pKI1_1 vector (V005243)

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LOCUS       40924_26679        9326 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pKI1_1 DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9326)
  AUTHORS   Tsutsui H, Higashiyama T.
  TITLE     pKAMA-ITACHI vectors for highly efficient CRISPR/Cas9-mediated gene 
            knockout in Arabidopsis thaliana
  JOURNAL   Plant Cell Physiol. (2016) In press
REFERENCE   2  (bases 1 to 9326)
  AUTHORS   Tsutsui H, Higashiyama T.
  TITLE     Direct Submission
  JOURNAL   Submitted (05-OCT-2016) Contact:Hiroki Tsutsui Nagoya University, 
            Graduate School of Science; 1 Furo-cho, Nagoya, Aichi 464-8602, 
            Japan
REFERENCE   3  (bases 1 to 9326)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 9326)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Plant Cell 
            Physiol. (2016) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (05-OCT-2016) Contact:Hiroki Tsutsui Nagoya University, Graduate 
            School of Science; 1 Furo-cho, Nagoya, Aichi 464-8602, Japan"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9326
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(387..473)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /label=attL1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        698..1121
                     /label=AtU6-26
                     /note="Arabidopsis U6-26 gene promoter"
     misc_feature    1122
                     /label=initial G for transcription
                     /note="initial G for transcription"
     misc_feature    complement(1127..1133)
                     /label=AarI recognition site
                     /note="AarI recognition site"
     misc_feature    1140..1146
                     /label=AarI recognition site
                     /note="AarI recognition site"
     misc_RNA        1151..1226
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      1227..1237
                     /regulatory_class="terminator"
     regulatory      1246..2909
                     /label=RPS5A promoter
                     /note="RPS5A promoter"
                     /regulatory_class="promoter"
     misc_feature    2912..2980
                     /label=3xFLAG
                     /note="3xFLAG"
     CDS             2915..2980
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    2981..3031
                     /label=NLS
                     /note="NLS"
     CDS             2987..3007
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3032..7132
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     misc_feature    7133..7180
                     /label=nucleoplasmin NLS peptide
                     /note="nucleoplasmin NLS peptide"
     CDS             7133..7180
                     /codon_start=1
                     /product="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     misc_feature    7181..7183
                     /label=stop codon
                     /note="stop codon"
     terminator      7194..7442
                     /label=HSP terminator
                     /note="efficient transcription terminator from the
                     Arabidopsis thaliana heat shock protein 18.2 gene (Nagaya 
                     et al., 2010)"
     protein_bind    complement(7451..7550)
                     /label=attL2
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(7568..7586)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7591..7607)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             7720..8526
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     rep_origin      8676..9264
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"