LOCUS       sequence-349623-        6191 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 6191)
  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 6191)
  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: pEE018
FEATURES             Location/Qualifiers
     source          1..6191
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     971..990
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1010..1087)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1293..1312
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1396..1415
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1396..1414
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1415..1439
                     /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             1454..1476
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2318..2335
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2388..2406)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2402..2449
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2486..2941
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2573..2592)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2783..2804
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2967..3071
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3072..3140,3141..3932)
                     /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(3290..3309)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4103..4691
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4592..4611
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4845..4862
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4877..5016
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4942..4961)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5102..5124
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5118..5309)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6118..6191,1..1009))
                     /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 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
       61 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
      121 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
      181 cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa
      241 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
      301 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
      361 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
      421 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
      481 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
      541 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
      601 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
      661 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
      721 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
      781 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
      841 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
      901 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
      961 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     1021 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     1081 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     1141 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     1201 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     1261 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     1321 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc
     1381 tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc
     1441 ctctagaaat aattttgttt aactttaaga aggagatata catatgctga ttcgcccggt
     1501 tgctttccgt aacatgaacc aacagatcat cggcatcctg cacacgccgg acaacattaa
     1561 acctggcgaa aaaaccccgg gtatcctgat gctgcacggc ttcaccggta acaaaaccga
     1621 agctcatcgt ctctttgttc atgtagcccg ttctctgagc gaatatggtt tcatcgtact
     1681 tcgtttcgac ttccgtggtt ccggcgattc tgatggtgaa ttcgaagata tgacccttcc
     1741 tggcgaagtc tccgacgcgg aacgtgctct gaccttcctg ctccgtcgcc gtaacatcga
     1801 ccgcgatcgc gtgggcgtca tcggtctgtc catgggtggc cgtgttgccg ccatcctggc
     1861 atctaaagat aagcgtgtta aatttgcggt cctgtactcc ccggcactgg gtccgttgcg
     1921 tgatcgttcc ctgtcgttca tgagccgtga gaaaatcgaa cgcctgaaca gcggtgaggc
     1981 ggtagaattt ttcgctgaag gctggtacat caaaaaaact ttcttcgaaa ccgtggacta
     2041 catcgttcca ctcgacatca tggactcgat ccgtgttccg gtgctcatcg tccatggtga
     2101 tcgtgatccg attattccgg tggaagaagc aattcgagcg tacgaaaaaa tcaaaggtgt
     2161 aaacaagaag aacgaactgt atattgttcg tggtggtgac cataccttct ccaaaaaaga
     2221 acacacccag gaagttatca aaaaaaccct ggactggatc cgtgccctct ccgtctccga
     2281 aggctccatc gttctgtttc gcctgctgga actcgagcac caccaccacc accactgaga
     2341 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata
     2401 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
     2461 aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2521 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     2581 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     2641 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     2701 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     2761 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     2821 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     2881 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     2941 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     3001 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     3061 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     3121 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     3181 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     3241 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     3301 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     3361 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     3421 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     3481 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     3541 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     3601 acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     3661 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     3721 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     3781 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     3841 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     3901 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     3961 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     4021 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     4081 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     4141 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     4201 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     4261 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     4321 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     4381 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     4441 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     4501 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     4561 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     4621 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     4681 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     4741 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     4801 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     4861 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     4921 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     4981 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     5041 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     5101 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     5161 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     5221 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     5281 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     5341 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     5401 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     5461 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     5521 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     5581 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     5641 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     5701 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     5761 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
     5821 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
     5881 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
     5941 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
     6001 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     6061 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     6121 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     6181 gcggggagag g
//
