LOCUS       sequence-413685-        8316 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Production of ent-isokaurene in E. coli MEV20.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8316)
  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 8316)
  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: pLRD18
FEATURES             Location/Qualifiers
     source          1..8316
                     /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        221..239
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    240..264
                     /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        2099..2117
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2118..2142
                     /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    4864..4884
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      4885..4942
                     /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(4966..4983)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      5517..5739
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             5787..6737
                     /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(7351..8142,8143..8211))
                     /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     7974..7993
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8212..8316)
                     /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 tcaaatccgt
      181 agcagtgcac aagcaagtga gtgctgacga gctcaggagc taatacgact cactataggg
      241 gaattgtgag cggataacaa ttcccctcta aacgccagcc actaaagcgg tcaacgcatg
      301 tgctttgcgt tctgatgaga cagtgatgtc gaaaccgcct ctacaaataa ttttgtttaa
      361 gagattattg attaatttta aggaggattt atatgctgga agttccggca cagccaacgc
      421 cagcgcctcg tgaagcagaa gcggcagcct tattggcagc gaccgttgct gatccttggg
      481 gcttagtcgc acctagtgtg tacgacacag ctcgtctggt aagccttgcc ccgtggctgg
      541 atggacaccg tgaacgcctg ggttacctcg tgaaagaaca gaatcaggat ggctcatggg
      601 gcgctccgga cggctatggt ctggtcccta ccttgtcggc cgttgaagcc ctgctgacgg
      661 aattagcgcg caccgatagt ggagcgccgc atcctccaca cgatgatctg gctgcggcct
      721 gcgcgggcgg acttggtgcg ttacaggatg gcctcttggc tgggccggtt cctgatacta
      781 tcggcgttga gtttgttgcg ccttcattac tggcggatat caacacgcgt ctggccgcac
      841 tgacagaaca agcccctggc aaactgggcg catggtccgg tacaaccctg acgtcaccag
      901 ctccggatct ggatggcgca cttctggccg gcgttcgtga gatgactgag caggcgccac
      961 tgccagagaa actgtggcat accttggaag ccatcacccg cgatggtacc cgtggggcac
     1021 gcccccatga aggtgcaccg ccacacaatg ggtctgttgg ttgtagtcca gcagctaccg
     1081 ctgcatggct tggtgcgtcg cctgatcctg cagcgccagg tgtagcatac ctgcgtgatg
     1141 ttcaggctcg ctttggcggc ccggtgccgt ccattacgcc gatcgtgtac tttgagcagg
     1201 catgggtgct gaatagttta gccgcatcgg gactgcgcta tgaagcgcct gcagcattac
     1261 tggacagtct ggaagccggc ttgactgatg aaggcatcgc ggcagcaccg ggcctccctt
     1321 ctgacagcga cgataccgcc gcagtattat tcgcgctggc ccagcacggt cgtacacatc
     1381 gcccagactc cctgatgcac ttccgccgtg atggctattt ttcatgtttt ggtgtcgagc
     1441 gcacgccatc aacctcaaca aacgcgcata tactggaagc gttgggacac catgttacag
     1501 tgcgccccga cgatgctggg cgttatggcg ccgaaatccg tatgatttct gactggctgc
     1561 tggataatca actgccagat ggctcttgga tggataaatg gcacgcgtca ccctattacg
     1621 ccaccgcctg ctgtgccctt gctctggccg aatttggcgg accgagcgct cgtgctgcag
     1681 tggatcgtgc ggcagcgtgg gctctggaaa ctcaacgtgc cgatggcagc tggggtcgct
     1741 ggcagggcac aacagaagaa accgcataca tggtgcagct cttgatgcgc acccgcacgc
     1801 ctggttcgcc aggtactgtg gctagatcgg ctgcgcgcgg ctgtgatgcc ctcctggctc
     1861 atgatgatcc ggcctcgtac ccgggccttt ggcatgataa agacatttat gccccggtaa
     1921 cggtgattcg agcggcacgt ttagccgcgc tggctctggg aggtgcagag tctgccgcga
     1981 gtggtggggc gtaataatta tgccttccgc taatcaactc ggtaccaaat tccagaaaag
     2041 agacgctttc gagcgtcttt tttcgttttg gtccgtggac aagcacagca tagccaagta
     2101 atacgactca ctatagggga attgtgagcg gataacaatt cccctctagg tccgaagata
     2161 cttagggtgt ctcaaggtgc gtaccttgac tgatgagtcc gaaaggacga aacacccctc
     2221 tacaaataat tttgtttaag agttttagtt caatttaagg aggagcttat gatgttaccg
     2281 atgtcatcgg catgcagcgg gggtcaattc cccggtgcgt ccccgcacgg catcattccg
     2341 aaacaatttt cgcgtgcacc gcgtatccgt gtctcaatac gtggagccgc aggcgtcgaa
     2401 aagagtctgg gactcggacg aaatgccggc tcgcaacaag gcatgcataa gaatgaactg
     2461 catgataaaa ttcgaaagca gctgcgggat gtgcaattac agccttcgtc gtatgacacc
     2521 gcatgggtgg ctatggttcc ggtgcagggt tctcatcaga cccccagatt tcctcaatcg
     2581 attgaatgga ttctgcaaaa ccaatatgat gacgggtcat gggggacgaa cctgcctggt
     2641 ctggtagtca acaaagatat tcttctctgt accttggcgt gcgttgttgc gctgaaacgt
     2701 tggaacacgg gtcgtgatca catttcccgc ggcttaaatt ttatcggacg caacttttcg
     2761 gttgctatgg acgagcagac agtagcaccg gtgggcttta acataacctt ttcagggctg
     2821 cttagcttag cgacgcgcac ggggctggag cttccggtaa tgcagacgga tatcgacgga
     2881 attattcata ttcgtaaaat tgagcttgaa cgcgatgcct acggcaccgc aagtagtcgt
     2941 cgcgccttta tggcttacgt gagcgaaggt ctgggtaatc tgcaagattg gaaccaagtt
     3001 atggcgtatc agcgtaaaaa tggcagcatt tttaactcgc cgagtgctac ggcggccacc
     3061 atcattcatg gtcacaatta cagtggatta gcgtacttag actttgtgac ctccaaattt
     3121 ggcggtccgg tgccagtaat gtatccccaa aatgcgtata gtcagttgtg tatggtcgat
     3181 actttagagc gcatgggtat atcggaatct tttgcctgcg agatttccga tattctggat
     3241 atgacgtatc gtctgtggat gcacaatgaa gaagaactca tgctggacat gcgtacctgc
     3301 gcaatggcct tccgtttact tcgcatgcac ggttatgata taacgtccga tggcatggcg
     3361 cagtttgtag aacagtctag ttttgatgac tccatccacg gctacctgaa cgataccaag
     3421 gcactgctgg agctctacaa aagcagccag ctgcgctgct tagaggacga tcttatcctg
     3481 gaagaaatcg gttcctggag cgcacgcgta ctgttggaaa agattagcag caaaatgatc
     3541 cacataagtg aactgccgga agtcgaatat gctctgaaat gtccagttta tgccatactg
     3601 gaacggctgg aacagaaacg caacatcgaa cagtttaaga ccaaggaaca gctcaaaatc
     3661 gaaggcttca agctgctgaa gagtggctac cgtggtgtga tcccgaatga cgaaatttta
     3721 gcgctggccg tagatgaatt ccatagttcg cagagtgtct accagcaaga actgcaggat
     3781 ctgaacagct gggtcgcaca tactcgcctg gatgaactga aattcgcccg cctgatgccg
     3841 agtattacct atttcagtgc agcggcagtc ctgctgccct cagagtctgc acggatagcg
     3901 tggacccaga actgcattct gactaccact gttgacgact tctttgacgg cgaaggtagc
     3961 aaggaggaaa tggaaaatct ggtcaagttg atcgaaaaat gggacgacca tggcgaaatt
     4021 ggcttctctt cggaatgcgt ggagattctg ttctatgcag tatataacac gagcaaacaa
     4081 attgcggaga aagcaatgcc gcttcaaaaa cgtaacgcag tggaccatat cgccgaatcc
     4141 tggtggttta ccgtacgtgg gatgctcacc gaagccgaat ggcgcatgga taaatacgtg
     4201 ccgacaacgg tggaagaata tatgagtgca gcggtcgact ccttcgccgt cggcccgatt
     4261 attacctctg ctgctctgtt cgtgggacct gaactcagcg aagaagtctt tcgctccgaa
     4321 gaatatattc atctcatgaa tttggcgaat accatcggcc gcctcctgaa tgacatgcag
     4381 acctatgaga aagaaataaa aatgggcaag gtgaacagcg tgatgctgca cgcgctgagt
     4441 cattcaggtg gtggcagagg gagtccggaa gcgagtatgg aagaggctaa acgtgagatg
     4501 cgccgcgtat tgcagggatg tcgctttgaa ctcttacgcc tggtaacccg cgacgctggt
     4561 gttgtgcccc cgccctgccg caagttgttc tggctgatga gcaaagtgct ccattttgtt
     4621 tacatggaaa aagaccgtta cttcacggca gaaggcatga tggcgagcgc gaatgcagta
     4681 attctcgacc cactgcaagt gactctgccg ccaagtgatt ccggcacact gtgataagcc
     4741 tcgttgctct tcataaatca tccctatgac cccttcaaaa aaaaaaaaca ccctaacggg
     4801 tgtttttttt tttttggtct gcctctcgct gacggtcaca atgcgcttac gaaagtcttc
     4861 ctgtactagt agcggccgct gcagtccggc aaaaaagggc aaggtgtcac caccctgccc
     4921 tttttcttta aaaccgaaaa gattacttcg cgttatgcag gcttcctcgc tcactgactc
     4981 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatgac
     5041 agtaagacgg gtaagcctgt tgatgatacc gctgccttac tgggtgcatt agccagtctg
     5101 aatgacctgt cacgggataa tccgaagtgg tcagactgga aaatcagagg gcaggaactg
     5161 ctgaacagca aaaagtcaga tagcaccaca tagcagaccc gccataaaac gccctgagaa
     5221 gcccgtgacg ggcttttctt gtattatggg tagtttcctt gcatgaatcc ataaaaggcg
     5281 cctgtagtgc catttacccc cattcactgc cagagccgtg agcgcagcga actgaatgtc
     5341 acgaaaaaga cagcgactca ggtgcctgat ggtcggagac aaaaggaata ttcagcgatt
     5401 tgcccgagct tgcgagggtg ctacttaagc ctttagggtt ttaaggtctg ttttgtagag
     5461 gagcaaacag cgtttgcgac atccttttgt aatactgcgg aactgactaa agtagtgagt
     5521 tatacacagg gctgggatct attcttttta tcttttttta ttctttcttt attctataaa
     5581 ttataaccac ttgaatataa acaaaaaaaa cacacaaagg tctagcggaa tttacagagg
     5641 gtctagcaga atttacaagt tttccagcaa aggtctagca gaatttacag atacccacaa
     5701 ctcaaaggaa aaggactagt aattatcatt gactagccca tctcaattgg tatagtgatt
     5761 aaaatcacct agaccaattg agatgtatgt ctgaattagt tgttttcaaa gcaaatgaac
     5821 tagcgattag tcgctatgac ttaacggagc atgaaaccaa gctaatttta tgctgtgtgg
     5881 cactactcaa ccccacgatt gaaaacccta caaggaaaga acggacggta tcgttcactt
     5941 ataaccaata cgctcagatg atgaacatca gtagggaaaa tgcttatggt gtattagcta
     6001 aagcaaccag agagctgatg acgagaactg tggaaatcag gaatcctttg gttaaaggct
     6061 tttggatttt ccagtggaca aactatgcca agttctcaag cgaaaaatta gaattagttt
     6121 ttagtgaaga gatattgcct tatcttttcc agttaaaaaa attcataaaa tataatctgg
     6181 aacatgttaa gtcttttgaa aacaaatact ctatgaggat ttatgagtgg ttattaaaag
     6241 aactaacaca aaagaaaact cacaaggcaa atatagagat tagccttgat gaatttaagt
     6301 tcatgttaat gcttgaaaat aactaccatg agtttaaaag gcttaaccaa tgggttttga
     6361 aaccaataag taaagattta aacacttaca gcaatatgaa attggtggtt gataagcgag
     6421 gccgcccgac tgatacgttg attttccaag ttgaactaga tagacaaatg gatctcgtaa
     6481 ccgaacttga gaacaaccag ataaaaatga atggtgacaa aataccaaca accattacat
     6541 cagattccta cctacataac ggactaagaa aaacactaca cgatgcttta actgcaaaaa
     6601 ttcagctcac cagttttgag gcaaaatttt tgagtgacat gcaaagtaag tatgatctca
     6661 atggttcgtt ctcatggctc acgcaaaaac aacgaaccac actagagaac atactggcta
     6721 aatacggaag gatctgaggt tcttatggct cttgtatcta tcagtgaagc atcaagacta
     6781 acaaacaaaa gtagaacaac tgttcaccgt tacatatcaa agggaaaact gtccatatgc
     6841 acagatgaaa acggtgtaaa aaagatagat acatcagagc ttttacgagt ttttggtgca
     6901 ttcaaagctg ttcaccatga acagatcgac aatgtaacag atgaacagca tgtaacacct
     6961 aatagaacag gtgaaaccag taaaacaaag caactagaac atgaaattga acacctgaga
     7021 caacttgtta cagctcaaca gtcacacata gacagcctga aacaggcgat gctgcttatc
     7081 gaatcaaagc tgccgacaac acgggagcca gtgacgcctc ccgtggggaa aaaatcatgg
     7141 caattctgga agaaatagcg ctttcagccg gcaaaccggc tgaagccgga tctgcgattc
     7201 tgataacaaa ctagcaacac cagaacagcc cgtttgcggg cagcaaaacc cgtaccgatt
     7261 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     7321 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     7381 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     7441 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagaaccacg
     7501 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     7561 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     7621 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     7681 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     7741 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     7801 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     7861 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     7921 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     7981 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     8041 actctcaagg atcttaccgc tgttgagatc cagttcgata taacccactc gtgcacccaa
     8101 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     8161 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     8221 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     8281 atgtatttag aaaaataaac aaataggggt tccgcg
//
