LOCUS       sequence-349681-        6158 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 6158)
  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 6158)
  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: pEE077
FEATURES             Location/Qualifiers
     source          1..6158
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     969..988
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1008..1085)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1291..1310
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1394..1413
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1394..1412
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1413..1437
                     /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             1452..1474
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2283..2300
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2353..2371)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2367..2414
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2451..2906
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2538..2557)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2748..2769
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2932..3036
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3037..3105,3106..3897)
                     /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(3255..3274)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4068..4656
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4557..4576
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4810..4827
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4842..4981
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4907..4926)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5067..5089
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5083..5274)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6083..6158,1..1007))
                     /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"
ORIGIN
        1 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
       61 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
      121 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
      181 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
      241 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
      301 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
      361 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
      421 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
      481 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
      541 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
      601 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
      661 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
      721 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
      781 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
      841 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
      901 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
      961 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     1021 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     1081 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     1141 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     1201 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     1261 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
     1321 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc
     1381 gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa caattcccct
     1441 ctagaaataa ttttgtttaa ctttaagaag gagatataca tatggcgaac ccatacgaac
     1501 gtggtccgaa cccgaccgat gcgctgttgg aagctcgtag cggtccgttc agcgtttctg
     1561 aggaaaacgt gagccgtttc ggcgcagacg gtttcggcgg cggcactatc tactaccctc
     1621 gcgaaaataa cacatatggc gcggttgcta tttctccggg ctacaccggt acccaagcga
     1681 gcgttgcgtg gctgggcgag cgtatcgcgt cacacggctt cgtggtaatc acaatcgaca
     1741 caaacactac tctggaccag ccggattcac gtgcccgcca gcttaacgcg gcgctggatt
     1801 acatgatcaa cgacgcgtcc tcagcggtgc gttcacgtat cgacagctcc cgtctggcgg
     1861 tgatgggtca ctccatgggc ggtggtggca ccctccgcct ggcgtcccag cgtccagatc
     1921 ttaaagcggc aatcccgctg acaccttggc acctgaacaa aaactggtcc tctgttcgcg
     1981 ttccaacttt gatcatcggc gccgatctgg acactatcgc accggtgctc acccacgctc
     2041 gcccgtttta taacagcctg ccaacttcga tttccaaagc ctacctggaa ttagacggtg
     2101 cgacccactt tgcgccgaac attccaaaca aaatcatcgg taaatattct gtcgcctggc
     2161 tcaaacgctt cgtggataac gatactcgtt acacgcaatt cctgtgtcca ggtccacgtg
     2221 atggtctttt cggcgaagtt gaagagtacc gtagcacttg cccgttcgca ctggaactcg
     2281 agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt
     2341 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     2401 tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg
     2461 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     2521 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     2581 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     2641 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     2701 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     2761 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     2821 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     2881 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     2941 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     3001 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     3061 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     3121 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     3181 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     3241 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     3301 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     3361 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     3421 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     3481 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     3541 gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg
     3601 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     3661 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     3721 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     3781 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     3841 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     3901 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     3961 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     4021 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     4081 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4141 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4201 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4261 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4321 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4381 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4441 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4501 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4561 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4621 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4681 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     4741 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     4801 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     4861 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     4921 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     4981 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     5041 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     5101 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     5161 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     5221 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     5281 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     5341 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     5401 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     5461 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     5521 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     5581 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     5641 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     5701 ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat
     5761 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag
     5821 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa
     5881 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
     5941 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
     6001 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     6061 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     6121 gctgcattaa tgaatcggcc aacgcgcggg gagaggcg
//
