LOCUS       sequence-349686-        6149 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 6149)
  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 6149)
  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: pEE071
FEATURES             Location/Qualifiers
     source          1..6149
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     930..949
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(969..1046)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1252..1271
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1355..1374
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1355..1373
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1374..1398
                     /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             1413..1435
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2235..2252
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2305..2323)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2319..2366
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2403..2858
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2490..2509)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2700..2721
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2884..2988
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2989..3057,3058..3849)
                     /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(3207..3226)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4020..4608
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4509..4528
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4762..4779
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4794..4933
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4859..4878)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5019..5041
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5035..5226)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6035..6149,1..968))
                     /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 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
       61 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
      121 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg
      181 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
      241 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
      301 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
      361 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
      421 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
      481 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
      541 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
      601 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
      661 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
      721 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
      781 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
      841 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
      901 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
      961 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     1021 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
     1081 cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg
     1141 tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac
     1201 gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc
     1261 ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc
     1321 ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat aggggaattg
     1381 tgagcggata acaattcccc tctagaaata attttgttta actttaagaa ggagatatac
     1441 atatggctaa cccgtacgaa cgtggcccga acccaaccaa cagcagcatc gaagcactgc
     1501 gaggtccgtt ccgcgtcgac gaagaacgcg tctctcgtct gcaagctcgt ggtttcggcg
     1561 gcggcactat ctactacccg actgataaca acaccttcgg cgctgttgct atctcgccgg
     1621 gttataccgg cacccagtcg tctattagct ggctgggcga gcgtctggcg tcccacggtt
     1681 tcgtcgtcat gactatcgat actaacacca ccttggacca gccggactct cgtgcatctc
     1741 agctggatgc ggcactggat tatatggtgg aagattctag ctattctgta cgtaaccgta
     1801 ttgattcttc tcgtctggcg gcgatgggtc attccatggg tggtggtggt accctgcgtc
     1861 tggctgaacg tcgcccggac ctgcaagctg caatcccgct gaccccttgg catacagaca
     1921 aaacttgggg ctctgtacgt gtaccgacgc tgatcattgg tgccgaaaat gataccattg
     1981 catctgtacg cagccatagc gaaccgttct ataatagcct gccgggatct ctggacaaag
     2041 cgtatctcga actggacggc gcaagccact tcgctccgaa cctttccaac actacgatcg
     2101 cgaaatactc catctcctgg ctgaaacgtt tcgtggatga tgacactcgc tacacccagt
     2161 tcctgtgccc aggcccgtca actggctggg gctcagacgt agaagaatat cgcagcactt
     2221 gcccgtttct cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa
     2281 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     2341 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
     2401 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     2461 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     2521 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     2581 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     2641 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     2701 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     2761 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     2821 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
     2881 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     2941 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     3001 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     3061 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     3121 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     3181 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     3241 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     3301 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     3361 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     3421 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     3481 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg
     3541 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     3601 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     3661 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     3721 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     3781 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     3841 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     3901 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     3961 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     4021 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     4081 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     4141 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     4201 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     4261 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     4321 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     4381 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     4441 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     4501 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     4561 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     4621 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     4681 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     4741 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     4801 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     4861 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     4921 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     4981 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     5041 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     5101 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     5161 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     5221 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     5281 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     5341 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     5401 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     5461 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     5521 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     5581 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     5641 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     5701 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     5761 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     5821 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     5881 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     5941 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     6001 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     6061 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     6121 tgggcgccag ggtggttttt cttttcacc
//
