LOCUS       sequence-331024-        5127 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express single gRNA with Aac.4 scaffold (with four MS2 binding 
            sites) under ZmUbi promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5127)
  AUTHORS   Pan C, Wu X, Markel K, Malzahn AA, Kundagrami N, Sretenovic S, Zhang
            Y, Cheng Y, Shih PM, Qi Y
  TITLE     CRISPR-Act3.0 for highly efficient multiplexed gene activation in 
            plants.
  JOURNAL   Nat Plants. 2021 Jul;7(7):942-953. doi: 10.1038/s41477-021-00953-7. 
            Epub 2021 Jun 24.
  PUBMED    34168320
REFERENCE   2  (bases 1 to 5127)
  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: 
            "10.1038/s41477-021-00953-7"; journalName: "Nat Plants"; date: 
            "2021-07"; volume: "7"; issue: "7"; pages: "942-953"
COMMENT     Sequence Label: pYPQ141-ZmUbi-RZ-Aac.4
FEATURES             Location/Qualifiers
     source          1..5127
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     4..26
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     18..35
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     19..35
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    50..145
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     ncRNA           2141..2177
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        2292..2310
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     ncRNA           2366..2433
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     protein_bind    complement(2453..2552)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(2570..2588)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2605..2623)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2628..2644)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2628..2644)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3075..3866
                     /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"
     rep_origin      3959..4547
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4448..4467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4701..4718
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4877..4904)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4996..5082)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 tttcccagtc acgacgttgt aaaacgacgg ccagtcttaa gctcgggccc aataatgatt
       61 ttattttgac tgatagtgac ctgttcgttg caacaaattg atgagcaatg cttttttata
      121 atgccaactt tgtatacaaa agttgcccca tggcgttccc tctagagaga taatgagcat
      181 tgcatgtcta agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc
      241 agtttatcta tctttataca tatatttaaa ctttactcta cgaataatat aatctatagt
      301 actacaataa tatcagtgtt ttagagaatc atataaatga acagttagac atggtctaaa
      361 ggacaattga gtattttgac aacaggactc tacagtttta tctttttagt gtgcatgtgt
      421 tctccttttt ttttgcaaat agcttcacct atataatact tcatccattt tattagtaca
      481 tccatttagg gtttagggtt aatggttttt atagactaat ttttttagta catctatttt
      541 attctatttt agcctctaaa ttaagaaaac taaaactcta ttttagtttt tttatttaat
      601 aatttagata taaaatagaa taaaataaag tgactaaaaa ttaaacaaat accctttaag
      661 aaattaaaaa aactaaggaa acatttttct tgtttcgagt agataatgcc agcctgttaa
      721 acgccgtcga cgagtctaac ggacaccaac cagcgaacca gcagcgtcgc gtcgggccaa
      781 gcgaagcaga cggcacggca tctctgtcgc tgcctctgga cccctctcga gagttccgct
      841 ccaccgttgg acttgctccg ctgtcggcat ccagaaattg cgtggcggag cggcagacgt
      901 gagccggcac ggcaggcggc ctcctcctcc tctcacggca ccggcagcta cgggggattc
      961 ctttcccacc gctccttcgc tttcccttcc tcgcccgccg taataaatag acaccccctc
     1021 cacaccctct ttccccaacc tcgtgttgtt cggagcgcac acacacacaa ccagatctcc
     1081 cccaaatcca cccgtcggca cctccgcttc aaggtacgcc gctcgtcctc cccccccccc
     1141 ccctctctac cttctctaga tcggcgttcc ggtccatggt tagggcccgg tagttctact
     1201 tctgttcatg tttgtgttag atccgtgttt gtgttagatc cgtgctgcta gcgttcgtac
     1261 acggatgcga cctgtacgtc agacacgttc tgattgctaa cttgccagtg tttctctttg
     1321 gggaatcctg ggatggctct agccgttccg cagacgggat cgatttcatg attttttttg
     1381 tttcgttgca tagggtttgg tttgcccttt tcctttattt caatatatgc cgtgcacttg
     1441 tttgtcgggt catcttttca tgcttttttt tgtcttggtt gtgatgatgt ggtctggttg
     1501 ggcggtcgtt ctagatcgga gtagaattaa ttctgtttca aactacctgg tggatttatt
     1561 aattttggat ctgtatgtgt gtgccataca tattcatagt tacgaattga agatgatgga
     1621 tggaaatatc gatctaggat aggtatacat gttgatgcgg gttttactga tgcatataca
     1681 gagatgcttt ttgttcgctt ggttgtgatg atgtggtgtg gttgggcggt cgttcattcg
     1741 ttctagatcg gagtagaata ctgtttcaaa ctacctggtg tatttattaa ttttggaact
     1801 gtatgtgtgt gtcatacatc ttcatagtta cgagtttaag atggatggaa atatcgatct
     1861 aggataggta tacatgttga tgtgggtttt actgatgcat atacatgatg gcatatgcag
     1921 catctattca tatgctctaa ccttgagtac ctatctatta taataaacaa gtatgtttta
     1981 taattatttt gatcttgata tacttggatg atggcatatg cagcagctat atgtggattt
     2041 ttttagccct gccttcatac gctatttatt tgcttggtac tgtttctttt gtcgatgctc
     2101 accctgttgt ttggtgttac ttctgcaggg atcctcggcc ctgatgagtc cgtgaggacg
     2161 aaacgagtaa gctcgtcggc cgacgtctgt acccactagg agtacaaggc cggtctagag
     2221 gacagaattt ttcaacgggt gtgccaatgg ccactttcca ggtggcaaag cccgttgagc
     2281 ttctcaggcc aacatgagga tcacccatgt ctgcagggcc tgagaagtgg cacgagacgg
     2341 agctcagtct gaccgcggcg tctctggccg gcatggtccc agcctcctcg ctggcgccgg
     2401 ctgggcaaca tgcttcggca tggcgaatgg gacggtaccc ggccggaatt cgacccagct
     2461 ttcttgtaca aagttggcat tataaaaaat aattgctcat caatttgttg caacgaacag
     2521 gtcactatca gtcaaaataa aatcattatt tgccatccag ctgatatccc ctatagtgag
     2581 tcgtattaca tggtcatagc atcccctata gtgagtcgta ttacatggtc atagctgttt
     2641 cctggcagct ctggcccgtg tctcaaaatc tctgatgtta cattgcacaa gataaaaata
     2701 tatcatcatg cctcctctgg accagccagg acagaaatgc ctcgacttcg ctgctaccca
     2761 aggttgccgg gtgacgcaca ccgtggaaac ggatgaaggc acgaacccag tggacataag
     2821 cctgttcggt tcgtaagctg taatgcaagt agcgtatgcg ctcacgcaac tggtccagaa
     2881 ccttgaccga acgcagcggt ggtaacggcg cagtggcggt tttcatggct tgttatgact
     2941 gtttttttgg ggtacagtct atgcctcggg catccaagca gcaagcgcgt tacgccgtgg
     3001 gtcgatgttt gatgttatgg agcagcaacg atgttacgca gcagggcagt cgccctaaaa
     3061 caaagttaaa cattatgagg gaagcggtga tcgccgaagt atcgactcaa ctatcagagg
     3121 tagttggcgt catcgagcgc catctcgaac cgacgttgct ggccgtacat ttgtacggct
     3181 ccgcagtgga tggcggcctg aagccacaca gtgatattga tttgctggtt acggtgaccg
     3241 taaggcttga tgaaacaacg cggcgagctt tgatcaacga ccttttggaa acttcggctt
     3301 cccctggaga gagcgagatt ctccgcgctg tagaagtcac cattgttgtg cacgacgaca
     3361 tcattccgtg gcgttatcca gctaagcgcg aactgcaatt tggagaatgg cagcgcaatg
     3421 acattcttgc aggtatcttc gagccagcca cgatcgacat tgatctggct atcttgctga
     3481 caaaagcaag agaacatagc gttgccttgg taggtccagc ggcggaggaa ctctttgatc
     3541 cggttcctga acaggatcta tttgaggcgc taaatgaaac cttaacgcta tggaactcgc
     3601 cgcccgactg ggctggcgat gagcgaaatg tagtgcttac gttgtcccgc atttggtaca
     3661 gcgcagtaac cggcaaaatc gcgccgaagg atgtcgctgc cgactgggca atggagcgcc
     3721 tgccggccca gtatcagccc gtcatacttg aagctagaca ggcttatctt ggacaagaag
     3781 aagatcgctt ggcctcgcgc gcagatcagt tggaagaatt tgtccactac gtgaaaggcg
     3841 agatcaccaa ggtagtcggc aaataaccct cgagccaccc atgaccaaaa tcccttaacg
     3901 tgagttacgc gtcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     3961 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     4021 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     4081 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     4141 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     4201 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     4261 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4321 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     4381 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     4441 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     4501 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     4561 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     4621 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     4681 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
     4741 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     4801 ccgactggaa agcgggcagt gagcgcaacg caattaatac gcgtaccgcg agccaggaag
     4861 agtttgtaga aacgcaaaaa ggccatccgt caggatggcc ttctgcttag tttgatgcct
     4921 ggcagtttat ggcgggcgtc ctgcccgcca ccctccgggc cgttgcttca caacgttcaa
     4981 atccgctccc ggcggatttg tcctactcag gagagcgttc accgacaaac aacagataaa
     5041 acgaaaggcc cagtcttccg actgagcctt tcgttttatt tgatgcctgg cagttcccta
     5101 ctctcgcgtt aacgcttgca tggatgt
//
