LOCUS       sequence-349074-        6140 bp    DNA     circular SYN 15-APR-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 6140)
  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 6140)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pEE046
FEATURES             Location/Qualifiers
     source          1..6140
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     545..564
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(584..661)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     867..886
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     970..989
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        970..988
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    989..1013
                     /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             1028..1050
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1841..1858
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1911..1929)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1925..1972
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2009..2464
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2096..2115)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2306..2327
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2490..2594
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2595..2663,2664..3455)
                     /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(2813..2832)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3626..4214
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4115..4134
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4368..4385
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4400..4539
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4465..4484)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4625..4647
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4641..4832)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5641..6140,1..583))
                     /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 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
       61 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
      121 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
      181 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
      241 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
      301 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
      361 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
      421 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
      481 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
      541 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
      601 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
      661 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
      721 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
      781 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
      841 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
      901 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
      961 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
     1021 aaataatttt gtttaacttt aagaaggaga tatacatatg cagaccagcc cgccgactag
     1081 cgctagcctt aacgcgactg caggcccgct gtctgtttcg acctccagcg tatctagctg
     1141 ggcagcacgc ggtttcggtg gtggcaccat ctattacccg aacgctaccg gccgctacgg
     1201 tgttgtggca atctcaccgg gctatactgc gcgccagtcc tcaattgctt ggctgggccg
     1261 tcgtctggct acccatggtt tcgtggtgat cactattgac accaactcca ccttggacca
     1321 gccgccgtcc cgtgctaccc agctcatggc ggcacttaac cacgtagtga acaacgcgaa
     1381 cgctacggtt cgttcgcgtg tcgatgcctc taaactcgcg gtagccggcc attccatggg
     1441 tggtggcggt tctctgatcg ctgctgaaaa caacccgagc ctgaaagcag cctacccgct
     1501 gactccgtgg agcgtctcta aaaactacag tagtgttcgt gtgccgacca tgatcatcgg
     1561 cgctgatggt gattccatcg caagtgtgtc cacccactct cgtctgttct acaactctct
     1621 gtctagcaac gttagcaaag cgtacggcga actgaacaac gcgtctcatt tcacccctaa
     1681 ctacaccaac actccgattg gccgctacgc tgttacctgg atgaaacgtt tcgtagacaa
     1741 cgatacccgt tattctccgt tcctttgcgg tgcaccgcac gacagctacg cgacccgtac
     1801 cgtgttcgac cgctatgaag ataactgtgc gtatctcgag caccaccacc accaccactg
     1861 agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     1921 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     1981 aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     2041 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     2101 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     2161 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     2221 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     2281 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     2341 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     2401 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     2461 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     2521 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     2581 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     2641 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     2701 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     2761 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     2821 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     2881 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     2941 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     3001 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     3061 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     3121 gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac
     3181 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     3241 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     3301 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     3361 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     3421 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     3481 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     3541 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     3601 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     3661 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     3721 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     3781 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     3841 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     3901 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     3961 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     4021 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     4081 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     4141 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     4201 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     4261 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     4321 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     4381 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     4441 cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     4501 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
     4561 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     4621 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     4681 ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc
     4741 gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc
     4801 atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg
     4861 ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat
     4921 gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg
     4981 cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt
     5041 gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc
     5101 gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt
     5161 gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat
     5221 tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc
     5281 acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa
     5341 cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc
     5401 gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc
     5461 cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg
     5521 gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     5581 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     5641 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     5701 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     5761 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     5821 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     5881 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag
     5941 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     6001 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     6061 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     6121 ccagccagcc agacgcagac
//
