LOCUS       sequence-349075-        6308 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 6308)
  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 6308)
  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: pEE044
FEATURES             Location/Qualifiers
     source          1..6308
                     /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             2385..2402
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2455..2473)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2469..2516
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2553..3008
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2640..2659)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2850..2871
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3034..3138
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3139..3207,3208..3999)
                     /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(3357..3376)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4170..4758
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4659..4678
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4912..4929
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4944..5083
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5009..5028)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5169..5191
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5185..5376)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6185..6308,1..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 catatgaaca
     1441 aggaaaagtc ctctttcgat cagactgcta aaatcaccac ccgctctaaa tccatcttca
     1501 aaactatctt cacttacttg ctggtactgg cgttcatcac tacgattttc ccgatgaacg
     1561 catttgccaa ctctccagct atcatccgta acgaagaagc gccgggcaaa tatgcgggga
     1621 acgggccgtt cagctataat tcctaccgtc tgccactcct ctctgtttat ggtaccggcg
     1681 gcgcgaccgt ttactaccct actagcggta ctgcgcctta cagcggtctg gtgtactgtc
     1741 cgccatatac ggccaaacaa tcggcactgg cagcttgggg cccgttcttc gcctcccacg
     1801 gtatcatcct ggtgactttc gacacgctga ccccgcttga cccggtctcc ctgcgtgcgc
     1861 tgcagcagcg taccgttctt aacgcactga aaactgaaaa ctcccgtctg aatagtccgc
     1921 tgtaccagaa ggtcgcaacc gaccgtatcg gcgctatggg ctggtccatg ggcggcggcg
     1981 caacttggat caactctgcg gagtattcgg gcttgaaaac cgcgatgacg atcgctggtc
     2041 acaacctgag ctccaccaac ctgaactcta agggctataa caccaagtgc ccgaccctga
     2101 ttatgaacgg tgcgatggac acgaccggcc tgggtggcct gggtcagtct aacggcgtct
     2161 acaaaaacat tccagcaaat gtgcctaagg tgttatacga ggttgccagc gcaggtcatc
     2221 tgaactggac ttccccgatc tcggcctcta atgatgtggc tgccatcgcg ctggcgttcc
     2281 aaaagacgta cctggacggc gactcacgtt ggttggcatt cattactcgt ccgaacagca
     2341 acgtgtctat ctgggaaacc agcaacctga tgaacccgct cgagcaccac caccaccacc
     2401 actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     2461 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     2521 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     2581 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     2641 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     2701 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     2761 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     2821 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     2881 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     2941 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     3001 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     3061 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     3121 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     3181 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     3241 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     3301 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     3361 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     3421 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     3481 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     3541 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     3601 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     3661 gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc
     3721 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     3781 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     3841 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     3901 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     3961 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     4021 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     4081 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     4141 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     4201 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     4261 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     4321 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     4381 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     4441 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     4501 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     4561 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     4621 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     4681 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     4741 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     4801 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     4861 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     4921 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     4981 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     5041 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     5101 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     5161 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     5221 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     5281 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     5341 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     5401 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     5461 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     5521 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     5581 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     5641 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     5701 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     5761 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     5821 gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc
     5881 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     5941 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     6001 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     6061 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     6121 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     6181 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     6241 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     6301 gtgagacg
//
