LOCUS       sequence-350564-        6215 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 6215)
  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 6215)
  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: pEE034
FEATURES             Location/Qualifiers
     source          1..6215
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1300..1491
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1485..1507)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1593..1732
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1648..1667
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1747..1764)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1918..2506)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1998..2017)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2677..3468,3469..3537))
                     /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     3300..3319
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3538..3642)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3668..4123)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3805..3826)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4017..4036
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4160..4207)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4203..4221
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4274..4291)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             5157..5179
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    5194..5218
                     /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)."
     primer_bind     complement(5218..5237)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5219..5237)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5321..5340)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5546..5623
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5624..6215,1..491)
                     /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     complement(5643..5662)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag
       61 gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg
      121 ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta
      181 ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag
      241 acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc
      301 aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc
      361 tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct
      421 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
      481 gcgggcagtg agcgcaacgc aattaatgta agttagctca ctcattaggc accgggatct
      541 cgaccgatgc ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg
      601 actatcgtcg ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg
      661 gcagcgctct gggtcatttt cggcgaggac cgctttcgct ggagcgcgac gatgatcggc
      721 ctgtcgcttg cggtattcgg aatcttgcac gccctcgctc aagccttcgt cactggtccc
      781 gccaccaaac gtttcggcga gaagcaggcc attatcgccg gcatggcggc cccacgggtg
      841 cgcatgatcg tgctcctgtc gttgaggacc cggctaggct ggcggggttg ccttactggt
      901 tagcagaatg aatcaccgat acgcgagcga acgtgaagcg actgctgctg caaaacgtct
      961 gcgacctgag caacaacatg aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg
     1021 cggaagtcag cgccctgcac cattatgttc cggatctgca tcgcaggatg ctgctggcta
     1081 ccctgtggaa cacctacatc tgtattaacg aagcgctggc attgaccctg agtgattttt
     1141 ctctggtccc gccgcatcca taccgccagt tgtttaccct cacaacgttc cagtaaccgg
     1201 gcatgttcat catcagtaac ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt
     1261 acccccatga acagaaatcc cccttacacg gaggcatcag tgaccaaaca ggaaaaaacc
     1321 gcccttaaca tggcccgctt tatcagaagc cagacattaa cgcttctgga gaaactcaac
     1381 gagctggacg cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag
     1441 ctttaccgca gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     1501 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     1561 ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat
     1621 agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     1681 attgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc
     1741 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     1801 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     1861 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     1921 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     1981 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     2041 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     2101 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     2161 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     2221 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     2281 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     2341 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     2401 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     2461 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     2521 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     2581 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     2641 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     2701 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     2761 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     2821 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     2881 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     2941 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat
     3001 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     3061 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     3121 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     3181 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     3241 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     3301 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     3361 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     3421 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     3481 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     3541 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     3601 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     3661 gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     3721 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     3781 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     3841 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     3901 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     3961 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     4021 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     4081 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccaatccgg
     4141 atatagttcc tcctttcagc aaaaaacccc tcaagacccg tttagaggcc ccaaggggtt
     4201 atgctagtta ttgctcagcg gtggcagcag ccaactcagc ttcctttcgg gctttgttag
     4261 cagccggatc tcagtggtgg tggtggtggt gctcgaggta attgcaagtg tcgcggtatt
     4321 cagagatatc ccattcagct tcgtggttcg gaccgcacag gaatgcatca taacgacggt
     4381 cgtcatcgat gaaacgtttc atccatgcaa ctccatattt acccaggatg tcctcatccg
     4441 ggtaaccgct gtttgcgcag aagtggtcac cgttacggat ctccaggaaa gcttttgcgg
     4501 tagagttcgg aatttcgttg tagaacggac tagcgtgttg cgccactggg gcgacagcat
     4561 ccagctggca agcgatgatg agggtagggg ttgtgatttc atcgaacggg ttaaagccgg
     4621 agtaccatgg cgcctgcgga atagcagcct tcagataacg gtccgtagac agtttcagag
     4681 taccgccgcc acccatggac cagccgattg cacccaggcg gttcggatca accatgccct
     4741 ggatcgcaga acggctggag ttgctctgcg cgatcacata atccagagca gcagacagct
     4801 ggttggcgcg ggagtccggc tgatcgtaga tggagttagt gtcgatcgta attacaacga
     4861 agccccatga cgcaagacgg ccgccccacc attcgatcga gttttcataa gaaacgtaac
     4921 ccgggatcac agcgatagcg cccatggtgc cctgtgtacc taccgggtag tggatagttc
     4981 caccaccgaa accagagacc cagcttgaaa cgttaatggt gcggactgaa tacggaccgc
     5041 ggtcagcttc cagcgccgct acggtcgggt caggcccacg ctgataacag tcaccgcaac
     5101 cgcccggcgg ttccggttcc ggttcaggtg tcggcgttgg ggtagtcata tgtatatctc
     5161 cttcttaaag ttaaacaaaa ttatttctag aggggaattg ttatccgctc acaattcccc
     5221 tatagtgagt cgtattaatt tcgcgggatc gagatctcga tcctctacgc cggacgcatc
     5281 gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc
     5341 gatggggaag atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg
     5401 gtggcaggcc ccgtggccgg gggactgttg ggcgccatct ccttgcatgc accattcctt
     5461 gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg
     5521 cataagggag agcgtcgaga tcccggacac catcgaatgg cgcaaaacct ttcgcggtat
     5581 ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt
     5641 atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca
     5701 ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa
     5761 ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt
     5821 tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg
     5881 cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc
     5941 ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta
     6001 tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt
     6061 atttcttgat gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg
     6121 tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc
     6181 gggcccatta agttctgtct cggcgcgtct gcgtc
//
