LOCUS       sequence-409624-        9180 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Production of miltiradiene in E. coli MEV20.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9180)
  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 9180)
  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: pLRD12
FEATURES             Location/Qualifiers
     source          1..9180
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     66..84
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(88..131)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    134..155
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     promoter        176..194
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    195..219
                     /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        1750..1768
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1769..1793
                     /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        3630..3648
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3649..3673
                     /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    5726..5746
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      5747..5804
                     /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(5828..5845)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      6379..6601
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             6649..7599
                     /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(8213..9004,9005..9073))
                     /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     8836..8855
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9074..9178)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
       61 ataaaaatag gcgtatcacg aggcagaatt tcagataaaa aaaatcctta gctttcgcta
      121 aggatgattt ctggaattcg cggccgcttc tagagcgcga agacttaatc aaatctaata
      181 cgactcacta taggggaatt gtgagcggat aacaattccc ctctacgttt tcaaggtggt
      241 agactgtcgc cggatgtgta tccgacctga cgatggccca aaagggccga aacagtcctc
      301 tacaaataat tttgtttaag agaatacccc gctaagaagg taattatgtc catgcaaagt
      361 ttagataaga attttcgaca tttatctcgt aaagaaaagt tacaacaatt ggttgataag
      421 caatggttat cagaagaaca attcgacatt ttactgaatc atccattaat cgatgaagaa
      481 gtagccaata gtttaattga aaatgtcatc gcgcaaggtg cattacccgt tggattatta
      541 ccgaatatca ttgtggacga taaggcatat gttgtaccta tgatggtgga agagccttca
      601 gttgtcgctg cagctagtta tggtgcaaag ctagtgaatc agactggcgg atttaaaacg
      661 gtatcttctg aacgtattat gataggtcaa atcgtctttg atggcgttga cgatactgaa
      721 aaattatcag cagacattaa agctttagaa aagcaaattc ataaaattgc ggatgaggca
      781 tatccttcta ttaaagcgcg tggtggtggt taccaacgta tagcgattga tacatttcct
      841 gagcaacagt tactatcttt aaaagtattt gttgatacga aagatgctat gggcgctaat
      901 atgcttaata cgattttaga ggccataact gcatttttaa aaaatgaatt tccgcaaagc
      961 gacattttaa tgagtatttt atccaatcat gcaacagcgt ccgttgttaa agttcaaggc
     1021 gaaattgatg ttaaagattt agcaaggggc gagagaactg gagaagaggt tgccaaacga
     1081 atggaacgtg cttctgtatt ggcccaagta gatattcatc gtgcagcaac acataataaa
     1141 ggtgttatga atggcataca tgctgttgtt ttagcaacag gaaatgatac gcgtggtgca
     1201 gaagcaagtg cgcatgcata cgcgagtcgt gacggacagt atcgtggtat tgctacatgg
     1261 cgttacgatc aagatcgtca acgattgatt ggtacaattg aagtgcctat gacattggca
     1321 atcgttggcg gtggtacaaa agtattacca attgctaaag cttcattaga gctactaaat
     1381 gtagagtcag cacaagaatt aggtcatgta gttgctgccg ttggtttagc gcaaaacttt
     1441 gcagcatgtc gcgcgcttgt gtcagaaggt attcaacaag gtcatatgag tttacaatat
     1501 aaatcattag ctatcgttgt aggggcaaaa ggtgatgaaa ttgctaaagt agctgaagct
     1561 ttgaaaaaag aaccccgtgc aaatacacaa gcagcggaac atattttaca agaaattaga
     1621 caacaataat aacggatgta aacagagaaa cgtctaacta aaaaggcctc ccaaatcggg
     1681 gggccttttt tattgataac aaaagcctaa tacacctgtt cgtgacgact acggatgcgg
     1741 gcaaggtgct aatacgactc actatagggg aattgtgagc ggataacaat tcccctctaa
     1801 acgccagcca ctaaagcggt caacgcatgt gctttgcgtt ctgatgagac agtgatgtcg
     1861 aaaccgcctc tacaaataat tttgtttaag aaggtcaaac acattagcaa cgataagggg
     1921 gtctttatgt cgtccatcag ccatcaagcg gctccgacgc gtgctcagca caccaattcc
     1981 tacgtgcagc tggcgcgcca attagtgact tcagtcgaca atgacccatg gggtgacgtc
     2041 ccgcctagtg tgtacgaaac cgcacgggtc acatcgtggg ctccgtggct ggaaggtcac
     2101 gagcgccgcc tggcatggtt actcgaacgc caaagtgcgg ccggttcctg gggtgaaggt
     2161 ccgacccctt atcgtctgct tcctacactg tctgttactg aagcacttct cagcaccctg
     2221 cgtcagaaca cagccgccgg tgtttctcgc gaacgcctgg ccgcggcagt cgacaatggt
     2281 cttgcagcgt tgcgtgatct gagcggtacc ggcggctggc cggataccgc cgccatcgag
     2341 attctggcac ctgatctggt ggttttaatc aatgaccatc tggatcaacc agaagttgcc
     2401 gcacttccgc gcctgggtcc ttgggcccgc ggtcaaagat tagcccaacc tcacggtttt
     2461 caagcggcgc tccctgaccg cgttgctgaa cgttgccagg tggctggggg cgttccgctg
     2521 aaacttcatc acacttttga aggcgtggca cgccgcctgc cgcgtatggt gcctggagtt
     2581 cccggtggac tcctgggctc aagtccagcg gctacggcag cgtggttagc aaccggtccg
     2641 gacgaagggc gtgaccaagc agttacggct cttacggcgg ttgcggaacg ctacgatggc
     2701 ctgttcccag aagcaacccc gattagtgtg ttcgaacgcc tgtggatctc tgtagcatta
     2761 gctcgcccgg gtctgcccgc cgcgtgcgta cctacaattc gtgcctgggc cgctgaaatc
     2821 tatgatgcga ccggagtgcg tggtgctccc ggtttactgc cagatactga tgatacagcg
     2881 atggcggtct tagcgtctgc actggccggt agcccgcgag atccttctcc actgtcggca
     2941 tttgaggcgg gtgatcacta tgattgttac gtgggcgagg acaccggtag ctctacggct
     3001 aatgcgcacg cactccaggc tcttacggcg tggcttagcc accgtccggc cactggcgac
     3061 gctttacagg ccagacgcga tctgacgcga gattggctgc tggcccagca ggaaagtgat
     3121 ggggcgtggc gtgataaatg gcatgcgagc ccatattatg caactgaacg ttgcgttact
     3181 gccctgtcag gccatacggg ccctacgacg cgtgatgcta tccgctcggc tgccgattgg
     3241 gtcctggatg cacagtccga tgatggcagt tggggtgtat ggggaggtac cgcggaagaa
     3301 accgcctacg ccgtgaacat cttgcttaac agcccggacc ataccgggac gccagaagcg
     3361 acacaggcgt taaaactggc agaaaatgta ttgcgtgaag cggtgcattc gtccggtcac
     3421 catcatcccg ctttatggca tgacaaaacc ctttacgcgc cccaagcgat ggcacaggcg
     3481 gaagtcattg cagccttgga actgctgcag gcacgtcgtc cgtaataatt atgccttccg
     3541 ctaatcaact cggtaccaaa ttccagaaaa gagacgcttt cgagcgtctt ttttcgtttt
     3601 ggtccgtgga caagcacagc atagccaagt aatacgactc actatagggg aattgtgagc
     3661 ggataacaat tcccctctag gtccgaagat acttagggtg tctcaaggtg cgtaccttga
     3721 ctgatgagtc cgaaaggacg aaacacccct ctacaaataa ttttgtttaa gagctcgaac
     3781 cacaattttc attaagtagg ttttttttat gtccctggct tttaacccgg ctgcaaccgc
     3841 gttttctggt aacggtgcgc gctctcgccg cgagaatttt cctgtcaagc atgtgactgt
     3901 aagaggcttt ccgatgatta ccaacaaatc ttcattcgcc gtaaaatgca atttgaccac
     3961 aaccgatctc atgggcaaaa ttgcagaaaa gtttaaggga gaggatagca acttcccagc
     4021 cgctgccgct gttcaacctg ctgcggacat gccgagcaat ctctgcatca tagacacgct
     4081 gcaacgtctg ggggtcgatc gttattttcg ttcagaaata gataccatcc tggaagatac
     4141 atatcgcttg tggcagcgca aagaacgcgc aatcttctcg gacacggcta ttcatgcgat
     4201 ggcctttcgt ctgctgcgag tcaagggcta tgaggtatcc agtgaagaac ttgcgccgta
     4261 tgccgatcag gagcatgtgg atctgcagac cattgaggta gcgacggtga tagaactgta
     4321 tcgtgctgcg caggaacgta cgggtgaaga tgaaagctca ctgaaaaagc tgcacgcgtg
     4381 gacgacgacc tttctgaaac agaaactgtt aacaaacagc atccccgata aaaagctcca
     4441 taaactggtg gaatattacc tgaaaaacta ccacggtatt ctggaccgaa tgggtgtgcg
     4501 tcagaatctg gacctgtacg atattagcta ctatagaacg agcaaagcag cgaatcgctt
     4561 ctcgaattta tgctccgaag attttcttgc atttgcccgc caggacttta atatttgcca
     4621 ggcccagcat cagaaagaac tgcagcagct gcagcgctgg tacgcggatt gtaaacttga
     4681 tacactgaaa tatggtcgtg atgttgtacg cgtcgcgaac ttcttaacga gcgcgatcat
     4741 cggcgatccc gaactttcag atgtgcgcat agtgttcgct caacacatcg tgttagtcac
     4801 acgaatcgat gatttctttg accaccgcgg ctcccgtgag gagtcttaca aaatcctgga
     4861 attaatcaaa gaatggaagg agaagccggc agcggagtat ggctccgaag aagtagaaat
     4921 tttgtttacc gccgtgtata acacagtgaa tgaactggct gaacgggcac atgtggaaca
     4981 aggacgctcg gtgaaagact ttttaattaa actctgggtg cagattctct ccattttcaa
     5041 gcgtgagctg gacacctggt ctgatgatac cgccctgact cttgatgatt atttgagcgc
     5101 gtcctgggtg tcgatcggtt gccgcatttg tatcttaatg agcatgcaat tcattggtat
     5161 caaactctca gatgaaatgc tgctctctga agagtgcatc gatctctgtc gccacgttag
     5221 catggtggat cgcctgctga atgacgttca aaccttcgaa aaagagcgca aagagaatac
     5281 cggcaatagt gtaactctgc tcttggccgc taacaaagat gactcctcct ttacagagga
     5341 agaagcaatt cggatagcca aagagatggc agaatgtaat cgtcgccagc tgatgcagat
     5401 agtttacaag actggtacaa tttttccccg ccaatgcaaa gacatgttct tgaaagtgtg
     5461 ccgtatcggc tgttatctgt atgcaagtgg ggacgaattt acttcaccgc aacagatgat
     5521 ggaggacatg aaatctctcg tatatgagcc gctgaccatc cacccgttgg ttgctaataa
     5581 cgtccgtggg aaataataag cctcgttgct cttcataaat catccctatg accccttcaa
     5641 aaaaaaaaaa caccctaacg ggtgtttttt tttttttggt ctgcctctcg ctgacggtca
     5701 caatgcgctt acgaaagtct tcctgtacta gtagcggccg ctgcagtccg gcaaaaaagg
     5761 gcaaggtgtc accaccctgc cctttttctt taaaaccgaa aagattactt cgcgttatgc
     5821 aggcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     5881 ctcactcaaa ggcggtaatg acagtaagac gggtaagcct gttgatgata ccgctgcctt
     5941 actgggtgca ttagccagtc tgaatgacct gtcacgggat aatccgaagt ggtcagactg
     6001 gaaaatcaga gggcaggaac tgctgaacag caaaaagtca gatagcacca catagcagac
     6061 ccgccataaa acgccctgag aagcccgtga cgggcttttc ttgtattatg ggtagtttcc
     6121 ttgcatgaat ccataaaagg cgcctgtagt gccatttacc cccattcact gccagagccg
     6181 tgagcgcagc gaactgaatg tcacgaaaaa gacagcgact caggtgcctg atggtcggag
     6241 acaaaaggaa tattcagcga tttgcccgag cttgcgaggg tgctacttaa gcctttaggg
     6301 ttttaaggtc tgttttgtag aggagcaaac agcgtttgcg acatcctttt gtaatactgc
     6361 ggaactgact aaagtagtga gttatacaca gggctgggat ctattctttt tatctttttt
     6421 tattctttct ttattctata aattataacc acttgaatat aaacaaaaaa aacacacaaa
     6481 ggtctagcgg aatttacaga gggtctagca gaatttacaa gttttccagc aaaggtctag
     6541 cagaatttac agatacccac aactcaaagg aaaaggacta gtaattatca ttgactagcc
     6601 catctcaatt ggtatagtga ttaaaatcac ctagaccaat tgagatgtat gtctgaatta
     6661 gttgttttca aagcaaatga actagcgatt agtcgctatg acttaacgga gcatgaaacc
     6721 aagctaattt tatgctgtgt ggcactactc aaccccacga ttgaaaaccc tacaaggaaa
     6781 gaacggacgg tatcgttcac ttataaccaa tacgctcaga tgatgaacat cagtagggaa
     6841 aatgcttatg gtgtattagc taaagcaacc agagagctga tgacgagaac tgtggaaatc
     6901 aggaatcctt tggttaaagg cttttggatt ttccagtgga caaactatgc caagttctca
     6961 agcgaaaaat tagaattagt ttttagtgaa gagatattgc cttatctttt ccagttaaaa
     7021 aaattcataa aatataatct ggaacatgtt aagtcttttg aaaacaaata ctctatgagg
     7081 atttatgagt ggttattaaa agaactaaca caaaagaaaa ctcacaaggc aaatatagag
     7141 attagccttg atgaatttaa gttcatgtta atgcttgaaa ataactacca tgagtttaaa
     7201 aggcttaacc aatgggtttt gaaaccaata agtaaagatt taaacactta cagcaatatg
     7261 aaattggtgg ttgataagcg aggccgcccg actgatacgt tgattttcca agttgaacta
     7321 gatagacaaa tggatctcgt aaccgaactt gagaacaacc agataaaaat gaatggtgac
     7381 aaaataccaa caaccattac atcagattcc tacctacata acggactaag aaaaacacta
     7441 cacgatgctt taactgcaaa aattcagctc accagttttg aggcaaaatt tttgagtgac
     7501 atgcaaagta agtatgatct caatggttcg ttctcatggc tcacgcaaaa acaacgaacc
     7561 acactagaga acatactggc taaatacgga aggatctgag gttcttatgg ctcttgtatc
     7621 tatcagtgaa gcatcaagac taacaaacaa aagtagaaca actgttcacc gttacatatc
     7681 aaagggaaaa ctgtccatat gcacagatga aaacggtgta aaaaagatag atacatcaga
     7741 gcttttacga gtttttggtg cattcaaagc tgttcaccat gaacagatcg acaatgtaac
     7801 agatgaacag catgtaacac ctaatagaac aggtgaaacc agtaaaacaa agcaactaga
     7861 acatgaaatt gaacacctga gacaacttgt tacagctcaa cagtcacaca tagacagcct
     7921 gaaacaggcg atgctgctta tcgaatcaaa gctgccgaca acacgggagc cagtgacgcc
     7981 tcccgtgggg aaaaaatcat ggcaattctg gaagaaatag cgctttcagc cggcaaaccg
     8041 gctgaagccg gatctgcgat tctgataaca aactagcaac accagaacag cccgtttgcg
     8101 ggcagcaaaa cccgtaccga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     8161 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     8221 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     8281 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     8341 caatgatacc gcgagaacca cgctcaccgg ctccagattt atcagcaata aaccagccag
     8401 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     8461 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     8521 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     8581 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     8641 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     8701 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     8761 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     8821 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     8881 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     8941 tataacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     9001 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     9061 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     9121 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
//
