LOCUS       sequence-413660-        8268 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Production of isopimara-8,15-diene in E. coli MEV20.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8268)
  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 8268)
  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: pLRD05
FEATURES             Location/Qualifiers
     source          1..8268
                     /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        3632..3650
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3651..3675
                     /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    4816..4836
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      4837..4894
                     /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(4918..4935)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      5469..5691
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             5739..6689
                     /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(7303..8094,8095..8163))
                     /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     7926..7945
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8164..8268)
                     /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 agaaggtcaa acacattagc aacgataagg
     1921 gggtctttat gtcgtccatc agccatcaag cggctccgac gcgtgctcag cacaccaatt
     1981 cctacgtgca gctggcgcgc caattagtga cttcagtcga caatgaccca tggggtgacg
     2041 tcccgcctag tgtgtacgaa accgcacggg tcacatcgtg ggctccgtgg ctggaaggtc
     2101 acgagcgccg cctggcatgg ttactcgaac gccaaagtgc ggccggttcc tggggtgaag
     2161 gtccgacccc ttatcgtctg cttcctacac tgtctgttac tgaagcactt ctcagcaccc
     2221 tgcgtcagaa cacagccgcc ggtgtttctc gcgaacgcct ggccgcggca gtcgacaatg
     2281 gtcttgcagc gttgcgtgat ctgagcggta ccggcggctg gccggatacc gccgccatcg
     2341 agattctggc acctgatctg gtggttttaa tcaatgacca tctggatcaa ccagaagttg
     2401 ccgcacttcc gcgcctgggt ccttgggccc gcggtcaaag attagcccaa cctcacggtt
     2461 ttcaagcggc gctccctgac cgcgttgctg aacgttgcca ggtggctggg ggcgttccgc
     2521 tgaaacttca tcacactttt gaaggcgtgg cacgccgcct gccgcgtatg gtgcctggag
     2581 ttcccggtgg actcctgggc tcaagtccag cggctacggc agcgtggtta gcaaccggtc
     2641 cggacgaagg gcgtgaccaa gcagttacgg ctcttacggc ggttgcggaa cgctacgatg
     2701 gcctgttccc agaagcaacc ccgattagtg tgttcgaacg cctgtggatc tctgtagcat
     2761 tagctcgccc gggtctgccc gccgcgtgcg tacctacaat tcgtgcctgg gccgctgaaa
     2821 tctatgatgc gaccggagtg cgtggtgctc ccggtttact gccagatact gatgatacag
     2881 cgatggcggt cttagcgtct gcactggccg gtagcccgcg agatccttct ccactgtcgg
     2941 catttgaggc gggtgatcac tatgattgtt acgtgggcga ggacaccggt agctctacgg
     3001 ctaatgcgca cgcactccag gctcttacgg cgtggcttag ccaccgtccg gccactggcg
     3061 acgctttaca ggccagacgc gatctgacgc gagattggct gctggcccag caggaaagtg
     3121 atggggcgtg gcgtgataaa tggcatgcga gcccatatta tgcaactgaa cgttgcgtta
     3181 ctgccctgtc aggccatacg ggccctacga cgcgtgatgc tatccgctcg gctgccgatt
     3241 gggtcctgga tgcacagtcc gatgatggca gttggggtgt atggggaggt accgcggaag
     3301 aaaccgccta cgccgtgaac atcttgctta acagcccgga ccataccggg acgccagaag
     3361 cgacacaggc gttaaaactg gcagaaaatg tattgcgtga agcggtgcat tcgtccggtc
     3421 accatcatcc cgctttatgg catgacaaaa ccctttacgc gccccaagcg atggcacagg
     3481 cggaagtcat tgcagccttg gaactgctgc aggcacgtcg tccgtaataa ttatgccttc
     3541 cgctaatcaa ctcggtacca aattccagaa aagagacgct ttcgagcgtc ttttttcgtt
     3601 ttggtccgtg gacaagcaca gcatagccaa gtaatacgac tcactatagg ggaattgtga
     3661 gcggataaca attcccctct aggtccgaag atacttaggg tgtctcaagg tgcgtacctt
     3721 gactgatgag tccgaaagga cgaaacaccc ctctacaaat aattttgttt aagaattcac
     3781 acttctttag gaggttagat gcccacccat cacgcaggca acggttttgc ggttgcgagt
     3841 gagcaggggc gtatttgtgc gttagcagcc cgtggccaac gcgatctgcg ccagtgcgtc
     3901 cgtgcgtatc catcgctgtt ccccaatcca cccgtggatg ataccatgct gagcgccctg
     3961 gcgttatcaa cggccttcat cgctccgtgg tgttccgcgg aacagctgcg cgtagcgaat
     4021 cgtgcatccc tgtgggtcac tgcggaggac tggcaggtcg atcgggttgc cacgagcgac
     4081 gatgccgtgc gtagcatcgt tagtgcatgc caggcggtgg ctgatggtgc ggcccctgat
     4141 gtcgattgcg ccttaggcca gctcctggct gaaatacgtg acgaactggc caccggagcc
     4201 ggctttaccg agtggcaacc ggtctggcgt gaggaagtac accgtatgct gactgccgat
     4261 attcgtgagt gggaatggcg tcacagcgct cgtccaccga gctttgcaga atatctggat
     4321 aacgcggata actacggtgc cagctttgtt aatgtatcac attggatcgt gaccggcgac
     4381 gcgcagacac gctcacactt accggaactg attgctgcgt cgcgcgaagt acagcgtatc
     4441 ctccgattgt ctaatgacct ggcgtcttat gagcgtgata tccgctcggg ggaccttaac
     4501 gcgctgttgc tggtcgatcg tgaagaagtc tcacgccagc tgcgagatgg aattcgtgca
     4561 tgccaggatc atttacatac cctggaagtt acgtgcccgc gagaagcgtt gtacttggcc
     4621 cgggaggctg gctttacgac tggcttctac catggggcgg actattgggg tgatagcgaa
     4681 cggtaataag cctcgttgct cttcataaat catccctatg accccttcaa aaaaaaaaaa
     4741 caccctaacg ggtgtttttt tttttttggt ctgcctctcg ctgacggtca caatgcgctt
     4801 acgaaagtct tcctgtacta gtagcggccg ctgcagtccg gcaaaaaagg gcaaggtgtc
     4861 accaccctgc cctttttctt taaaaccgaa aagattactt cgcgttatgc aggcttcctc
     4921 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4981 ggcggtaatg acagtaagac gggtaagcct gttgatgata ccgctgcctt actgggtgca
     5041 ttagccagtc tgaatgacct gtcacgggat aatccgaagt ggtcagactg gaaaatcaga
     5101 gggcaggaac tgctgaacag caaaaagtca gatagcacca catagcagac ccgccataaa
     5161 acgccctgag aagcccgtga cgggcttttc ttgtattatg ggtagtttcc ttgcatgaat
     5221 ccataaaagg cgcctgtagt gccatttacc cccattcact gccagagccg tgagcgcagc
     5281 gaactgaatg tcacgaaaaa gacagcgact caggtgcctg atggtcggag acaaaaggaa
     5341 tattcagcga tttgcccgag cttgcgaggg tgctacttaa gcctttaggg ttttaaggtc
     5401 tgttttgtag aggagcaaac agcgtttgcg acatcctttt gtaatactgc ggaactgact
     5461 aaagtagtga gttatacaca gggctgggat ctattctttt tatctttttt tattctttct
     5521 ttattctata aattataacc acttgaatat aaacaaaaaa aacacacaaa ggtctagcgg
     5581 aatttacaga gggtctagca gaatttacaa gttttccagc aaaggtctag cagaatttac
     5641 agatacccac aactcaaagg aaaaggacta gtaattatca ttgactagcc catctcaatt
     5701 ggtatagtga ttaaaatcac ctagaccaat tgagatgtat gtctgaatta gttgttttca
     5761 aagcaaatga actagcgatt agtcgctatg acttaacgga gcatgaaacc aagctaattt
     5821 tatgctgtgt ggcactactc aaccccacga ttgaaaaccc tacaaggaaa gaacggacgg
     5881 tatcgttcac ttataaccaa tacgctcaga tgatgaacat cagtagggaa aatgcttatg
     5941 gtgtattagc taaagcaacc agagagctga tgacgagaac tgtggaaatc aggaatcctt
     6001 tggttaaagg cttttggatt ttccagtgga caaactatgc caagttctca agcgaaaaat
     6061 tagaattagt ttttagtgaa gagatattgc cttatctttt ccagttaaaa aaattcataa
     6121 aatataatct ggaacatgtt aagtcttttg aaaacaaata ctctatgagg atttatgagt
     6181 ggttattaaa agaactaaca caaaagaaaa ctcacaaggc aaatatagag attagccttg
     6241 atgaatttaa gttcatgtta atgcttgaaa ataactacca tgagtttaaa aggcttaacc
     6301 aatgggtttt gaaaccaata agtaaagatt taaacactta cagcaatatg aaattggtgg
     6361 ttgataagcg aggccgcccg actgatacgt tgattttcca agttgaacta gatagacaaa
     6421 tggatctcgt aaccgaactt gagaacaacc agataaaaat gaatggtgac aaaataccaa
     6481 caaccattac atcagattcc tacctacata acggactaag aaaaacacta cacgatgctt
     6541 taactgcaaa aattcagctc accagttttg aggcaaaatt tttgagtgac atgcaaagta
     6601 agtatgatct caatggttcg ttctcatggc tcacgcaaaa acaacgaacc acactagaga
     6661 acatactggc taaatacgga aggatctgag gttcttatgg ctcttgtatc tatcagtgaa
     6721 gcatcaagac taacaaacaa aagtagaaca actgttcacc gttacatatc aaagggaaaa
     6781 ctgtccatat gcacagatga aaacggtgta aaaaagatag atacatcaga gcttttacga
     6841 gtttttggtg cattcaaagc tgttcaccat gaacagatcg acaatgtaac agatgaacag
     6901 catgtaacac ctaatagaac aggtgaaacc agtaaaacaa agcaactaga acatgaaatt
     6961 gaacacctga gacaacttgt tacagctcaa cagtcacaca tagacagcct gaaacaggcg
     7021 atgctgctta tcgaatcaaa gctgccgaca acacgggagc cagtgacgcc tcccgtgggg
     7081 aaaaaatcat ggcaattctg gaagaaatag cgctttcagc cggcaaaccg gctgaagccg
     7141 gatctgcgat tctgataaca aactagcaac accagaacag cccgtttgcg ggcagcaaaa
     7201 cccgtaccga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     7261 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     7321 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     7381 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     7441 gcgagaacca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     7501 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     7561 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     7621 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     7681 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     7741 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     7801 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     7861 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     7921 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     7981 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tataacccac
     8041 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     8101 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     8161 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     8221 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcg
//
