LOCUS       sequence-203143-        3553 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express single gRNA with gRNA2.0 scaffold (with four MS2 binding 
            sites) under AtU3 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3553)
  AUTHORS   Lowder LG, Zhou J, Zhang Y, Malzahn A, Zhong Z, Hsieh TF, Voytas DF,
            Zhang Y, Qi Y
  TITLE     Robust transcriptional activation in plants using multiplexed 
            CRISPR-Act2.0 and mTALE-Act systems.
  JOURNAL   Mol Plant. 2017 Nov 29. pii: S1674-2052(17)30344-1. doi: 
            10.1016/j.molp.2017.11.010.
  PUBMED    29197638
REFERENCE   2  (bases 1 to 3553)
  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: "Mol Plant. 
            2017 Nov 29. pii: S1674-2052(17)30344-1. doi: 
            10.1016/j.molp.2017.11.010."
COMMENT     Sequence Label: pYPQ141B2.0
FEATURES             Location/Qualifiers
     source          1..3553
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     3..25
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     17..34
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     18..34
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    49..144
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_RNA        557..575
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        627..645
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     protein_bind    complement(878..977)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(995..1013)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1030..1048)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1053..1069)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1053..1069)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1500..2291
                     /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      2384..2972
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2873..2892
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3126..3143
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3302..3329)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3421..3507)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ttcccagtca cgacgttgta aaacgacggc cagtcttaag ctcgggccca ataatgattt
       61 tattttgact gatagtgacc tgttcgttgc aacaaattga tgagcaatgc ttttttataa
      121 tgccaacttt gtatacaaaa gttgccccat ggcgttccct ctagataacg caggatccta
      181 tttactttaa atttttctta tggctcagcc tgtgatggat aactgaatca aacaaatggc
      241 gtctgggttt aagaacatct gttttggcta tgttggacga aacaagtgaa cttttaggat
      301 caacttccgt ttatatacgg agcttatatc gagcaataag ataagtgggc tttttatgta
      361 atttaatggg ctatcgtcca tatattcact aatacccatg cccagtaccc atgtatgcgt
      421 ttcatataag ctcctaattt ctcccacatc gctcaaatct aaacaaatct tgttgtatat
      481 ataacactga gggagcacca ttggtcagag acgagatcta gtctgagtcg accgtctctg
      541 ttttagagct aggccaacat gaggatcacc catgtctgca gggcctagca agttaaaata
      601 aggctagtcc gttatcaact tggccaacat gaggatcacc catgtctgca gggccaagtg
      661 gcaccgagtc ggtgcttttt tttccctttc cttttttctt ttttttgcca taaacttaaa
      721 tttgtatatc gatcattgta gatattgaaa acctagaaca aaccaacatc catgtgaatg
      781 tctttcatga ctgatttaga gataattctt gaattttgga actagaatct ataatgagcc
      841 taaattaaaa cattgtgaac tagtaagggc gaattcgacc cagctttctt gtacaaagtt
      901 ggcattataa aaaataattg ctcatcaatt tgttgcaacg aacaggtcac tatcagtcaa
      961 aataaaatca ttatttgcca tccagctgat atcccctata gtgagtcgta ttacatggtc
     1021 atagcatccc ctatagtgag tcgtattaca tggtcatagc tgtttcctgg cagctctggc
     1081 ccgtgtctca aaatctctga tgttacattg cacaagataa aaatatatca tcatgcctcc
     1141 tctggaccag ccaggacaga aatgcctcga cttcgctgct acccaaggtt gccgggtgac
     1201 gcacaccgtg gaaacggatg aaggcacgaa cccagtggac ataagcctgt tcggttcgta
     1261 agctgtaatg caagtagcgt atgcgctcac gcaactggtc cagaaccttg accgaacgca
     1321 gcggtggtaa cggcgcagtg gcggttttca tggcttgtta tgactgtttt tttggggtac
     1381 agtctatgcc tcgggcatcc aagcagcaag cgcgttacgc cgtgggtcga tgtttgatgt
     1441 tatggagcag caacgatgtt acgcagcagg gcagtcgccc taaaacaaag ttaaacatta
     1501 tgagggaagc ggtgatcgcc gaagtatcga ctcaactatc agaggtagtt ggcgtcatcg
     1561 agcgccatct cgaaccgacg ttgctggccg tacatttgta cggctccgca gtggatggcg
     1621 gcctgaagcc acacagtgat attgatttgc tggttacggt gaccgtaagg cttgatgaaa
     1681 caacgcggcg agctttgatc aacgaccttt tggaaacttc ggcttcccct ggagagagcg
     1741 agattctccg cgctgtagaa gtcaccattg ttgtgcacga cgacatcatt ccgtggcgtt
     1801 atccagctaa gcgcgaactg caatttggag aatggcagcg caatgacatt cttgcaggta
     1861 tcttcgagcc agccacgatc gacattgatc tggctatctt gctgacaaaa gcaagagaac
     1921 atagcgttgc cttggtaggt ccagcggcgg aggaactctt tgatccggtt cctgaacagg
     1981 atctatttga ggcgctaaat gaaaccttaa cgctatggaa ctcgccgccc gactgggctg
     2041 gcgatgagcg aaatgtagtg cttacgttgt cccgcatttg gtacagcgca gtaaccggca
     2101 aaatcgcgcc gaaggatgtc gctgccgact gggcaatgga gcgcctgccg gcccagtatc
     2161 agcccgtcat acttgaagct agacaggctt atcttggaca agaagaagat cgcttggcct
     2221 cgcgcgcaga tcagttggaa gaatttgtcc actacgtgaa aggcgagatc accaaggtag
     2281 tcggcaaata accctcgagc cacccatgac caaaatccct taacgtgagt tacgcgtcgt
     2341 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2401 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     2461 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     2521 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     2581 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     2641 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     2701 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     2761 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     2821 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     2881 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     2941 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3001 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     3061 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     3121 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
     3181 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     3241 gcagtgagcg caacgcaatt aatacgcgta ccgcgagcca ggaagagttt gtagaaacgc
     3301 aaaaaggcca tccgtcagga tggccttctg cttagtttga tgcctggcag tttatggcgg
     3361 gcgtcctgcc cgccaccctc cgggccgttg cttcacaacg ttcaaatccg ctcccggcgg
     3421 atttgtccta ctcaggagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc
     3481 ttccgactga gcctttcgtt ttatttgatg cctggcagtt ccctactctc gcgttaacgc
     3541 ttgcatggat gtt
//
