LOCUS       sequence-342590-        5957 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 5957)
  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 5957)
  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: pEE011
FEATURES             Location/Qualifiers
     source          1..5957
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             598..615
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(668..686)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      682..729
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      766..1221
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(853..872)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1063..1084
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1247..1351
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1352..1420,1421..2212)
                     /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(1570..1589)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2383..2971
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2872..2891
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3125..3142
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3157..3296
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3222..3241)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3382..3404
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3398..3589)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4398..5480)
                     /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     5442..5461
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5481..5558)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5764..5783
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5867..5886
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5867..5885
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5886..5910
                     /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             5925..5947
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 gttccgctga tcctgaacgt gcatggtggt ccggcaggcg ttttccagca gacctttacc
       61 ggtggtcgca gcatctatcc gatcgctacc ttcgcagcgc gcggttacgc tgttctgcgt
      121 ccgaacccgc gcggttcctc tggttatggt gtggaattcc gccgtgccaa cctcaaagat
      181 tggggtggca tggactacca ggacctcatg gctggtgttg acaaagtaat cgaaatgggt
      241 gtggcggact cctcccgtct gggtgttatg ggttggtcct atggtggttt tatgacgtct
      301 tggatcgtaa ctcagacaaa ccgtttcaaa gcggcttcag caggcgctcc ggtcaccaac
      361 ctgacgtcat tcactactac cgccgacatc ccggcgttca tcccggacta tttcggcggc
      421 cagttctggg actctccgga agtgtaccgt gcacactccc caatttcgtt cgtgaaatct
      481 gtcaccactc ctactatgat ccagcacggt accgcagaca tgcgtgttcc gatcagccag
      541 ggtttcgaat tctacaacgc tctcaaagct cgtggcatcc cgacccgcat gctcgagcac
      601 caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct
      661 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
      721 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
      781 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
      841 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
      901 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
      961 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     1021 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     1081 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     1141 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     1201 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
     1261 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     1321 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     1381 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     1441 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     1501 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     1561 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     1621 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     1681 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     1741 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     1801 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     1861 cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa
     1921 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     1981 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     2041 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     2101 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     2161 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     2221 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     2281 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     2341 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     2401 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     2461 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     2521 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     2581 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     2641 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     2701 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     2761 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     2821 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     2881 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     2941 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     3001 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     3061 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     3121 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     3181 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc
     3241 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     3301 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     3361 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     3421 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     3481 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     3541 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     3601 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     3661 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     3721 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     3781 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     3841 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     3901 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     3961 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     4021 tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg
     4081 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
     4141 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt
     4201 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
     4261 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
     4321 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     4381 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4441 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     4501 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     4561 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     4621 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
     4681 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     4741 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     4801 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     4861 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     4921 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     4981 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     5041 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     5101 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     5161 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     5221 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     5281 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     5341 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     5401 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     5461 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     5521 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     5581 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     5641 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
     5701 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
     5761 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
     5821 cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact
     5881 ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag
     5941 aaggagatat acatatg
//
