LOCUS       sequence-413687-        9835 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Production of 13R-manoyl oxide in E. coli MEV20.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9835)
  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 9835)
  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: pLRD20
FEATURES             Location/Qualifiers
     source          1..9835
                     /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        4375..4393
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4394..4418
                     /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)."
     RBS             4552..4560
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     misc_feature    6383..6403
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      6404..6461
                     /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(6485..6502)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      7036..7258
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             7306..8256
                     /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(8870..9661,9662..9730))
                     /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     9493..9512
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9731..9835)
                     /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 agagctctat ctaataagga gaactataat
     1921 gaatcgtgaa ttcccgccga gcttttggaa caatgacatt ataaacagca taacagcgag
     1981 ccataaagta cagactggtg atcgcaagcg cattcagacg ctgatttccg aaattaaaaa
     2041 tgtgtttaat agcatggggg acggggaaac aagcccgtcg gcatacgata cggcatgggt
     2101 ggcgcgcatt cctgctgttg atggttccga acagccgcag ttcccgcaga cgctggagtg
     2161 gattttacag aaccaactta aagatggatc gtggggcgag gaattttatt tcttagcgta
     2221 cgatcggtta cttgcaacgc tggcgtgcat cattaccctg accatctggc gcaccggcaa
     2281 cgtccaatta cacaaaggga ttgagttctt tcgcaaacag gtagtacgga tggacgacga
     2341 ggcggacaat catcgcccca gcggctttga aattgtcttt ccggcgatgc tgaacgaagc
     2401 aaaatctctg ggtctggacc tgccttatga acttccgttc atcgaacaga tggtgaagaa
     2461 acgcgaagcg aaattgaaga tgattacgac caatgtactc tataccattc agaccacgct
     2521 gttatacagt ttggaaggcc tccacgagat cgtggacttt gataaaatta ttaaactcca
     2581 atccaaggat gggagctttc tcggtagtcc ggcgtctacc gcggctgtgt tcatgcaaac
     2641 cggtaatacc aaatgccttg aatttctgga gttcgttctg cgaaagtttc gtaatcatgt
     2701 gccttcagat tatccgctgg atctgttcga gcgcctctgg gtggtggata ccgttgaacg
     2761 tctgggtatt gaccgtcact ttaagaaaga aatcaaagac gcgctggact atgtctattc
     2821 ttgttgggat gaacgaggaa ttggctgggc gaaggattcc cctatcgcag atattgatga
     2881 tactgccatg ggtctgcgca tcctgcgtct gcacgggtat aatgttagtc cggatgttct
     2941 gaaaaccttt aaggacgaga acggtgaatt cttttgcttt atgggccaga cgcaaagagg
     3001 tgtgaccgat atgctgaacg tgtaccgttg cagtcaagtg gcattccccg gggaaaccat
     3061 catggaggaa gcgaaattat gcacagaacg ttacttgcgc aacgctttag aaaacgctga
     3121 tgccttcgat aagtgggcga tcaagaaaaa cattcgcgga gaggttgaat atgccttaaa
     3181 atatccttgg catcgaagca tgccacggct ggaagtccgt tcgtacattg gaaattatgg
     3241 tccaaacgat gtttggctgg gtaaaagtct gtatatgatg ccgtatattt ccaacgagaa
     3301 atacctggaa cttgcgaaac ttgatttcaa tagcgtccag tctctgcacc aggaagaaat
     3361 ccgcgaatta gtgcgctggt gtaaatcatc aggatttacg gaactgaagt ttacgcgtga
     3421 ccgcgttgtg gaaacgtact ttgccgtggc atcaagcatg tttgaaccag aattctccac
     3481 ttgccgtgct gtgtatacga aaatcagcgt gctccttgta atcctggctg acctgtatga
     3541 tggttacggc tccccggatg agattaaact gttctctgag gcggtgaaac gttgggacct
     3601 gagcctgctg gaacaaatgc cggatcacat gaaaatttgc ttcctgggcc tttacaacac
     3661 cgtaaatgag gtggcggaag aggggcgtaa gacccagggt catgatgtac ttggttacat
     3721 ccggaatctg tgggagattc aactggcagc atttacccgt gaagcagaat ggtcccaggg
     3781 aaaatatgtg ccatcgtttg acgaatatat tgagaacgcg caagtaagca ttggtgtggc
     3841 tacaatcctg ctgattacca tcttgtttac tgaagaagat gacatcctga gccacattga
     3901 ttatggttcc aaatttcttc gactggcgag tctcactgcg cgtttggcga atgacattaa
     3961 aacttatcaa gaagaacggg cgcacggcga agtggttagc gcaattcaat gctacatgaa
     4021 agatcgcccg gaaatcacgg aagaagaagc cctgaaatat gtgtacggcc gcatggtcaa
     4081 cgatctggcc gaactgaata gcgaatacct gaaaagtaac gaaatgcccc agaactgcaa
     4141 gcgcttagta ttcgacaccg cgcgcgtcgc acagctgttt accatggagg gcgacgggct
     4201 cacgtattcg gataccatgg agatcaagga acacatcaag aaatgcttat tcgaaccggc
     4261 aacttaataa taattatgcc ttccgctaat caactcggta ccaaattcca gaaaagagac
     4321 gctttcgagc gtcttttttc gttttggtcc gtggacaagc acagcatagc caagtaatac
     4381 gactcactat aggggaattg tgagcggata acaattcccc tctaggtccg aagatactta
     4441 gggtgtctca aggtgcgtac cttgactgat gagtccgaaa ggacgaaaca cccctctaca
     4501 aataattttg tttaagagaa gaataattaa gttaacaatt tataccccca ataaggaggt
     4561 ttttatgagt gccgcggtta aatgcagcct tacgacaccg acggacctga tgggcaaaat
     4621 taaagaagta tttaaccgcg aggttgacac ctccccggcc gcgatgacga cccactcaac
     4681 ggacatcccc tcaaatcttt gtatcatcga cactttgcag cgcctgggaa ttgatcagta
     4741 cttccagagc gaaattgatg ccgtactcca cgacacctat cgcctgtggc aactgaagaa
     4801 gaaagacata ttttcggaca ttaccaccca tgccatggca ttccgcttgc tccgcgtgaa
     4861 agggtacgaa gttgcgagcg acgagttagc cccgtacgct gatcaggaac gcattaatct
     4921 gcagacgatc gacgtcccaa ctgtcgttga gctctaccgc gcagcccagg aacgcctgac
     4981 agaggaggat tccaccctgg agaagctgta cgtgtggacg tcagcgtttt tgaaacagca
     5041 gctgctcact gatgcgatcc cggataagaa actgcataaa caggtggagt actacttgaa
     5101 gaattatcat ggcattctgg accgcatggg ggttcggcgt aacttggacc tgtacgatat
     5161 tagccactac aaatctctta aagcggcaca tcgtttttac aatctgtcga acgaagatat
     5221 ccttgcgttt gcacgtcagg actttaacat ctcacaggcc caacaccaaa aggaactgca
     5281 gcagcttcag cgttggtatg ctgattgccg cctggatacc ttaaaatttg gacgtgacgt
     5341 cgtgcgtatc ggtaattttc tgacctcagc tatgattggt gaccccgaac tgtcagatct
     5401 gcgactggcc ttcgcaaagc atatcgtgtt ggtgacccgc attgatgatt tcttcgatca
     5461 tggaggcccc aaggaagaaa gttatgagat cttagaactg gtgaaagaat ggaaagaaaa
     5521 acccgctggc gagtatgtat ccgaagaagt ggaaattctg ttcaccgccg tgtataatac
     5581 cgtgaatgag ctggcagaaa tggcccacat tgaacagggt cgttctgtaa aagatctgtt
     5641 ggtgaaactg tgggttgaaa tactcagtgt tttccggatt gaattggaca cctggacgaa
     5701 tgataccgct ctgacgttag aggaatacct gtcccagagc tgggtgagta ttggttgccg
     5761 gatttgtatc ttgataagta tgcagttcca gggtgtgaaa ttgagcgatg agatgctgca
     5821 gagtgaagaa tgcacggatt tatgccgtta tgtgtcaatg gtcgaccgcc tgttgaatga
     5881 tgtccaaaca ttcgaaaagg aacgtaaaga aaataccgga aatagcgtaa gcctcctgca
     5941 ggcagcgcac aaagatgaac gcgttataaa cgaggaagaa gcctgtatta aagtcaaaga
     6001 actggctgag tacaaccgac gcaaactgat gcagattgtt tataaaacgg gcaccatctt
     6061 tccccgcaag tgcaaagacc tgttcttgaa agcctgccgg atcggctgtt atctttacag
     6121 cagtggtgac gaatttacct ccccacagca aatgatggag gacatgaaaa gtttggtgta
     6181 tgaacccctc ccgatttcgc ctccggaagc caacaacgcg tcaggcgaaa agatgtcgtg
     6241 cgtatcgaac tgataagcct cgttgctctt cataaatcat ccctatgacc ccttcaaaaa
     6301 aaaaaaacac cctaacgggt gttttttttt ttttggtctg cctctcgctg acggtcacaa
     6361 tgcgcttacg aaagtcttcc tgtactagta gcggccgctg cagtccggca aaaaagggca
     6421 aggtgtcacc accctgccct ttttctttaa aaccgaaaag attacttcgc gttatgcagg
     6481 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     6541 actcaaaggc ggtaatgaca gtaagacggg taagcctgtt gatgataccg ctgccttact
     6601 gggtgcatta gccagtctga atgacctgtc acgggataat ccgaagtggt cagactggaa
     6661 aatcagaggg caggaactgc tgaacagcaa aaagtcagat agcaccacat agcagacccg
     6721 ccataaaacg ccctgagaag cccgtgacgg gcttttcttg tattatgggt agtttccttg
     6781 catgaatcca taaaaggcgc ctgtagtgcc atttaccccc attcactgcc agagccgtga
     6841 gcgcagcgaa ctgaatgtca cgaaaaagac agcgactcag gtgcctgatg gtcggagaca
     6901 aaaggaatat tcagcgattt gcccgagctt gcgagggtgc tacttaagcc tttagggttt
     6961 taaggtctgt tttgtagagg agcaaacagc gtttgcgaca tccttttgta atactgcgga
     7021 actgactaaa gtagtgagtt atacacaggg ctgggatcta ttctttttat ctttttttat
     7081 tctttcttta ttctataaat tataaccact tgaatataaa caaaaaaaac acacaaaggt
     7141 ctagcggaat ttacagaggg tctagcagaa tttacaagtt ttccagcaaa ggtctagcag
     7201 aatttacaga tacccacaac tcaaaggaaa aggactagta attatcattg actagcccat
     7261 ctcaattggt atagtgatta aaatcaccta gaccaattga gatgtatgtc tgaattagtt
     7321 gttttcaaag caaatgaact agcgattagt cgctatgact taacggagca tgaaaccaag
     7381 ctaattttat gctgtgtggc actactcaac cccacgattg aaaaccctac aaggaaagaa
     7441 cggacggtat cgttcactta taaccaatac gctcagatga tgaacatcag tagggaaaat
     7501 gcttatggtg tattagctaa agcaaccaga gagctgatga cgagaactgt ggaaatcagg
     7561 aatcctttgg ttaaaggctt ttggattttc cagtggacaa actatgccaa gttctcaagc
     7621 gaaaaattag aattagtttt tagtgaagag atattgcctt atcttttcca gttaaaaaaa
     7681 ttcataaaat ataatctgga acatgttaag tcttttgaaa acaaatactc tatgaggatt
     7741 tatgagtggt tattaaaaga actaacacaa aagaaaactc acaaggcaaa tatagagatt
     7801 agccttgatg aatttaagtt catgttaatg cttgaaaata actaccatga gtttaaaagg
     7861 cttaaccaat gggttttgaa accaataagt aaagatttaa acacttacag caatatgaaa
     7921 ttggtggttg ataagcgagg ccgcccgact gatacgttga ttttccaagt tgaactagat
     7981 agacaaatgg atctcgtaac cgaacttgag aacaaccaga taaaaatgaa tggtgacaaa
     8041 ataccaacaa ccattacatc agattcctac ctacataacg gactaagaaa aacactacac
     8101 gatgctttaa ctgcaaaaat tcagctcacc agttttgagg caaaattttt gagtgacatg
     8161 caaagtaagt atgatctcaa tggttcgttc tcatggctca cgcaaaaaca acgaaccaca
     8221 ctagagaaca tactggctaa atacggaagg atctgaggtt cttatggctc ttgtatctat
     8281 cagtgaagca tcaagactaa caaacaaaag tagaacaact gttcaccgtt acatatcaaa
     8341 gggaaaactg tccatatgca cagatgaaaa cggtgtaaaa aagatagata catcagagct
     8401 tttacgagtt tttggtgcat tcaaagctgt tcaccatgaa cagatcgaca atgtaacaga
     8461 tgaacagcat gtaacaccta atagaacagg tgaaaccagt aaaacaaagc aactagaaca
     8521 tgaaattgaa cacctgagac aacttgttac agctcaacag tcacacatag acagcctgaa
     8581 acaggcgatg ctgcttatcg aatcaaagct gccgacaaca cgggagccag tgacgcctcc
     8641 cgtggggaaa aaatcatggc aattctggaa gaaatagcgc tttcagccgg caaaccggct
     8701 gaagccggat ctgcgattct gataacaaac tagcaacacc agaacagccc gtttgcgggc
     8761 agcaaaaccc gtaccgatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     8821 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     8881 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     8941 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     9001 tgataccgcg agaaccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     9061 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     9121 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     9181 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     9241 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     9301 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     9361 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     9421 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     9481 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     9541 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatat
     9601 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     9661 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     9721 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     9781 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcg
//
