LOCUS       sequence-349641-        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: pEE066
FEATURES             Location/Qualifiers
     source          1..6149
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     950..969
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(989..1066)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1272..1291
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1375..1394
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1375..1393
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1394..1418
                     /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             1433..1455
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2255..2272
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2325..2343)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2339..2386
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2423..2878
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2510..2529)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2720..2741
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2904..3008
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3009..3077,3078..3869)
                     /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(3227..3246)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4040..4628
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4529..4548
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4782..4799
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4814..4953
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4879..4898)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5039..5061
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5055..5246)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6055..6149,1..988))
                     /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 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
       61 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
      121 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
      181 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
      241 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
      301 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
      361 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
      421 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
      481 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
      541 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
      601 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
      661 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
      721 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
      781 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
      841 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
      901 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
      961 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     1021 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     1081 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     1141 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     1201 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     1261 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     1321 atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac
     1381 gactcactat aggggaattg tgagcggata acaattcccc tctagaaata attttgttta
     1441 actttaagaa ggagatatac atatggctaa cccatacgaa cgcggtccga atccgacgga
     1501 cgccctgctg gaggcgcgtt ctggtccttt cagcgttagc gaagaacgtg catcccgttt
     1561 cggtgctgat ggcttcggtg gtgggaccat ctactacccg cgtgaaaaca acacatacgg
     1621 cgcggtcgct atctccccgg gctatacggg cacacaagct tctgtggctt ggctgggtga
     1681 gcgtatcgcg tctcatggct tcgttgtcat cacgattgac actaacacca ccctggacca
     1741 gccggattca cgtgcccgtc agctgaacgc agcgctcgat tacatgatta acgatgcctc
     1801 gtccgctgtg cgttcccgta tcgattcttc tcgtctggca gttatgggtc actctatggg
     1861 tggcggcggt acactgcgcc tcgccagcca gcgtccggac ctgaaggctg ccatcccact
     1921 gaccccgtgg cacctgaaca aaaactggtc ttcagtacgc gtgccgactc tgatcatcgg
     1981 tgctgacctg gacaccatcg cgccggttct gactcatgcg cgtccgttct acaactctct
     2041 gccgacctct atttcgaaag cctatttaga gctggatggt gcaacccact ttgcaccgaa
     2101 catccctaac aaaattattg ggaagtattc tgttgcatgg ctgaaacgct tcgtggacaa
     2161 cgacacccgc tatactcagt ttctgtgtcc ggggccgcgc gacggtcttt tcggtgaggt
     2221 tgaagaatac cgttcgactt gcccgttcct cgagcaccac caccaccacc actgagatcc
     2281 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     2341 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     2401 tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     2461 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     2521 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     2581 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     2641 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     2701 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     2761 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     2821 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     2881 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     2941 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     3001 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     3061 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     3121 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     3181 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     3241 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     3301 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     3361 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     3421 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     3481 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     3541 ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     3601 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     3661 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     3721 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     3781 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     3841 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     3901 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     3961 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     4021 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     4081 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     4141 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     4201 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     4261 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     4321 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     4381 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     4441 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     4501 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     4561 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     4621 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     4681 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     4741 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     4801 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4861 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     4921 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     4981 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     5041 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     5101 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     5161 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     5221 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     5281 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     5341 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     5401 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     5461 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     5521 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
     5581 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     5641 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     5701 atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg
     5761 gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc
     5821 gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc
     5881 gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg
     5941 atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc
     6001 ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     6061 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     6121 gggagaggcg gtttgcgtat tgggcgcca
//
