LOCUS       sequence-330817-        3486 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express single gRNA with gRNA2.1 scaffold (with four MS2 binding 
            sites) under OsU3 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3486)
  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 3486)
  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: pYPQ141D-gRNA2.1
FEATURES             Location/Qualifiers
     source          1..3486
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(50..136)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     185..207
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     199..216
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     200..216
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    231..326
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        363..740
                     /label=OsU3 promoter
                     /note="Oryza sativa (rice) snRNA U3 promoter"
     misc_RNA        773..848
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(993..1092)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(1110..1128)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1145..1163)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1168..1184)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1168..1184)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1615..2406
                     /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      2499..3087
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2988..3007
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3241..3258
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3417..3444)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gccaccctcc gggccgttgc ttcacaacgt tcaaatccgc tcccggcgga tttgtcctac
       61 tcaggagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct tccgactgag
      121 cctttcgttt tatttgatgc ctggcagttc cctactctcg cgttaacgct tgcatggatg
      181 ttttcccagt cacgacgttg taaaacgacg gccagtctta agctcgggcc caataatgat
      241 tttattttga ctgatagtga cctgttcgtt gcaacaaatt gatgagcaat gcttttttat
      301 aatgccaact ttgtatacaa aagttgcccc atggcgttcc ctctagataa cgcaggatcc
      361 taaagggatc tttaaacata cgaacagatc acttaaagtt cttctgaagc aacttaaagt
      421 tatcaggcat gcatggatct tggaggaatc agatgtgcag tcagggacca tagcacagga
      481 caggcgtctt ctactggtgc taccagcaaa tgctggaagc cgggaacact gggtacgttg
      541 gaaaccacgt gatgtggagt aagataaact gtaggagaaa agcatttcgt agtgggccat
      601 gaagcctttc aggacatgta ttgcagtatg ggccggccca ttacgcaatt ggacgacaac
      661 aaagactagt attagtacca cctcggctat ccacatagat caaagctggt ttaaaagagt
      721 tgtgcagatg atccgtggca gagacgagat ctagtctgag tcgaccgtct ctgttttaga
      781 gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga
      841 gtcggtgcgg gagcacatga ggaatcaccc atgtgcgact cccacagtca ctggggagtc
      901 ttcccttttt tgtcccttcg aagggcaatt ctgcagatat ccatcacact ggcggccgct
      961 cgaggtcgag ggtatcgata agcttgaatt cgacccagct ttcttgtaca aagttggcat
     1021 tataaaaaat aattgctcat caatttgttg caacgaacag gtcactatca gtcaaaataa
     1081 aatcattatt tgccatccag ctgatatccc ctatagtgag tcgtattaca tggtcatagc
     1141 atcccctata gtgagtcgta ttacatggtc atagctgttt cctggcagct ctggcccgtg
     1201 tctcaaaatc tctgatgtta cattgcacaa gataaaaata tatcatcatg cctcctctgg
     1261 accagccagg acagaaatgc ctcgacttcg ctgctaccca aggttgccgg gtgacgcaca
     1321 ccgtggaaac ggatgaaggc acgaacccag tggacataag cctgttcggt tcgtaagctg
     1381 taatgcaagt agcgtatgcg ctcacgcaac tggtccagaa ccttgaccga acgcagcggt
     1441 ggtaacggcg cagtggcggt tttcatggct tgttatgact gtttttttgg ggtacagtct
     1501 atgcctcggg catccaagca gcaagcgcgt tacgccgtgg gtcgatgttt gatgttatgg
     1561 agcagcaacg atgttacgca gcagggcagt cgccctaaaa caaagttaaa cattatgagg
     1621 gaagcggtga tcgccgaagt atcgactcaa ctatcagagg tagttggcgt catcgagcgc
     1681 catctcgaac cgacgttgct ggccgtacat ttgtacggct ccgcagtgga tggcggcctg
     1741 aagccacaca gtgatattga tttgctggtt acggtgaccg taaggcttga tgaaacaacg
     1801 cggcgagctt tgatcaacga ccttttggaa acttcggctt cccctggaga gagcgagatt
     1861 ctccgcgctg tagaagtcac cattgttgtg cacgacgaca tcattccgtg gcgttatcca
     1921 gctaagcgcg aactgcaatt tggagaatgg cagcgcaatg acattcttgc aggtatcttc
     1981 gagccagcca cgatcgacat tgatctggct atcttgctga caaaagcaag agaacatagc
     2041 gttgccttgg taggtccagc ggcggaggaa ctctttgatc cggttcctga acaggatcta
     2101 tttgaggcgc taaatgaaac cttaacgcta tggaactcgc cgcccgactg ggctggcgat
     2161 gagcgaaatg tagtgcttac gttgtcccgc atttggtaca gcgcagtaac cggcaaaatc
     2221 gcgccgaagg atgtcgctgc cgactgggca atggagcgcc tgccggccca gtatcagccc
     2281 gtcatacttg aagctagaca ggcttatctt ggacaagaag aagatcgctt ggcctcgcgc
     2341 gcagatcagt tggaagaatt tgtccactac gtgaaaggcg agatcaccaa ggtagtcggc
     2401 aaataaccct cgagccaccc atgaccaaaa tcccttaacg tgagttacgc gtcgttccac
     2461 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     2521 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     2581 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     2641 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     2701 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     2761 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     2821 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     2881 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     2941 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     3001 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     3061 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     3121 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     3181 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     3241 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     3301 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     3361 gagcgcaacg caattaatac gcgtaccgcg agccaggaag agtttgtaga aacgcaaaaa
     3421 ggccatccgt caggatggcc ttctgcttag tttgatgcct ggcagtttat ggcgggcgtc
     3481 ctgccc
//
