LOCUS       sequence-349195-        6278 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 6278)
  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 6278)
  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: pEE037
FEATURES             Location/Qualifiers
     source          1..6278
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(70..1152)
                     /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     1114..1133
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1153..1230)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1436..1455
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1539..1558
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1539..1557
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1558..1582
                     /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             1597..1619
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2548..2565
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2618..2636)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2632..2679
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2716..3171
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2803..2822)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3013..3034
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3197..3301
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3302..3370,3371..4162)
                     /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(3520..3539)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4333..4921
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4822..4841
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5075..5092
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5107..5246
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5172..5191)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5332..5354
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5348..5539)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
       61 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
      121 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
      181 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
      241 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
      301 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc
      361 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
      421 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
      481 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
      541 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
      601 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
      661 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
      721 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
      781 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
      841 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
      901 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
      961 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     1021 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     1081 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     1141 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     1201 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
     1261 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     1321 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     1381 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     1441 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     1501 gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga
     1561 attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta agaaggagat
     1621 atacatatgc gttttcgtgt tgcagcgtct ctgccggctg agtatctggc tgtggacaac
     1681 gttgtcctgg aaggtaccgc tcagccaccg gcaccgggtg ggtcgggtta tcagaaaggc
     1741 ccagagccaa ccgcggccct gctggaagcg ggtaccggtc cattcgcaac cgctagcgtt
     1801 accctgtccc gctccgcggc ctccggcttc ggtgggggta ccatccatta cccgcaaggt
     1861 gtagccggcc ctttcgcggc agttgcggtc gttccgggct acctggctgc agagtcgacc
     1921 atcgcttggt ggggtccgcg cctggccagc cacggtttcg ttgttatcac tatggcgacg
     1981 aacaacacct tggacctgcc ggccagccgc agcgctcagc tgactgcagc tttaaaccag
     2041 ttaaaaaccc tgtccgcaac tccgggtcat gcagtttttg gcctggttga cccgaaccgc
     2101 ctgggtgtgg taggttggtc ttacggtggc ggtggtactc tgctgaacgc tcaggctaac
     2161 ccgcagctga aagcagctat ggccctggct ccaaaaaccc tcctccaggg tgatttcact
     2221 ggtaccaccg tcccgaccct tgttgttggt tgtcaggcgg atactaccgc tgctccggcg
     2281 ttttgggcaa tcccattcta caacaaggtg tccgcttcta cgggcaaagc atacctggaa
     2341 gttcgtggtg ggtcacactt ctgcgtaacc agcagcacct ctgatgcaga caaaaaggcg
     2401 ctgggtaaat acggtgtagc gtggctgaaa cgcttcatgg acgaagatac ccgctatgct
     2461 ccattcctgt gcggtgctcc acgccaggct gacgtcgcag gcaatgcggc aattagcgac
     2521 tatcgtgata actgcccgta tctcgagcac caccaccacc accactgaga tccggctgct
     2581 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     2641 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     2701 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2761 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2821 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     2881 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     2941 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     3001 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     3061 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     3121 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     3181 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3241 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     3301 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     3361 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     3421 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     3481 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     3541 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     3601 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     3661 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     3721 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     3781 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     3841 gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     3901 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     3961 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     4021 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     4081 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     4141 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     4201 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     4261 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     4321 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     4381 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     4441 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     4501 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     4561 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     4621 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     4681 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     4741 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     4801 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     4861 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     4921 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     4981 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     5041 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     5101 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     5161 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     5221 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     5281 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     5341 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     5401 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     5461 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     5521 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     5581 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     5641 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     5701 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     5761 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     5821 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     5881 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     5941 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     6001 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     6061 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     6121 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     6181 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     6241 tgccccgcgc ccaccggaag gagctgactg ggttgaag
//
