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"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctcc gcggccgctc gttgaacaac ggaaactcga cttgccttcc gcacaataca
      721 tcatttcttc ttagcttttt ttcttcttct tcgttcatac agtttttttt tgtttatcag
      781 cttacatttt cttgaaccgt agctttcgtt ttcttctttt taactttcca ttcggagttt
      841 ttgtatcttg tttcatagtt tgtcccagga ttagaatgat taggcatcga accttcaaga
      901 atttgattga ataaaacatc ttcattctta agatatgaag ataatcttca aaaggcccct
      961 gggaatctga aagaagagaa gcaggcccat ttatatggga aagaacaata gtatttctta
     1021 tataggccca tttaagttga aaacaatctt caaaagtccc acatcgctta gataagaaaa
     1081 cgaagctgag tttatataca gctagagtcg aagtagtgat tgaaatgcag gtgatgactc
     1141 acctgcataa gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac
     1201 ttgaaaaagt ggcaccgagt cggtgctttt tttttttgcg gccgcgatcc ctcaactttt
     1261 gattcgctat ttgcagtgca cctgtggcgt tcatcacatc ttttgtgaca ctgtttgcac
     1321 tggtcattgc tattacaaag gaccttcctg atgttgaagg agatcgaaag taagtaactg
     1381 cacgcataac cattttcttt ccgctctttg gctcaatcca tttgacagtc aaagacaatg
     1441 tttaaccagc tccgtttgat atattgtctt tatgtgtttg ttcaagcatg tttagttaat
     1501 catgcctttg attgatcttg aataggttcc aaatatcaac cctggcaaca aaacttggag
     1561 tgagaaacat tgcattcctc ggttctggac ttctgctagt aaattatgtt tcagccatat
     1621 cactagcttt ctacatgcct caggtgaatt catctatttc cgtcttaact atttcggtta
     1681 attaaagcac gaacaccatt actgcatgta gaagcttgat aaactatcgc caccaattta
     1741 tttttgttgc gatattgtta ctttcctcag tatgcagctt tgaaaagacc aaccctctta
     1801 tcctttaaca atgaacaggt ttttagaggt agcttgatga ttcctgcaca tgtgatcttg
     1861 gcttcaggct taattttcca ggtaaagcat tatgagatac tcttatatct cttacatact
     1921 tttgagataa tgcacaagaa cttcataact atatgcttta gtttctgcat ttgacactgc
     1981 caaattcatt aatctctaat atctttgttg ttgatctttg gtagacatgg gtactagaaa
     2041 aagcaaacta caccaaggta aaatactttt gtacaaacat aaactcgtta tcacggaaca
     2101 tcaatggagt gtatatctaa cggagtgtag aaacatttga ttattgcagg aagctatctc
     2161 aggatattat cggtttatat ggaatctctt ctacgcagag tatctgttat tccccttcct
     2221 ctagctttca atttcatggt gaggatatgc agttttcttt gtatatcatt cttcttcttc
     2281 tttgtagctt ggagtcaaaa tcggttcctt catgtacata catcaaggat atgtccttct
     2341 gaatttttat atcttgcaat aaaaatgctt gtaccaattg aaacaccagc tttttgagtt
     2401 ctatgatcac tgacttggtt ctaaccaaaa aaaaaaaatg tttaatttac atatctaaaa
     2461 gtaggtttag ggaaacctaa acagtaaaat atttgtatat tattcgaatt tcactcatca
     2521 taaaaactta aattgcacca taaaattttg ttttactatt aatgatgtaa tttgtgtaac
     2581 ttaagataaa aataatattc cgtaagttaa ccggctaaaa ccacgtataa accagggaac
     2641 ctgttaaacc ggttctttac tggataaaga aatgaaagcc catgtagaca gctccattag
     2701 agcccaaacc ctaaatttct catctatata aaaggagtga cattagggtt tttgttcgtc
     2761 ctcttaaagc ttctcgtttt ctctgccgtc tctctcattc gcgcgacgca aacgatcttc
     2821 aggtgatctt ctttctccaa atcctctctc ataactctga tttcgtactt gtgtatttga
     2881 gctcacgctc tgtttctctc accacagccc catggactat aaggaccacg acggagacta
     2941 caaggatcat gatattgatt acaaagacga tgacgataag atggccccaa agaagaagcg
     3001 gaaggtcggt atccacggag tcccagcagc cgacaagaag tacagcatcg gcctggacat
     3061 cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa
     3121 attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct
     3181 gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag
     3241 atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc
     3301 caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa
     3361 gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa
     3421 gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct
     3481 gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga
     3541 gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac
     3601 ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat
     3661 cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg
     3721 cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc tgacccccaa
     3781 cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta
     3841 cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct
     3901 ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga
     3961 gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga
     4021 cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt
     4081 cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga
     4141 gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt
     4201 gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc
     4261 ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc
     4321 attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta
     4381 cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga
     4441 aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt
     4501 catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca
     4561 cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac
     4621 cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct
     4681 gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa
     4741 aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct
     4801 gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga
     4861 aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat
     4921 gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct
     4981 gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg
     5041 ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag
     5101 aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc
     5161 ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc
     5221 cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat
     5281 gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca
     5341 gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct
     5401 gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct
     5461 gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa
     5521 ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga aggacgactc
     5581 catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc
     5641 ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct
     5701 gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact
     5761 ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt
     5821 ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg
     5881 ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca
     5941 gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc
     6001 cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg
     6061 cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa
     6121 ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac
     6181 cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaaccgggga
     6241 gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca
     6301 agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct
     6361 gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta
     6421 cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa
     6481 gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag
     6541 aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa
     6601 aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa
     6661 gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa
     6721 atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga
     6781 taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga
     6841 gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct
     6901 gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca
     6961 cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat
     7021 cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag
     7081 catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acaaaaggcc
     7141 ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag taaaagggtg ggatatgaag
     7201 atgaagatga aatatttggt gtgtcaaata aaaagcttgt gtgcttaagt ttgtgttttt
     7261 ttcttggctt gttgtgttat gaatttgtgg ctttttctaa tattaaatga atgtaagatc
     7321 tcattataat gaataaacaa atgtttctat aatccattgt gaatgttttg ttggatctct
     7381 tctgcagcat ataactactg tatgtgctat ggtatggact atggaatatg attaaagata
     7441 aggcgcgccg acccagcttt cttgtacaaa gttggcatta taagaaagca ttgcttatca
     7501 atttgttgca acgaacaggt cactatcagt caaaataaaa tcattatttg ccatccagct
     7561 gatatcccct atagtgagtc gtattacatg gtcatagctg tttcctggca gctctggccc
     7621 gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata
     7681 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
     7741 acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct
     7801 cgcgataatg tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg
     7861 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg
     7921 gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt
     7981 actcctgatg atgcatggtt actcaccact gcgatccccg gaaaaacagc attccaggta
     8041 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc
     8101 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc
     8161 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag
     8221 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca
     8281 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
     8341 aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt
     8401 gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa
     8461 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag
     8521 tttttctaat cagaattggt taattggttg taacactggc agagcattac gctgacttga
     8581 cgggacggcg caagctcatg accaaaatcc cttaacgtga gttacgcgtc gttccactga
     8641 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     8701 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     8761 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     8821 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     8881 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     8941 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     9001 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     9061 cgtgagcatt gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     9121 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     9181 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     9241 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     9301 ttttgctggc cttttgctca catgtt
//
