LOCUS       62056_4900        8581 bp DNA     circular SYN 18-APR-2020
DEFINITION  Gateway adapted binary vector pCas9_P4P1r DNA, complete sequence.
ACCESSION   LC477681
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8581)
  AUTHORS   Yamamoto A, Ishida T, Yoshimura M, Kimura Y, Sawa S.
  TITLE     Developing heritable mutations in Arabidopsis thaliana using a 
            modified CRISPR/Cas9 toolkit comprising PAM-altered Cas9 variants 
            and gRNAs
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8581)
  AUTHORS   Ishida T.
  TITLE     Direct Submission
  JOURNAL   Submitted (18-APR-2019) Contact:Takashi Ishida Kumamoto University, 
            International Research Organization for Advanced Science and 
            Technology (IROAST); Kurokami 2-39-1, Kumamoto, Kumamoto 860-8555, 
            Japan
REFERENCE   3  (bases 1 to 8581)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (18-APR-2019) Contact:Takashi Ishida Kumamoto University, 
            International Research Organization for Advanced Science and 
            Technology (IROAST); Kurokami 2-39-1, Kumamoto, Kumamoto 860-8555, 
            Japan"
FEATURES             Location/Qualifiers
     source          1..8581
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    424..519
                     /label=attL4
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      525..1496
                     /label=PcUbi promoter
                     /note="PcUbi promoter"
                     /regulatory_class="promoter"
     CDS             1511..5614
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     regulatory      5665..6133
                     /label=rbcS-3A terminator
                     /note="rbcS-3A terminator"
                     /regulatory_class="terminator"
     protein_bind    6152..6276
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(6360..6376)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(6384..6400)
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(6408..6438)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(6453..6474)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(6762..7350)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7524..8381)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(8382..8486)
                     /label=AmpR promoter
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccggggat
      421 cccaaataat gattttattt tgactgatag tgacctgttc gttgcaacaa attgataagc
      481 aatgcttttt tataatgcca actttgtata gaaaagttga attcaaaaat tacggatatg
      541 aatataggca tatccgtatc cgaattatcc gtttgacagc tagcaacgat tgtacaattg
      601 cttctttaaa aaaggaagaa agaaagaaag aaaagaatca acatcagcgt taacaaacgg
      661 ccccgttacg gcccaaacgg tcatatagag taacggcgtt aagcgttgaa agactcctat
      721 cgaaatacgt aaccgcaaac gtgtcatagt cagatcccct cttccttcac cgcctcaaac
      781 acaaaaataa tcttctacag cctatatata caaccccccc ttctatctct cctttctcac
      841 aattcatcat ctttctttct ctacccccaa ttttaagaaa tcctctcttc tcctcttcat
      901 tttcaaggta aatctctctc tctctctctc tctctgttat tccttgtttt aattaggtat
      961 gtattattgc tagtttgtta atctgcttat cttatgtatg ccttatgtga atatctttat
     1021 cttgttcatc tcatccgttt agaagctata aatttgttga tttgactgtg tatctacacg
     1081 tggttatgtt tatatctaat cagatatgaa tttcttcata ttgttgcgtt tgtgtgtacc
     1141 aatccgaaat cgttgatttt tttcatttaa tcgtgtagct aattgtacgt atacatatgg
     1201 atctacgtat caattgttca tctgtttgtg tttgtatgta tacagatctg aaaacatcac
     1261 ttctctcatc tgattgtgtt gttacataca tagatataga tctgttatat catttttttt
     1321 attaattgtg tatatatata tgtgcataga tctggattac atgattgtga ttatttacat
     1381 gattttgtta tttacgtatg tatatatgta gatctggact ttttggagtt gttgacttga
     1441 ttgtatttgt gtgtgtatat gtgtgttctg atcttgatat gttatgtatg tgcagcgaat
     1501 tcggcgcgcc atggataaga agtactctat cggactcgat atcggaacta actctgtggg
     1561 atgggctgtg atcaccgatg agtacaaggt gccatctaag aagttcaagg ttctcggaaa
     1621 caccgatagg cactctatca agaaaaacct tatcggtgct ctcctcttcg attctggtga
     1681 aactgctgag gctaccagac tcaagagaac cgctagaaga aggtacacca gaagaaagaa
     1741 caggatctgc tacctccaag agatcttctc taacgagatg gctaaagtgg atgattcatt
     1801 cttccacagg ctcgaagagt cattcctcgt ggaagaagat aagaagcacg agaggcaccc
     1861 tatcttcgga aacatcgttg atgaggtggc ataccacgag aagtacccta ctatctacca
     1921 cctcagaaag aagctcgttg attctactga taaggctgat ctcaggctca tctacctcgc
     1981 tctcgctcac atgatcaagt tcagaggaca cttcctcatc gagggtgatc tcaaccctga
     2041 taactctgat gtggataagt tgttcatcca gctcgtgcag acctacaacc agcttttcga
     2101 agagaaccct atcaacgctt caggtgtgga tgctaaggct atcctctctg ctaggctctc
     2161 taagtcaaga aggcttgaga acctcattgc tcagctccct ggtgagaaga agaacggact
     2221 tttcggaaac ttgatcgctc tctctctcgg actcacccct aacttcaagt ctaacttcga
     2281 tctcgctgag gatgcaaagc tccagctctc aaaggatacc tacgatgatg atctcgataa
     2341 cctcctcgct cagatcggag atcagtacgc tgatttgttc ctcgctgcta agaacctctc
     2401 tgatgctatc ctcctcagtg atatcctcag agtgaacacc gagatcacca aggctccact
     2461 ctcagcttct atgatcaaga gatacgatga gcaccaccag gatctcacac ttctcaaggc
     2521 tcttgttaga cagcagctcc cagagaagta caaagagatt ttcttcgatc agtctaagaa
     2581 cggatacgct ggttacatcg atggtggtgc atctcaagaa gagttctaca agttcatcaa
     2641 gcctatcctc gagaagatgg atggaaccga ggaactcctc gtgaagctca atagagagga
     2701 tcttctcaga aagcagagga ccttcgataa cggatctatc cctcatcaga tccacctcgg
     2761 agagttgcac gctatcctta gaaggcaaga ggatttctac ccattcctca aggataacag
     2821 ggaaaagatt gagaagattc tcaccttcag aatcccttac tacgtgggac ctctcgctag
     2881 aggaaactca agattcgctt ggatgaccag aaagtctgag gaaaccatca ccccttggaa
     2941 cttcgaagag gtggtggata agggtgctag tgctcagtct ttcatcgaga ggatgaccaa
     3001 cttcgataag aaccttccaa acgagaaggt gctccctaag cactctttgc tctacgagta
     3061 cttcaccgtg tacaacgagt tgaccaaggt taagtacgtg accgagggaa tgaggaagcc
     3121 tgcttttttg tcaggtgagc aaaagaaggc tatcgttgat ctcttgttca agaccaacag
     3181 aaaggtgacc gtgaagcagc tcaaagagga ttacttcaag aaaatcgagt gcttcgattc
     3241 agttgagatt tctggtgttg aggataggtt caacgcatct ctcggaacct accacgatct
     3301 cctcaagatc attaaggata aggatttctt ggataacgag gaaaacgagg atatcttgga
     3361 ggatatcgtt cttaccctca ccctctttga agatagagag atgattgaag aaaggctcaa
     3421 gacctacgct catctcttcg atgataaggt gatgaagcag ttgaagagaa gaagatacac
     3481 tggttgggga aggctctcaa gaaagctcat taacggaatc agggataagc agtctggaaa
     3541 gacaatcctt gatttcctca agtctgatgg attcgctaac agaaacttca tgcagctcat
     3601 ccacgatgat tctctcacct ttaaagagga tatccagaag gctcaggttt caggacaggg
     3661 tgatagtctc catgagcata tcgctaacct cgctggatct cctgcaatca agaagggaat
     3721 cctccagact gtgaaggttg tggatgagtt ggtgaaggtg atgggaaggc ataagcctga
     3781 gaacatcgtg atcgaaatgg ctagagagaa ccagaccact cagaagggac agaagaactc
     3841 tagggaaagg atgaagagga tcgaggaagg tatcaaagag cttggatctc agatcctcaa
     3901 agagcaccct gttgagaaca ctcagctcca gaatgagaag ctctacctct actacctcca
     3961 gaacggaagg gatatgtatg tggatcaaga gttggatatc aacaggctct ctgattacga
     4021 tgttgatcat atcgtgccac agtcattctt gaaggatgat tctatcgata acaaggtgct
     4081 caccaggtct gataagaaca ggggtaagag tgataacgtg ccaagtgaag aggttgtgaa
     4141 gaaaatgaag aactattgga ggcagctcct caacgctaag ctcatcactc agagaaagtt
     4201 cgataacttg actaaggctg agaggggagg actctctgaa ttggataagg caggattcat
     4261 caagaggcag cttgtggaaa ccaggcagat cactaagcac gttgcacaga tcctcgattc
     4321 taggatgaac accaagtacg atgagaacga taagttgatc agggaagtga aggttatcac
     4381 cctcaagtca aagctcgtgt ctgatttcag aaaggatttc caattctaca aggtgaggga
     4441 aatcaacaac taccaccacg ctcacgatgc ttaccttaac gctgttgttg gaaccgctct
     4501 catcaagaag tatcctaagc tcgagtcaga gttcgtgtac ggtgattaca aggtgtacga
     4561 tgtgaggaag atgatcgcta agtctgagca agagatcgga aaggctaccg ctaagtattt
     4621 cttctactct aacatcatga atttcttcaa gaccgagatt accctcgcta acggtgagat
     4681 cagaaagagg ccactcatcg agacaaacgg tgaaacaggt gagatcgtgt gggataaggg
     4741 aagggatttc gctaccgtta gaaaggtgct ctctatgcca caggtgaaca tcgttaagaa
     4801 aaccgaggtg cagaccggtg gattctctaa agagtctatc ctccctaaga ggaactctga
     4861 taagctcatt gctaggaaga aggattggga ccctaagaaa tacggtggtt tcgattctcc
     4921 taccgtggct tactctgttc tcgttgtggc taaggttgag aagggaaaga gtaagaagct
     4981 caagtctgtt aaggaacttc tcggaatcac tatcatggaa aggtcatctt tcgagaagaa
     5041 cccaatcgat ttcctcgagg ctaagggata caaagaggtt aagaaggatc tcatcatcaa
     5101 gctcccaaag tactcactct tcgaactcga gaacggtaga aagaggatgc tcgcttctgc
     5161 tggtgagctt caaaagggaa acgagcttgc tctcccatct aagtacgtta actttcttta
     5221 cctcgcttct cactacgaga agttgaaggg atctccagaa gataacgagc agaagcaact
     5281 tttcgttgag cagcacaagc actacttgga tgagatcatc gagcagatct ctgagttctc
     5341 taaaagggtg atcctcgctg atgcaaacct cgataaggtg ttgtctgctt acaacaagca
     5401 cagagataag cctatcaggg aacaggcaga gaacatcatc catctcttca cccttaccaa
     5461 cctcggtgct cctgctgctt tcaagtactt cgatacaacc atcgatagga agagatacac
     5521 ctctaccaaa gaagtgctcg atgctaccct catccatcag tctatcactg gactctacga
     5581 gactaggatc gatctctcac agctcggtgg tgattcaagg gctgatccta agaagaagag
     5641 gaaggtttga ggcgcgccga gctccaggcc tcccagcttt cgtccgtatc atcggtttcg
     5701 acaacgttcg tcaagttcaa tgcatcagtt tcattgccca cacaccagaa tcctactaag
     5761 tttgagtatt atggcattgg aaaagctgtt ttcttctatc atttgttctg cttgtaattt
     5821 actgtgttct ttcagttttt gttttcggac atcaaaatgc aaatggatgg ataagagtta
     5881 ataaatgata tggtcctttt gttcattctc aaattattat tatctgttgt ttttacttta
     5941 atgggttgaa tttaagtaag aaaggaacta acagtgtgat attaaggtgc aatgttagac
     6001 atataaaaca gtctttcacc tctctttggt tatgtcttga attggtttgt ttcttcactt
     6061 atctgtgtaa tcaagtttac tatgagtcta tgatcaagta attatgcaat caagttaagt
     6121 acagtatagg cttgagctcc ctaggggcgc gccaagtttg tacaaaaaag ttgaacgaga
     6181 aacgtaaaat gatataaata tcaatatatt aaattagatt ttgcataaaa aacagactac
     6241 ataatactgt aaaacacaac atatgcagtc actatgaatc aactacttag atggtattag
     6301 tgacctgtag aagatcctct agagtcgacc tgcaggcatg caagcttggc gtaatcatgg
     6361 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     6421 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     6481 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     6541 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     6601 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     6661 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     6721 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     6781 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     6841 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     6901 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     6961 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     7021 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     7081 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     7141 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     7201 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     7261 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     7321 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     7381 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     7441 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     7501 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     7561 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     7621 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     7681 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     7741 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     7801 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     7861 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     7921 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     7981 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     8041 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     8101 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     8161 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     8221 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     8281 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     8341 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     8401 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     8461 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     8521 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     8581 c
//
