LOCUS       sequence-342654-        6212 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 6212)
  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 6212)
  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: pEE043
FEATURES             Location/Qualifiers
     source          1..6212
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             853..870
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(923..941)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      937..984
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1021..1476
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1108..1127)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1318..1339
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1502..1606
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1607..1675,1676..2467)
                     /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(1825..1844)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2638..3226
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3127..3146
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3380..3397
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3412..3551
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3477..3496)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3637..3659
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3653..3844)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4653..5735)
                     /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     5697..5716
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5736..5813)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6019..6038
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6122..6141
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6122..6140
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6141..6165
                     /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             6180..6202
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 tcccaggttc cgccgacccc gccgaccgat gatccgatgg gtgattgccc gtctacagct
       61 atctgccgag gcgaggcgcc gggtagctat tctggtaacg gcccgtatgg ttcccggagc
      121 tacaccctgt ctcgtttcca gaccccgggc ggcgcaaccg tatactaccc gtctaacgcc
      181 gaaccaccgt acagcggtct ggttttcact ccgccgtaca ccggtactca ggctatgttt
      241 cgcgcatggg gcccattttt tgcatctcac ggtatcgttc tggtaaccat ggacacgtcc
      301 actacagtgg acaccgttga tcagcgtgcg agccagcaga aacgcgtact ggacgttctg
      361 aaacaggaaa acacgcgttc gggctctccg ctccgtggta agctggacac ttcccgtctg
      421 ggtgccgtgg gctggagtat gggtggcgga gctacctgga tcaactctgc ggagtacaac
      481 ggtctcaaaa cggctatgag cctcgcaggt cacaatatga ccgctatcga tctggacagc
      541 aaaggtggta acacccgtgt tccgaccctc ctgttcaacg gcgcgctgga cctgaccatg
      601 ctgggtggcc tgggccagtc tatcggtgtt tacaacgcta tcccgcgcgg tattccgaaa
      661 gttatctacg aagttgccag cgctgggcac ttcgactggg gttccccaac cgcagcgaat
      721 cgttccgttg cgggtatcgc actggcgttc cacaaaacgt ttctggatgg cgacacccgt
      781 tgggtttcct acatcaaacg tccatcctcc gatgtggcta cctggcgtac cgaaaacctg
      841 ccgcagctcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag
      901 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
      961 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
     1021 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     1081 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     1141 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     1201 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     1261 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     1321 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     1381 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     1441 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
     1501 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     1561 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     1621 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     1681 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     1741 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     1801 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     1861 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     1921 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     1981 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     2041 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     2101 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc
     2161 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     2221 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     2281 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     2341 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     2401 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     2461 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     2521 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     2581 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     2641 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     2701 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     2761 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     2821 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     2881 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     2941 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     3001 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     3061 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     3121 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     3181 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     3241 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     3301 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     3361 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     3421 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca
     3481 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     3541 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     3601 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     3661 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
     3721 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
     3781 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
     3841 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
     3901 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
     3961 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
     4021 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
     4081 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg
     4141 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
     4201 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
     4261 gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca
     4321 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     4381 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     4441 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     4501 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     4561 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
     4621 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     4681 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     4741 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     4801 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     4861 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     4921 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     4981 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     5041 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     5101 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     5161 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     5221 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     5281 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     5341 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     5401 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     5461 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     5521 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     5581 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     5641 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     5701 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     5761 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
     5821 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     5881 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
     5941 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
     6001 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
     6061 gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa
     6121 ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt
     6181 ttgtttaact ttaagaagga gatatacata tg
//
