LOCUS       sequence-349203-        6146 bp    DNA     circular SYN 15-APR-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 6146)
  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 6146)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pEE059
FEATURES             Location/Qualifiers
     source          1..6146
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..25
                     /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             40..62
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             859..876
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(929..947)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      943..990
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1027..1482
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1114..1133)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1324..1345
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1508..1612
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1613..1681,1682..2473)
                     /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(1831..1850)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2644..3232
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3133..3152
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3386..3403
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3418..3557
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3483..3502)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3643..3665
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3659..3850)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4659..5741)
                     /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     5703..5722
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5742..5819)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6025..6044
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(6128..6146,1)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6128..6146
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
       61 gatatacata tggttgaagc caccgatgtt cacggcccgg acccgacgga agaaacgatt
      121 accgctccgc gtggcccttt cgacgttgag caggaatcag tttcccgctt cgaagttgaa
      181 ggcttcggtg gcggtaccat ctattacccg accgacacca ccgacggtct gtttagcgct
      241 gtgtccatct ccccgggtta tactggcacc caggaaagca tggcctggta tggcccacgc
      301 ctcgcatcac acggttttgt ggtttttacc attgatacga tcaccaccac ggatcagccg
      361 gattcccgtg cccgccagct gcaggcatcg ctggaccatc tcgtagacga ttcctccgta
      421 cgtgaccgcg ttgacccggc acgtctgggt gttatgggcc actccatggg cggcggtggt
      481 tcactgaaag cggctctgga taacccagcg ctgcaggcag cgatcccact gacgccgtgg
      541 catactacta aggatttctc cggcgtacgc actccaaccc tgatcatcgg tgctcaaaac
      601 gatacggtgg caccggttag ccaacatgca gaaccgttct acgaaagttt accggacgac
      661 ccgggcaaag cgtatctgga actggcgggc gcgggtcacc tggcgccgaa caccccggac
      721 actaccattg cgaagtattc cctggcgtgg ctgaaacgct tcctggacga tgacactcgt
      781 tacgaccaat ttctgtgccc gccaccgcag gacgacccgg agatcgcaga acatcgttct
      841 acttgcccgt acctcgagca ccaccaccac caccactgag atccggctgc taacaaagcc
      901 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
      961 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
     1021 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     1081 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     1141 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     1201 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     1261 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     1321 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     1381 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     1441 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     1501 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     1561 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     1621 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     1681 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     1741 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     1801 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     1861 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     1921 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     1981 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     2041 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     2101 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc
     2161 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     2221 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     2281 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     2341 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     2401 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     2461 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     2521 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     2581 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     2641 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     2701 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     2761 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     2821 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     2881 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     2941 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     3001 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     3061 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     3121 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     3181 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     3241 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     3301 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     3361 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     3421 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc
     3481 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
     3541 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     3601 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     3661 agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt
     3721 ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct
     3781 ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct
     3841 gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga
     3901 tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg
     3961 aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc
     4021 agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc
     4081 atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac
     4141 tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc
     4201 agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc
     4261 aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg
     4321 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     4381 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     4441 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     4501 ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg
     4561 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     4621 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4681 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4741 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     4801 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     4861 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     4921 tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc
     4981 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     5041 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     5101 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     5161 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     5221 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     5281 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     5341 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     5401 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     5461 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     5521 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     5581 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     5641 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     5701 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     5761 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     5821 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
     5881 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
     5941 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     6001 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
     6061 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc
     6121 gcgaaattaa tacgactcac tatagg
//
