LOCUS       sequence-349199-        6212 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 6212)
  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 6212)
  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: pEE029
FEATURES             Location/Qualifiers
     source          1..6212
                     /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             2333..2350
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2403..2421)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2417..2464
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2501..2956
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2588..2607)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2798..2819
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2982..3086
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3087..3155,3156..3947)
                     /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(3305..3324)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4118..4706
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4607..4626
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4860..4877
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4892..5031
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4957..4976)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5117..5139
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5133..5324)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6133..6212,1..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 aaggtgaagt ctaaacccct
     1501 gactctgtac aacgtttctg gcgaccgtat tactgcggat gtccactttg ttgaatcctt
     1561 tctgccggcg ccggttgtga tctacagcca cggctttctt ggtttcaaag actggggctt
     1621 catcccgtac gtggcagaac gtttcgcgga aaacggcttc gtcttcgttc gttttaactt
     1681 ttcccacaac ggcatcggtg agaacccgaa caaaattacc gaatttgata aactggcgaa
     1741 aaacaccatt agcaagcaga tcgaagatct gactgcagtt atcgaatacg tattctccga
     1801 tgaatttggt gttctcaacg atggtcaact gttcctcctg ggccactctg gcggcggtgg
     1861 catctccatc atcaaggcag tcgaagacga acgtgttcgt gctctggctc tgtgggcaag
     1921 catctccact ttccgtcggt actctaagca tcagattgaa gaactggaaa aaaacggcta
     1981 tatcttcgtt cgtgtcccgg actctgttat tcaggtcaaa attgaaaaga ttgtttacga
     2041 tgatttcgtc gagaactctg aacgctacga tatcatcaaa gccatttcta agctgaaaat
     2101 cccaatcctg atcgtgcacg gcactgcgga cgctattgtc ccgctggcag aggcggaaaa
     2161 actgcgtaat tccaacccgg aatataccaa actggtcctg atctccggtg ccaatcacct
     2221 gttcaacgta aaacatccga tggagcacag cactgaccaa ctggataaag cgatcgacga
     2281 gaccgttctg ttcttcaaga agattatcga aaacaaaaag gcggatctcg agcaccacca
     2341 ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     2401 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     2461 tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca
     2521 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2581 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2641 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2701 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     2761 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     2821 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     2881 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     2941 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     3001 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     3061 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     3121 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     3181 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     3241 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     3301 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     3361 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     3421 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     3481 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     3541 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     3601 caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat
     3661 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     3721 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     3781 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     3841 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     3901 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     3961 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     4021 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     4081 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     4141 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4201 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4261 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4321 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4381 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4441 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4501 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4561 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4621 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4681 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4741 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     4801 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     4861 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     4921 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     4981 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     5041 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     5101 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     5161 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     5221 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     5281 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     5341 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     5401 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     5461 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     5521 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     5581 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     5641 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     5701 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     5761 aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc
     5821 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag
     5881 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg
     5941 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     6001 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     6061 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     6121 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     6181 tgaatcggcc aacgcgcggg gagaggcggt tt
//
