LOCUS       sequence-203126-        3722 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express single gRNA with gRNA2.0 scaffold (with four MS2 binding 
            sites) under AtU6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3722)
  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 3722)
  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: pYPQ141A2.0
FEATURES             Location/Qualifiers
     source          1..3722
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        21..324
                     /label=AtU6-1 promoter
                     /note="promoter for the Arabidopsis thaliana U6-1 snRNA 
                     gene (Waibel and Filipowicz, 1990)"
     misc_RNA        375..393
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        445..463
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     protein_bind    complement(887..986)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(1004..1022)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1039..1057)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1062..1078)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1062..1078)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1509..2300
                     /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      2393..2981
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2882..2901
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3135..3152
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3311..3338)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3430..3516)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3565..3587
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3579..3596
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3580..3596
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3611..3706
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 ctagataacg caggatccta agaaatctca aaattccggc agaacaattt tgaatctcga
       61 tccgtagaaa ccagacggtc attgttttag ttccaccacg attatatttg aaatttacgt
      121 gagtgtgagt gagacttgca taagaaaata aaatctttag ttgggaaaaa attcaataat
      181 ataaatgggc ttgagaagga agcgagggat aggccttttt ctaaaatagg cccatttaag
      241 ctattaacaa tcttcaaaag taccacagcg cttaggtaaa gaaagcagct gagtttatat
      301 atggttagac acgaagtagt gattggagac gagatctagt ctgagtcgac cgtctctgtt
      361 ttagagctag gccaacatga ggatcaccca tgtctgcagg gcctagcaag ttaaaataag
      421 gctagtccgt tatcaacttg gccaacatga ggatcaccca tgtctgcagg gccaagtggc
      481 accgagtcgg tgcttttttt ggcaaaaatt ttcagatttt ttcttcatct gtagatttct
      541 gggttttttt ttccgtttcg tgaatcataa gtgaagtttt ggatgcaaat ctgcgcgaaa
      601 aaagttggac ctgcaatgag cttatttaga tagctaagac aaagtgattg gtccgttgtt
      661 tcagttctga ttgtcagaga gtttgtttcg agtcggcgac accaatgcgt tttgttaacc
      721 agatttcggg taagaaatgt atcgagagtt tgtttccaga cggctacatc attttcttat
      781 gaagggtgaa attagataga ccaaagattg aaacacaaca tttctttcac aaaaatataa
      841 taaacttgat agcatttagg atcagcaact agtaagggcg aattcgaccc agctttcttg
      901 tacaaagttg gcattataaa aaataattgc tcatcaattt gttgcaacga acaggtcact
      961 atcagtcaaa ataaaatcat tatttgccat ccagctgata tcccctatag tgagtcgtat
     1021 tacatggtca tagcatcccc tatagtgagt cgtattacat ggtcatagct gtttcctggc
     1081 agctctggcc cgtgtctcaa aatctctgat gttacattgc acaagataaa aatatatcat
     1141 catgcctcct ctggaccagc caggacagaa atgcctcgac ttcgctgcta cccaaggttg
     1201 ccgggtgacg cacaccgtgg aaacggatga aggcacgaac ccagtggaca taagcctgtt
     1261 cggttcgtaa gctgtaatgc aagtagcgta tgcgctcacg caactggtcc agaaccttga
     1321 ccgaacgcag cggtggtaac ggcgcagtgg cggttttcat ggcttgttat gactgttttt
     1381 ttggggtaca gtctatgcct cgggcatcca agcagcaagc gcgttacgcc gtgggtcgat
     1441 gtttgatgtt atggagcagc aacgatgtta cgcagcaggg cagtcgccct aaaacaaagt
     1501 taaacattat gagggaagcg gtgatcgccg aagtatcgac tcaactatca gaggtagttg
     1561 gcgtcatcga gcgccatctc gaaccgacgt tgctggccgt acatttgtac ggctccgcag
     1621 tggatggcgg cctgaagcca cacagtgata ttgatttgct ggttacggtg accgtaaggc
     1681 ttgatgaaac aacgcggcga gctttgatca acgacctttt ggaaacttcg gcttcccctg
     1741 gagagagcga gattctccgc gctgtagaag tcaccattgt tgtgcacgac gacatcattc
     1801 cgtggcgtta tccagctaag cgcgaactgc aatttggaga atggcagcgc aatgacattc
     1861 ttgcaggtat cttcgagcca gccacgatcg acattgatct ggctatcttg ctgacaaaag
     1921 caagagaaca tagcgttgcc ttggtaggtc cagcggcgga ggaactcttt gatccggttc
     1981 ctgaacagga tctatttgag gcgctaaatg aaaccttaac gctatggaac tcgccgcccg
     2041 actgggctgg cgatgagcga aatgtagtgc ttacgttgtc ccgcatttgg tacagcgcag
     2101 taaccggcaa aatcgcgccg aaggatgtcg ctgccgactg ggcaatggag cgcctgccgg
     2161 cccagtatca gcccgtcata cttgaagcta gacaggctta tcttggacaa gaagaagatc
     2221 gcttggcctc gcgcgcagat cagttggaag aatttgtcca ctacgtgaaa ggcgagatca
     2281 ccaaggtagt cggcaaataa ccctcgagcc acccatgacc aaaatccctt aacgtgagtt
     2341 acgcgtcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     2401 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     2461 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     2521 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     2581 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     2641 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     2701 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     2761 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     2821 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     2881 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     2941 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3001 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3061 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     3121 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     3181 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     3241 ggaaagcggg cagtgagcgc aacgcaatta atacgcgtac cgcgagccag gaagagtttg
     3301 tagaaacgca aaaaggccat ccgtcaggat ggccttctgc ttagtttgat gcctggcagt
     3361 ttatggcggg cgtcctgccc gccaccctcc gggccgttgc ttcacaacgt tcaaatccgc
     3421 tcccggcgga tttgtcctac tcaggagagc gttcaccgac aaacaacaga taaaacgaaa
     3481 ggcccagtct tccgactgag cctttcgttt tatttgatgc ctggcagttc cctactctcg
     3541 cgttaacgct tgcatggatg ttttcccagt cacgacgttg taaaacgacg gccagtctta
     3601 agctcgggcc caataatgat tttattttga ctgatagtga cctgttcgtt gcaacaaatt
     3661 gatgagcaat gcttttttat aatgccaact ttgtatacaa aagttgcccc atggcgttcc
     3721 ct
//
