LOCUS       sequence-349625-        6185 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 6185)
  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 6185)
  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: pEE021
FEATURES             Location/Qualifiers
     source          1..6185
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     564..583
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(603..680)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     886..905
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     989..1008
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        989..1007
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1008..1032
                     /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             1047..1069
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1905..1922
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1975..1993)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1989..2036
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2073..2528
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2160..2179)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2370..2391
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2554..2658
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2659..2727,2728..3519)
                     /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(2877..2896)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3690..4278
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4179..4198
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4432..4449
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4464..4603
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4529..4548)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4689..4711
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4705..4896)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5705..6185,1..602))
                     /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 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
       61 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
      121 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
      181 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
      241 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
      301 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
      361 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
      421 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
      481 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
      541 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
      601 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
      661 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
      721 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
      781 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
      841 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      901 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      961 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
     1021 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacatatga
     1081 gcgaactgga gctgaacgaa gtgttcaaac tgattaaact ggtctcgttc atgaacaagg
     1141 gtcaacaaat cattggtgtg ctgcacaaac cggataaaat caaaccgcac gaaaaagtac
     1201 cggggatcgt tatgttccac ggttttactg gtaacaaatc tgaagctcac cgtctgttcg
     1261 tgcacatcgc acgtggtctg tcttcccgtg gcttcatggt actgcgtttt gatttccgtg
     1321 gtagcggcga ttctgatggc gacttcgaag atatgacact gccggaagaa gtttctgacg
     1381 cggagcgtgc catcaccttc gtactgcgtc agcgtaacgt cgaccgtgaa aaaatcgggg
     1441 tcattggtct gtccattggt ggccgtgtag cggccatcct ggcgtctcgc gacgaacgta
     1501 ttaaattcgc agtgctgtac tccccggcgc tgggccgtct gaaagaacgc ttcctgtccc
     1561 tcatgggcga agaggccctg cgtcggctga actgtggcga accgatcgaa gtaagctctg
     1621 gctggtacct gaagaaagcc ttcttcgaaa ccgtggacta catcgtaccg gttgaagtta
     1681 tgtccaacat tcgcgttccg gttctgatca ttcacggtga tcgtgatgaa atcatccctg
     1741 ttgaagagag catgaaagca tacgagcgca taaaaggtct gaacgaaaaa aacgaactgt
     1801 acatcgttaa aggcggtgac cataccttct ctaagcgcga acacactctg gaagtgctga
     1861 ataaaacgat cgaatggctg agctctctga acctgatgct cgagcaccac caccaccacc
     1921 actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     1981 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     2041 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     2101 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     2161 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     2221 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     2281 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     2341 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     2401 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     2461 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     2521 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     2581 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     2641 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     2701 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     2761 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     2821 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     2881 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     2941 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     3001 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     3061 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     3121 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     3181 gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc
     3241 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     3301 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     3361 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     3421 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     3481 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     3541 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     3601 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     3661 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     3721 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     3781 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     3841 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     3901 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     3961 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     4021 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     4081 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     4141 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     4201 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     4261 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     4321 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     4381 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     4441 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     4501 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     4561 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     4621 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     4681 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     4741 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     4801 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     4861 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     4921 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     4981 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     5041 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     5101 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     5161 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     5221 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     5281 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     5341 gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc
     5401 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     5461 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     5521 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     5581 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     5641 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     5701 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     5761 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     5821 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     5881 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     5941 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     6001 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     6061 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     6121 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     6181 tatgc
//
