LOCUS       sequence-455514-        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: pEPDG1CB0010
FEATURES             Location/Qualifiers
     source          1..6569
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    42..66
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(222..1370)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMTNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     rep_origin      complement(1706..2294)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1786..1805)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2465..3256,3257..3325))
                     /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     3088..3107
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3326..3430)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3517..4148
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    4240..4264
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     promoter        4450..4795
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     4738..4758
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     polyA_signal    6392..6566
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
ORIGIN
        1 actattgtct tctgcacgaa gtggtttaaa ctatcagtgt ttgacaggat atattggcgg
       61 gtaaacctaa gagaaaagag cgtttattag aataatcgga tatttaaaag ggcgtgaaaa
      121 ggtttatccg ttcgtccatt tgtatgtgca tgccaaccac agggttcccc agatcaggcg
      181 ctggctgctg aacccccagc cggaactgac cccacaaggc cctagcgttt gcaatgcacc
      241 aggtcatcat tgacccaggc gtgttccacc aggccgctgc ctcgcaactc ttcgcaggct
      301 tcgccgacct gctcgcgcca cttcttcacg cgggtggaat ccgatccgca catgaggcgg
      361 aaggtttcca gcttgagcgg gtacggctcc cggtgcgagc tgaaatagtc gaacatccgt
      421 cgggccgtcg gcgacagctt gcggtacttc tcccatatga atttcgtgta gtggtcgcca
      481 gcaaacagca cgacgatttc ctcgtcgatc aggacctggc aacgggacgt tttcttgcca
      541 cggtccagga cgcggaagcg gtgcagcagc gacaccgatt ccaggtgccc aacgcggtcg
      601 gacgtgaagc ccatcgccgt cgcctgtagg cgcgacaggc attcctcggc cttcgtgtaa
      661 taccggccat tgatcgacca gcccaggtcc tggcaaagct cgtagaacgt gaaggtgatc
      721 ggctcgccga taggggtgcg cttcgcgtac tccaacacct gctgccacac cagttcgtca
      781 tcgtcggccc gcagctcgac gccggtgtag gtgatcttca cgtccttgtt gacgtggaaa
      841 atgaccttgt tttgcagcgc ctcgcgcggg attttcttgt tgcgcgtggt gaacagggca
      901 gagcgggccg tgtcgtttgt catcgctcgc atcgtgtccg gccacggcgc aatatcgaac
      961 aaggaaagct gcatttcctt gatctgctgc ttcgtgtgtt tcagcaacgc ggcctgcttg
     1021 gcctcgctga cctgttttgc caggtcctcg ccggcggttt ttcgcttctt ggtcgtcata
     1081 gttcctcgcg tgtcgatggt catcgacttc gccaaacctg ccgcctcctg ttcaagacga
     1141 cgcgaacgct ccacggcggc cgatggcgcg ggcagggcag ggggagccag ttgcacgctg
     1201 tcgcgctcga tcttggccgt agcttgctgg accatcgagc cgacggactg gaaggtttcg
     1261 cggggcgcac gcatgacggt gcggcttgcg atggtttcgg catcctcggc ggaaaacccc
     1321 gcgtcgatca gttcttgcct gtatgccttc cggtcaaacg tccgattcat tcaccctcct
     1381 tgcgggattg ccccgactca cgccggggca atgtgccctt attcctgatt tgacccgcct
     1441 ggtgccttgg tgtccagata atccacctta tcggcaatga agtcggtccc gtagaccgtc
     1501 tggccgtcct tctcgtactt ggtattccga atcttgccct gcacgaatac cagcgacccc
     1561 ttgcccaaat acttgccgtg ggcctcggcc tgagagccaa aacacttgat gcggaagaag
     1621 tcggtgcgct cctgcttgtc gccggcatcg ttgcgccaca tctaggatct gccaggaacc
     1681 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     1741 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     1801 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     1861 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     1921 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     1981 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     2041 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     2101 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     2161 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     2221 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     2281 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     2341 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     2401 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     2461 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     2521 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     2581 gccccagtgc tgcaatgata ccgcgagaac cacgctcacc ggctccagat ttatcagcaa
     2641 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     2701 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     2761 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     2821 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     2881 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     2941 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     3001 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     3061 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     3121 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     3181 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     3241 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     3301 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     3361 aaggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     3421 gggttccgcg cacgaattgg ccagcgctgc catttttggg gtgaggccgt tcgcggccga
     3481 ggggcgcagc ccctgggggg atgggaggcc cgcgttagcg ggccgggagg gttcgagaag
     3541 ggggggcacc ccccttcggc gtgcgcggtc acgcgcacag ggcgcagccc tggttaaaaa
     3601 caaggtttat aaatattggt ttaaaagcag gttaaaagac aggttagcgg tggccgaaaa
     3661 acgggcggaa acccttgcaa atgctggatt ttctgcctgt ggacagcccc tcaaatgtca
     3721 ataggtgcgc ccctcatctg tcagcactct gcccctcaag tgtcaaggat cgcgcccctc
     3781 atctgtcagt agtcgcgccc ctcaagtgtc aataccgcag ggcacttatc cccaggcttg
     3841 tccacatcat ctgtgggaaa ctcgcgtaaa atcaggcgtt ttcgccgatt tgcgaggctg
     3901 gccagctcca cgtcgccggc cgaaatcgag cctgcccctc atctgtcaac gccgcgccgg
     3961 gtgagtcggc ccctcaagtg tcaacgtccg cccctcatct gtcagtgagg gccaagtttt
     4021 ccgcgaggta tccacaacgc cggcggccgc ggtgtctcgc acacggcttc gacggcgttt
     4081 ctggcgcgtt tgcagggcca tagacggccg ccagcccagc ggcgagggca accagcccgg
     4141 tgagcgtcgc aaaggagatc ctgatctgac tgatgggctg cctgtatcga gtggtgattt
     4201 tgtgccgagc tgccggtcgg ggagctgttg gctggctggt ggcaggatat attgtggtgt
     4261 aaacaaattg acgcttagac aacttaataa cacattgcgg acgtttttaa tgtactgggg
     4321 tggatgcagt gggccccact ctgtgaagac aagcaagaat tcaagcttgg aggtcaacat
     4381 ggtggagcac gacactctgg tctactccaa aaatgtcaaa gatacagtct cagaagatca
     4441 aagggctatt gagacttttc aacaaaggat aatttcggga aacctcctcg gattccattg
     4501 cccagctatc tgtcacttca tcgaaaggac agtagaaaag gaaggtggct cctacaaatg
     4561 ccatcattgc gataaaggaa aggctatcat tcaagatctc tctgccgaca gtggtcccaa
     4621 agatggaccc ccacccacga ggagcatcgt ggaaaaagaa gaggttccaa ccacgtctac
     4681 aaagcaagtg gattgatgtg acatctccac tgacgtaagg gatgacgcac aatcccacta
     4741 tccttcgcaa gacccttcct ctatataagg aagttcattt catttggaga ggacacgctc
     4801 gagtataaga gctcattttt acaacaatta ccaacaacaa caaacaacaa acaacattac
     4861 aattacattt acaattatcg atacaatgga aatcttctat gcttctattc tctctctctt
     4921 tgttcttgtt gtttcttttt ctcttcattt tttcttctac aattcaaaac aacccgaaga
     4981 tcagaaacta cttccaccgg gtagaacggg ctggcccata atcggggaaa gtctcgagtt
     5041 tctcggaagc ggttggtacg gtcatcccga aaagttcata cttgaccgaa tgtctaagtt
     5101 ttcgcctata gtatttaggt catctctctt gctagaggat gctgcagtga tgtgtggtgc
     5161 tcaaggtaac aaattcttgt tctcaaatga gaacaaacta gtccaagtat ggtggcccgc
     5221 ttcggtcgac aaaatctttc catcggctaa caaaacctac aaaaccgaaa cgattaaaat
     5281 gcgaaaaatg ctcccaaatt tcctcaaacc cgaagccttg caacgataca ttccaatcat
     5341 ggatattacc acacaaaaac actttgccgc gggatgggaa gggaagaaag aagttgtgac
     5401 ttatgaactt actagaaact ttgctttttg gcttgcgtgc aagatctttt tagatatcga
     5461 tgatcctgaa cgggtcagat atttatctgg cccgtttgag agcatcgtta cggggatcct
     5521 gtcaatcccc attgatcttc ctgggacgcc gtttagaaga ggaatcaatg ggtccaactt
     5581 cataataaag gaacttgtgg ctattgtgaa gcaaaggaaa attgatttgg ctcaagggaa
     5641 agcaacacca acccaagata tattgtcaca tatgattcta tttagcgacg aagatggcga
     5701 gttcatgacc gaacacgaca tttcgggcaa gattcttgcg ttgttgatcg gtgggcatga
     5761 cactgcgagc tccgctacgg cttttattgt gaagtatctt gccgaattgc ctgaaatcta
     5821 cgatggagtc tacaatgaac aaatggaaat tgcaaaatct aaagcagctg gagaattatt
     5881 gaattgggag gatctatcaa agatgaaata ctcatggaaa gttgcatgtg aagttcttag
     5941 attagtccca ccggcccaag gtgcttttag agaagccctt acagatttca tgtacaatgg
     6001 ttactctatc cccaagggct ggaagttata ttggagtgca agctcaaccc ataaaaaccc
     6061 cgaggtgttc ccggaacccg aaaaatttga cccgtcaaga tttgatggca gcgggccagc
     6121 accttacacg ttcgtgccat ttggaggagg cccgagaatg tgtcccggaa aagagtacgc
     6181 ccgattagaa atactcgtgt ttatgcatca tctcgtgaca aaattcaaat gggagaaaat
     6241 gattcccgat gagcccgttg tgttcaaccc gatgcctagc cctgccaagg gacttcctat
     6301 tcgtatttac cctcataaag ctggttcgta agtcgaccgg ctgctaacaa agcccgagct
     6361 tctctagcta gagtcgatcg acaagctcga gtttctccat aataatgtgt gagtagttcc
     6421 cagataaggg aattagggtt cctatagggt ttcgctcatg tgttgagcat ataagaaacc
     6481 cttagtatgt atttgtattt gtaaaatact tctatcaata aaatttctaa ttcctaaaac
     6541 caaaatccag tactaaaatc cagatcgct
//
