LOCUS       sequence-349677-        6152 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 6152)
  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 6152)
  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: pEE056
FEATURES             Location/Qualifiers
     source          1..6152
                     /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             865..882
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(935..953)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      949..996
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1033..1488
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1120..1139)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1330..1351
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1514..1618
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1619..1687,1688..2479)
                     /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(1837..1856)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2650..3238
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3139..3158
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3392..3409
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3424..3563
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3489..3508)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3649..3671
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3665..3856)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4665..5747)
                     /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     5709..5728
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5748..5825)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6031..6050
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(6134..6152,1)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6134..6152
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
       61 gatatacata tggccgacaa tccgtacgaa cgtggcccgg cgccgactac cgcaagcatc
      121 gaagccgctc gtggtccgta cgctgtgtcc cagaccaccg tttctagcct ggctgtaact
      181 ggttttggcg gtggtacgat ttattaccca acttctaccg gtgatggtac cttcggtgcc
      241 attgcggtgt ccccgggcta caccgctacg cagtcctcta tcgcatggct ggggccgcgc
      301 cttgcgagcc aggggtttgt agtattcacc attgacaccc tgaccaccct ggaccaaccg
      361 gactcacgtg gccgtcagct tctggcggca ctggaccatc tcacccaggt ttctagcgtg
      421 cgcacgcgcg tcgacggttc tcgtctgggt gttatgggcc attccatggg cggcggtggc
      481 tctctggaag cagcaaaggc gcgtccgtcg ctgcaggcag ctattccgct gactccgtgg
      541 aacctggaca aaagctggcc ggaggttggc accccgactc tgatcgtagg cgcagatggc
      601 gataccgttg cgccggtcgc ttctcacgcg gaacctttct actcttccct gccgtcctct
      661 ctggaccgtg cgtacctgga actcaacaac gcgactcact tcactccgaa ctctagcaat
      721 actaccattg cgaagtatgg tatctcttgg ctgaagcgtt ttgtggacaa cgacactcgt
      781 tacgaacagt ttctgtgccc gctcccgcag ccgagcacta ccatcgatga gtatcgcggt
      841 aactgcccgc ataccagcct cgagcaccac caccaccacc actgagatcc ggctgctaac
      901 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
      961 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
     1021 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     1081 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     1141 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     1201 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     1261 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     1321 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     1381 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     1441 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     1501 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     1561 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     1621 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     1681 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     1741 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     1801 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     1861 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     1921 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     1981 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     2041 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     2101 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     2161 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     2221 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     2281 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     2341 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     2401 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     2461 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     2521 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     2581 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     2641 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     2701 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     2761 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     2821 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     2881 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     2941 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     3001 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     3061 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     3121 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     3181 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     3241 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     3301 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     3361 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     3421 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
     3481 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
     3541 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
     3601 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     3661 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     3721 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     3781 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
     3841 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
     3901 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
     3961 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
     4021 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
     4081 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
     4141 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
     4201 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
     4261 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
     4321 gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac
     4381 cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
     4441 tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca
     4501 cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
     4561 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     4621 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     4681 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4741 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     4801 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     4861 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     4921 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
     4981 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     5041 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     5101 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     5161 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     5221 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     5281 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     5341 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     5401 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     5461 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     5521 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     5581 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     5641 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     5701 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     5761 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     5821 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     5881 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     5941 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     6001 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
     6061 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc
     6121 gatcccgcga aattaatacg actcactata gg
//
