LOCUS       sequence-413653-        8252 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Production of terpentetriene in E. coli MEV20.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8252)
  AUTHORS   Lin GM, Voigt CA
  TITLE     Design of a redox-proficient Escherichia coli for screening 
            terpenoids and modifying cytochrome P450s
  JOURNAL   Nature Catalysis. 6, 1016–1029 (2023). 
            https://doi.org/10.1038/s41929-023-01049-5
REFERENCE   2  (bases 1 to 8252)
  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: "Nature 
            Catalysis. 6, 1016–1029 (2023). 
            https://doi.org/10.1038/s41929-023-01049-5"
COMMENT     Sequence Label: pLRD01
FEATURES             Location/Qualifiers
     source          1..8252
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     68..86
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(90..133)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    136..157
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     promoter        178..196
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    197..221
                     /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)."
     promoter        1752..1770
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1771..1795
                     /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)."
     promoter        3561..3579
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3580..3604
                     /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)."
     misc_feature    4800..4820
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      4821..4878
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     primer_bind     complement(4902..4919)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      5453..5675
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             5723..6673
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFWIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     CDS             complement(join(7287..8078,8079..8147))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7910..7929
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8148..8252)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
       61 ctataaaaat aggcgtatca cgaggcagaa tttcagataa aaaaaatcct tagctttcgc
      121 taaggatgat ttctggaatt cgcggccgct tctagagcgc gaagacttaa tcaaatctaa
      181 tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctacgt tttcaaggtg
      241 gtagactgtc gccggatgtg tatccgacct gacgatggcc caaaagggcc gaaacagtcc
      301 tctacaaata attttgttta agagaatacc ccgctaagaa ggtaattatg tccatgcaaa
      361 gtttagataa gaattttcga catttatctc gtaaagaaaa gttacaacaa ttggttgata
      421 agcaatggtt atcagaagaa caattcgaca ttttactgaa tcatccatta atcgatgaag
      481 aagtagccaa tagtttaatt gaaaatgtca tcgcgcaagg tgcattaccc gttggattat
      541 taccgaatat cattgtggac gataaggcat atgttgtacc tatgatggtg gaagagcctt
      601 cagttgtcgc tgcagctagt tatggtgcaa agctagtgaa tcagactggc ggatttaaaa
      661 cggtatcttc tgaacgtatt atgataggtc aaatcgtctt tgatggcgtt gacgatactg
      721 aaaaattatc agcagacatt aaagctttag aaaagcaaat tcataaaatt gcggatgagg
      781 catatccttc tattaaagcg cgtggtggtg gttaccaacg tatagcgatt gatacatttc
      841 ctgagcaaca gttactatct ttaaaagtat ttgttgatac gaaagatgct atgggcgcta
      901 atatgcttaa tacgatttta gaggccataa ctgcattttt aaaaaatgaa tttccgcaaa
      961 gcgacatttt aatgagtatt ttatccaatc atgcaacagc gtccgttgtt aaagttcaag
     1021 gcgaaattga tgttaaagat ttagcaaggg gcgagagaac tggagaagag gttgccaaac
     1081 gaatggaacg tgcttctgta ttggcccaag tagatattca tcgtgcagca acacataata
     1141 aaggtgttat gaatggcata catgctgttg ttttagcaac aggaaatgat acgcgtggtg
     1201 cagaagcaag tgcgcatgca tacgcgagtc gtgacggaca gtatcgtggt attgctacat
     1261 ggcgttacga tcaagatcgt caacgattga ttggtacaat tgaagtgcct atgacattgg
     1321 caatcgttgg cggtggtaca aaagtattac caattgctaa agcttcatta gagctactaa
     1381 atgtagagtc agcacaagaa ttaggtcatg tagttgctgc cgttggttta gcgcaaaact
     1441 ttgcagcatg tcgcgcgctt gtgtcagaag gtattcaaca aggtcatatg agtttacaat
     1501 ataaatcatt agctatcgtt gtaggggcaa aaggtgatga aattgctaaa gtagctgaag
     1561 ctttgaaaaa agaaccccgt gcaaatacac aagcagcgga acatatttta caagaaatta
     1621 gacaacaata ataacggatg taaacagaga aacgtctaac taaaaaggcc tcccaaatcg
     1681 gggggccttt tttattgata acaaaagcct aatacacctg ttcgtgacga ctacggatgc
     1741 gggcaaggtg ctaatacgac tcactatagg ggaattgtga gcggataaca attcccctct
     1801 aaacgccagc cactaaagcg gtcaacgcat gtgctttgcg ttctgatgag acagtgatgt
     1861 cgaaaccgcc tctacaaata attttgttta agataaccaa ctataaggat agacaatatg
     1921 ccagccatga gtgatgccga ccggattgca gccttgctga aagatcgcgc tgcagatcca
     1981 gtcaccaagt ttagcccgag cccctacgaa acggggcaat ttttgcgcat cagcgaacgt
     2041 gcagatgtgg gcaccccgca gatcgattat cttctggcga cgcaacgtcc ggatggcctg
     2101 tgggggagcg ttggcttcga actggtgcca accctgggtg ctgttgctgg tctgtcgtca
     2161 cggcctgagt acgctgatcg tgcgggtgta accgatgcgg tggctcgcgc ttgcgaaaaa
     2221 ctttgggaac tggcgctggg cgagggcgga ctcccgaaac tccccgatac agtggcctca
     2281 gaaatcattg taccgtcgct gatagacctg ctgtcggaag tgttacaacg acatcgtccg
     2341 gccgtgggcg gcaaagcagg acaggaacag gagtttccgt caccgccggg ggcaaaaccg
     2401 gagttgtggc ggcagttaag tgatcgtatc gcacgcgggc aggcgatccc gaaaaccgct
     2461 tggcacacgc tggaagcctt tcatcccctc cccaagcagt ttgcagctac cgtaacgccg
     2521 gcggccgatg gtgccgtcac atgcagtcct tcctctactg cagcgtggct gtccgctgtg
     2581 ggtaccgatg ccggtgcgtc tacacgcgcc tatttggatg aagcacaatc ccgctatggt
     2641 ggtgcgatcc ctatgggatc atccatgccg tattttgaag tcctgtgggt ccttaatctc
     2701 gtcctgaaat actttcctga tgtgcctatc cctagagaga tcattgaaga gattgctgcg
     2761 ggtttcagtg attctggtat cggtggtggt ccgggccttc cgccggatgg tgatgacacg
     2821 gcgtatgcga acctggcggg cgataaactg ggcgcaccta cccatccgga aatcttaatg
     2881 aaattctggg cggaagatca ctttgtttcc tacccaggtg aacaaacccc atctgagact
     2941 gtcaacgccc atgcgctgga atatttaaac catttgcgta tgcgacgtgg tatcacggaa
     3001 ttcggtgccg tggaggatgc gtgcgccgaa tgggtgatct cacagcagac tgaggacggc
     3061 tgctggtacg ataaatggaa tgtctctcct tactattcaa ccgccgcctg cgtcgaggcc
     3121 ctgctggatg cgcgtaaaca agatgaaccg cagttagact cgctgcgtcg cgcccgcgag
     3181 tggctgctgc gtcatcagac tgatagcgga ggctggggta tggctgagcc gtcccctgaa
     3241 gaaaccgcgt atgctgtcct cgcgctggat ttatttgcaa gccgcggcgg cgagggagcc
     3301 gaagaatgtg cggcagctat ttcccgtgca aaagaattct ttaccgatga aagtcgcgaa
     3361 aatcctccgc tgtggatggg caaagattta tacacaccgt ttcgtattgt tgatgtaact
     3421 gttatgtgcg ggcgtgcagt ggttggtcgg tactaataat tatgccttcc gctaatcaac
     3481 tcggtaccaa attccagaaa agagacgctt tcgagcgtct tttttcgttt tggtccgtgg
     3541 acaagcacag catagccaag taatacgact cactataggg gaattgtgag cggataacaa
     3601 ttcccctcta ggtccgaaga tacttagggt gtctcaaggt gcgtaccttg actgatgagt
     3661 ccgaaaggac gaaacacccc tctacaaata attttgttta agaaccgcag tcactacttt
     3721 taaaggagga ttttatgcca gacgcgatcg aattcgagca cgaaggccgc cgcaacccga
     3781 actcagcgga ggcagagtca gcgtattcct cgatcatcgc ggcgttggat ctgcaggaat
     3841 cagattatgc cgttatttcg ggtcattccc gtatcgtggg ggccgccgcg ttggtttatc
     3901 cggatgctga cgcggaaacc ttactggcgg ccagtctctg gacggcgtgc ctgattgtga
     3961 atgatgaccg ttgggattat gtccaggagg atggaggccg tctggcaccg ggcgagtggt
     4021 tcgatggcgt aaccgaagtc gttgatacgt ggcgcacagc gggtccacgc ttgcccgatc
     4081 cattctttga attggttcga acaacgatgt cgagactgga cgccgctctt ggagcagagg
     4141 ccgcagacga gatcggtcac gaaattaaac gagcgataac ggcgatgaaa tgggaaggag
     4201 tctggaacga atacaccaag aaaactagtc tcgcgactta tctgagtttt cgtcgtggat
     4261 attgtacgat ggatgttcag gtcgtactcg ataaatggat caacggtggt cgctcttttg
     4321 ccgccctgcg agatgacccg gtccgtagag caatcgatga cgttgtggtc cgttttggct
     4381 gcttgagcaa tgactactac tcttggggcc gtgaaaagaa agcagtggat aaaagtaatg
     4441 cagttcgcat cctgatggac catgcggggt atgatgaatc cactgcgttg gctcatgtgc
     4501 gtgacgattg cgtccaagcg attaccgatc tcgattgtat tgaagagtcg ataaaacgtt
     4561 ctggccacct gggcagtcat gcacaggagt tactggatta tttagcctgt caccgtccgc
     4621 tgatctacgc agcagcgaca tggccgaccg aaacaaaccg ctatcgataa taagcctcgt
     4681 tgctcttcat aaatcatccc tatgacccct tcaaaaaaaa aaaacaccct aacgggtgtt
     4741 tttttttttt tggtctgcct ctcgctgacg gtcacaatgc gcttacgaaa gtcttcctgt
     4801 actagtagcg gccgctgcag tccggcaaaa aagggcaagg tgtcaccacc ctgccctttt
     4861 tctttaaaac cgaaaagatt acttcgcgtt atgcaggctt cctcgctcac tgactcgctg
     4921 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatgacagta
     4981 agacgggtaa gcctgttgat gataccgctg ccttactggg tgcattagcc agtctgaatg
     5041 acctgtcacg ggataatccg aagtggtcag actggaaaat cagagggcag gaactgctga
     5101 acagcaaaaa gtcagatagc accacatagc agacccgcca taaaacgccc tgagaagccc
     5161 gtgacgggct tttcttgtat tatgggtagt ttccttgcat gaatccataa aaggcgcctg
     5221 tagtgccatt tacccccatt cactgccaga gccgtgagcg cagcgaactg aatgtcacga
     5281 aaaagacagc gactcaggtg cctgatggtc ggagacaaaa ggaatattca gcgatttgcc
     5341 cgagcttgcg agggtgctac ttaagccttt agggttttaa ggtctgtttt gtagaggagc
     5401 aaacagcgtt tgcgacatcc ttttgtaata ctgcggaact gactaaagta gtgagttata
     5461 cacagggctg ggatctattc tttttatctt tttttattct ttctttattc tataaattat
     5521 aaccacttga atataaacaa aaaaaacaca caaaggtcta gcggaattta cagagggtct
     5581 agcagaattt acaagttttc cagcaaaggt ctagcagaat ttacagatac ccacaactca
     5641 aaggaaaagg actagtaatt atcattgact agcccatctc aattggtata gtgattaaaa
     5701 tcacctagac caattgagat gtatgtctga attagttgtt ttcaaagcaa atgaactagc
     5761 gattagtcgc tatgacttaa cggagcatga aaccaagcta attttatgct gtgtggcact
     5821 actcaacccc acgattgaaa accctacaag gaaagaacgg acggtatcgt tcacttataa
     5881 ccaatacgct cagatgatga acatcagtag ggaaaatgct tatggtgtat tagctaaagc
     5941 aaccagagag ctgatgacga gaactgtgga aatcaggaat cctttggtta aaggcttttg
     6001 gattttccag tggacaaact atgccaagtt ctcaagcgaa aaattagaat tagtttttag
     6061 tgaagagata ttgccttatc ttttccagtt aaaaaaattc ataaaatata atctggaaca
     6121 tgttaagtct tttgaaaaca aatactctat gaggatttat gagtggttat taaaagaact
     6181 aacacaaaag aaaactcaca aggcaaatat agagattagc cttgatgaat ttaagttcat
     6241 gttaatgctt gaaaataact accatgagtt taaaaggctt aaccaatggg ttttgaaacc
     6301 aataagtaaa gatttaaaca cttacagcaa tatgaaattg gtggttgata agcgaggccg
     6361 cccgactgat acgttgattt tccaagttga actagataga caaatggatc tcgtaaccga
     6421 acttgagaac aaccagataa aaatgaatgg tgacaaaata ccaacaacca ttacatcaga
     6481 ttcctaccta cataacggac taagaaaaac actacacgat gctttaactg caaaaattca
     6541 gctcaccagt tttgaggcaa aatttttgag tgacatgcaa agtaagtatg atctcaatgg
     6601 ttcgttctca tggctcacgc aaaaacaacg aaccacacta gagaacatac tggctaaata
     6661 cggaaggatc tgaggttctt atggctcttg tatctatcag tgaagcatca agactaacaa
     6721 acaaaagtag aacaactgtt caccgttaca tatcaaaggg aaaactgtcc atatgcacag
     6781 atgaaaacgg tgtaaaaaag atagatacat cagagctttt acgagttttt ggtgcattca
     6841 aagctgttca ccatgaacag atcgacaatg taacagatga acagcatgta acacctaata
     6901 gaacaggtga aaccagtaaa acaaagcaac tagaacatga aattgaacac ctgagacaac
     6961 ttgttacagc tcaacagtca cacatagaca gcctgaaaca ggcgatgctg cttatcgaat
     7021 caaagctgcc gacaacacgg gagccagtga cgcctcccgt ggggaaaaaa tcatggcaat
     7081 tctggaagaa atagcgcttt cagccggcaa accggctgaa gccggatctg cgattctgat
     7141 aacaaactag caacaccaga acagcccgtt tgcgggcagc aaaacccgta ccgattatca
     7201 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     7261 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     7321 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     7381 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga accacgctca
     7441 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     7501 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     7561 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     7621 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     7681 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     7741 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     7801 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     7861 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     7921 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     7981 tcaaggatct taccgctgtt gagatccagt tcgatataac ccactcgtgc acccaactga
     8041 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     8101 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     8161 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     8221 atttagaaaa ataaacaaat aggggttccg cg
//
