LOCUS       sequence-349076-        6173 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 6173)
  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 6173)
  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: pEE048
FEATURES             Location/Qualifiers
     source          1..6173
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     921..940
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(960..1037)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1243..1262
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1346..1365
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1346..1364
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1365..1389
                     /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             1404..1426
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2250..2267
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2320..2338)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2334..2381
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2418..2873
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2505..2524)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2715..2736
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2899..3003
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3004..3072,3073..3864)
                     /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(3222..3241)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4035..4623
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4524..4543
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4777..4794
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4809..4948
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4874..4893)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5034..5056
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5050..5241)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6050..6173,1..959))
                     /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 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
       61 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
      121 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
      181 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
      241 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
      301 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
      361 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
      421 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
      481 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
      541 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
      601 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
      661 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
      721 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
      781 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
      841 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
      901 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
      961 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     1021 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     1081 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     1141 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     1201 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     1261 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     1321 gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat
     1381 aacaattccc ctctagaaat aattttgttt aactttaaga aggagatata catatgcagg
     1441 cgcagtacca gaaaggtccg gatccgactg cttctgctct ggagcgcaac ggtccgttcg
     1501 ctatccgttc aaccagcgtt agccgtacta gcgtaagcgg ctttggtggt ggccgtctgt
     1561 actacccgac ggccagcggc acgtatggtg cgattgccgt tagccctggt tttaccggca
     1621 ctagctctac tatgaccttt tggggtgaac gtctggcctc tcacggcttc gtagtacttg
     1681 taatcgatac aatcactctg tacgatcagc cggactcccg cgcacgccag ctgaaagcag
     1741 cactggacta cctggccacc cagaacggtc gctcctcatc tccgatctat cgtaaagtcg
     1801 acacttctcg tcgtgcggtt gccggccaca gcatgggtgg tggcggcagt ctgctggcag
     1861 cacgtgacaa tccatcttac aaagccgcga tcccaatggc gccgtggaac acctcctcta
     1921 ccgcctttcg taccgtttct gtcccgacca tgatcttcgg ctgtcaggat gactctatcg
     1981 ccccagtatt ctctcatgct atcccgttct acaacgcgat cccgaacagc acgcgcaaaa
     2041 actacgttga aatccgtaac gacgaccact tctgtgtgat gaacggcggt ggccacgatg
     2101 caactctggg taaattgggc atctcttgga tgaaacgctt cgtggacaat gatacccgtt
     2161 acagcccgtt cgtgtgtggt gcggagtaca accgtgttgt ttcatcttac gaagtgtccc
     2221 gttcttataa caactgtccg tatctcgagc accaccacca ccaccactga gatccggctg
     2281 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
     2341 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     2401 ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2461 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     2521 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2581 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     2641 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     2701 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     2761 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     2821 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     2881 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     2941 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     3001 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     3061 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     3121 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     3181 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     3241 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     3301 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     3361 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     3421 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     3481 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     3541 atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     3601 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     3661 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     3721 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     3781 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     3841 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     3901 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     3961 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     4021 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     4081 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     4141 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     4201 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     4261 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     4321 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     4381 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     4441 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     4501 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     4561 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     4621 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     4681 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     4741 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     4801 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat
     4861 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     4921 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     4981 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     5041 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     5101 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     5161 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     5221 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
     5281 aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc
     5341 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
     5401 aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg
     5461 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
     5521 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
     5581 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
     5641 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg
     5701 cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
     5761 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
     5821 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
     5881 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
     5941 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     6001 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     6061 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     6121 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acg
//
