LOCUS       sequence-349680-        6146 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 6146)
  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 6146)
  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: pEE072
FEATURES             Location/Qualifiers
     source          1..6146
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1341..1532
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1526..1548)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1634..1773
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1689..1708
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1788..1805)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1959..2547)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2039..2058)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2718..3509,3510..3578))
                     /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     3341..3360
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3579..3683)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3709..4164)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3846..3867)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4058..4077
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4201..4248)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4244..4262
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4315..4332)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             5129..5151
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    5166..5190
                     /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)."
     primer_bind     complement(5190..5209)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5191..5209)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5293..5312)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5518..5595
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5596..6146,1..532)
                     /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"
     primer_bind     complement(5615..5634)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata
       61 tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc
      121 cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt
      181 tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt
      241 tggtgcggat atctcggtag tgggatacga cgataccgaa gacagctcat gttatatccc
      301 gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt
      361 gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg tctcactggt
      421 gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
      481 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
      541 caattaatgt aagttagctc actcattagg caccgggatc tcgaccgatg cccttgagag
      601 ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta
      661 tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc tgggtcattt
      721 tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt gcggtattcg
      781 gaatcttgca cgccctcgct caagccttcg tcactggtcc cgccaccaaa cgtttcggcg
      841 agaagcaggc cattatcgcc ggcatggcgg ccccacgggt gcgcatgatc gtgctcctgt
      901 cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat gaatcaccga
      961 tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga gcaacaacat
     1021 gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca
     1081 ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga acacctacat
     1141 ctgtattaac gaagcgctgg cattgaccct gagtgatttt tctctggtcc cgccgcatcc
     1201 ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa
     1261 cccgtatcgt gagcatcctc tctcgtttca tcggtatcat tacccccatg aacagaaatc
     1321 ccccttacac ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac atggcccgct
     1381 ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac gcggatgaac
     1441 aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc agctgcctcg
     1501 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     1561 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     1621 gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct
     1681 taactatgcg gcatcagagc agattgtact gagagtgcac cattgcggtg tgaaataccg
     1741 cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac
     1801 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     1861 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     1921 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     1981 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     2041 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     2101 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     2161 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     2221 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     2281 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     2341 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     2401 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     2461 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     2521 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     2581 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     2641 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     2701 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     2761 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     2821 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     2881 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     2941 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     3001 gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg
     3061 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     3121 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     3181 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     3241 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     3301 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     3361 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     3421 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     3481 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     3541 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     3601 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     3661 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg
     3721 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     3781 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     3841 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     3901 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     3961 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     4021 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     4081 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     4141 ttaatgcgcc gctacagggc gcgtcccatt cgccaatccg gatatagttc ctcctttcag
     4201 caaaaaaccc ctcaagaccc gtttagaggc cccaaggggt tatgctagtt attgctcagc
     4261 ggtggcagca gccaactcag cttcctttcg ggctttgtta gcagccggat ctcagtggtg
     4321 gtggtggtgg tgctcgagga atgggcaagt gctacggtat tcttccacat cggagaacag
     4381 gccggtacgc ggaccaggac acaggaaacg ggtgtaacgt gtgtcttcat cgacaaagcg
     4441 tttcagccac gcaacaccgt acatgccaat agtagcgtta gcgacactcg gcgcaaagtg
     4501 agacgcaccg ttcagttcca gataagcttt acgggtggag gttgggatgg aggtgtagga
     4561 cggttccgcg tgcagtgcta ccggggcaac cgtgtcattc tcggcgccaa tgatcagggt
     4621 agggacctga acgttggacc agcttttgtt caggtgccat ggggtcagcg ggatcgccgc
     4681 tttcagatcc gggcgacgag atgccaggcg cagagtaccg ccgccaccca tgctatgacc
     4741 cattactgcc aggcggttac ggtcgatgcg agagcgaacg gtagaggacg cgccatctac
     4801 catgtgatcc agcgcagctt ccagttggtc tgcacgcgaa tccggctggt ccagagtggt
     4861 gttggtgtcg atcgtgataa caacaaagcc gtgtgatgca atgcgttctc ccagccaagc
     4921 cacagaggac tgggtagcag tatagcccgg agagattgcc accgcgccgt aggtgttgtt
     4981 ttcctgcggg taataaattg tgccgccccc gaagccgctt actggagaag aaacacgctc
     5041 ttcagaaacc gggaacgggc cacgagaggc ttccagggac gcctgagtcg ggtccggacc
     5101 acgctcgtac gggttcgcca tatgtatatc tccttcttaa agttaaacaa aattatttct
     5161 agaggggaat tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcgcggga
     5221 tcgagatctc gatcctctac gccggacgca tcgtggccgg catcaccggc gccacaggtg
     5281 cggttgctgg cgcctatatc gccgacatca ccgatgggga agatcgggct cgccacttcg
     5341 ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc gggggactgt
     5401 tgggcgccat ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc
     5461 tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgtcga gatcccggac
     5521 accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa
     5581 ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc
     5641 tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg
     5701 gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa
     5761 ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg
     5821 ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg
     5881 gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc
     5941 gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct
     6001 gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc
     6061 atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc
     6121 gcattgggtc accagcaaat cgcgct
//
