LOCUS       sequence-124435-        7316 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry vector with AteCas9D10A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7316)
  AUTHORS   Lowder LG, Paul JW, Baltes NJ, Voytas DF, Zhang Y, Zhang D, Tang X, 
            Zheng X, Hsieh TF, Qi Y
  TITLE     A CRISPR/Cas9 toolbox for multiplexed plant genome editing and 
            transcriptional regulation.
  JOURNAL   Plant Physiol. 2015 Aug 21. pii: pp.00636.2015.
  PUBMED    26297141
REFERENCE   2  (bases 1 to 7316)
  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: "Plant 
            Physiol. 2015 Aug 21. pii: pp.00636.2015."
COMMENT     Sequence Label: pYPQ155
FEATURES             Location/Qualifiers
     source          1..7316
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             358..1149
                     /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      1242..1830
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1731..1750
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1984..2001
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2160..2187)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2279..2365)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2414..2436
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2428..2445
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2429..2445
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2461..2560
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             join(2579..2605,2606..2608,2609..6682)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6695..6715
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      6766..7018
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    7063..7186
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(7203..7222)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7204..7222)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7227..7243)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7227..7243)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 tggaccagcc aggacagaaa tgcctcgact tcgctgctac ccaaggttgc cgggtgacgc
       61 acaccgtgga aacggatgaa ggcacgaacc cagtggacat aagcctgttc ggttcgtaag
      121 ctgtaatgca agtagcgtat gcgctcacgc aactggtcca gaaccttgac cgaacgcagc
      181 ggtggtaacg gcgcagtggc ggttttcatg gcttgttatg actgtttttt tggggtacag
      241 tctatgcctc gggcatccaa gcagcaagcg cgttacgccg tgggtcgatg tttgatgtta
      301 tggagcagca acgatgttac gcagcagggc agtcgcccta aaacaaagtt aaacatcatg
      361 agggaagcgg tgatcgccga agtatcgact caactatcag aggtagttgg cgtcatcgag
      421 cgccatctcg aaccgacgtt gctggccgta catttgtacg gctccgcagt ggatggcggc
      481 ctgaagccac acagtgatat tgatttgctg gttacggtga ccgtaaggct tgatgaaaca
      541 acgcggcgag ctttgatcaa cgaccttttg gaaacttcgg cttcccctgg agagagcgag
      601 attctccgcg ctgtagaagt caccattgtt gtgcacgacg acatcattcc gtggcgttat
      661 ccagctaagc gcgaactgca atttggagaa tggcagcgca atgacattct tgcaggtatc
      721 ttcgagccag ccacgatcga cattgatctg gctatcttgc tgacaaaagc aagagaacat
      781 agcgttgcct tggtaggtcc agcggcggag gaactctttg atccggttcc tgaacaggat
      841 ctatttgagg cgctaaatga aaccttaacg ctatggaact cgccgcccga ctgggctggc
      901 gatgagcgaa atgtagtgct tacgttgtcc cgcatttggt acagcgcagt aaccggcaaa
      961 atcgcgccga aggatgtcgc tgccgactgg gcaatggagc gcctgccggc ccagtatcag
     1021 cccgtcatac ttgaagctag acaggcttat cttggacaag aagaagatcg cttggcctcg
     1081 cgcgcagatc agttggaaga atttgtccac tacgtgaaag gcgagatcac caaggtagtc
     1141 ggcaaataac cctcgagcca cccatgacca aaatccctta acgtgagtta cgcgtcgttc
     1201 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     1261 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     1321 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     1381 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     1441 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     1501 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     1561 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     1621 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     1681 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     1741 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     1801 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     1861 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     1921 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     1981 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     2041 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     2101 agtgagcgca acgcaattaa tacgcgtacc gcgagccagg aagagtttgt agaaacgcaa
     2161 aaaggccatc cgtcaggatg gccttctgct tagtttgatg cctggcagtt tatggcgggc
     2221 gtcctgcccg ccaccctccg ggccgttgct tcacaacgtt caaatccgct cccggcggat
     2281 ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt
     2341 ccgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc gttaacgctt
     2401 gcatggatgt tttcccagtc acgacgttgt aaaacgacgg ccagtcttaa gctcgggccc
     2461 caaataatga ttttattttg actgatagtg acctgttcgt tgcaacaaat tgatgagcaa
     2521 tgctttttta taatgccaac tttgtacaaa aaagcaggct ccgaattcgc ccttcaccat
     2581 ggataagaag tactctatcg gactcgctat cggaactaac tctgtgggat gggctgtgat
     2641 caccgatgag tacaaggtgc catctaagaa gttcaaggtt ctcggaaaca ccgataggca
     2701 ctctatcaag aaaaacctta tcggtgctct cctcttcgat tctggtgaaa ctgctgaggc
     2761 taccagactc aagagaaccg ctagaagaag gtacaccaga agaaagaaca ggatctgcta
     2821 cctccaagag atcttctcta acgagatggc taaagtggat gattcattct tccacaggct
     2881 cgaagagtca ttcctcgtgg aagaagataa gaagcacgag aggcacccta tcttcggaaa
     2941 catcgttgat gaggtggcat accacgagaa gtaccctact atctaccacc tcagaaagaa
     3001 gctcgttgat tctactgata aggctgatct caggctcatc tacctcgctc tcgctcacat
     3061 gatcaagttc agaggacact tcctcatcga gggtgatctc aaccctgata actctgatgt
     3121 ggataagttg ttcatccagc tcgtgcagac ctacaaccag cttttcgaag agaaccctat
     3181 caacgcttca ggtgtggatg ctaaggctat cctctctgct aggctctcta agtcaagaag
     3241 gcttgagaac ctcattgctc agctccctgg tgagaagaag aacggacttt tcggaaactt
     3301 gatcgctctc tctctcggac tcacccctaa cttcaagtct aacttcgatc tcgctgagga
     3361 tgcaaagctc cagctctcaa aggataccta cgatgatgat ctcgataacc tcctcgctca
     3421 gatcggagat cagtacgctg atttgttcct cgctgctaag aacctctctg atgctatcct
     3481 cctcagtgat atcctcagag tgaacaccga gatcaccaag gctccactct cagcttctat
     3541 gatcaagaga tacgatgagc accaccagga tctcacactt ctcaaggctc ttgttagaca
     3601 gcagctccca gagaagtaca aagagatttt cttcgatcag tctaagaacg gatacgctgg
     3661 ttacatcgat ggtggtgcat ctcaagaaga gttctacaag ttcatcaagc ctatcctcga
     3721 gaagatggat ggaaccgagg aactcctcgt gaagctcaat agagaggatc ttctcagaaa
     3781 gcagaggacc ttcgataacg gatctatccc tcatcagatc cacctcggag agttgcacgc
     3841 tatccttaga aggcaagagg atttctaccc attcctcaag gataacaggg aaaagattga
     3901 gaagattctc accttcagaa tcccttacta cgtgggacct ctcgctagag gaaactcaag
     3961 attcgcttgg atgaccagaa agtctgagga aaccatcacc ccttggaact tcgaagaggt
     4021 ggtggataag ggtgctagtg ctcagtcttt catcgagagg atgaccaact tcgataagaa
     4081 ccttccaaac gagaaggtgc tccctaagca ctctttgctc tacgagtact tcaccgtgta
     4141 caacgagttg accaaggtta agtacgtgac cgagggaatg aggaagcctg cttttttgtc
     4201 aggtgagcaa aagaaggcta tcgttgatct cttgttcaag accaacagaa aggtgaccgt
     4261 gaagcagctc aaagaggatt acttcaagaa aatcgagtgc ttcgattcag ttgagatttc
     4321 tggtgttgag gataggttca acgcatctct cggaacctac cacgatctcc tcaagatcat
     4381 taaggataag gatttcttgg ataacgagga aaacgaggat atcttggagg atatcgttct
     4441 taccctcacc ctctttgaag atagagagat gattgaagaa aggctcaaga cctacgctca
     4501 tctcttcgat gataaggtga tgaagcagtt gaagagaaga agatacactg gttggggaag
     4561 gctctcaaga aagctcatta acggaatcag ggataagcag tctggaaaga caatccttga
     4621 tttcctcaag tctgatggat tcgctaacag aaacttcatg cagctcatcc acgatgattc
     4681 tctcaccttt aaagaggata tccagaaggc tcaggtttca ggacagggtg atagtctcca
     4741 tgagcatatc gctaacctcg ctggatctcc tgcaatcaag aagggaatcc tccagactgt
     4801 gaaggttgtg gatgagttgg tgaaggtgat gggaaggcat aagcctgaga acatcgtgat
     4861 cgaaatggct agagagaacc agaccactca gaagggacag aagaactcta gggaaaggat
     4921 gaagaggatc gaggaaggta tcaaagagct tggatctcag atcctcaaag agcaccctgt
     4981 tgagaacact cagctccaga atgagaagct ctacctctac tacctccaga acggaaggga
     5041 tatgtatgtg gatcaagagt tggatatcaa caggctctct gattacgatg ttgatcatat
     5101 cgtgccacag tcattcttga aggatgattc tatcgataac aaggtgctca ccaggtctga
     5161 taagaacagg ggtaagagtg ataacgtgcc aagtgaagag gttgtgaaga aaatgaagaa
     5221 ctattggagg cagctcctca acgctaagct catcactcag agaaagttcg ataacttgac
     5281 taaggctgag aggggaggac tctctgaatt ggataaggca ggattcatca agaggcagct
     5341 tgtggaaacc aggcagatca ctaagcacgt tgcacagatc ctcgattcta ggatgaacac
     5401 caagtacgat gagaacgata agttgatcag ggaagtgaag gttatcaccc tcaagtcaaa
     5461 gctcgtgtct gatttcagaa aggatttcca attctacaag gtgagggaaa tcaacaacta
     5521 ccaccacgct cacgatgctt accttaacgc tgttgttgga accgctctca tcaagaagta
     5581 tcctaagctc gagtcagagt tcgtgtacgg tgattacaag gtgtacgatg tgaggaagat
     5641 gatcgctaag tctgagcaag agatcggaaa ggctaccgct aagtatttct tctactctaa
     5701 catcatgaat ttcttcaaga ccgagattac cctcgctaac ggtgagatca gaaagaggcc
     5761 actcatcgag acaaacggtg aaacaggtga gatcgtgtgg gataagggaa gggatttcgc
     5821 taccgttaga aaggtgctct ctatgccaca ggtgaacatc gttaagaaaa ccgaggtgca
     5881 gaccggtgga ttctctaaag agtctatcct ccctaagagg aactctgata agctcattgc
     5941 taggaagaag gattgggacc ctaagaaata cggtggtttc gattctccta ccgtggctta
     6001 ctctgttctc gttgtggcta aggttgagaa gggaaagagt aagaagctca agtctgttaa
     6061 ggaacttctc ggaatcacta tcatggaaag gtcatctttc gagaagaacc caatcgattt
     6121 cctcgaggct aagggataca aagaggttaa gaaggatctc atcatcaagc tcccaaagta
     6181 ctcactcttc gaactcgaga acggtagaaa gaggatgctc gcttctgctg gtgagcttca
     6241 aaagggaaac gagcttgctc tcccatctaa gtacgttaac tttctttacc tcgcttctca
     6301 ctacgagaag ttgaagggat ctccagaaga taacgagcag aagcaacttt tcgttgagca
     6361 gcacaagcac tacttggatg agatcatcga gcagatctct gagttctcta aaagggtgat
     6421 cctcgctgat gcaaacctcg ataaggtgtt gtctgcttac aacaagcaca gagataagcc
     6481 tatcagggaa caggcagaga acatcatcca tctcttcacc cttaccaacc tcggtgctcc
     6541 tgctgctttc aagtacttcg atacaaccat cgataggaag agatacacct ctaccaaaga
     6601 agtgctcgat gctaccctca tccatcagtc tatcactgga ctctacgaga ctaggatcga
     6661 tctctcacag ctcggtggtg attcaagggc tgatcctaag aagaagagga aggtttgagg
     6721 cgcgccgagc tctctagagc tagcgtttaa acaccggtga cgtccgatcg ttcaaacatt
     6781 tggcaataaa gtttcttaag attgaatcct gttgccggtc ttgcgatgat tatcatataa
     6841 tttctgttga attacgttaa gcatgtaata attaacatgt aatgcatgac gttatttatg
     6901 agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat agaaaacaaa
     6961 atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt actagatcgg
     7021 gaattgatcc cccctcgaca gcttccggaa agggcgaatt cgcaactttg tatacaaaag
     7081 ttgaacgaga aacgtaaaat gatataaata tcaatatatt aaattagatt ttgcataaaa
     7141 aacagactac ataatactgt aaaacacaac atatccagtc actatgccat ccagctgata
     7201 tcccctatag tgagtcgtat tacatggtca tagctgtttc ctggcagctc tggcccgtgt
     7261 ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatgc ctcctc
//
