LOCUS       sequence-455231-        6200 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 1 - Position 3 with 35S promoter and terminator for plant 
            expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6200)
  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 6200)
  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: pEPCT1CB0066
FEATURES             Location/Qualifiers
     source          1..6200
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    38..62
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(218..1366)
                     /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(1702..2290)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1782..1801)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2461..3252,3253..3321))
                     /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     3084..3103
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3322..3426)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3513..4144
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    4236..4260
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     promoter        4446..4791
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     4734..4754
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     polyA_signal    6019..6193
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
ORIGIN
        1 ttgtcttctg cacgaagtgg tttaaactat cagtgtttga caggatatat tggcgggtaa
       61 acctaagaga aaagagcgtt tattagaata atcggatatt taaaagggcg tgaaaaggtt
      121 tatccgttcg tccatttgta tgtgcatgcc aaccacaggg ttccccagat caggcgctgg
      181 ctgctgaacc cccagccgga actgacccca caaggcccta gcgtttgcaa tgcaccaggt
      241 catcattgac ccaggcgtgt tccaccaggc cgctgcctcg caactcttcg caggcttcgc
      301 cgacctgctc gcgccacttc ttcacgcggg tggaatccga tccgcacatg aggcggaagg
      361 tttccagctt gagcgggtac ggctcccggt gcgagctgaa atagtcgaac atccgtcggg
      421 ccgtcggcga cagcttgcgg tacttctccc atatgaattt cgtgtagtgg tcgccagcaa
      481 acagcacgac gatttcctcg tcgatcagga cctggcaacg ggacgttttc ttgccacggt
      541 ccaggacgcg gaagcggtgc agcagcgaca ccgattccag gtgcccaacg cggtcggacg
      601 tgaagcccat cgccgtcgcc tgtaggcgcg acaggcattc ctcggccttc gtgtaatacc
      661 ggccattgat cgaccagccc aggtcctggc aaagctcgta gaacgtgaag gtgatcggct
      721 cgccgatagg ggtgcgcttc gcgtactcca acacctgctg ccacaccagt tcgtcatcgt
      781 cggcccgcag ctcgacgccg gtgtaggtga tcttcacgtc cttgttgacg tggaaaatga
      841 ccttgttttg cagcgcctcg cgcgggattt tcttgttgcg cgtggtgaac agggcagagc
      901 gggccgtgtc gtttggcatc gctcgcatcg tgtccggcca cggcgcaata tcgaacaagg
      961 aaagctgcat ttccttgatc tgctgcttcg tgtgtttcag caacgcggcc tgcttggcct
     1021 cgctgacctg ttttgccagg tcctcgccgg cggtttttcg cttcttggtc gtcatagttc
     1081 ctcgcgtgtc gatggtcatc gacttcgcca aacctgccgc ctcctgttca agacgacgcg
     1141 aacgctccac ggcggccgat ggcgcgggca gggcaggggg agccagttgc acgctgtcgc
     1201 gctcgatctt ggccgtagct tgctggacca tcgagccgac ggactggaag gtttcgcggg
     1261 gcgcacgcat gacggtgcgg cttgcgatgg tttcggcatc ctcggcggaa aaccccgcgt
     1321 cgatcagttc ttgcctgtat gccttccggt caaacgtccg attcattcac cctccttgcg
     1381 ggattgcccc gactcacgcc ggggcaatgt gcccttattc ctgatttgac ccgcctggtg
     1441 ccttggtgtc cagataatcc accttatcgg caatgaagtc ggtcccgtag accgtctggc
     1501 cgtccttctc gtacttggta ttccgaatct tgccctgcac gaataccagc gaccccttgc
     1561 ccaaatactt gccgtgggcc tcggcctgag agccaaaaca cttgatgcgg aagaagtcgg
     1621 tgcgctcctg cttgtcgccg gcatcgttgc gccacatcta ggatctgcca ggaaccgtaa
     1681 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     1741 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     1801 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     1861 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     1921 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     1981 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     2041 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     2101 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     2161 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     2221 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     2281 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     2341 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     2401 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     2461 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     2521 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     2581 cagtgctgca atgataccgc gagaaccacg ctcaccggct ccagatttat cagcaataaa
     2641 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     2701 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     2761 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     2821 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     2881 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     2941 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     3001 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     3061 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     3121 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     3181 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     3241 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     3301 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaagg
     3361 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     3421 tccgcgcacg aattggccag cgctgccatt tttggggtga ggccgttcgc ggccgagggg
     3481 cgcagcccct ggggggatgg gaggcccgcg ttagcgggcc gggagggttc gagaaggggg
     3541 ggcacccccc ttcggcgtgc gcggtcacgc gcacagggcg cagccctggt taaaaacaag
     3601 gtttataaat attggtttaa aagcaggtta aaagacaggt tagcggtggc cgaaaaacgg
     3661 gcggaaaccc ttgcaaatgc tggattttct gcctgtggac agcccctcaa atgtcaatag
     3721 gtgcgcccct catctgtcag cactctgccc ctcaagtgtc aaggatcgcg cccctcatct
     3781 gtcagtagtc gcgcccctca agtgtcaata ccgcagggca cttatcccca ggcttgtcca
     3841 catcatctgt gggaaactcg cgtaaaatca ggcgttttcg ccgatttgcg aggctggcca
     3901 gctccacgtc gccggccgaa atcgagcctg cccctcatct gtcaacgccg cgccgggtga
     3961 gtcggcccct caagtgtcaa cgtccgcccc tcatctgtca gtgagggcca agttttccgc
     4021 gaggtatcca caacgccggc ggccgcggtg tctcgcacac ggcttcgacg gcgtttctgg
     4081 cgcgtttgca gggccataga cggccgccag cccagcggcg agggcaacca gcccggtgag
     4141 cgtcgcaaag gagatcctga tctgactgat gggctgcctg tatcgagtgg tgattttgtg
     4201 ccgagctgcc ggtcggggag ctgttggctg gctggtggca ggatatattg tggtgtaaac
     4261 aaattgacgc ttagacaact taataacaca ttgcggacgt ttttaatgta ctggggtgga
     4321 tgcagtgggc cccactctgt gaagacaagc aagaattcaa gcttggaggt caacatggtg
     4381 gagcacgaca ctctggtcta ctccaaaaat gtcaaagata cagtctcaga agatcaaagg
     4441 gctattgaga cttttcaaca aaggataatt tcgggaaacc tcctcggatt ccattgccca
     4501 gctatctgtc acttcatcga aaggacagta gaaaaggaag gtggctccta caaatgccat
     4561 cattgcgata aaggaaaggc tatcattcaa gatctctctg ccgacagtgg tcccaaagat
     4621 ggacccccac ccacgaggag catcgtggaa aaagaagagg ttccaaccac gtctacaaag
     4681 caagtggatt gatgtgacat ctccactgac gtaagggatg acgcacaatc ccactatcct
     4741 tcgcaagacc cttcctctat ataaggaagt tcatttcatt tggagaggac acgctcgagt
     4801 ataagagctc atttttacaa caattaccaa caacaacaaa caacaaacaa cattacaatt
     4861 acatttacaa ttatcgatac aatggatgga gaggtgcaca acttcatagt ggtttggata
     4921 atagtgttgc taggacttct ctattcttac accataggca agttcatccc aaaaggtaca
     4981 attagacttt tggctttatc cattgttata tgcctctttc tatggtttcc actcaatcta
     5041 accaccatac accttggtgg tttaacctct ttctttatat catggcttgc aaacttcaaa
     5101 cttcttctct atgcttatgg aaaaggtccc ctttcttcgg atccatcgat ctcgttgtct
     5161 agctttatcc tcctggcgtg cctgccaatt cggtccgtcc atacttcatc ttcttcatac
     5221 cctgaaaagc cgactcaaaa aaccaagata aatgagaaaa tagttttatc gaaggtttta
     5281 ttgtttggga tgttacttaa agggtatgat tattatgaat atattcatcc gaagttgata
     5341 atggtgttgt atagcatcca tatctacttc atgcttgagt tcgtgcttgc catgctcgca
     5401 tttgtggctc gaaccctagt gcaaatggat ttagagccac aatttgacga gccttatctt
     5461 gctacttccc tccaagactt ttggggcaaa aggtggaacc taatggtcac tagaattcta
     5521 catccgacta tatatgaacc tattcattca atttctacgc gttttatacc aagaaaatgg
     5581 gcatctctag ttgcggtttt tgcatccttt cttgtttcgg gtttgatgca cgagttcata
     5641 ttctataaca ttggtagact gaaaccgacc ggagaagtaa catgtttttt cttattacat
     5701 ggagtattga tgagcattga agttgttatc aagagagcaa caaagggaaa attacatttg
     5761 catcccgttg tgtcgaggcc aataactttg ggatgcgtgc tcattacatg tttttggttg
     5821 ttctttccgc cgtttttgcg gtttaaacca gacatccgtg gttgtcaaga aaccgttgca
     5881 tttctaaagt ttgtaacaaa cggtcaacta attagcccta gtaacgcttc ttgtccgtat
     5941 ctatcaagtg gttcgtaagt cgaccggctg ctaacaaagc ccgagcttct ctagctagag
     6001 tcgatcgaca agctcgagtt tctccataat aatgtgtgag tagttcccag ataagggaat
     6061 tagggttcct atagggtttc gctcatgtgt tgagcatata agaaaccctt agtatgtatt
     6121 tgtatttgta aaatacttct atcaataaaa tttctaattc ctaaaaccaa aatccagtac
     6181 taaaatccag atcgctacta
//
