LOCUS       sequence-245054-        7390 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry vector with pcoCas9D10A-x3.7 (Plant codon optimized).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7390)
  AUTHORS   Zhong Z, Sretenovic S, Ren Q, Yang L, Bao Y, Qi C, Yuan M, He Y, Liu
            S, Liu X, Wang J, Huang L, Wang Y, Baby D, Wang D, Zhang T, Qi Y, 
            Zhang Y
  TITLE     Improving Plant Genome Editing with High-Fidelity xCas9 and 
            Non-canonical PAM-Targeting Cas9-NG.
  JOURNAL   Mol Plant. 2019 Mar 27. pii: S1674-2052(19)30124-8. doi: 
            10.1016/j.molp.2019.03.011.
  PUBMED    30928637
REFERENCE   2  (bases 1 to 7390)
  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: "Mol Plant. 
            2019 Mar 27. pii: S1674-2052(19)30124-8. doi: 
            10.1016/j.molp.2019.03.011."
COMMENT     Sequence Label: pYPQ151D10A-x3.7
FEATURES             Location/Qualifiers
     source          1..7390
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             37..57
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             82..4182
                     /codon_start=1
                     /product="Streptococcus pyogenes Cas9 mutant with broad PAM
                     compatibility (Hu et al., 2018)"
                     /label=xCas9-3.7
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDTKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKLYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGIIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEKVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGDQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFIQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4183..4230
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      4241..4493
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4538..4661
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4678..4697)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4679..4697)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4702..4718)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4702..4718)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5149..5940
                     /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      6033..6621
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6522..6541
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6775..6792
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(6951..6978)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(7070..7156)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     7205..7227
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7219..7236
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7220..7236
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    7252..7351
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             join(7373..7390,1..30)
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
ORIGIN
        1 gataaggatt acaaggatga tgatgataag atggctccaa agaagaagag aaaggttgga
       61 atccacggag ttccagctgc tgataagaag tactctatcg gacttgccat cggaaccaac
      121 tctgttggat gggctgttat caccgatgag tacaaggttc catctaagaa gttcaaggtt
      181 cttggaaaca ccgatagaca ctctatcaag aagaacctta tcggtgctct tcttttcgat
      241 tctggagaga ccgctgaggc taccagattg aagagaaccg ctagaagaag atacaccaga
      301 agaaagaaca gaatctgcta ccttcaggaa atcttctcta acgagatggc taaggttgat
      361 gattctttct tccacagact tgaggagtct ttccttgttg aggaggataa gaagcacgag
      421 agacacccaa tcttcggaaa catcgttgat gaggttgctt accacgagaa gtacccaacc
      481 atctaccacc ttagaaagaa gttggttgat tctaccgata aggctgatct tagacttatc
      541 taccttgctc ttgctcacat gatcaagttc agaggacact tccttatcga gggagacctt
      601 aacccagata actctgatgt tgataagttg ttcatccagc ttgttcagac ctacaaccag
      661 cttttcgagg agaacccaat caacgcttct ggagttgatg ctaaggctat cctttctgct
      721 agactttcta agtctcgtag acttgagaac cttatcgctc agcttccagg agagaagaag
      781 aacggacttt tcggaaacct tatcgctctt tctcttggac ttaccccaaa cttcaagtct
      841 aacttcgatc ttgctgagga taccaagttg cagctttcta aggataccta cgatgatgat
      901 cttgataacc ttcttgctca gatcggagat cagtacgctg atcttttcct tgctgctaag
      961 aacctttctg atgctatcct tctttctgac atccttagag ttaacaccga gatcaccaag
     1021 gctccacttt ctgcttctat gatcaagctc tacgatgagc accaccagga tcttaccctt
     1081 ttgaaggctc ttgttagaca gcagcttcca gagaagtaca aggaaatctt cttcgatcag
     1141 tctaagaacg gatacgctgg atacatcgat ggaggagctt ctcaggagga gttctacaag
     1201 ttcatcaagc caatccttga gaagatggat ggaaccgagg agcttcttgt taagttgaac
     1261 agagaggatc ttcttagaaa gcagagaacc ttcgataacg gaatcatccc acaccagatc
     1321 caccttggag agcttcacgc tatccttcgt agacaggagg atttctaccc attcttgaag
     1381 gataacagag agaagatcga gaagatcctt accttcagaa tcccatacta cgttggacca
     1441 cttgctagag gaaactctcg tttcgcttgg atgaccagaa agtctgagga gaccatcacc
     1501 ccttggaact tcgagaaggt tgttgataag ggagcttctg ctcagtcttt catcgagaga
     1561 atgaccaact tcgataagaa ccttccaaac gagaaggttc ttccaaagca ctctcttctt
     1621 tacgagtact tcaccgttta caacgagctt accaaggtta agtacgttac cgagggaatg
     1681 agaaagccag ctttcctttc tggagaccag aagaaggcta tcgttgatct tcttttcaag
     1741 accaacagaa aggttaccgt taagcagttg aaggaggatt acttcaagaa gatcgagtgc
     1801 ttcgattctg ttgaaatctc tggagttgag gatagattca acgcttctct tggaacctac
     1861 cacgatcttt tgaagatcat caaagataag gacttccttg ataacgaaga gaacgaggat
     1921 atcctcgagg acatcgttct taccttgacc cttttcgagg atagagagat gatcgaggag
     1981 agactcaaga cctacgctca ccttttcgat gataaggtta tgaagcagtt gaagagaaga
     2041 agatacaccg gatggggtag actttctcgt aagttgatca acggaatcag agataagcag
     2101 tctggaaaga ccatccttga tttcttgaag tctgatggat tcgctaacag aaacttcatc
     2161 cagcttatcc acgatgattc tcttaccttc aaggaggaca tccagaaggc tcaggtttct
     2221 ggacagggag attctcttca cgagcacatc gctaaccttg ctggatctcc agctatcaag
     2281 aagggaatcc ttcagaccgt taaggttgtt gatgagcttg ttaaggttat gggtagacac
     2341 aagccagaga acatcgttat cgagatggct agagagaacc agaccaccca gaagggacag
     2401 aagaactctc gtgagagaat gaagagaatc gaggagggaa tcaaggagct tggatctcaa
     2461 atcttgaagg agcacccagt tgagaacacc cagcttcaga acgagaagtt gtacctttac
     2521 taccttcaga acggaagaga tatgtacgtt gatcaggagc ttgacatcaa cagactttct
     2581 gattacgatg ttgatcacat cgttccacag tctttcttga aggatgattc tatcgataac
     2641 aaggttctta cccgttctga taagaacaga ggaaagtctg ataacgttcc atctgaggag
     2701 gttgttaaga agatgaagaa ctactggaga cagcttctta acgctaagtt gatcacccag
     2761 agaaagttcg ataaccttac caaggctgag agaggaggac tttctgagct tgataaggct
     2821 ggattcatca agagacagct tgttgagacc agacagatca ccaagcacgt tgctcagatc
     2881 cttgattctc gtatgaacac caagtacgat gagaacgata agttgatcag agaggttaag
     2941 gttatcacct tgaagtctaa gttggtttct gatttcagaa aggatttcca gttctacaag
     3001 gttagagaga tcaacaacta ccaccacgct cacgatgctt accttaacgc tgttgttgga
     3061 accgctctta tcaagaagta cccaaagttg gagtctgagt tcgtttacgg agattacaag
     3121 gtttacgatg ttagaaagat gatcgctaag tctgagcagg agatcggaaa ggctaccgct
     3181 aagtacttct tctactctaa catcatgaac ttcttcaaga ccgagatcac ccttgctaac
     3241 ggagagatca gaaagagacc acttatcgag accaacggag agaccggaga gatcgtttgg
     3301 gataagggaa gagatttcgc taccgttaga aaggttcttt ctatgccaca ggttaacatc
     3361 gttaagaaaa ccgaggttca gaccggagga ttctctaagg agtctatcct tccaaagaga
     3421 aactctgata agttgatcgc tagaaagaag gattgggacc caaagaagta cggaggattc
     3481 gattctccaa ccgttgctta ctctgttctt gttgttgcta aggttgagaa gggaaagtct
     3541 aagaagttga agtctgttaa ggagcttctt ggaatcacca tcatggagcg ttcttctttc
     3601 gagaagaacc caatcgattt ccttgaggct aagggataca aggaggttaa gaaggatctt
     3661 atcatcaagt tgccaaagta ctctcttttc gagcttgaga acggaagaaa gagaatgctt
     3721 gcttctgctg gagtgcttca gaagggaaac gagcttgctc ttccatctaa gtacgttaac
     3781 ttcctttacc ttgcttctca ctacgagaag ttgaagggat ctccagagga taacgagcag
     3841 aagcagcttt tcgttgagca gcacaagcac taccttgatg agatcatcga gcaaatctct
     3901 gagttctcta agagagttat ccttgctgat gctaaccttg ataaggttct ttctgcttac
     3961 aacaagcaca gagataagcc aatcagagag caggctgaga acatcatcca ccttttcacc
     4021 cttaccaacc ttggtgctcc agctgctttc aagtacttcg ataccaccat cgatagaaaa
     4081 agatacacct ctaccaagga ggttcttgat gctaccctta tccaccagtc tatcaccgga
     4141 ctttacgaga ccagaatcga tctttctcag cttggaggag ataagagacc agctgctacc
     4201 aagaaggctg gacaggctaa gaagaagaag tgagacgtcc gatcgttcaa acatttggca
     4261 ataaagtttc ttaagattga atcctgttgc cggtcttgcg atgattatca tataatttct
     4321 gttgaattac gttaagcatg taataattaa catgtaatgc atgacgttat ttatgagatg
     4381 ggtttttatg attagagtcc cgcaattata catttaatac gcgatagaaa acaaaatata
     4441 gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct atgttactag atcgggaatt
     4501 gatcccccct cgacagcttc cggaaagggc gaattcgcaa ctttgtatac aaaagttgaa
     4561 cgagaaacgt aaaatgatat aaatatcaat atattaaatt agattttgca taaaaaacag
     4621 actacataat actgtaaaac acaacatatc cagtcactat gccatccagc tgatatcccc
     4681 tatagtgagt cgtattacat ggtcatagct gtttcctggc agctctggcc cgtgtctcaa
     4741 aatctctgat gttacattgc acaagataaa aatatatcat catgcctcct ctggaccagc
     4801 caggacagaa atgcctcgac ttcgctgcta cccaaggttg ccgggtgacg cacaccgtgg
     4861 aaacggatga aggcacgaac ccagtggaca taagcctgtt cggttcgtaa gctgtaatgc
     4921 aagtagcgta tgcgctcacg caactggtcc agaaccttga ccgaacgcag cggtggtaac
     4981 ggcgcagtgg cggttttcat ggcttgttat gactgttttt ttggggtaca gtctatgcct
     5041 cgggcatcca agcagcaagc gcgttacgcc gtgggtcgat gtttgatgtt atggagcagc
     5101 aacgatgtta cgcagcaggg cagtcgccct aaaacaaagt taaacattat gagggaagcg
     5161 gtgatcgccg aagtatcgac tcaactatca gaggtagttg gcgtcatcga gcgccatctc
     5221 gaaccgacgt tgctggccgt acatttgtac ggctccgcag tggatggcgg cctgaagcca
     5281 cacagtgata ttgatttgct ggttacggtg accgtaaggc ttgatgaaac aacgcggcga
     5341 gctttgatca acgacctttt ggaaacttcg gcttcccctg gagagagcga gattctccgc
     5401 gctgtagaag tcaccattgt tgtgcacgac gacatcattc cgtggcgtta tccagctaag
     5461 cgcgaactgc aatttggaga atggcagcgc aatgacattc ttgcaggtat cttcgagcca
     5521 gccacgatcg acattgatct ggctatcttg ctgacaaaag caagagaaca tagcgttgcc
     5581 ttggtaggtc cagcggcgga ggaactcttt gatccggttc ctgaacagga tctatttgag
     5641 gcgctaaatg aaaccttaac gctatggaac tcgccgcccg actgggctgg cgatgagcga
     5701 aatgtagtgc ttacgttgtc ccgcatttgg tacagcgcag taaccggcaa aatcgcgccg
     5761 aaggatgtcg ctgccgactg ggcaatggag cgcctgccgg cccagtatca gcccgtcata
     5821 cttgaagcta gacaggctta tcttggacaa gaagaagatc gcttggcctc gcgcgcagat
     5881 cagttggaag aatttgtcca ctacgtgaaa ggcgagatca ccaaggtagt cggcaaataa
     5941 ccctcgagcc acccatgacc aaaatccctt aacgtgagtt acgcgtcgtt ccactgagcg
     6001 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     6061 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     6121 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     6181 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     6241 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     6301 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     6361 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     6421 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     6481 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     6541 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     6601 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     6661 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     6721 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     6781 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg
     6841 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     6901 aacgcaatta atacgcgtac cgcgagccag gaagagtttg tagaaacgca aaaaggccat
     6961 ccgtcaggat ggccttctgc ttagtttgat gcctggcagt ttatggcggg cgtcctgccc
     7021 gccaccctcc gggccgttgc ttcacaacgt tcaaatccgc tcccggcgga tttgtcctac
     7081 tcaggagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct tccgactgag
     7141 cctttcgttt tatttgatgc ctggcagttc cctactctcg cgttaacgct tgcatggatg
     7201 ttttcccagt cacgacgttg taaaacgacg gccagtctta agctcgggcc ccaaataatg
     7261 attttatttt gactgatagt gacctgttcg ttgcaacaaa ttgatgagca atgctttttt
     7321 ataatgccaa ctttgtacaa aaaagcaggc tccgaattcg cccttcacca tggattacaa
     7381 ggatgatgat
//
