LOCUS       sequence-455292-        6569 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 1 - Position 2 with 35S promoter and terminator for plant 
            expression, with mutations in CDS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6569)
  AUTHORS   Golubova D, Salmon M, Su H, Tansley C, Kaithakottil GG, Linsmith G, 
            Schudoma C, Swarbreck D, O'Connell MA, Patron NJ
  TITLE     Biosynthesis and bioactivity of anti-inflammatory triterpenoids in 
            Calendula officinalis.
  JOURNAL   Nat Commun. 2025 Jul 28;16(1):6941. doi: 10.1038/s41467-025-62269-w.
  PUBMED    40721615
REFERENCE   2  (bases 1 to 6569)
  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: "Nat 
            Commun."; date: "2025-07-28"; volume: "16(1):6941. doi"; pages: " 
            10.1038/s41467-025-62269-w"
COMMENT     Sequence Label: pEPDG1CB0018
FEATURES             Location/Qualifiers
     source          1..6569
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    135..159
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     promoter        345..690
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     633..653
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     polyA_signal    2287..2461
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     misc_feature    2506..2530
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(2686..3834)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     rep_origin      complement(4170..4758)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4250..4269)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4929..5720,5721..5789))
                     /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     5552..5571
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5790..5894)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(5981..6569,1..43)
                     /label=oriV
                     /note="incP origin of replication"
ORIGIN
        1 ggccgccagc ccagcggcga gggcaaccag cccggtgagc gtcgcaaagg agatcctgat
       61 ctgactgatg ggctgcctgt atcgagtggt gattttgtgc cgagctgccg gtcggggagc
      121 tgttggctgg ctggtggcag gatatattgt ggtgtaaaca aattgacgct tagacaactt
      181 aataacacat tgcggacgtt tttaatgtac tggggtggat gcagtgggcc ccactctgtg
      241 aagacaagca agaattcaag cttggaggtc aacatggtgg agcacgacac tctggtctac
      301 tccaaaaatg tcaaagatac agtctcagaa gatcaaaggg ctattgagac ttttcaacaa
      361 aggataattt cgggaaacct cctcggattc cattgcccag ctatctgtca cttcatcgaa
      421 aggacagtag aaaaggaagg tggctcctac aaatgccatc attgcgataa aggaaaggct
      481 atcattcaag atctctctgc cgacagtggt cccaaagatg gacccccacc cacgaggagc
      541 atcgtggaaa aagaagaggt tccaaccacg tctacaaagc aagtggattg atgtgacatc
      601 tccactgacg taagggatga cgcacaatcc cactatcctt cgcaagaccc ttcctctata
      661 taaggaagtt catttcattt ggagaggaca cgctcgagta taagagctca tttttacaac
      721 aattaccaac aacaacaaac aacaaacaac attacaatta catttacaat tatcgataca
      781 atggaaatct tctatgcttc tattctctct ctctttgttc ttgttgtttc tttttctctt
      841 cattttttct tctacaattc aaaacaaccc gaagatcaga aactacttcc accgggtaga
      901 acgggctggc ccataatcgg ggaaagtctc gagtttctcg gaagcggttg gtacggtcat
      961 cccgaaaagt tcatacttga ccgaatgtct aagttttcgc ctatagtatt taggtcatct
     1021 ctcttgctag aggatgctgc agtgatgtgt ggtgctcaag gtaacaaatt cttgttctca
     1081 aatgagaaca aactagtcca agtatggtgg cccgcttcgg tcgacaaaat ctttccatcg
     1141 gctaacaaaa cctacaaaac cgaaacgatt aaaatgcgaa aaatgctccc aaatttcctc
     1201 aaacccgaag ccttgcaacg atacattcca atcatggata ttaccacaca aaaacacttt
     1261 gccgcgggat gggaagggaa gaaagaagtt gtgacttatg aacttactag aaactttgct
     1321 ttttggcttg cgtgcaagat ctttttagat atcgatgatc ctgaacgggt cagatattta
     1381 tctggcccgt ttgagagcat cgttacgggg atcctgtcaa tccccattga tcttcctggg
     1441 acgccgttta gaagaggaat caatgggtcc aacttcataa taaaggaact tgtggctatt
     1501 gtgaagcaaa ggaaaattga tttggctcaa gggaaagcaa caccaaccca agatatattg
     1561 tcacatatga ttctatttag cgacgaagat ggcgagttca tgaccgaaca cgacatttcg
     1621 ggcaagattc ttgtgttgtt gatcggtggg catgacactg cgagctccgc tacggctttt
     1681 attgtgaagt atcttgccga attgcctgaa atctacgatg gagtctacaa tgaacaaatg
     1741 gaaattgcaa aatctaaagc agctggagaa ttattgaatt gggaggatct atcaaagatg
     1801 aaatactcat ggaaagttgc atgtgaagtt cttagattag tcccaccggc ccaaggtgct
     1861 tttagagaag cccttacaga tttcatgtac aatggttact ctatccccaa gggctggaag
     1921 ttatattgga gtgcaagctc aacccataaa aaccccgagg tgttcccgga acccgaaaaa
     1981 tttgacccgt caagatttga tggcagcggg ccagcacctt acacgttcgt gccatttgga
     2041 ggaggcccgc acatgtgtcc cggaaaagag tacgcccgat tagaaatact cgtgtttatg
     2101 catcatctcg tgacaaaatt caaatgggag aaaatgattc ccgatgagcc cgttgtgttc
     2161 aacccgatgc ctagccctgc caagggactt cctattcgta tttaccctca taaagctggt
     2221 tcgtaagtcg accggctgct aacaaagccc gagcttctct agctagagtc gatcgacaag
     2281 ctcgagtttc tccataataa tgtgtgagta gttcccagat aagggaatta gggttcctat
     2341 agggtttcgc tcatgtgttg agcatataag aaacccttag tatgtatttg tatttgtaaa
     2401 atacttctat caataaaatt tctaattcct aaaaccaaaa tccagtacta aaatccagat
     2461 cgctactatt gtcttctgca cgaagtggtt taaactatca gtgtttgaca ggatatattg
     2521 gcgggtaaac ctaagagaaa agagcgttta ttagaataat cggatattta aaagggcgtg
     2581 aaaaggttta tccgttcgtc catttgtatg tgcatgccaa ccacagggtt ccccagatca
     2641 ggcgctggct gctgaacccc cagccggaac tgaccccaca aggccctagc gtttgcaatg
     2701 caccaggtca tcattgaccc aggcgtgttc caccaggccg ctgcctcgca actcttcgca
     2761 ggcttcgccg acctgctcgc gccacttctt cacgcgggtg gaatccgatc cgcacatgag
     2821 gcggaaggtt tccagcttga gcgggtacgg ctcccggtgc gagctgaaat agtcgaacat
     2881 ccgtcgggcc gtcggcgaca gcttgcggta cttctcccat atgaatttcg tgtagtggtc
     2941 gccagcaaac agcacgacga tttcctcgtc gatcaggacc tggcaacggg acgttttctt
     3001 gccacggtcc aggacgcgga agcggtgcag cagcgacacc gattccaggt gcccaacgcg
     3061 gtcggacgtg aagcccatcg ccgtcgcctg taggcgcgac aggcattcct cggccttcgt
     3121 gtaataccgg ccattgatcg accagcccag gtcctggcaa agctcgtaga acgtgaaggt
     3181 gatcggctcg ccgatagggg tgcgcttcgc gtactccaac acctgctgcc acaccagttc
     3241 gtcatcgtcg gcccgcagct cgacgccggt gtaggtgatc ttcacgtcct tgttgacgtg
     3301 gaaaatgacc ttgttttgca gcgcctcgcg cgggattttc ttgttgcgcg tggtgaacag
     3361 ggcagagcgg gccgtgtcgt ttggcatcgc tcgcatcgtg tccggccacg gcgcaatatc
     3421 gaacaaggaa agctgcattt ccttgatctg ctgcttcgtg tgtttcagca acgcggcctg
     3481 cttggcctcg ctgacctgtt ttgccaggtc ctcgccggcg gtttttcgct tcttggtcgt
     3541 catagttcct cgcgtgtcga tggtcatcga cttcgccaaa cctgccgcct cctgttcaag
     3601 acgacgcgaa cgctccacgg cggccgatgg cgcgggcagg gcagggggag ccagttgcac
     3661 gctgtcgcgc tcgatcttgg ccgtagcttg ctggaccatc gagccgacgg actggaaggt
     3721 ttcgcggggc gcacgcatga cggtgcggct tgcgatggtt tcggcatcct cggcggaaaa
     3781 ccccgcgtcg atcagttctt gcctgtatgc cttccggtca aacgtccgat tcattcaccc
     3841 tccttgcggg attgccccga ctcacgccgg ggcaatgtgc ccttattcct gatttgaccc
     3901 gcctggtgcc ttggtgtcca gataatccac cttatcggca atgaagtcgg tcccgtagac
     3961 cgtctggccg tccttctcgt acttggtatt ccgaatcttg ccctgcacga ataccagcga
     4021 ccccttgccc aaatacttgc cgtgggcctc ggcctgagag ccaaaacact tgatgcggaa
     4081 gaagtcggtg cgctcctgct tgtcgccggc atcgttgcgc cacatctagg atctgccagg
     4141 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4201 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4261 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4321 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4381 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4441 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4501 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4561 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     4621 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4681 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4741 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4801 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4861 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     4921 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     4981 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     5041 tctggcccca gtgctgcaat gataccgcga gaaccacgct caccggctcc agatttatca
     5101 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     5161 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     5221 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     5281 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     5341 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     5401 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     5461 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     5521 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     5581 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     5641 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     5701 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     5761 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     5821 tatcaaggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     5881 ataggggttc cgcgcacgaa ttggccagcg ctgccatttt tggggtgagg ccgttcgcgg
     5941 ccgaggggcg cagcccctgg ggggatggga ggcccgcgtt agcgggccgg gagggttcga
     6001 gaaggggggg cacccccctt cggcgtgcgc ggtcacgcgc acagggcgca gccctggtta
     6061 aaaacaaggt ttataaatat tggtttaaaa gcaggttaaa agacaggtta gcggtggccg
     6121 aaaaacgggc ggaaaccctt gcaaatgctg gattttctgc ctgtggacag cccctcaaat
     6181 gtcaataggt gcgcccctca tctgtcagca ctctgcccct caagtgtcaa ggatcgcgcc
     6241 cctcatctgt cagtagtcgc gcccctcaag tgtcaatacc gcagggcact tatccccagg
     6301 cttgtccaca tcatctgtgg gaaactcgcg taaaatcagg cgttttcgcc gatttgcgag
     6361 gctggccagc tccacgtcgc cggccgaaat cgagcctgcc cctcatctgt caacgccgcg
     6421 ccgggtgagt cggcccctca agtgtcaacg tccgcccctc atctgtcagt gagggccaag
     6481 ttttccgcga ggtatccaca acgccggcgg ccgcggtgtc tcgcacacgg cttcgacggc
     6541 gtttctggcg cgtttgcagg gccatagac
//
