LOCUS       sequence-447731-        9758 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BeYDV LIR:2nd intron + 3'eGFP +T35S:BeYDV SIR + p35S + 5'eGFP + 1st 
            intron:BeYDV LIR for INPACT configuration..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9758)
  AUTHORS   Garcia-Perez E, Vazquez-Vilar M, Lozano-Duran R, Orzaez D
  TITLE     CuBe: a geminivirus-based copper-regulated expression system 
            suitable for post-harvest activation.
  JOURNAL   Plant Biotechnol J. 2025 Jan;23(1):141-155. doi: 10.1111/pbi.14485. 
            Epub 2024 Oct 22.
  PUBMED    39435699
REFERENCE   2  (bases 1 to 9758)
  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"; doi: "10.1111/pbi.14485";
            journalName: "Plant Biotechnol J"; date: "2025-01"; volume: "23"; 
            issue: "1"; pages: "141-155"
COMMENT     Sequence Label: pDGB3_o1_LIR-2nd
            intron-3'eGFP-T35S-SIR-p35S-5...
FEATURES             Location/Qualifiers
     source          1..9758
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    18..42
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             1343..1972
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             2401..3474
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     rep_origin      3540..3734
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(3970..3992)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4078..4218
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4133..4152
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4233..4250)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4404..4992)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4484..4503)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5080..5871)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     misc_feature    6657..6681
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     primer_bind     7451..7472
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        8539..8883
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     8827..8847
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     primer_bind     complement(9002..9023)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(9263..9282)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
ORIGIN
        1 gtcatgagac cggatcctga caggatatat tggcgggtaa acctaagaga aaagagcgtt
       61 tattagaata atcggatatt taaaagggcg tgaaaaggtt tatccgttcg tccatttgta
      121 tgtgcatgcc aaccacaggg ttcccctcgg gatcaaagta ctttgatcca acccctccgc
      181 tgctatagtg cagtcggctt ctgacgttca gtgcagccgt catctgaaaa cgacatgtcg
      241 cacaagtcct aagttacgcg acaggctgcc gccctgccct tttcctggcg ttttcttgtc
      301 gcgtgtttta gtcgcataaa gtagaatact tgcgactaga accggagaca ttacgccatg
      361 aacaagagcg ccgccgctgg cctgctgggc tatgcccgcg tcagcaccga cgaccaggac
      421 ttgaccaacc aacgggccga actgcacgcg gccggctgca ccaagctgtt ttccgagaag
      481 atcaccggca ccaggcgcga ccgcccggag ctggccagga tgcttgacca cctacgccct
      541 ggcgacgttg tgacagtgac caggctagac cgcctggccc gcagcacccg cgacctactg
      601 gacattgccg agcgcatcca ggaggccggc gcgggcctgc gtagcctggc agagccgtgg
      661 gccgacacca ccacgccggc cggccgcatg gtgttgaccg tgttcgccgg cattgccgag
      721 ttcgagcgtt ccctaatcat cgaccgcacc cggagcgggc gcgaggccgc caaggcccga
      781 ggcgtgaagt ttggcccccg ccctaccctc accccggcac agatcgcgca cgcccgcgag
      841 ctgatcgacc aggaaggccg caccgtgaaa gaggcggctg cactgcttgg cgtgcatcgc
      901 tcgaccctgt accgcgcact tgagcgcagc gaggaagtga cgcccaccga ggccaggcgg
      961 cgcggtgcct tccgtgagga cgcattgacc gaggccgacg ccctggcggc cgccgagaat
     1021 gaacgccaag aggaacaagc atgaaaccgc accaggacgg ccaggacgaa ccgtttttca
     1081 ttaccgaaga gatcgaggcg gagatgatcg cggccgggta cgtgttcgag ccgcccgcgc
     1141 acctctcaac cgtgcggctg catgaaatcc tggccggttt gtctgatgcc aagctggcgg
     1201 cctggccggc cagcttggcc gctgaagaaa ccgagcgccg ccgtctaaaa aggtgatgtg
     1261 tatttgagta aaacagcttg cgtcatgcgg tcgctgcgta tatgatgcga tgagtaaata
     1321 aacaaatacg caaggggaac gcatgaaggt tatcgctgta cttaaccaga aaggcgggtc
     1381 aggcaagacg accatcgcaa cccatctagc ccgcgccctg caactcgccg gggccgatgt
     1441 tctgttagtc gattccgatc cccagggcag tgcccgcgat tgggcggccg tgcgggaaga
     1501 tcaaccgcta accgttgtcg gcatcgaccg cccgacgatt gaccgcgacg tgaaggccat
     1561 cggccggcgc gacttcgtag tgatcgacgg agcgccccag gcggcggact tggctgtgtc
     1621 cgcgatcaag gcagccgact tcgtgctgat tccggtgcag ccaagccctt acgacatatg
     1681 ggccaccgcc gacctggtgg agctggttaa gcagcgcatt gaggtcacgg atggaaggct
     1741 acaagcggcc tttgtcgtgt cgcgggcgat caaaggcacg cgcatcggcg gtgaggttgc
     1801 cgaggcgctg gccgggtacg agctgcccat tcttgagtcc cgtatcacgc agcgcgtgag
     1861 ctacccaggc actgccgccg ccggcacaac cgttcttgaa tcagaacccg agggcgacgc
     1921 tgcccgcgag gtccaggcgc tggccgctga aattaaatca aaactcattt gagttaatga
     1981 ggtaaagaga aaatgagcaa aagcacaaac acgctaagtg ccggccgtcc gagcgcacgc
     2041 agcagcaagg ctgcaacgtt ggccagcctg gcagacacgc cagccatgaa gcgggtcaac
     2101 tttcagttgc cggcggagga tcacaccaag ctgaagatgt acgcggtacg ccaaggcaag
     2161 accattaccg agctgctatc tgaatacatc gcgcagctac cagagtaaat gagcaaatga
     2221 ataaatgagt agatgaattt tagcggctaa aggaggcggc atggaaaatc aagaacaacc
     2281 aggcaccgac gccgtggaat gccccatgtg tggaggaacg ggcggttggc caggcgtaag
     2341 cggctgggtt gcctgccggc cctgcaatgg cactggaacc cccaagcccg aggaatcggc
     2401 gtgagcggtc gcaaaccatc cggcccggta caaatcggcg cggcgctggg tgatgacctg
     2461 gtggagaagt tgaaggccgc gcaggccgcc cagcggcaac gcatcgaggc agaagcacgc
     2521 cccggtgaat cgtggcaagc ggccgctgat cgaatccgca aagaatcccg gcaaccgccg
     2581 gcagccggtg cgccgtcgat taggaagccg cccaagggcg acgagcaacc agattttttc
     2641 gttccgatgc tctatgacgt gggcacccgc gatagtcgca gcatcatgga cgtggccgtt
     2701 ttccgtctgt cgaagcgtga ccgacgagct ggcgaggtga tccgctacga gcttccagac
     2761 gggcacgtag aggtttccgc agggccggcc ggcatggcca gtgtgtggga ttacgacctg
     2821 gtactgatgg cggtttccca tctaaccgaa tccatgaacc gataccggga agggaaggga
     2881 gacaagcccg gccgcgtgtt ccgtccacac gttgcggacg tactcaagtt ctgccggcga
     2941 gccgatggcg gaaagcagaa agacgacctg gtagaaacct gcattcggtt aaacaccacg
     3001 cacgttgcca tgcagcgtac gaagaaggcc aagaacggcc gcctggtgac ggtatccgag
     3061 ggtgaagcct tgattagccg ctacaagatc gtaaagagcg aaaccgggcg gccggagtac
     3121 atcgagatcg agctagctga ttggatgtac cgcgagatca cagaaggcaa gaacccggac
     3181 gtgctgacgg ttcaccccga ttactttttg atcgatcccg gcatcggccg ttttctctac
     3241 cgcctggcac gccgcgccgc aggcaaggca gaagccagat ggttgttcaa gacgatctac
     3301 gaacgcagtg gcagcgccgg agagttcaag aagttctgtt tcaccgtgcg caagctgatc
     3361 gggtcaaatg acctgccgga gtacgatttg aaggaggagg cggggcaggc tggcccgatc
     3421 ctagtcatgc gctaccgcaa cctgatcgag ggcgaagcat ccgccggttc ctaatgtacg
     3481 gagcagatgc tagggcaaat tgccctagca ggggaaaaag gtcgaaaagg actctttcct
     3541 gtggatagca cgtacattgg gaacccaaag ccgtacattg ggaaccggaa cccgtacatt
     3601 gggaacccaa agccgtacat tgggaaccgg tcacacatgt aagtgactga tataaaagag
     3661 aaaaaaggcg atttttccgc ctaaaactct ttaaaactta ttaaaactct taaaacccgc
     3721 ctggcctgtg cataactgtc tggccagcgc acagccgaag agctgcaaaa agcgcctacc
     3781 cttcggtcgc tgcgctccct acgccccgcc gcttcgcgtc ggcctatcgc ggccgctggc
     3841 cgctcaaaaa tggctggcct acggccaggc aatctaccag ggcgcggaca agccgcgccg
     3901 tcgccactcg accgccggcg cccacatcaa ggcaccctgc ctcgcgcgtt tcggtgatga
     3961 cggtgaaaac ctctgacaca tgcagctccc ggtgacggtc acagcttgtc tgtaagcgga
     4021 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc
     4081 agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca
     4141 gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg
     4201 agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4261 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4321 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4381 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4441 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4501 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4561 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     4621 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4681 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4741 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     4801 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     4861 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4921 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     4981 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5041 aactcacgtt aagggatttt ggtcatgcat tctaggtgat tatttgccga ctaccttggt
     5101 gatctcgcct ttcacgtagt ggacaaattc ttccaactga tctgcgcgcg aggccaagcg
     5161 atcttcttct tgtccaagat aagcctgtct agcttcaagt atgacgggct gatactgggc
     5221 cggcaggcgc tccattgccc agtcggcagc gacatccttc ggcgcgattt tgccggttac
     5281 tgcgctgtac caaatgcggg acaacgtaag cactacattt cgctcatcac cagcccagtc
     5341 gggcggcgag ttccatagcg ttaaggtttc atttagcgcc tcaaatagat cctgttcagg
     5401 aaccggatca aagagttcct ccgccgctgg acctaccaag gcaacgctat gttctcttgc
     5461 ttttgtcagc aagatagcca gatcaatgtc gatcgtggct ggctcgaaga tacctgcaag
     5521 aatgtcattg cgctgccatt ctccaaattg cagttcgcgc ttagctggat aacgccacgg
     5581 aatgatgtcg tcgtgcacaa caatggtgac ttctacagcg cggagaatct cgctctctcc
     5641 aggggaagcc gaagtttcca aaaggtcgtt gatcaaagct cgccgcgttg tttcatcaag
     5701 ccttacggtc accgtaacca gcaaatcaat atcactgtgt ggcttcaggc cgccatccac
     5761 tgcggagccg tacaaatgta cggccagcaa cgtcggttcg agatggcgct cgatgacgcc
     5821 aactacctct gatagttgag tcgatacttc ggcgatcacc gcttccctca taatgtttaa
     5881 ctttgtttta gggcgactgc cctgctgcgt aacatcgttg ctgctccata acatcaaaca
     5941 tcgacccacg gcgtaacgcg cttgctgctt ggatgcccga ggcatagact gtaccccaaa
     6001 aaaacagtca taacaagcca tgaaaaccgc cactgcgccg ttaccaccgc tgcgttcggt
     6061 caaggttctg gaccagttgc gtgagcgcat acgctacttg cattacagct tacgaaccga
     6121 acaggcttat gtccactggg ttcgtgcctt catccgtttc cacggtgtgc gtcacccggc
     6181 aaccttgggt agcagcgaag tcgaggcatt tctgtcctgg ctggaacaga acttattatt
     6241 tccttcctct tttctacagt atttaaagat accccaagaa gctaattata acaagacgaa
     6301 ctccaattca ctgttccttg cattctaaaa ccttaaatac cagaaaacag ctttttcaaa
     6361 gttgttttca aagttggcgt ataacatagt atcgacggag ccgattttga aaccgcggtg
     6421 atcacaggca gcaacgctct gtcatcgtta caatcaacat gctaccctcc gcgagatcat
     6481 ccgtgtttca aacccggcag cttagttgcc gttcttccga atagcatcgg taacatgagc
     6541 aaagtctgcc gccttacaac ggctctcccg ctgacgccgt cccggactga tgggctgcct
     6601 gtatcgagtg gtgattttgt gccgagctgc cggtcgggga gctgttggct ggctggtggc
     6661 aggatatatt gtggtgtaaa cataacggat ccggtctcag gagtagcaga aggcatgttg
     6721 ttgtgactcc gaggggttgc ctcaaactct atcttataac cggcgtggag gcatggaggc
     6781 aggggtattt tggtcatttt aatagatagt ggaaaatgac gtggaattta cttaaagacg
     6841 aagtctttgc gacaaggggg ggcccacgcc gaatttaata ttaccggcgt ggccccccct
     6901 tatcgcgagt gctttagcac gagcggtcca gatttaaagt agaaaatttc ccgcccacta
     6961 gggttaaagg tgttcacact ataaaagcat atacgatgtg atggtatttg atggagcgta
     7021 tattgtatca ggtatttccg ttggatacga attattcgta cgaccctcct aattaacctg
     7081 actcaaaatc taccctcata attgttgttt gatattggtc ttgtattttg caggtgaagt
     7141 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     7201 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     7261 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     7321 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     7381 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     7441 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     7501 acgagctgta caaggacgag ctgtaagctt cggccatgct agagtccgca aaaatcacca
     7561 gtctctctct acaaatctat ctctctctat ttttctccag aataatgtgt gagtagttcc
     7621 cagataaggg aattagggtt cttatagggt ttcgctcatg tgttgagcat ataagaaacc
     7681 cttagtatgt atttgtattt gtaaaatact tctatcaata aaatttctaa ttcctaaaac
     7741 caaaatccag tgacctataa aatgattatt ttatgaatat atttcattgt gcaagtagat
     7801 agaaattaca tatgttacat aacacacgaa ataaacaaaa aaagacaatc caaaaacaaa
     7861 caccccaaaa aaaataatca ctttagataa actcgtatga ggagaggcac gttcacgctg
     7921 tcaggagact agagccaagc tgatctcctt tgccccggag atcaccatgg acgactttct
     7981 ctatctctac gatctaggaa gaaagttcga cggagaaggt gacgatacca tgttcaccac
     8041 cgataatgag aagattagcc tcttcaattt cagaaagaat gctgacccac agatggttag
     8101 agaggcctac gcggcaggtc tgatcaagac gatctacccg agtaataatc tccaggagat
     8161 caaatacctt cccaagaagg ttaaagatgc agtcaaaaga ttcaggacta actgcatcaa
     8221 gaacacagag aaagatatat ttctcaagat cagaagtact attccagtat ggacgattca
     8281 aggcttgctt cataaaccaa ggcaagtaat agagattgga gtctctaaga aagtagttcc
     8341 tactgaatca aaggccatgg agtcaaaaat tcagatcgag gatctaacag aactcgccgt
     8401 gaagactggc gaacagttca tacagagtct tttacgactc aatgacaaga agaaaatctt
     8461 cgtcaacatg gtggagcacg acactctcgt ctactccaag aatatcaaag atacagtctc
     8521 agaagaccaa agggctattg agacttttca acaaagggta atatcgggaa acctcctcgg
     8581 attccattgc ccagctatct gtcacttcat caaaaggaca gtagaaaagg aaggtggcac
     8641 ctacaaatgc catcattgcg ataaaggaaa ggctatcgtt caagatgccc ctgccgacag
     8701 tggtcccaaa gatggacccc cacccacgag gagcatcgtg gaaaaagaag acgttccaac
     8761 cacgtcttca aagcaagtgg attgatgtga tatctccact gacgtaaggg atgacgcaca
     8821 atcccactat ccttcgcaag acccttcctc tatataagga agttcatttc atttggagag
     8881 gactccggta tttttacaac aattaccaca acaaaacaaa caacaaacaa cattacaatt
     8941 tactattcta gtcgaaatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     9001 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     9061 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     9121 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     9181 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     9241 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtaagcca
     9301 tcttcctgct tatttttata atgaacatag aaataggaag ttgtgcagag aaacgctgtc
     9361 aggaggtgcc aagcttgcat gcctgcaggt agcagaaggc atgttgttgt gactccgagg
     9421 ggttgcctca aactctatct tataaccggc gtggaggcat ggaggcaggg gtattttggt
     9481 cattttaata gatagtggaa aatgacgtgg aatttactta aagacgaagt ctttgcgaca
     9541 agggggggcc cacgccgaat ttaatattac cggcgtggcc cccccttatc gcgagtgctt
     9601 tagcacgagc ggtccagatt taaagtagaa aatttcccgc ccactagggt taaaggtgtt
     9661 cacactataa aagcatatac gatgtgatgg tatttgatgg agcgtatatt gtatcaggta
     9721 tttccgttgg atacgaatta ttcgtacgac cctccgct
//
