LOCUS       sequence-413691-        7955 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Production of levopimaradiene in E. coli MEV20.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7955)
  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 7955)
  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: pLRD26
FEATURES             Location/Qualifiers
     source          1..7955
                     /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)."
     misc_feature    4503..4523
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      4524..4581
                     /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(4605..4622)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      5156..5378
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             5426..6376
                     /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(6990..7781,7782..7850))
                     /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     7613..7632
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7851..7955)
                     /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 gggcaagcaa gtaatacgac tcactatagg ggaattgtga gcggataaca attcccctct
     1801 aggtccgaag atacttaggg tgtctcaagg tgcgtacctt gactgatgag tccgaaagga
     1861 cgaaacaccc ctctacaaat aattttgttt aagagcaact atttaataaa actcaaggag
     1921 gttaatttat gtctgctgcg gaaacgcggc ctgatcagct gccccaggaa gaacgttttg
     1981 tgtcacgtct gaatgcggat taccacccag ccgtttggaa agacgatttt attgactcgc
     2041 tgacgtcccc gaactcacat gccacgtcca aatcctctgt tgatgaaacg attaataaac
     2101 gcattcagac gttggtcaag gaaattcaat gcatgtttca gtcgatgggg gacggagaaa
     2161 ctaaccctag cgcttatgac actgcgtggg tagcgcgcat cccatctatt gacggcagtg
     2221 gagcgcctca atttccgcag acactgcagt ggattctcaa taaccagctg ccggatggtt
     2281 cctggggcga agaatgtatc tttttagcgt atgatcgtgt tctgaatacc ctggcctgtt
     2341 tactgacgct gaaaatctgg aacaaggggg acattcaagt gcaaaaaggc gtggaatttg
     2401 tgcgtaagca catggaagag atgaaagacg aggcagataa tcaccgtcct tctgggttcg
     2461 aggtggtgtt ccccgcaatg ctggatgaag ccaaatctct ggggctggat ctgccgtacc
     2521 acttaccgtt tattagtcag atccaccaga aacgacagaa gaaattgcag aaaatacctc
     2581 tgaacgtgct gcacaaccat cagacagcgt tgctttattc gcttgaaggt ctgcaggatg
     2641 tggttgactg gcaggaaatt acgaaccttc agagccggga tggctcgttt ctgtcgtcac
     2701 cggcaagcac cgcctgcgtg ttcatgcaca ctcagaataa gcgctgctta cattttctga
     2761 attttgtcct gtcgaagttt ggcgactacg tgccttgcca ttacccgttg gacttgtttg
     2821 aacgcctctg ggcagtcgac accgttgagc gtctgggcat cgatcgttac tttaaaaagg
     2881 aaatcaagga gtccttagac tacgtctacc gctactggga tgcggaacgt ggcgttggtt
     2941 gggcccgttg caatccgatc cctgacgtag atgataccgc gatggggctg cgcatactgc
     3001 gattgcacgg gtacaatgtg tccagcgatg tcttagagaa ctttcgagat gaaaaaggtg
     3061 acttcttctg ttttgcgggt cagacccaga tcggtgtgac agacaatttg aacttgtatc
     3121 gctgttcgca ggtatgcttt ccgggagaga aaatcatgga agaggccaaa acgtttacca
     3181 cgaaccacct tcagaacgcc ctcgcgaaaa acaatgcctt cgacaaatgg gcagttaaga
     3241 aagatctgcc gggcgaagtg gagtatgcga ttaaatatcc atggcaccgt tcaatgccac
     3301 ggctcgaagc acggtcgtat attgaacaat ttggcagtaa cgatgtttgg ctgggcaaga
     3361 cagtatataa aatgctgtat gtcagtaacg agaagtacct ggaactggcc aaactggact
     3421 ttaatatggt tcaggccttg catcagaaag agacacagca tattgttagt tggtggcgtg
     3481 agagcggatt caatgacctg accttcacgc gacaacgccc ggttgaaatg tacttcagcg
     3541 tcgcggtcag catgtttgaa ccggagtttg cagcttgtcg tatcgcttat gccaaaacga
     3601 gttgtctggc agtgattctg gacgatctgt atgacaccca cggttctctc gatgacctga
     3661 agctgtttag cgaggccgta cgccgttggg acatcagtgt gctggacagc gttcgagata
     3721 accaattaaa agtgtgcttc cttgggttat acaacactgt caatggcttc ggcaaagatg
     3781 gtttaaaaga acaaggacgt gatgttctgg gttatttacg gaaagtgtgg gaaggtctgc
     3841 tggcgagcta tacgaaagaa gccgaatgga gcgcggcaaa gtatgtacca acctttaacg
     3901 aatatgtgga gaacgccaag gtatccattg cactggcgac cgttgtcctg aattcgatct
     3961 tctttaccgg cgaactgtta ccagattata tcctgcagca agttgatctg agatcgaagt
     4021 ttctgcatct ggtaagtctt accggtcgcc tgataaacga tactaaaacc tatcaggctg
     4081 aacgaaatcg cggcgaactt gtgtcctcgg tccagtgtta tatgcgcgag aacccggaat
     4141 gtacggaaga agaagcgtta tctcacgtgt atggcatcat tgataatgca ctgaaagagt
     4201 tgaattggga actggcgaac ccggcctcta acgcgcccct ttgcgtccgc cgtctgcttt
     4261 tcaacacggc acgtgttatg cagctgtttt acatgtatcg ggatggcttc ggtatcagtg
     4321 ataaagaaat gaaagatcat gtgagccgca cgctgtttga tccggtggca taataagcct
     4381 cgttgctctt cataaatcat ccctatgacc ccttcaaaaa aaaaaaacac cctaacgggt
     4441 gttttttttt ttttggtctg cctctcgctg acggtcacaa tgcgcttacg aaagtcttcc
     4501 tgtactagta gcggccgctg cagtccggca aaaaagggca aggtgtcacc accctgccct
     4561 ttttctttaa aaccgaaaag attacttcgc gttatgcagg cttcctcgct cactgactcg
     4621 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatgaca
     4681 gtaagacggg taagcctgtt gatgataccg ctgccttact gggtgcatta gccagtctga
     4741 atgacctgtc acgggataat ccgaagtggt cagactggaa aatcagaggg caggaactgc
     4801 tgaacagcaa aaagtcagat agcaccacat agcagacccg ccataaaacg ccctgagaag
     4861 cccgtgacgg gcttttcttg tattatgggt agtttccttg catgaatcca taaaaggcgc
     4921 ctgtagtgcc atttaccccc attcactgcc agagccgtga gcgcagcgaa ctgaatgtca
     4981 cgaaaaagac agcgactcag gtgcctgatg gtcggagaca aaaggaatat tcagcgattt
     5041 gcccgagctt gcgagggtgc tacttaagcc tttagggttt taaggtctgt tttgtagagg
     5101 agcaaacagc gtttgcgaca tccttttgta atactgcgga actgactaaa gtagtgagtt
     5161 atacacaggg ctgggatcta ttctttttat ctttttttat tctttcttta ttctataaat
     5221 tataaccact tgaatataaa caaaaaaaac acacaaaggt ctagcggaat ttacagaggg
     5281 tctagcagaa tttacaagtt ttccagcaaa ggtctagcag aatttacaga tacccacaac
     5341 tcaaaggaaa aggactagta attatcattg actagcccat ctcaattggt atagtgatta
     5401 aaatcaccta gaccaattga gatgtatgtc tgaattagtt gttttcaaag caaatgaact
     5461 agcgattagt cgctatgact taacggagca tgaaaccaag ctaattttat gctgtgtggc
     5521 actactcaac cccacgattg aaaaccctac aaggaaagaa cggacggtat cgttcactta
     5581 taaccaatac gctcagatga tgaacatcag tagggaaaat gcttatggtg tattagctaa
     5641 agcaaccaga gagctgatga cgagaactgt ggaaatcagg aatcctttgg ttaaaggctt
     5701 ttggattttc cagtggacaa actatgccaa gttctcaagc gaaaaattag aattagtttt
     5761 tagtgaagag atattgcctt atcttttcca gttaaaaaaa ttcataaaat ataatctgga
     5821 acatgttaag tcttttgaaa acaaatactc tatgaggatt tatgagtggt tattaaaaga
     5881 actaacacaa aagaaaactc acaaggcaaa tatagagatt agccttgatg aatttaagtt
     5941 catgttaatg cttgaaaata actaccatga gtttaaaagg cttaaccaat gggttttgaa
     6001 accaataagt aaagatttaa acacttacag caatatgaaa ttggtggttg ataagcgagg
     6061 ccgcccgact gatacgttga ttttccaagt tgaactagat agacaaatgg atctcgtaac
     6121 cgaacttgag aacaaccaga taaaaatgaa tggtgacaaa ataccaacaa ccattacatc
     6181 agattcctac ctacataacg gactaagaaa aacactacac gatgctttaa ctgcaaaaat
     6241 tcagctcacc agttttgagg caaaattttt gagtgacatg caaagtaagt atgatctcaa
     6301 tggttcgttc tcatggctca cgcaaaaaca acgaaccaca ctagagaaca tactggctaa
     6361 atacggaagg atctgaggtt cttatggctc ttgtatctat cagtgaagca tcaagactaa
     6421 caaacaaaag tagaacaact gttcaccgtt acatatcaaa gggaaaactg tccatatgca
     6481 cagatgaaaa cggtgtaaaa aagatagata catcagagct tttacgagtt tttggtgcat
     6541 tcaaagctgt tcaccatgaa cagatcgaca atgtaacaga tgaacagcat gtaacaccta
     6601 atagaacagg tgaaaccagt aaaacaaagc aactagaaca tgaaattgaa cacctgagac
     6661 aacttgttac agctcaacag tcacacatag acagcctgaa acaggcgatg ctgcttatcg
     6721 aatcaaagct gccgacaaca cgggagccag tgacgcctcc cgtggggaaa aaatcatggc
     6781 aattctggaa gaaatagcgc tttcagccgg caaaccggct gaagccggat ctgcgattct
     6841 gataacaaac tagcaacacc agaacagccc gtttgcgggc agcaaaaccc gtaccgatta
     6901 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     6961 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     7021 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     7081 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agaaccacgc
     7141 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     7201 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     7261 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     7321 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     7381 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     7441 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     7501 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     7561 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     7621 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     7681 ctctcaagga tcttaccgct gttgagatcc agttcgatat aacccactcg tgcacccaac
     7741 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     7801 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     7861 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     7921 tgtatttaga aaaataaaca aataggggtt ccgcg
//
