LOCUS       sequence-349637-        6245 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 6245)
  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 6245)
  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: pEE033
FEATURES             Location/Qualifiers
     source          1..6245
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     964..983
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1003..1080)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1286..1305
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1389..1408
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1389..1407
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1408..1432
                     /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             1447..1469
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2365..2382
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2435..2453)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2449..2496
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2533..2988
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2620..2639)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2830..2851
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3014..3118
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3119..3187,3188..3979)
                     /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(3337..3356)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4150..4738
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4639..4658
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4892..4909
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4924..5063
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4989..5008)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5149..5171
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5165..5356)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6165..6245,1..1002))
                     /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 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
       61 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
      121 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
      181 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
      241 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
      301 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
      361 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
      421 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
      481 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
      541 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
      601 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
      661 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
      721 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
      781 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
      841 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
      901 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
      961 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     1021 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     1081 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
     1141 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
     1201 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
     1261 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
     1321 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc
     1381 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
     1441 aataattttg tttaacttta agaaggagat atacatatgg catttgcgat cactccgtct
     1501 ccgaccccaa ccccggatcc gaccccgaat ccatccccgg atccgggctc ctgttccggc
     1561 gccgagtgct acatccgcgg tcctaaccct actgtacgtg ccctggaagc agacgatggt
     1621 ccgtactcgg tgcgtaccac caacgtatct tccttcgttt ctggcttcgg tggtggcaca
     1681 attcactacc cggtgggtac cgaaggcaag atgggtgcca tcgccgtgat tccgggctac
     1741 gtttcctacg aatcatccat ccgttggtgg ggtagccgcc tggcgtcatg gggttttgtt
     1801 gttattacca tcgacactaa caccatttat gatcaaccgg attctcgtgc aaaccagctg
     1861 tcagccgctc tggattacgt gatcgctcaa agcaactctc gtaactcgtc catttccggc
     1921 atggtggact ccaaccgcct gggtgttatc ggctggtcta tgggtggtgg cggttctctg
     1981 aaactgtcta ctcagcgcac gctgaaagcc gcaatccctc aggctccgtg gtactctggt
     2041 ttcaacagct tcaaccgcat tactactcca acgctcatta ttgcctgcga gctggacgtt
     2101 gtagctcctg taggtcagca cgcttctccg ttttacaacc gcattccgag ctccactgcg
     2161 aaagcgtttc tggaaatcaa tggtggcgac catttctgcg ccaacagcgg ctacccgaac
     2221 gaagacatcc ttggcaaata tggcgtttct tggatgaaac gctttattga cggtgatcgt
     2281 cgctacgacc agttcctgtg tggtccaaat cacgaatctg atcgctctat cagcgactac
     2341 cgtgaaacct gtaactacct cgagcaccac caccaccacc actgagatcc ggctgctaac
     2401 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
     2461 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
     2521 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     2581 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     2641 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     2701 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     2761 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     2821 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     2881 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     2941 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     3001 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     3061 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     3121 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     3181 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     3241 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     3301 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     3361 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     3421 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     3481 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     3541 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     3601 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     3661 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     3721 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     3781 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     3841 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     3901 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     3961 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     4021 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     4081 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4141 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     4201 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     4261 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     4321 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     4381 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     4441 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     4501 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     4561 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     4621 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     4681 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     4741 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     4801 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     4861 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     4921 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
     4981 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
     5041 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
     5101 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     5161 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     5221 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     5281 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
     5341 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
     5401 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
     5461 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
     5521 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
     5581 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
     5641 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
     5701 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
     5761 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
     5821 gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac
     5881 cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
     5941 tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca
     6001 cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
     6061 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     6121 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     6181 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     6241 gtttg
//
