LOCUS       sequence-409625-        9696 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Production of ent-sandaracopimaradiene in E. coli MEV20.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9696)
  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 9696)
  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: pLRD15
FEATURES             Location/Qualifiers
     source          1..9696
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     72..90
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(94..137)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    140..161
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /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        1790..1808
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1809..1833
                     /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        3668..3686
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3687..3711
                     /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    6248..6268
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      6269..6326
                     /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(6350..6367)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      6901..7123
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             7171..8121
                     /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(8735..9526,9527..9595))
                     /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     9358..9377
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(9596..9696,1..4))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
       61 taacctataa aaataggcgt atcacgaggc agaatttcag ataaaaaaaa tccttagctt
      121 tcgctaagga tgatttctgg aattcgcggc cgcttctaga gcgcgaagac ttaatcaaat
      181 ccgtagcagt gcacaagcaa gtgaaatcta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctacg ttttcaaggt ggtagactgt cgccggatgt gtatccgacc
      301 tgacgatggc ccaaaagggc cgaaacagtc ctctacaaat aattttgttt aagagaatac
      361 cccgctaaga aggtaattat gtccatgcaa agtttagata agaattttcg acatttatct
      421 cgtaaagaaa agttacaaca attggttgat aagcaatggt tatcagaaga acaattcgac
      481 attttactga atcatccatt aatcgatgaa gaagtagcca atagtttaat tgaaaatgtc
      541 atcgcgcaag gtgcattacc cgttggatta ttaccgaata tcattgtgga cgataaggca
      601 tatgttgtac ctatgatggt ggaagagcct tcagttgtcg ctgcagctag ttatggtgca
      661 aagctagtga atcagactgg cggatttaaa acggtatctt ctgaacgtat tatgataggt
      721 caaatcgtct ttgatggcgt tgacgatact gaaaaattat cagcagacat taaagcttta
      781 gaaaagcaaa ttcataaaat tgcggatgag gcatatcctt ctattaaagc gcgtggtggt
      841 ggttaccaac gtatagcgat tgatacattt cctgagcaac agttactatc tttaaaagta
      901 tttgttgata cgaaagatgc tatgggcgct aatatgctta atacgatttt agaggccata
      961 actgcatttt taaaaaatga atttccgcaa agcgacattt taatgagtat tttatccaat
     1021 catgcaacag cgtccgttgt taaagttcaa ggcgaaattg atgttaaaga tttagcaagg
     1081 ggcgagagaa ctggagaaga ggttgccaaa cgaatggaac gtgcttctgt attggcccaa
     1141 gtagatattc atcgtgcagc aacacataat aaaggtgtta tgaatggcat acatgctgtt
     1201 gttttagcaa caggaaatga tacgcgtggt gcagaagcaa gtgcgcatgc atacgcgagt
     1261 cgtgacggac agtatcgtgg tattgctaca tggcgttacg atcaagatcg tcaacgattg
     1321 attggtacaa ttgaagtgcc tatgacattg gcaatcgttg gcggtggtac aaaagtatta
     1381 ccaattgcta aagcttcatt agagctacta aatgtagagt cagcacaaga attaggtcat
     1441 gtagttgctg ccgttggttt agcgcaaaac tttgcagcat gtcgcgcgct tgtgtcagaa
     1501 ggtattcaac aaggtcatat gagtttacaa tataaatcat tagctatcgt tgtaggggca
     1561 aaaggtgatg aaattgctaa agtagctgaa gctttgaaaa aagaaccccg tgcaaataca
     1621 caagcagcgg aacatatttt acaagaaatt agacaacaat aataacggat gtaaacagag
     1681 aaacgtctaa ctaaaaaggc ctcccaaatc ggggggcctt ttttattgat aacaaaagcc
     1741 taatacacct gttcgtgacg atgagaccta gccgcaatac acagtgtgct aatacgactc
     1801 actatagggg aattgtgagc ggataacaat tcccctctaa acgccagcca ctaaagcggt
     1861 caacgcatgt gctttgcgtt ctgatgagac agtgatgtcg aaaccgcctc tacaaataat
     1921 tttgtttaag agattattga ttaattttaa ggaggattta tatgctggaa gttccggcac
     1981 agccaacgcc agcgcctcgt gaagcagaag cggcagcctt attggcagcg accgttgctg
     2041 atccttgggg cttagtcgca cctagtgtgt acgacacagc tcgtctggta agccttgccc
     2101 cgtggctgga tggacaccgt gaacgcctgg gttacctcgt gaaagaacag aatcaggatg
     2161 gctcatgggg cgctccggac ggctatggtc tggtccctac cttgtcggcc gttgaagccc
     2221 tgctgacgga attagcgcgc accgatagtg gagcgccgca tcctccacac gatgatctgg
     2281 ctgcggcctg cgcgggcgga cttggtgcgt tacaggatgg cctcttggct gggccggttc
     2341 ctgatactat cggcgttgag tttgttgcgc cttcattact ggcggatatc aacacgcgtc
     2401 tggccgcact gacagaacaa gcccctggca aactgggcgc atggtccggt acaaccctga
     2461 cgtcaccagc tccggatctg gatggcgcac ttctggccgg cgttcgtgag atgactgagc
     2521 aggcgccact gccagagaaa ctgtggcata ccttggaagc catcacccgc gatggtaccc
     2581 gtggggcacg cccccatgaa ggtgcaccgc cacacaatgg gtctgttggt tgtagtccag
     2641 cagctaccgc tgcatggctt ggtgcgtcgc ctgatcctgc agcgccaggt gtagcatacc
     2701 tgcgtgatgt tcaggctcgc tttggcggcc cggtgccgtc cattacgccg atcgtgtact
     2761 ttgagcaggc atgggtgctg aatagtttag ccgcatcggg actgcgctat gaagcgcctg
     2821 cagcattact ggacagtctg gaagccggct tgactgatga aggcatcgcg gcagcaccgg
     2881 gcctcccttc tgacagcgac gataccgccg cagtattatt cgcgctggcc cagcacggtc
     2941 gtacacatcg cccagactcc ctgatgcact tccgccgtga tggctatttt tcatgttttg
     3001 gtgtcgagcg cacgccatca acctcaacaa acgcgcatat actggaagcg ttgggacacc
     3061 atgttacagt gcgccccgac gatgctgggc gttatggcgc cgaaatccgt atgatttctg
     3121 actggctgct ggataatcaa ctgccagatg gctcttggat ggataaatgg cacgcgtcac
     3181 cctattacgc caccgcctgc tgtgcccttg ctctggccga atttggcgga ccgagcgctc
     3241 gtgctgcagt ggatcgtgcg gcagcgtggg ctctggaaac tcaacgtgcc gatggcagct
     3301 ggggtcgctg gcagggcaca acagaagaaa ccgcatacat ggtgcagctc ttgatgcgca
     3361 cccgcacgcc tggttcgcca ggtactgtgg ctagatcggc tgcgcgcggc tgtgatgccc
     3421 tcctggctca tgatgatccg gcctcgtacc cgggcctttg gcatgataaa gacatttatg
     3481 ccccggtaac ggtgattcga gcggcacgtt tagccgcgct ggctctggga ggtgcagagt
     3541 ctgccgcgag tggtggggcg taataattat gccttccgct aatcaactcg gtaccaaatt
     3601 ccagaaaaga gacgctttcg agcgtctttt ttcgttttgg tccgtggaca agcacagcat
     3661 agccaagtaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctaggt
     3721 ccgaagatac ttagggtgtc tcaaggtgcg taccttgact gatgagtccg aaaggacgaa
     3781 acacccctct acaaataatt ttgtttaaga atacacaaga agctagggag aataacgatg
     3841 cactctaagg aattacaggc taaaatacgc cgacagctgc agcgtgtgga gctctcacca
     3901 tctttgtatg ataccgcgtg ggttgcgatg gtcccggagc gcagttctag tcaggctccc
     3961 tgctacccgc agtgtattga atggattttg cagaaccagc atgacgacgg gagctggggt
     4021 attaactctt cgtcactctc tgttaataaa gatatcctgc tgtctacgtt ggcgtgcgtc
     4081 gttgcgctca agaaatggaa cgcaggtagt tatcacatta agagaggcct gaactttgtt
     4141 ggacgcaact ttagcgtagc tatggacgtc caaaatattg caccggttgg tttcaatgtt
     4201 accttctctg gtctgattac cttggcctcc gggatgggtc tgcagctgcc ggtttggcag
     4261 accgatattg atgaaatttt ccaccttcgc aaaattgaac tggaacgaga tagtggcgga
     4321 accatttcag cccgcaaagc gtttatggcc tatgtggcgg agggatttgg ttcgttacag
     4381 gactgggatc aggttatggc ataccaacgc aaaaacggct ccttgtttaa ttcaccgtcc
     4441 accaccgccg ctgcggctat acacaccttc aatgatcgta cccttaatta tttggatagc
     4501 ctcacaaaca aatttggcgg tccggttccg gcaatgtacc cccagaatat ttactcacag
     4561 ctctgtaccg tggacgcgct ggaacgcacg ggtatcagcc agaaattcgc gcgcgaaatc
     4621 agagacatcc tggataccac gtaccgctcc tggcttcata atgaagaaga agtcatgtta
     4681 gatattccta cctgcgcgat ggcctttcgt ctgctccgca cacacggcta tgatattacg
     4741 agtgatgaaa tggcgcactt ttctgaacag agctctttcg atgatagcat ccacggctat
     4801 ctgaatgaca ccaaaacttt gcttgaactg ttcaaaacct cgcagattcg tttttcttgc
     4861 gaagatcttg tgctggaaaa cattggaaca tggtcagcaa aactgctgaa acagcaactg
     4921 ctgtcaaaca aactgagcac atcagcccag agtgaggtag aatacgtact caagtttccg
     4981 ttacatagta ccctggaccg tctggaacac cgtcgcaaca ttgaacagtt taaagttgag
     5041 ggctccaaag tattaaaatc aggttattgc ggttctcatt cgaacgagga aattctggca
     5101 ctggctgtag attattttca ttcatctcaa agcgtatacc agcaggaact gaaatatttt
     5161 gaaagctggg tcaaacagtg tcgtttggat gagctgaaat tcgcgcgcgt gatgccgctg
     5221 atcgttcatt tcagcagtgc ggcgacaatc ttcgcgccgg aattggcgga tgcacgaatg
     5281 gttctgtcgc agacctgtat gttaattacg gtgtacgatg atttctttga ttgcccggag
     5341 atcagtcgcg aagagaagga gaactatata gcgttaattg agaaatggga caatcacgcg
     5401 gaaatcgggt tttgcagcaa aaatgtagaa atcgttttct atgcggtata caacacgtac
     5461 aaacagattg gtgaaaaagc agcgctgaaa cagaaccggt ccattatgga tcaattagtg
     5521 gaagatctgg tgagcagtgc gaaagcgatg atggttgaag cagattggac agccacgaaa
     5581 tatatcccag cgacaatgga agagtatatg tcaaacgcag aggtaagtgg tgcgtttgcc
     5641 tcgtttgtat gcccacctct gtactttctg ggtctgaaac tgtccgaaga ggacgttaaa
     5701 agtcatgagt atacgcagct tctgaagttg actaacgtga ttggacgctt acagaacgac
     5761 tctcagacct atcgtaaaga aatcctggca ggtaaagtga atagcgtgct tctgcgcgcg
     5821 ctgaccgatt ccgggaatac ttcgccggaa agtattgaag cagcaaaaga aattgtcaat
     5881 cgggacgcgg aatctagcat ggtagaaatg cgctcgcttg tgttttcgga gggcggcccg
     5941 attccgcggc cttgtaaaga cagattctgg gaaatgtgca aaattgtttt ctacttctat
     6001 agcgaggacg acgcctatcg cactcctaaa gaaacgatgt caagcgcgcg tgcagttatt
     6061 cttgacccgc tgcgactgat accgcccccg tcctgtccgg aaaccttatc tagttaataa
     6121 gcctcgttgc tcttcataaa tcatccctat gaccccttca aaaaaaaaaa acaccctaac
     6181 gggtgttttt tttttttttg gtctgcctct cgctgacggt cacaatgcgc ttacgaaagt
     6241 cttcctgtac tagtagcggc cgctgcagtc cggcaaaaaa gggcaaggtg tcaccaccct
     6301 gccctttttc tttaaaaccg aaaagattac ttcgcgttat gcaggcttcc tcgctcactg
     6361 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     6421 tgacagtaag acgggtaagc ctgttgatga taccgctgcc ttactgggtg cattagccag
     6481 tctgaatgac ctgtcacggg ataatccgaa gtggtcagac tggaaaatca gagggcagga
     6541 actgctgaac agcaaaaagt cagatagcac cacatagcag acccgccata aaacgccctg
     6601 agaagcccgt gacgggcttt tcttgtatta tgggtagttt ccttgcatga atccataaaa
     6661 ggcgcctgta gtgccattta cccccattca ctgccagagc cgtgagcgca gcgaactgaa
     6721 tgtcacgaaa aagacagcga ctcaggtgcc tgatggtcgg agacaaaagg aatattcagc
     6781 gatttgcccg agcttgcgag ggtgctactt aagcctttag ggttttaagg tctgttttgt
     6841 agaggagcaa acagcgtttg cgacatcctt ttgtaatact gcggaactga ctaaagtagt
     6901 gagttataca cagggctggg atctattctt tttatctttt tttattcttt ctttattcta
     6961 taaattataa ccacttgaat ataaacaaaa aaaacacaca aaggtctagc ggaatttaca
     7021 gagggtctag cagaatttac aagttttcca gcaaaggtct agcagaattt acagataccc
     7081 acaactcaaa ggaaaaggac tagtaattat cattgactag cccatctcaa ttggtatagt
     7141 gattaaaatc acctagacca attgagatgt atgtctgaat tagttgtttt caaagcaaat
     7201 gaactagcga ttagtcgcta tgacttaacg gagcatgaaa ccaagctaat tttatgctgt
     7261 gtggcactac tcaaccccac gattgaaaac cctacaagga aagaacggac ggtatcgttc
     7321 acttataacc aatacgctca gatgatgaac atcagtaggg aaaatgctta tggtgtatta
     7381 gctaaagcaa ccagagagct gatgacgaga actgtggaaa tcaggaatcc tttggttaaa
     7441 ggcttttgga ttttccagtg gacaaactat gccaagttct caagcgaaaa attagaatta
     7501 gtttttagtg aagagatatt gccttatctt ttccagttaa aaaaattcat aaaatataat
     7561 ctggaacatg ttaagtcttt tgaaaacaaa tactctatga ggatttatga gtggttatta
     7621 aaagaactaa cacaaaagaa aactcacaag gcaaatatag agattagcct tgatgaattt
     7681 aagttcatgt taatgcttga aaataactac catgagttta aaaggcttaa ccaatgggtt
     7741 ttgaaaccaa taagtaaaga tttaaacact tacagcaata tgaaattggt ggttgataag
     7801 cgaggccgcc cgactgatac gttgattttc caagttgaac tagatagaca aatggatctc
     7861 gtaaccgaac ttgagaacaa ccagataaaa atgaatggtg acaaaatacc aacaaccatt
     7921 acatcagatt cctacctaca taacggacta agaaaaacac tacacgatgc tttaactgca
     7981 aaaattcagc tcaccagttt tgaggcaaaa tttttgagtg acatgcaaag taagtatgat
     8041 ctcaatggtt cgttctcatg gctcacgcaa aaacaacgaa ccacactaga gaacatactg
     8101 gctaaatacg gaaggatctg aggttcttat ggctcttgta tctatcagtg aagcatcaag
     8161 actaacaaac aaaagtagaa caactgttca ccgttacata tcaaagggaa aactgtccat
     8221 atgcacagat gaaaacggtg taaaaaagat agatacatca gagcttttac gagtttttgg
     8281 tgcattcaaa gctgttcacc atgaacagat cgacaatgta acagatgaac agcatgtaac
     8341 acctaataga acaggtgaaa ccagtaaaac aaagcaacta gaacatgaaa ttgaacacct
     8401 gagacaactt gttacagctc aacagtcaca catagacagc ctgaaacagg cgatgctgct
     8461 tatcgaatca aagctgccga caacacggga gccagtgacg cctcccgtgg ggaaaaaatc
     8521 atggcaattc tggaagaaat agcgctttca gccggcaaac cggctgaagc cggatctgcg
     8581 attctgataa caaactagca acaccagaac agcccgtttg cgggcagcaa aacccgtacc
     8641 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     8701 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     8761 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     8821 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagaac
     8881 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     8941 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     9001 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     9061 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     9121 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     9181 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     9241 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     9301 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     9361 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     9421 gaaaactctc aaggatctta ccgctgttga gatccagttc gatataaccc actcgtgcac
     9481 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     9541 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     9601 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     9661 ttgaatgtat ttagaaaaat aaacaaatag gggttc
//
