LOCUS       sequence-413656-        8282 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Production of ent-kaurene in E. coli MEV20.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8282)
  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 8282)
  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: pLRD04
FEATURES             Location/Qualifiers
     source          1..8282
                     /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        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)."
     regulatory      4609..4618
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    4830..4850
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      4851..4908
                     /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(4932..4949)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      5483..5705
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             5753..6703
                     /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(7317..8108,8109..8177))
                     /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     7940..7959
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8178..8282)
                     /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 agagattatt gattaatttt aaggaggatt
     1921 tatatgctgg aagttccggc acagccaacg ccagcgcctc gtgaagcaga agcggcagcc
     1981 ttattggcag cgaccgttgc tgatccttgg ggcttagtcg cacctagtgt gtacgacaca
     2041 gctcgtctgg taagccttgc cccgtggctg gatggacacc gtgaacgcct gggttacctc
     2101 gtgaaagaac agaatcagga tggctcatgg ggcgctccgg acggctatgg tctggtccct
     2161 accttgtcgg ccgttgaagc cctgctgacg gaattagcgc gcaccgatag tggagcgccg
     2221 catcctccac acgatgatct ggctgcggcc tgcgcgggcg gacttggtgc gttacaggat
     2281 ggcctcttgg ctgggccggt tcctgatact atcggcgttg agtttgttgc gccttcatta
     2341 ctggcggata tcaacacgcg tctggccgca ctgacagaac aagcccctgg caaactgggc
     2401 gcatggtccg gtacaaccct gacgtcacca gctccggatc tggatggcgc acttctggcc
     2461 ggcgttcgtg agatgactga gcaggcgcca ctgccagaga aactgtggca taccttggaa
     2521 gccatcaccc gcgatggtac ccgtggggca cgcccccatg aaggtgcacc gccacacaat
     2581 gggtctgttg gttgtagtcc agcagctacc gctgcatggc ttggtgcgtc gcctgatcct
     2641 gcagcgccag gtgtagcata cctgcgtgat gttcaggctc gctttggcgg cccggtgccg
     2701 tccattacgc cgatcgtgta ctttgagcag gcatgggtgc tgaatagttt agccgcatcg
     2761 ggactgcgct atgaagcgcc tgcagcatta ctggacagtc tggaagccgg cttgactgat
     2821 gaaggcatcg cggcagcacc gggcctccct tctgacagcg acgataccgc cgcagtatta
     2881 ttcgcgctgg cccagcacgg tcgtacacat cgcccagact ccctgatgca cttccgccgt
     2941 gatggctatt tttcatgttt tggtgtcgag cgcacgccat caacctcaac aaacgcgcat
     3001 atactggaag cgttgggaca ccatgttaca gtgcgccccg acgatgctgg gcgttatggc
     3061 gccgaaatcc gtatgatttc tgactggctg ctggataatc aactgccaga tggctcttgg
     3121 atggataaat ggcacgcgtc accctattac gccaccgcct gctgtgccct tgctctggcc
     3181 gaatttggcg gaccgagcgc tcgtgctgca gtggatcgtg cggcagcgtg ggctctggaa
     3241 actcaacgtg ccgatggcag ctggggtcgc tggcagggca caacagaaga aaccgcatac
     3301 atggtgcagc tcttgatgcg cacccgcacg cctggttcgc caggtactgt ggctagatcg
     3361 gctgcgcgcg gctgtgatgc cctcctggct catgatgatc cggcctcgta cccgggcctt
     3421 tggcatgata aagacattta tgccccggta acggtgattc gagcggcacg tttagccgcg
     3481 ctggctctgg gaggtgcaga gtctgccgcg agtggtgggg 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 gaggcattat
     3781 acaaaggagg aacaaaaatg attcagacgg agcgggcggt gcagcaggtc ttggaatggg
     3841 gccggtcact gaccggtttc gccgatgaac acgccgtgga ggcagttaga ggcggtcagt
     3901 acattctgca acgtatccat ccttcactgc gcggtaccag tgcacgcacc ggtcgtgatc
     3961 cacaagatga aacgcttatt gtgaccttct atcgggaatt agcgctgctc ttttggctgg
     4021 acgactgtaa cgatctcggt ttgattagcc cggaacaatt agcggccgta gaacaggcat
     4081 tgggccaagg ggtaccatgc gctctgccgg gatttgaagg ctgtgccgtc ctgcgggcaa
     4141 gcctggccac tctggcctat gatcgacgcg actatgccca acttctggac gatacacgct
     4201 gttacagtgc ggcgctgaga gcgggtcacg cacaagcggt cgctgcggag cgttggagct
     4261 atgccgaata cctgcacaat ggcatagact ccattgcgta cgccaacgtg ttttgctgtc
     4321 tgagtttatt gtggggtctg gatatggcca cccttcgtgc tcgtccggcg tttcgccaag
     4381 tactgcgact gatcagtgcc atcggtcgtc tgcaaaatga cctgcacggc tgcgacaaag
     4441 accggtcagc tggggaggca gataatgccg ttattcttct tttacagaga tacccggcaa
     4501 tgccggtagt agaattcctg aacgatgaac tggcagggca tacccgcatg ctgcaccggg
     4561 ttatggctga ggagcgtttt ccggccccgt ggggcccgct gatcgaagcc atggcggcca
     4621 ttcgcgtaca atattatcgt acgtccacct cccgttatcg gagcgatgca gtccgtggtg
     4681 gccagcgggc cccagcataa taagcctcgt tgctcttcat aaatcatccc tatgacccct
     4741 tcaaaaaaaa aaaacaccct aacgggtgtt tttttttttt tggtctgcct ctcgctgacg
     4801 gtcacaatgc gcttacgaaa gtcttcctgt actagtagcg gccgctgcag tccggcaaaa
     4861 aagggcaagg tgtcaccacc ctgccctttt tctttaaaac cgaaaagatt acttcgcgtt
     4921 atgcaggctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     4981 tcagctcact caaaggcggt aatgacagta agacgggtaa gcctgttgat gataccgctg
     5041 ccttactggg tgcattagcc agtctgaatg acctgtcacg ggataatccg aagtggtcag
     5101 actggaaaat cagagggcag gaactgctga acagcaaaaa gtcagatagc accacatagc
     5161 agacccgcca taaaacgccc tgagaagccc gtgacgggct tttcttgtat tatgggtagt
     5221 ttccttgcat gaatccataa aaggcgcctg tagtgccatt tacccccatt cactgccaga
     5281 gccgtgagcg cagcgaactg aatgtcacga aaaagacagc gactcaggtg cctgatggtc
     5341 ggagacaaaa ggaatattca gcgatttgcc cgagcttgcg agggtgctac ttaagccttt
     5401 agggttttaa ggtctgtttt gtagaggagc aaacagcgtt tgcgacatcc ttttgtaata
     5461 ctgcggaact gactaaagta gtgagttata cacagggctg ggatctattc tttttatctt
     5521 tttttattct ttctttattc tataaattat aaccacttga atataaacaa aaaaaacaca
     5581 caaaggtcta gcggaattta cagagggtct agcagaattt acaagttttc cagcaaaggt
     5641 ctagcagaat ttacagatac ccacaactca aaggaaaagg actagtaatt atcattgact
     5701 agcccatctc aattggtata gtgattaaaa tcacctagac caattgagat gtatgtctga
     5761 attagttgtt ttcaaagcaa atgaactagc gattagtcgc tatgacttaa cggagcatga
     5821 aaccaagcta attttatgct gtgtggcact actcaacccc acgattgaaa accctacaag
     5881 gaaagaacgg acggtatcgt tcacttataa ccaatacgct cagatgatga acatcagtag
     5941 ggaaaatgct tatggtgtat tagctaaagc aaccagagag ctgatgacga gaactgtgga
     6001 aatcaggaat cctttggtta aaggcttttg gattttccag tggacaaact atgccaagtt
     6061 ctcaagcgaa aaattagaat tagtttttag tgaagagata ttgccttatc ttttccagtt
     6121 aaaaaaattc ataaaatata atctggaaca tgttaagtct tttgaaaaca aatactctat
     6181 gaggatttat gagtggttat taaaagaact aacacaaaag aaaactcaca aggcaaatat
     6241 agagattagc cttgatgaat ttaagttcat gttaatgctt gaaaataact accatgagtt
     6301 taaaaggctt aaccaatggg ttttgaaacc aataagtaaa gatttaaaca cttacagcaa
     6361 tatgaaattg gtggttgata agcgaggccg cccgactgat acgttgattt tccaagttga
     6421 actagataga caaatggatc tcgtaaccga acttgagaac aaccagataa aaatgaatgg
     6481 tgacaaaata ccaacaacca ttacatcaga ttcctaccta cataacggac taagaaaaac
     6541 actacacgat gctttaactg caaaaattca gctcaccagt tttgaggcaa aatttttgag
     6601 tgacatgcaa agtaagtatg atctcaatgg ttcgttctca tggctcacgc aaaaacaacg
     6661 aaccacacta gagaacatac tggctaaata cggaaggatc tgaggttctt atggctcttg
     6721 tatctatcag tgaagcatca agactaacaa acaaaagtag aacaactgtt caccgttaca
     6781 tatcaaaggg aaaactgtcc atatgcacag atgaaaacgg tgtaaaaaag atagatacat
     6841 cagagctttt acgagttttt ggtgcattca aagctgttca ccatgaacag atcgacaatg
     6901 taacagatga acagcatgta acacctaata gaacaggtga aaccagtaaa acaaagcaac
     6961 tagaacatga aattgaacac ctgagacaac ttgttacagc tcaacagtca cacatagaca
     7021 gcctgaaaca ggcgatgctg cttatcgaat caaagctgcc gacaacacgg gagccagtga
     7081 cgcctcccgt ggggaaaaaa tcatggcaat tctggaagaa atagcgcttt cagccggcaa
     7141 accggctgaa gccggatctg cgattctgat aacaaactag caacaccaga acagcccgtt
     7201 tgcgggcagc aaaacccgta ccgattatca aaaaggatct tcacctagat ccttttaaat
     7261 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     7321 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     7381 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     7441 gctgcaatga taccgcgaga accacgctca ccggctccag atttatcagc aataaaccag
     7501 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     7561 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     7621 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     7681 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     7741 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     7801 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     7861 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     7921 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     7981 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     8041 tcgatataac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     8101 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     8161 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     8221 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     8281 cg
//
