LOCUS       sequence-349639-        6854 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 6854)
  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 6854)
  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: pEE007
FEATURES             Location/Qualifiers
     source          1..6854
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     965..984
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1004..1081)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1287..1306
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1390..1409
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1390..1408
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1409..1433
                     /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             1448..1470
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2975..2992
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3045..3063)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3059..3106
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3143..3598
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3230..3249)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3440..3461
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3624..3728
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3729..3797,3798..4589)
                     /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(3947..3966)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4760..5348
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5249..5268
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5502..5519
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5534..5673
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5599..5618)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5759..5781
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5775..5966)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6775..6854,1..1003))
                     /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 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
       61 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
      121 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
      181 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
      241 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
      301 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
      361 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
      421 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
      481 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
      541 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
      601 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
      661 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
      721 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
      781 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
      841 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
      901 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
      961 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     1021 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     1081 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     1141 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     1201 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     1261 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     1321 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     1381 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
     1441 aaataatttt gtttaacttt aagaaggaga tatacatatg caaggcatca ctgtgcgcac
     1501 cccgctgggt ccggcgttag gtcaaatgga aaaaggtgcg atcgcgttct acggtctgcc
     1561 gtatgctcag gcgtctcgtt tcgaagctcc acgcccagta gcggcttggc cgccaggcgt
     1621 aggccgtgaa cgtgttgcgt gcccgcagac tccgggtact accgcgcgtc tgggtggcta
     1681 tatcccgccg caacgtgaag actgtctggt cgcaaatctg ttcctgccgc tggaaccgcc
     1741 gccacctgag ggtttcccgg taatggtgta tctgcatggc ggcggtttca cgtctggctc
     1801 cgcagcagaa ccaatttacg gtggacaccg tatggctcag gaaggtgtgg ttgttgtttc
     1861 tgttaactac cgcctgggcc cgctgggctt cctggctctt ccggccctgg aaaaagaaaa
     1921 cccgaaggca gtaggtaact acggtctgct ggacctggta gaggctttac gtttcgtcca
     1981 gcgtcacatc cgttacttcg ggggtaaccc gcagaacgtt actctgttcg gtgaatctgc
     2041 tggtggcatg ctggtctgca cccttctggc taccccggaa gcgcagggtc tgttccacaa
     2101 agcgattctg caatccggcg gctgccacca ggtccgcccg ctggaacgcg atttcccttt
     2161 cggggaacag tgggctaaaa acctggggtg ctctccggag gacttggctt gcctgcgcaa
     2221 cctgccactg tctcgcctgt tcccaactat ggaaccgaaa gcgccgccgg atatcacagc
     2281 tagcgctttg ggcttcccaa actccccgtt caaaccgcac ctgggcgcac tgctgccgga
     2341 atccccgacc gaggcactgc gtaagggcca agctcgcgat attccgttgc tcgtcggtgc
     2401 taatctcgaa gagctggctt tcccgggcct ggcttggctg ctgggtccgc gccgttggga
     2461 agaattcggt cagcgcctgg cggcgcaagg tcttactcag cagcagcgtg aggcattaaa
     2521 aggcgtttat cagaaacgtt tctccgagcc gcgtgccgcc tggggtcaag cgcagactga
     2581 tctgcttctg ctgtgcccat ccctgaaagc agcacgtctg caggcctcct tcgccccgac
     2641 ctacgcgtat cttttcactt ttcgtgtgcc aggtttcgaa ggcctcggtg cgttccacgg
     2701 tctggaactg gcgccgctgt ttggcaactt tgaagaaatg ccgttcctgc cgctgttcct
     2761 gagcgctgaa gctcgcgaaa aagcggaagc tctcggcaaa cgtatgcgtc gttactgggt
     2821 tagcttcgca cgcgaaggtg aaccgcgctc ttggccgcac tggcctactt atgaagaggg
     2881 ttacttgctg cgcctcgatg aaccgccggg tctgatcccg gatctgtatg aagaacgttg
     2941 cggtgtcctg gaagcgctgg gcttgctgct cgagcaccac caccaccacc actgagatcc
     3001 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     3061 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     3121 tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     3181 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     3241 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     3301 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     3361 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     3421 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     3481 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     3541 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     3601 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     3661 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     3721 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     3781 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     3841 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     3901 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     3961 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     4021 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     4081 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     4141 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     4201 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     4261 ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     4321 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     4381 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     4441 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     4501 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     4561 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     4621 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     4681 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     4741 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     4801 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     4861 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     4921 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     4981 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     5041 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     5101 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     5161 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     5221 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     5281 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     5341 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     5401 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     5461 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     5521 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     5581 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     5641 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     5701 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     5761 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     5821 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     5881 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     5941 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     6001 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     6061 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     6121 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     6181 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     6241 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
     6301 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     6361 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     6421 atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg
     6481 gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc
     6541 gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc
     6601 gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg
     6661 atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc
     6721 ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     6781 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     6841 gggagaggcg gttt
//
