LOCUS       sequence-240989-        5581 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Binary expression vector containing 35S:MS2-GFP:tRBCS followed by 
            GreenGate A-G sequences for insertion of additional modules.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5581)
  AUTHORS   Lee JE, Neumann M, Duro DI, Schmid M
  TITLE     CRISPR-based tools for targeted transcriptional and epigenetic 
            regulation in plants.
  JOURNAL   PLoS One. 2019 Sep 26;14(9):e0222778. doi: 
            10.1371/journal.pone.0222778. eCollection 2019.
  PUBMED    31557222
REFERENCE   2  (bases 1 to 5581)
  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: "PLoS One.";
            date: "2019-09-26"; volume: "14(9):e0222778. doi"; pages: " 
            10.1371/journal.pone.0222778. eCollection 2019"
COMMENT     Sequence Label: pJL003
FEATURES             Location/Qualifiers
     source          1..5581
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        130..475
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     418..438
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             526..912
                     /codon_start=1
                     /product="bacteriophage MS2 coat protein"
                     /label=MS2-N55K
                     /note="binds with high affinity to a specific stem-loop 
                     structure in the viral RNA (Lim et al., 1994)"
                     /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
                     CSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI
                     VKAMQGLLKDGNPIPSAIAANSGIY"
     CDS             967..987
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1108..1110,1111..1113,1114..1827)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(1153..1174)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1414..1433)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1761..1782
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     terminator      1834..2475
                     /label=E9 terminator
                     /note="terminator and polyadenylation signal from the pea 
                     rbcS-E9 gene"
     misc_feature    2581..2605
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(2696..3284)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2776..2795)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3455..4243)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      4534..4969
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     misc_feature    5096..5118
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA 
                     (truncated)"
ORIGIN
        1 cgaacagttc atacagagtc tcttacgact caatgacaag aagaaaatct tcgtcaacat
       61 ggtggagcac gacacacttg tctactccaa aaatatcaaa gatacagtct cagaagacca
      121 aagggcaatt gagacttttc aacaaagggt aatatccgga aacctcctcg gattccattg
      181 cccagctatc tgtcacttta ttgtgaagat agtggaaaag gaaggtggct cctacaaatg
      241 ccatcattgc gataaaggaa aggccatcgt tgaagatgcc tctgccgaca gtggtcccaa
      301 agatggaccc ccacccacga ggagcatcgt ggaaaaagaa gacgttccaa ccacgtcttc
      361 aaagcaagtg gattgatgtg atatctccac tgacgtaagg gatgacgcac aatcccacta
      421 tccttcgcaa gacccttcct ctatataagg aagttcattt catttggaga gaacacgggg
      481 gacgagctaa cagtattcag tcgactggta ccaacaggct ccatggcttc taattttact
      541 caatttgttt tggttgataa tggaggaact ggagatgtta ctgttgctcc ttctaatttt
      601 gctaatggag ttgctgaatg gatttcttct aattcaagat ctcaggctta taaggttact
      661 tgttctgtta gacaatcttc tgctcaaaag agaaagtata ctattaaggt tgaagttcct
      721 aaggttgcta ctcaaactgt tggaggagtt gaattgcctg ttgctgcttg gagatcttat
      781 ttgaatatgg aattgactat tcctattttt gctactaatt ctgattgtga attgattgtt
      841 aaggctatgc aaggattgtt gaaggatgga aatcctattc cttctgctat tgctgctaat
      901 tctggaattt attctgctgg aggaggagga tctggaggag gaggatctgg aggaggagga
      961 tctggaccta agaagaagag aaaggttgct gctgctggat cttcaggtgc cgctagtgcg
     1021 atcgcatcag ggagtggttc cggaagcggc tctggatcgg gctcagggag tggttccggc
     1081 agcggctctg gatcggcggc cgctgcaatg gtgagcaagg gcgaggagct gttcaccggg
     1141 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     1201 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
     1261 ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct tcacctacgg cgtgcagtgc
     1321 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     1381 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     1441 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     1501 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc
     1561 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
     1621 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
     1681 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac
     1741 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
     1801 cacggcatgg acgagctgta caagtagctg caaagctttc gttcgtatca tcggtttcga
     1861 caacgttcgt caagttcaat gcatcagttt cattgcgcac acaccagaat cctactgagt
     1921 ttgagtatta tggcattggg aaaactgttt ttcttgtacc atttgttgtg cttgtaattt
     1981 actgtgtttt ttattcggtt ttcgctatcg aactgtgaaa tggaaatgga tggagaagag
     2041 ttaatgaatg atatggtcct tttgttcatt ctcaaattaa tattatttgt tttttctctt
     2101 atttgttgtg tgttgaattt gaaattataa gagatatgca aacattttgt tttgagtaaa
     2161 aatgtgtcaa atcgtggcct ctaatgaccg aagttaatat gaggagtaaa acacttgtag
     2221 ttgtaccatt atgcttattc actaggcaac aaatatattt tcagacctag aaaagctgca
     2281 aatgttactg aatacaagta tgtcctcttg tgttttagac atttatgaac tttcctttat
     2341 gtaattttcc agaatccttg tcagattcta atcattgctt tataattata gttatactca
     2401 tggatttgta gttgagtatg aaaatatttt ttaatgcatt ttatgacttg ccaattgatt
     2461 gacaacatgc atcaatcgac tatgagtagg caagatgttc tgtaggacct tgagaccgaa
     2521 aaggtggtct cagtattcta gaccaagcat gcacagctgg cgttctcagc cagatcccct
     2581 tgacaggata tattggcggg taaactaagt cgctgtatgt gtttgtttga gatctcatgt
     2641 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     2701 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     2761 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     2821 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     2881 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     2941 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3001 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3061 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3121 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3181 gaagaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3241 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     3301 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     3361 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     3421 tctaaagtat atatgtgtaa cattggtcta gtgattattt gccgactacc ttggtgatct
     3481 cgcctttcac gtagtgaaca aattcttcca actgatctgc gcgcgaggcc aagcgatctt
     3541 cttgtccaag ataagcctgc ctagcttcaa gtatgacggg ctgatactgg gccggcaggc
     3601 gctccattgc ccagtcggca gcgacatcct tcggcgcgat tttgccggtt actgcgctgt
     3661 accaaatgcg ggacaacgta agcactacat ttcgctcatc gccagcccag tcgggcggcg
     3721 agttccatag cgttaaggtt tcatttagcg cctcaaatag atcctgttca ggaaccggat
     3781 caaagagttc ctccgccgct ggacctacca aggcaacgct atgttctctt gcttttgtca
     3841 gcaagatagc cagatcaatg tcgatcgtgg ctggctcgaa gatacctgca agaatgtcat
     3901 tgcgctgcca ttctccaaat tgcagttcgc gcttagctgg ataacgccac ggaatgatgt
     3961 cgtcgtgcac aacaatggtg acttctacag cgcggagaat ctcgctctct ccaggggaag
     4021 ccgaagtttc caaaaggtcg ttgatcaaag ctcgccgcgt tgtttcatca agccttacgg
     4081 tcaccgtaac cagcaaatca atatcactgt gtggcttcag gccgccatcc actgcggagc
     4141 cgtacaaatg tacggccagc aacgtcggtt cgagatggcg ctcgatgacg ccaactacct
     4201 ctgatagttg agtcgatact tcggcgatca ccgcttccct cataacaccc cttgtattac
     4261 tgtttatgta agcagacagt tttattgttc atgatgatat atttttatct tgtgcaatgt
     4321 aacatcagag attttgagac acaacgtggc tttgttgaat aaatcgaact tttgctgagt
     4381 tgaaggatca gatcacgcat cttcccgaca acgcagaccg ttccgtggca aagcaaaagt
     4441 tcaaaatcac caactggtcc acctacaaca aagctctcat caaccgtggc tccctcactt
     4501 tctggctgga tgatggggcg attcaggcga tccccatcca acagcccgcc gtcgagcggg
     4561 cttttttatc cccggaagcc tgtggataga gggtagttat ccacgtgaaa ccgctaatgc
     4621 cccgcaaagc cttgattcac ggggctttcc ggcccgctcc aaaaactatc cacgtgaaat
     4681 cgctaatcag ggtacgtgaa atcgctaatc ggagtacgtg aaatcgctaa taaggtcacg
     4741 tgaaatcgct aatcaaaaag gcacgtgaga acgctaatag ccctttcaga tcaacagctt
     4801 gcaaacaccc ctcgctccgg caagtagtta cagcaagtag tatgttcaat tagcttttca
     4861 attatgaata tatatatcaa ttattggtcg cccttggctt gtggacaatg cgctacgcgc
     4921 accggctccg cccgtggaca accgcaagcg gttgcccacc gtcgagcgcc tttgcccaca
     4981 acccggcggc cggccgcaac agatcgtttt ataaattttt ttttttgaaa aagaaaaagc
     5041 ccgaaaggcg gcaacctctc gggcttctgg atttccgatc cccggaatta gatcttggca
     5101 ggatatattg tggtgtaacg ttctcgaact aggctcggac gaagtaagcg gccgcaacct
     5161 taattaaaca cggcgcgcca acagcgatcg caccaggtac cacctccgat ccattcggtc
     5221 cccagattag ccttttcaat ttcagaaaga atgctaaccc acagatggtt agagaggctt
     5281 acgcagcacg tctcatcaag acgatctacc cgagcaataa tctccaggaa atcaaatacc
     5341 ttcccaagaa ggttaaagat gcagtcaaaa gattcaggac taactgcatc aagaacacag
     5401 agaaagatat atttctcaag atcagaagta ctattccagt atggacgatt caaggcttgc
     5461 ttcacaaacc aaggcaagta atagagattg gagtctctaa aaaggtagtt cccactgaat
     5521 caaaggccat ggagtcaaag attcaaatag aggacctaac agaactcgcc gtaaagactg
     5581 g
//
