LOCUS       sequence-342616-        5996 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 5996)
  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 5996)
  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: pEE024
FEATURES             Location/Qualifiers
     source          1..5996
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             637..654
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(707..725)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      721..768
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      805..1260
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(892..911)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1102..1123
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1286..1390
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1391..1459,1460..2251)
                     /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(1609..1628)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2422..3010
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2911..2930
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3164..3181
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3196..3335
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3261..3280)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3421..3443
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3437..3628)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4437..5519)
                     /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     5481..5500
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5520..5597)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5803..5822
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5906..5925
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5906..5924
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5925..5949
                     /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             5964..5986
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 caaccgtacc cggtcggtac tcgtaccatt acttaccagg atccggtacg taacaaccgc
       61 aacatccaga cgtacctgta ctatccggcg accgcagccg gtgctaacca gcctgttgct
      121 ggtggtcagt ttccggtcgt agtggtgggg cacggtttca ctatgaatta cgcgccgtat
      181 gcgttttggg gtaacgcgct ggctgagtct ggttatatcg tagctatccc gaacacggaa
      241 accggctttt ctccgtccca tagcgccttc gctgctgata tggctttcct ggtggcgaaa
      301 ctgtacaccg aaaacaccaa ctcctcctcc cctttttatc agcatgttca gtacaattct
      361 tgcattattg gtcactctat gggtggtgga tgcacttacc tggcggccca aaacaacgca
      421 gacgtgagcg ctacggttac cttcgcagcc gcagaaacca acccgtctgc taccgcggct
      481 gcagcaaacg ttaactgtcc gtctctggtt ttctctggtt ccgccgactg catcaccccg
      541 ccggctcagc accaggtacc gatgtataac gctctgccgg actgtaaagc gtacggcggt
      601 tcttcccgcg ttgacctgca agcatgcaaa ctcgagcacc accaccacca ccactgagat
      661 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
      721 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
      781 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
      841 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
      901 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
      961 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     1021 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     1081 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     1141 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     1201 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     1261 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     1321 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     1381 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     1441 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     1501 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     1561 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     1621 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     1681 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     1741 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     1801 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     1861 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     1921 caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     1981 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     2041 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     2101 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     2161 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     2221 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     2281 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     2341 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     2401 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     2461 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     2521 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     2581 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     2641 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     2701 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     2761 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     2821 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     2881 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     2941 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     3001 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     3061 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     3121 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     3181 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3241 ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     3301 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     3361 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     3421 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     3481 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
     3541 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     3601 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     3661 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     3721 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     3781 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     3841 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     3901 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     3961 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     4021 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     4081 tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
     4141 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
     4201 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
     4261 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     4321 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     4381 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     4441 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4501 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     4561 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     4621 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     4681 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
     4741 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     4801 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     4861 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     4921 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     4981 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     5041 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     5101 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     5161 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     5221 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     5281 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     5341 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     5401 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     5461 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     5521 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     5581 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     5641 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     5701 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     5761 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     5821 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     5881 gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat
     5941 aacaattccc ctctagaaat aattttgttt aactttaaga aggagatata catatg
//
