LOCUS       sequence-234581-        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"
     misc_feature    534..556
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA 
                     (truncated)"
     promoter        1149..1494
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     1437..1457
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             1545..1931
                     /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             1986..2006
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2127..2129,2130..2132,2133..2846)
                     /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(2172..2193)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2433..2452)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2780..2801
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     terminator      2853..3494
                     /label=E9 terminator
                     /note="terminator and polyadenylation signal from the pea 
                     rbcS-E9 gene"
     misc_feature    3600..3624
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(3715..4303)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3795..3814)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4474..5262)
                     /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      join(5553..5581,1..407)
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
ORIGIN
        1 tttttatccc cggaagcctg tggatagagg gtagttatcc acgtgaaacc gctaatgccc
       61 cgcaaagcct tgattcacgg ggctttccgg cccgctccaa aaactatcca cgtgaaatcg
      121 ctaatcaggg tacgtgaaat cgctaatcgg agtacgtgaa atcgctaata aggtcacgtg
      181 aaatcgctaa tcaaaaaggc acgtgagaac gctaatagcc ctttcagatc aacagcttgc
      241 aaacacccct cgctccggca agtagttaca gcaagtagta tgttcaatta gcttttcaat
      301 tatgaatata tatatcaatt attggtcgcc cttggcttgt ggacaatgcg ctacgcgcac
      361 cggctccgcc cgtggacaac cgcaagcggt tgcccaccgt cgagcgcctt tgcccacaac
      421 ccggcggccg gccgcaacag atcgttttat aaattttttt ttttgaaaaa gaaaaagccc
      481 gaaaggcggc aacctctcgg gcttctggat ttccgatccc cggaattaga tcttggcagg
      541 atatattgtg gtgtaacgtt ctcgaactag gctcggacga agtaagcggc cgcaacctta
      601 attaaacacg gcgcgccaac agcgatcgca ccaggtacca cctccgatcc attcggtccc
      661 cagattagcc ttttcaattt cagaaagaat gctaacccac agatggttag agaggcttac
      721 gcagcacgtc tcatcaagac gatctacccg agcaataatc tccaggaaat caaatacctt
      781 cccaagaagg ttaaagatgc agtcaaaaga ttcaggacta actgcatcaa gaacacagag
      841 aaagatatat ttctcaagat cagaagtact attccagtat ggacgattca aggcttgctt
      901 cacaaaccaa ggcaagtaat agagattgga gtctctaaaa aggtagttcc cactgaatca
      961 aaggccatgg agtcaaagat tcaaatagag gacctaacag aactcgccgt aaagactggc
     1021 gaacagttca tacagagtct cttacgactc aatgacaaga agaaaatctt cgtcaacatg
     1081 gtggagcacg acacacttgt ctactccaaa aatatcaaag atacagtctc agaagaccaa
     1141 agggcaattg agacttttca acaaagggta atatccggaa acctcctcgg attccattgc
     1201 ccagctatct gtcactttat tgtgaagata gtggaaaagg aaggtggctc ctacaaatgc
     1261 catcattgcg ataaaggaaa ggccatcgtt gaagatgcct ctgccgacag tggtcccaaa
     1321 gatggacccc cacccacgag gagcatcgtg gaaaaagaag acgttccaac cacgtcttca
     1381 aagcaagtgg attgatgtga tatctccact gacgtaaggg atgacgcaca atcccactat
     1441 ccttcgcaag acccttcctc tatataagga agttcatttc atttggagag aacacggggg
     1501 acgagctaac agtattcagt cgactggtac caacaggctc catggcttct aattttactc
     1561 aatttgtttt ggttgataat ggaggaactg gagatgttac tgttgctcct tctaattttg
     1621 ctaatggagt tgctgaatgg atttcttcta attcaagatc tcaggcttat aaggttactt
     1681 gttctgttag acaatcttct gctcaaaaga gaaagtatac tattaaggtt gaagttccta
     1741 aggttgctac tcaaactgtt ggaggagttg aattgcctgt tgctgcttgg agatcttatt
     1801 tgaatatgga attgactatt cctatttttg ctactaattc tgattgtgaa ttgattgtta
     1861 aggctatgca aggattgttg aaggatggaa atcctattcc ttctgctatt gctgctaatt
     1921 ctggaattta ttctgctgga ggaggaggat ctggaggagg aggatctgga ggaggaggat
     1981 ctggacctaa gaagaagaga aaggttgctg ctgctggatc ttcaggtgcc gctagtgcga
     2041 tcgcatcagg gagtggttcc ggaagcggct ctggatcggg ctcagggagt ggttccggca
     2101 gcggctctgg atcggcggcc gctgcaatgg tgagcaaggg cgaggagctg ttcaccgggg
     2161 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     2221 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     2281 gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt cacctacggc gtgcagtgct
     2341 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     2401 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     2461 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     2521 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     2581 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     2641 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     2701 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     2761 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     2821 acggcatgga cgagctgtac aagtagctgc aaagctttcg ttcgtatcat cggtttcgac
     2881 aacgttcgtc aagttcaatg catcagtttc attgcgcaca caccagaatc ctactgagtt
     2941 tgagtattat ggcattggga aaactgtttt tcttgtacca tttgttgtgc ttgtaattta
     3001 ctgtgttttt tattcggttt tcgctatcga actgtgaaat ggaaatggat ggagaagagt
     3061 taatgaatga tatggtcctt ttgttcattc tcaaattaat attatttgtt ttttctctta
     3121 tttgttgtgt gttgaatttg aaattataag agatatgcaa acattttgtt ttgagtaaaa
     3181 atgtgtcaaa tcgtggcctc taatgaccga agttaatatg aggagtaaaa cacttgtagt
     3241 tgtaccatta tgcttattca ctaggcaaca aatatatttt cagacctaga aaagctgcaa
     3301 atgttactga atacaagtat gtcctcttgt gttttagaca tttatgaact ttcctttatg
     3361 taattttcca gaatccttgt cagattctaa tcattgcttt ataattatag ttatactcat
     3421 ggatttgtag ttgagtatga aaatattttt taatgcattt tatgacttgc caattgattg
     3481 acaacatgca tcaatcgact atgagtaggc aagatgttct gtaggacctt gagaccgaaa
     3541 aggtggtctc agtattctag accaagcatg cacagctggc gttctcagcc agatcccctt
     3601 gacaggatat attggcgggt aaactaagtc gctgtatgtg tttgtttgag atctcatgtg
     3661 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     3721 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     3781 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     3841 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     3901 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     3961 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     4021 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     4081 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     4141 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     4201 aagaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     4261 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     4321 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     4381 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     4441 ctaaagtata tatgtgtaac attggtctag tgattatttg ccgactacct tggtgatctc
     4501 gcctttcacg tagtgaacaa attcttccaa ctgatctgcg cgcgaggcca agcgatcttc
     4561 ttgtccaaga taagcctgcc tagcttcaag tatgacgggc tgatactggg ccggcaggcg
     4621 ctccattgcc cagtcggcag cgacatcctt cggcgcgatt ttgccggtta ctgcgctgta
     4681 ccaaatgcgg gacaacgtaa gcactacatt tcgctcatcg ccagcccagt cgggcggcga
     4741 gttccatagc gttaaggttt catttagcgc ctcaaataga tcctgttcag gaaccggatc
     4801 aaagagttcc tccgccgctg gacctaccaa ggcaacgcta tgttctcttg cttttgtcag
     4861 caagatagcc agatcaatgt cgatcgtggc tggctcgaag atacctgcaa gaatgtcatt
     4921 gcgctgccat tctccaaatt gcagttcgcg cttagctgga taacgccacg gaatgatgtc
     4981 gtcgtgcaca acaatggtga cttctacagc gcggagaatc tcgctctctc caggggaagc
     5041 cgaagtttcc aaaaggtcgt tgatcaaagc tcgccgcgtt gtttcatcaa gccttacggt
     5101 caccgtaacc agcaaatcaa tatcactgtg tggcttcagg ccgccatcca ctgcggagcc
     5161 gtacaaatgt acggccagca acgtcggttc gagatggcgc tcgatgacgc caactacctc
     5221 tgatagttga gtcgatactt cggcgatcac cgcttccctc ataacacccc ttgtattact
     5281 gtttatgtaa gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta
     5341 acatcagaga ttttgagaca caacgtggct ttgttgaata aatcgaactt ttgctgagtt
     5401 gaaggatcag atcacgcatc ttcccgacaa cgcagaccgt tccgtggcaa agcaaaagtt
     5461 caaaatcacc aactggtcca cctacaacaa agctctcatc aaccgtggct ccctcacttt
     5521 ctggctggat gatggggcga ttcaggcgat ccccatccaa cagcccgccg tcgagcgggc
     5581 t
//
