LOCUS       sequence-122142-        7729 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry vector with pco-dCas9-3X(SRDX).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7729)
  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 7729)
  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: pYPQ153
FEATURES             Location/Qualifiers
     source          1..7729
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      3..255
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    300..423
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(440..459)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(441..459)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(464..480)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(464..480)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             911..1702
                     /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      1795..2383
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2284..2303
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2537..2554
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2713..2740)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2832..2918)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2967..2989
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2981..2998
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2982..2998
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3014..3113
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             3135..3182
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             3189..3209
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3234..4670,4860..7523)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system"
                     /label=pcoCas9
                     /note="plant codon-optimized Cas9 gene containing the 
                     potato IV2 intron"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     intron          4671..4859
                     /label=IV2 intron
                     /note="modified second intron of the potato ST-LS1 gene 
                     (Vancanneyt et al., 1990)"
     CDS             7524..7571
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
ORIGIN
        1 ccgatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg
       61 cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat
      121 gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat
      181 acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat
      241 ctatgttact agatcgggaa ttgatccccc ctcgacagct tccggaaagg gcgaattcgc
      301 aactttgtat acaaaagttg aacgagaaac gtaaaatgat ataaatatca atatattaaa
      361 ttagattttg cataaaaaac agactacata atactgtaaa acacaacata tccagtcact
      421 atgccatcca gctgatatcc cctatagtga gtcgtattac atggtcatag ctgtttcctg
      481 gcagctctgg cccgtgtctc aaaatctctg atgttacatt gcacaagata aaaatatatc
      541 atcatgcctc ctctggacca gccaggacag aaatgcctcg acttcgctgc tacccaaggt
      601 tgccgggtga cgcacaccgt ggaaacggat gaaggcacga acccagtgga cataagcctg
      661 ttcggttcgt aagctgtaat gcaagtagcg tatgcgctca cgcaactggt ccagaacctt
      721 gaccgaacgc agcggtggta acggcgcagt ggcggttttc atggcttgtt atgactgttt
      781 ttttggggta cagtctatgc ctcgggcatc caagcagcaa gcgcgttacg ccgtgggtcg
      841 atgtttgatg ttatggagca gcaacgatgt tacgcagcag ggcagtcgcc ctaaaacaaa
      901 gttaaacatc atgagggaag cggtgatcgc cgaagtatcg actcaactat cagaggtagt
      961 tggcgtcatc gagcgccatc tcgaaccgac gttgctggcc gtacatttgt acggctccgc
     1021 agtggatggc ggcctgaagc cacacagtga tattgatttg ctggttacgg tgaccgtaag
     1081 gcttgatgaa acaacgcggc gagctttgat caacgacctt ttggaaactt cggcttcccc
     1141 tggagagagc gagattctcc gcgctgtaga agtcaccatt gttgtgcacg acgacatcat
     1201 tccgtggcgt tatccagcta agcgcgaact gcaatttgga gaatggcagc gcaatgacat
     1261 tcttgcaggt atcttcgagc cagccacgat cgacattgat ctggctatct tgctgacaaa
     1321 agcaagagaa catagcgttg ccttggtagg tccagcggcg gaggaactct ttgatccggt
     1381 tcctgaacag gatctatttg aggcgctaaa tgaaacctta acgctatgga actcgccgcc
     1441 cgactgggct ggcgatgagc gaaatgtagt gcttacgttg tcccgcattt ggtacagcgc
     1501 agtaaccggc aaaatcgcgc cgaaggatgt cgctgccgac tgggcaatgg agcgcctgcc
     1561 ggcccagtat cagcccgtca tacttgaagc tagacaggct tatcttggac aagaagaaga
     1621 tcgcttggcc tcgcgcgcag atcagttgga agaatttgtc cactacgtga aaggcgagat
     1681 caccaaggta gtcggcaaat aaccctcgag ccacccatga ccaaaatccc ttaacgtgag
     1741 ttacgcgtcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     1801 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     1861 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     1921 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     1981 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     2041 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     2101 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     2161 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     2221 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     2281 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     2341 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     2401 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     2461 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     2521 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     2581 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     2641 ctggaaagcg ggcagtgagc gcaacgcaat taatacgcgt accgcgagcc aggaagagtt
     2701 tgtagaaacg caaaaaggcc atccgtcagg atggccttct gcttagtttg atgcctggca
     2761 gtttatggcg ggcgtcctgc ccgccaccct ccgggccgtt gcttcacaac gttcaaatcc
     2821 gctcccggcg gatttgtcct actcaggaga gcgttcaccg acaaacaaca gataaaacga
     2881 aaggcccagt cttccgactg agcctttcgt tttatttgat gcctggcagt tccctactct
     2941 cgcgttaacg cttgcatgga tgttttccca gtcacgacgt tgtaaaacga cggccagtct
     3001 taagctcggg ccccaaataa tgattttatt ttgactgata gtgacctgtt cgttgcaaca
     3061 aattgatgag caatgctttt ttataatgcc aactttgtac aaaaaagcag gctccgaatt
     3121 cgcccttcac catggattac aaggatgatg atgataagga ttacaaggat gatgatgata
     3181 agatggctcc aaagaagaag agaaaggttg gaatccacgg agttccagct gctgataaga
     3241 agtactctat cggacttgcc atcggaacca actctgttgg atgggctgtt atcaccgatg
     3301 agtacaaggt tccatctaag aagttcaagg ttcttggaaa caccgataga cactctatca
     3361 agaagaacct tatcggtgct cttcttttcg attctggaga gaccgctgag gctaccagat
     3421 tgaagagaac cgctagaaga agatacacca gaagaaagaa cagaatctgc taccttcagg
     3481 aaatcttctc taacgagatg gctaaggttg atgattcttt cttccacaga cttgaggagt
     3541 ctttccttgt tgaggaggat aagaagcacg agagacaccc aatcttcgga aacatcgttg
     3601 atgaggttgc ttaccacgag aagtacccaa ccatctacca ccttagaaag aagttggttg
     3661 attctaccga taaggctgat cttagactta tctaccttgc tcttgctcac atgatcaagt
     3721 tcagaggaca cttccttatc gagggagacc ttaacccaga taactctgat gttgataagt
     3781 tgttcatcca gcttgttcag acctacaacc agcttttcga ggagaaccca atcaacgctt
     3841 ctggagttga tgctaaggct atcctttctg ctagactttc taagtctcgt agacttgaga
     3901 accttatcgc tcagcttcca ggagagaaga agaacggact tttcggaaac cttatcgctc
     3961 tttctcttgg acttacccca aacttcaagt ctaacttcga tcttgctgag gatgctaagt
     4021 tgcagctttc taaggatacc tacgatgatg atcttgataa ccttcttgct cagatcggag
     4081 atcagtacgc tgatcttttc cttgctgcta agaacctttc tgatgctatc cttctttctg
     4141 acatccttag agttaacacc gagatcacca aggctccact ttctgcttct atgatcaaga
     4201 gatacgatga gcaccaccag gatcttaccc ttttgaaggc tcttgttaga cagcagcttc
     4261 cagagaagta caaggaaatc ttcttcgatc agtctaagaa cggatacgct ggatacatcg
     4321 atggaggagc ttctcaggag gagttctaca agttcatcaa gccaatcctt gagaagatgg
     4381 atggaaccga ggagcttctt gttaagttga acagagagga tcttcttaga aagcagagaa
     4441 ccttcgataa cggatctatc ccacaccaga tccaccttgg agagcttcac gctatccttc
     4501 gtagacagga ggatttctac ccattcttga aggataacag agagaagatc gagaagatcc
     4561 ttaccttcag aatcccatac tacgttggac cacttgctag aggaaactct cgtttcgctt
     4621 ggatgaccag aaagtctgag gagaccatca ccccttggaa cttcgaggag gtaagtttct
     4681 gcttctacct ttgatatata tataataatt atcattaatt agtagtaata taatatttca
     4741 aatatttttt tcaaaataaa agaatgtagt atatagcaat tgcttttctg tagtttataa
     4801 gtgtgtatat tttaatttat aacttttcta atatatgacc aaaatttgtt gatgtgcagg
     4861 ttgttgataa gggagcttct gctcagtctt tcatcgagag aatgaccaac ttcgataaga
     4921 accttccaaa cgagaaggtt cttccaaagc actctcttct ttacgagtac ttcaccgttt
     4981 acaacgagct taccaaggtt aagtacgtta ccgagggaat gagaaagcca gctttccttt
     5041 ctggagagca gaagaaggct atcgttgatc ttcttttcaa gaccaacaga aaggttaccg
     5101 ttaagcagtt gaaggaggat tacttcaaga agatcgagtg cttcgattct gttgaaatct
     5161 ctggagttga ggatagattc aacgcttctc ttggaaccta ccacgatctt ttgaagatca
     5221 tcaaggataa ggatttcctt gataacgagg agaacgagga catccttgag gacatcgttc
     5281 ttacccttac ccttttcgag gatagagaga tgatcgagga gagactcaag acctacgctc
     5341 accttttcga tgataaggtt atgaagcagt tgaagagaag aagatacacc ggatggggta
     5401 gactttctcg taagttgatc aacggaatca gagataagca gtctggaaag accatccttg
     5461 atttcttgaa gtctgatgga ttcgctaaca gaaacttcat gcagcttatc cacgatgatt
     5521 ctcttacctt caaggaggac atccagaagg ctcaggtttc tggacaggga gattctcttc
     5581 acgagcacat cgctaacctt gctggatctc cagctatcaa gaagggaatc cttcagaccg
     5641 ttaaggttgt tgatgagctt gttaaggtta tgggtagaca caagccagag aacatcgtta
     5701 tcgagatggc tagagagaac cagaccaccc agaagggaca gaagaactct cgtgagagaa
     5761 tgaagagaat cgaggaggga atcaaggagc ttggatctca aatcttgaag gagcacccag
     5821 ttgagaacac ccagcttcag aacgagaagt tgtaccttta ctaccttcag aacggaagag
     5881 atatgtacgt tgatcaggag cttgacatca acagactttc tgattacgat gttgatgcca
     5941 tcgttccaca gtctttcttg aaggatgatt ctatcgataa caaggttctt acccgttctg
     6001 ataagaacag aggaaagtct gataacgttc catctgagga ggttgttaag aagatgaaga
     6061 actactggag acagcttctt aacgctaagt tgatcaccca gagaaagttc gataacctta
     6121 ccaaggctga gagaggagga ctttctgagc ttgataaggc tggattcatc aagagacagc
     6181 ttgttgagac cagacagatc accaagcacg ttgctcagat ccttgattct cgtatgaaca
     6241 ccaagtacga tgagaacgat aagttgatca gagaggttaa ggttatcacc ttgaagtcta
     6301 agttggtttc tgatttcaga aaggatttcc agttctacaa ggttagagag atcaacaact
     6361 accaccacgc tcacgatgct taccttaacg ctgttgttgg aaccgctctt atcaagaagt
     6421 acccaaagtt ggagtctgag ttcgtttacg gagattacaa ggtttacgat gttagaaaga
     6481 tgatcgctaa gtctgagcag gagatcggaa aggctaccgc taagtacttc ttctactcta
     6541 acatcatgaa cttcttcaag accgagatca cccttgctaa cggagagatc agaaagagac
     6601 cacttatcga gaccaacgga gagaccggag agatcgtttg ggataaggga agagatttcg
     6661 ctaccgttag aaaggttctt tctatgccac aggttaacat cgttaagaaa accgaggttc
     6721 agaccggagg attctctaag gagtctatcc ttccaaagag aaactctgat aagttgatcg
     6781 ctagaaagaa ggattgggac ccaaagaagt acggaggatt cgattctcca accgttgctt
     6841 actctgttct tgttgttgct aaggttgaga agggaaagtc taagaagttg aagtctgtta
     6901 aggagcttct tggaatcacc atcatggagc gttcttcttt cgagaagaac ccaatcgatt
     6961 tccttgaggc taagggatac aaggaggtta agaaggatct tatcatcaag ttgccaaagt
     7021 actctctttt cgagcttgag aacggaagaa agagaatgct tgcttctgct ggagagcttc
     7081 agaagggaaa cgagcttgct cttccatcta agtacgttaa cttcctttac cttgcttctc
     7141 actacgagaa gttgaaggga tctccagagg ataacgagca gaagcagctt ttcgttgagc
     7201 agcacaagca ctaccttgat gagatcatcg agcaaatctc tgagttctct aagagagtta
     7261 tccttgctga tgctaacctt gataaggttc tttctgctta caacaagcac agagataagc
     7321 caatcagaga gcaggctgag aacatcatcc accttttcac ccttaccaac cttggtgctc
     7381 cagctgcttt caagtacttc gataccacca tcgatagaaa aagatacacc tctaccaagg
     7441 aggttcttga tgctaccctt atccaccagt ctatcaccgg actttacgag accagaatcg
     7501 atctttctca gcttggagga gataagagac cagctgctac caagaaggct ggacaggcta
     7561 agaagaagaa gggagacggc tctggatcgg ggtcgggttc tggctcagtc gaccttgatc
     7621 ttgacctcga actcagactt ggatttgctc tcgatctcga ccttgaactt agactcggat
     7681 ttgctcttga cctcgatctt gagcttagac tcggattcgc ttaggacgt
//
