LOCUS       sequence-413675-        9691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Production of ent-cassa-12,15-diene in E. coli MEV20.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9691)
  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 9691)
  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: pLRD13
FEATURES             Location/Qualifiers
     source          1..9691
                     /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)."
     misc_feature    6239..6259
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      6260..6317
                     /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(6341..6358)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      6892..7114
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             7162..8112
                     /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(8726..9517,9518..9586))
                     /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     9349..9368
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9587..9691)
                     /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 gagagacgaa
     3781 ggaggtatta aatggcggca gcggttggtg ggctggaaat gaaaaagcca gaagcggaag
     3841 gaattgccga gagtctccag gcaactcatc gtaaagaact ggaagcgagc atccgcaaac
     3901 aattacaggg agttgaatta tcgccgtcgc cgtacgatac tgcctgggtg gcgatggtgc
     3961 cattgcgggg gtcaagtcat aatccgagct tcccgcagtg tgtggattgg attttagaaa
     4021 accagtggga cgatggctcc tggagcattg acggcagtat cagcaccgcc aataaagacg
     4081 ttctgagctc gacgcttgct tgtgtgctgg cattaaacaa atggaatgtc gggcgtgagc
     4141 acattcgacg cggcctctct ttcatcggcc ggaacttctc tattgccatg gacgaccaag
     4201 ccgtggctcc gattggcttc ggcatcacgt ttcccgccat gctgaccctc gcgaatggct
     4261 ctggcctcga agttcctgtg cgccaaaacg atattgattc actgaaccat ctgcgcgaaa
     4321 tgaaaatcca acgggaagct ggtaaccact cgagaggccg caaagcatac atggcatatc
     4381 tggcagaagg ttttggtaac ttgcttgaat gggatgagat catgatgttt cagcgtaaaa
     4441 acgggtccct gtttaactgt ccgtctagca cggctggcgc gttggcgaac taccacgacg
     4501 ataaagctct gcagtactta caatcgctgg taaacaaatt cgatggggtt gtgccgaccc
     4561 tgtatccgct gaacatttat tgccaactgt ctatggtgga tgcattagag aatatgggca
     4621 ttagccagta ttttgccagc gaaatcaaaa gtatccttga tatgacctat agcagctggt
     4681 tgggaaaaga tgaagagatt atgctggatg tcacgacctg cgccatggcg tttcgcctgc
     4741 tgcgcatgaa tggttatgat gtctcgtcgg acgagctgtc acacgttgcc ggggctagcg
     4801 gttttcgtga ttcccttcaa ggctatctga acgatcgtaa aagcgtgtta gaggtctata
     4861 agacgtcaaa acatagcata tcggaaaacg acctgatttt agattctatc ggttcgtggt
     4921 cgggcggctc tttactgaaa gagatgctta gttcgaacgg gaaggggacc cctggtcgtg
     4981 aggagattga attcgcgctg aagtacccgt tttacagtac cctggaacga ctggttcacc
     5041 gtaaaaacat tgtcctgttc gacgcaaaag gctcgcagat gctgaaaacg gcttgtatgc
     5101 ctgtccacga tagtcaagac ttcttagccc tggccgtgga tgatttttgt atctctcagt
     5161 caaactacca aaacgagttg aactatttgg aatcatgggt gaaagataac cgtttggacc
     5221 agttacattt tgcccgccag aaaataacgt actgctatct gagcggcgcg gcgaccacgt
     5281 ttcgtcctga aatgggatac gccagaacta gttgggcccg aaccgcatgg ctgaccgctg
     5341 tcatcgacga tctgtttgat gttggcggcc ttgagcaaga gcaggaaaac cttcttgctc
     5401 tgatggagaa atgggaagaa ccgggcgaag atgaatacta cagtgaggac gtaaaaatcg
     5461 tgtttcaagc gctctacaac accgtgaacg aaatcggcgc caaagccagc gcattacagg
     5521 gccacgatgt cactaagtac ttagtcgatg tctggctgca cgtagtacgt tgcatgaagg
     5581 tagaagctga gtggcagcgt agtcagcact taccgacctt tgaagaatat atggaaagtg
     5641 gtatggtgtc gctgggccaa ggctgtacgg tgatgtcggc cctgtttctg ataggcgaaa
     5701 aattaccaga aggtattgtt gaacttgagg aatacgatga actgtttcgt ctgatgggaa
     5761 cctgtgggcg cttgttaaac gatatccgag gaatcgaacg agaagaatcg gacggtaaga
     5821 tgacaaacgg cgtgtctctg ttagtgcatg catcgggtgg ttcaatgtct gttgatgagg
     5881 caaagacgga agtcatgaaa cgcatcgacg ccagtcgtcg caaacttctc tccttagtgg
     5941 tatccgagca ggagggtcca attcctcgtc cttgtaaaca gctgttctgg aaaatgtgca
     6001 aaattctcca tcttttctat tatcagaccg acggcttttc tagtcctaaa gaaatggtga
     6061 gtgcggtgga cgcggtaatt aatgaaccac tgcagctgag actgctgtga taagcctcgt
     6121 tgctcttcat aaatcatccc tatgacccct tcaaaaaaaa aaaacaccct aacgggtgtt
     6181 tttttttttt ggtctgcctc tcgctgacgg tcacaatgcg cttacgaaag tcttcctgta
     6241 ctagtagcgg ccgctgcagt ccggcaaaaa agggcaaggt gtcaccaccc tgcccttttt
     6301 ctttaaaacc gaaaagatta cttcgcgtta tgcaggcttc ctcgctcact gactcgctgc
     6361 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atgacagtaa
     6421 gacgggtaag cctgttgatg ataccgctgc cttactgggt gcattagcca gtctgaatga
     6481 cctgtcacgg gataatccga agtggtcaga ctggaaaatc agagggcagg aactgctgaa
     6541 cagcaaaaag tcagatagca ccacatagca gacccgccat aaaacgccct gagaagcccg
     6601 tgacgggctt ttcttgtatt atgggtagtt tccttgcatg aatccataaa aggcgcctgt
     6661 agtgccattt acccccattc actgccagag ccgtgagcgc agcgaactga atgtcacgaa
     6721 aaagacagcg actcaggtgc ctgatggtcg gagacaaaag gaatattcag cgatttgccc
     6781 gagcttgcga gggtgctact taagccttta gggttttaag gtctgttttg tagaggagca
     6841 aacagcgttt gcgacatcct tttgtaatac tgcggaactg actaaagtag tgagttatac
     6901 acagggctgg gatctattct ttttatcttt ttttattctt tctttattct ataaattata
     6961 accacttgaa tataaacaaa aaaaacacac aaaggtctag cggaatttac agagggtcta
     7021 gcagaattta caagttttcc agcaaaggtc tagcagaatt tacagatacc cacaactcaa
     7081 aggaaaagga ctagtaatta tcattgacta gcccatctca attggtatag tgattaaaat
     7141 cacctagacc aattgagatg tatgtctgaa ttagttgttt tcaaagcaaa tgaactagcg
     7201 attagtcgct atgacttaac ggagcatgaa accaagctaa ttttatgctg tgtggcacta
     7261 ctcaacccca cgattgaaaa ccctacaagg aaagaacgga cggtatcgtt cacttataac
     7321 caatacgctc agatgatgaa catcagtagg gaaaatgctt atggtgtatt agctaaagca
     7381 accagagagc tgatgacgag aactgtggaa atcaggaatc ctttggttaa aggcttttgg
     7441 attttccagt ggacaaacta tgccaagttc tcaagcgaaa aattagaatt agtttttagt
     7501 gaagagatat tgccttatct tttccagtta aaaaaattca taaaatataa tctggaacat
     7561 gttaagtctt ttgaaaacaa atactctatg aggatttatg agtggttatt aaaagaacta
     7621 acacaaaaga aaactcacaa ggcaaatata gagattagcc ttgatgaatt taagttcatg
     7681 ttaatgcttg aaaataacta ccatgagttt aaaaggctta accaatgggt tttgaaacca
     7741 ataagtaaag atttaaacac ttacagcaat atgaaattgg tggttgataa gcgaggccgc
     7801 ccgactgata cgttgatttt ccaagttgaa ctagatagac aaatggatct cgtaaccgaa
     7861 cttgagaaca accagataaa aatgaatggt gacaaaatac caacaaccat tacatcagat
     7921 tcctacctac ataacggact aagaaaaaca ctacacgatg ctttaactgc aaaaattcag
     7981 ctcaccagtt ttgaggcaaa atttttgagt gacatgcaaa gtaagtatga tctcaatggt
     8041 tcgttctcat ggctcacgca aaaacaacga accacactag agaacatact ggctaaatac
     8101 ggaaggatct gaggttctta tggctcttgt atctatcagt gaagcatcaa gactaacaaa
     8161 caaaagtaga acaactgttc accgttacat atcaaaggga aaactgtcca tatgcacaga
     8221 tgaaaacggt gtaaaaaaga tagatacatc agagctttta cgagtttttg gtgcattcaa
     8281 agctgttcac catgaacaga tcgacaatgt aacagatgaa cagcatgtaa cacctaatag
     8341 aacaggtgaa accagtaaaa caaagcaact agaacatgaa attgaacacc tgagacaact
     8401 tgttacagct caacagtcac acatagacag cctgaaacag gcgatgctgc ttatcgaatc
     8461 aaagctgccg acaacacggg agccagtgac gcctcccgtg gggaaaaaat catggcaatt
     8521 ctggaagaaa tagcgctttc agccggcaaa ccggctgaag ccggatctgc gattctgata
     8581 acaaactagc aacaccagaa cagcccgttt gcgggcagca aaacccgtac cgattatcaa
     8641 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     8701 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     8761 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     8821 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagaa ccacgctcac
     8881 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     8941 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     9001 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     9061 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     9121 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     9181 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     9241 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     9301 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     9361 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     9421 caaggatctt accgctgttg agatccagtt cgatataacc cactcgtgca cccaactgat
     9481 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     9541 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     9601 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     9661 tttagaaaaa taaacaaata ggggttccgc g
//
