggcactc aggcgatgtt cgctgcttgg ggcccattct tcgcgtctca
      241 cggcttcgtt ctggttacca tggacacgag caccacactg gactccgtcg accagcgtgc
      301 tgctcagcag aaagaagtac tgaacgcact gaaatctgag aacacccgtt ccggctctcc
      361 actgcgcggt aaactggata ccgcacgtct gggcgctgtt ggctggtcca tgggtggtgg
      421 cgcaacttgg atcaatagcg cagaatactc cggcctgaaa accgctatgt ctctggctgg
      481 tcacaacctg acggcagttg atattgatag caagggctat aatacccgtg tgccgaccct
      541 gctgttcaac ggtgcacagg atctgactta cctgggcggt ttgggccagt ctgatggcgt
      601 atacaacaac atcccggcgg gaatcccgaa agttttttat gaagtcagca gcgcgggcca
      661 ctttgattgg ggttccccga ctgcggccaa ccgttctgtg gcgtctctgg cgcttgcctt
      721 ccacaaagca tacctggatg gcgacacccg ttggctgcag tacattactc gtccgagcag
      781 cgatgttact acttggcgta ccgcgaacat tcgtctcgag caccaccacc accaccactg
      841 agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
      901 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
      961 aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     1021 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     1081 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     1141 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     1201 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     1261 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     1321 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     1381 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     1441 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     1501 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     1561 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     1621 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     1681 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     1741 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     1801 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     1861 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     1921 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     1981 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     2041 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     2101 gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac
     2161 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     2221 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     2281 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     2341 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     2401 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     2461 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     2521 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     2581 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2641 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2701 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     2761 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     2821 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     2881 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     2941 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     3001 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     3061 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     3121 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3181 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3241 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     3301 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     3361 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     3421 cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     3481 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
     3541 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     3601 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     3661 ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc
     3721 gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc
     3781 atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg
     3841 ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat
     3901 gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg
     3961 cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt
     4021 gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc
     4081 gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt
     4141 gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat
     4201 tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc
     4261 acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa
     4321 cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc
     4381 gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc
     4441 cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg
     4501 gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     4561 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     4621 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     4681 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     4741 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     4801 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     4861 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag
     4921 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     4981 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     5041 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     5101 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     5161 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
     5221 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     5281 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     5341 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     5401 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     5461 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
     5521 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     5581 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     5641 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     5701 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     5761 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta
     5821 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
     5881 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     5941 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
     6001 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
     6061 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
     6121 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga
//
