LOCUS       sequence-237782-        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             364..1155
                     /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      1248..1836
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1737..1756
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1990..2007
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2166..2193)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2285..2371)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2420..2442
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2434..2451
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2435..2451
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2467..2566
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2588..2635
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             2642..2662
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2687..6787
                     /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             6788..6835
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      6846..7098
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    7143..7266
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(7283..7302)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7284..7302)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7307..7323)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7307..7323)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ctcctctgga ccagccagga cagaaatgcc tcgacttcgc tgctacccaa ggttgccggg
       61 tgacgcacac cgtggaaacg gatgaaggca cgaacccagt ggacataagc ctgttcggtt
      121 cgtaagctgt aatgcaagta gcgtatgcgc tcacgcaact ggtccagaac cttgaccgaa
      181 cgcagcggtg gtaacggcgc agtggcggtt ttcatggctt gttatgactg tttttttggg
      241 gtacagtcta tgcctcgggc atccaagcag caagcgcgtt acgccgtggg tcgatgtttg
      301 atgttatgga gcagcaacga tgttacgcag cagggcagtc gccctaaaac aaagttaaac
      361 atcatgaggg aagcggtgat cgccgaagta tcgactcaac tatcagaggt agttggcgtc
      421 atcgagcgcc atctcgaacc gacgttgctg gccgtacatt tgtacggctc cgcagtggat
      481 ggcggcctga agccacacag tgatattgat ttgctggtta cggtgaccgt aaggcttgat
      541 gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa cttcggcttc ccctggagag
      601 agcgagattc tccgcgctgt agaagtcacc attgttgtgc acgacgacat cattccgtgg
      661 cgttatccag ctaagcgcga actgcaattt ggagaatggc agcgcaatga cattcttgca
      721 ggtatcttcg agccagccac gatcgacatt gatctggcta tcttgctgac aaaagcaaga
      781 gaacatagcg ttgccttggt aggtccagcg gcggaggaac tctttgatcc ggttcctgaa
      841 caggatctat ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg
      901 gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca tttggtacag cgcagtaacc
      961 ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct gccggcccag
     1021 tatcagcccg tcatacttga agctagacag gcttatcttg gacaagaaga agatcgcttg
     1081 gcctcgcgcg cagatcagtt ggaagaattt gtccactacg tgaaaggcga gatcaccaag
     1141 gtagtcggca aataaccctc gagccaccca tgaccaaaat cccttaacgt gagttacgcg
     1201 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     1261 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     1321 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     1381 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     1441 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     1501 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     1561 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     1621 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     1681 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     1741 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     1801 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     1861 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     1921 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     1981 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     2041 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     2101 gcgggcagtg agcgcaacgc aattaatacg cgtaccgcga gccaggaaga gtttgtagaa
     2161 acgcaaaaag gccatccgtc aggatggcct tctgcttagt ttgatgcctg gcagtttatg
     2221 gcgggcgtcc tgcccgccac cctccgggcc gttgcttcac aacgttcaaa tccgctcccg
     2281 gcggatttgt cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc
     2341 agtcttccga ctgagccttt cgttttattt gatgcctggc agttccctac tctcgcgtta
     2401 acgcttgcat ggatgttttc ccagtcacga cgttgtaaaa cgacggccag tcttaagctc
     2461 gggccccaaa taatgatttt attttgactg atagtgacct gttcgttgca acaaattgat
     2521 gagcaatgct tttttataat gccaactttg tacaaaaaag caggctccga attcgccctt
     2581 caccatggat tacaaggatg atgatgataa ggattacaag gatgatgatg ataagatggc
     2641 tccaaagaag aagagaaagg ttggaatcca cggagttcca gctgctgata agaagtactc
     2701 tatcggactt gccatcggaa ccaactctgt tggatgggct gttatcaccg atgagtacaa
     2761 ggttccatct aagaagttca aggttcttgg aaacaccgat agacactcta tcaagaagaa
     2821 ccttatcggt gctcttcttt tcgattctgg agagaccgct gaggctacca gattgaagag
     2881 aaccgctaga agaagataca ccagaagaaa gaacagaatc tgctaccttc aggaaatctt
     2941 ctctaacgag atggctaagg ttgatgattc tttcttccac agacttgagg agtctttcct
     3001 tgttgaggag gataagaagc acgagagaca cccaatcttc ggaaacatcg ttgatgaggt
     3061 tgcttaccac gagaagtacc caaccatcta ccaccttaga aagaagttgg ttgattctac
     3121 cgataaggct gatcttagac ttatctacct tgctcttgct cacatgatca agttcagagg
     3181 acacttcctt atcgagggag accttaaccc agataactct gatgttgata agttgttcat
     3241 ccagcttgtt cagacctaca accagctttt cgaggagaac ccaatcaacg cttctggagt
     3301 tgatgctaag gctatccttt ctgctagact ttctaagtct cgtagacttg agaaccttat
     3361 cgctcagctt ccaggagaga agaagaacgg acttttcgga aaccttatcg ctctttctct
     3421 tggacttacc ccaaacttca agtctaactt cgatcttgct gaggatacca agttgcagct
     3481 ttctaaggat acctacgatg atgatcttga taaccttctt gctcagatcg gagatcagta
     3541 cgctgatctt ttccttgctg ctaagaacct ttctgatgct atccttcttt ctgacatcct
     3601 tagagttaac accgagatca ccaaggctcc actttctgct tctatgatca agctctacga
     3661 tgagcaccac caggatctta cccttttgaa ggctcttgtt agacagcagc ttccagagaa
     3721 gtacaaggaa atcttcttcg atcagtctaa gaacggatac gctggataca tcgatggagg
     3781 agcttctcag gaggagttct acaagttcat caagccaatc cttgagaaga tggatggaac
     3841 cgaggagctt cttgttaagt tgaacagaga ggatcttctt agaaagcaga gaaccttcga
     3901 taacggaatc atcccacacc agatccacct tggagagctt cacgctatcc ttcgtagaca
     3961 ggaggatttc tacccattct tgaaggataa cagagagaag atcgagaaga tccttacctt
     4021 cagaatccca tactacgttg gaccacttgc tagaggaaac tctcgtttcg cttggatgac
     4081 cagaaagtct gaggagacca tcaccccttg gaacttcgag aaggttgttg ataagggagc
     4141 ttctgctcag tctttcatcg agagaatgac caacttcgat aagaaccttc caaacgagaa
     4201 ggttcttcca aagcactctc ttctttacga gtacttcacc gtttacaacg agcttaccaa
     4261 ggttaagtac gttaccgagg gaatgagaaa gccagctttc ctttctggag accagaagaa
     4321 ggctatcgtt gatcttcttt tcaagaccaa cagaaaggtt accgttaagc agttgaagga
     4381 ggattacttc aagaagatcg agtgcttcga ttctgttgaa atctctggag ttgaggatag
     4441 attcaacgct tctcttggaa cctaccacga tcttttgaag atcatcaaag ataaggactt
     4501 ccttgataac gaagagaacg aggatatcct cgaggacatc gttcttacct tgaccctttt
     4561 cgaggataga gagatgatcg aggagagact caagacctac gctcaccttt tcgatgataa
     4621 ggttatgaag cagttgaaga gaagaagata caccggatgg ggtagacttt ctcgtaagtt
     4681 gatcaacgga atcagagata agcagtctgg aaagaccatc cttgatttct tgaagtctga
     4741 tggattcgct aacagaaact tcatccagct tatccacgat gattctctta ccttcaagga
     4801 ggacatccag aaggctcagg tttctggaca gggagattct cttcacgagc acatcgctaa
     4861 ccttgctgga tctccagcta tcaagaaggg aatccttcag accgttaagg ttgttgatga
     4921 gcttgttaag gttatgggta gacacaagcc agagaacatc gttatcgaga tggctagaga
     4981 gaaccagacc acccagaagg gacagaagaa ctctcgtgag agaatgaaga gaatcgagga
     5041 gggaatcaag gagcttggat ctcaaatctt gaaggagcac ccagttgaga acacccagct
     5101 tcagaacgag aagttgtacc tttactacct tcagaacgga agagatatgt acgttgatca
     5161 ggagcttgac atcaacagac tttctgatta cgatgttgat cacatcgttc cacagtcttt
     5221 cttgaaggat gattctatcg ataacaaggt tcttacccgt tctgataaga acagaggaaa
     5281 gtctgataac gttccatctg aggaggttgt taagaagatg aagaactact ggagacagct
     5341 tcttaacgct aagttgatca cccagagaaa gttcgataac cttaccaagg ctgagagagg
     5401 aggactttct gagcttgata aggctggatt catcaagaga cagcttgttg agaccagaca
     5461 gatcaccaag cacgttgctc agatccttga ttctcgtatg aacaccaagt acgatgagaa
     5521 cgataagttg atcagagagg ttaaggttat caccttgaag tctaagttgg tttctgattt
     5581 cagaaaggat ttccagttct acaaggttag agagatcaac aactaccacc acgctcacga
     5641 tgcttacctt aacgctgttg ttggaaccgc tcttatcaag aagtacccaa agttggagtc
     5701 tgagttcgtt tacggagatt acaaggttta cgatgttaga aagatgatcg ctaagtctga
     5761 gcaggagatc ggaaaggcta ccgctaagta cttcttctac tctaacatca tgaacttctt
     5821 caagaccgag atcacccttg ctaacggaga gatcagaaag agaccactta tcgagaccaa
     5881 cggagagacc ggagagatcg tttgggataa gggaagagat ttcgctaccg ttagaaaggt
     5941 tctttctatg ccacaggtta acatcgttaa gaaaaccgag gttcagaccg gaggattctc
     6001 taaggagtct atccttccaa agagaaactc tgataagttg atcgctagaa agaaggattg
     6061 ggacccaaag aagtacggag gattcgattc tccaaccgtt gcttactctg ttcttgttgt
     6121 tgctaaggtt gagaagggaa agtctaagaa gttgaagtct gttaaggagc ttcttggaat
     6181 caccatcatg gagcgttctt ctttcgagaa gaacccaatc gatttccttg aggctaaggg
     6241 atacaaggag gttaagaagg atcttatcat caagttgcca aagtactctc ttttcgagct
     6301 tgagaacgga agaaagagaa tgcttgcttc tgctggagtg cttcagaagg gaaacgagct
     6361 tgctcttcca tctaagtacg ttaacttcct ttaccttgct tctcactacg agaagttgaa
     6421 gggatctcca gaggataacg agcagaagca gcttttcgtt gagcagcaca agcactacct
     6481 tgatgagatc atcgagcaaa tctctgagtt ctctaagaga gttatccttg ctgatgctaa
     6541 ccttgataag gttctttctg cttacaacaa gcacagagat aagccaatca gagagcaggc
     6601 tgagaacatc atccaccttt tcacccttac caaccttggt gctccagctg ctttcaagta
     6661 cttcgatacc accatcgata gaaaaagata cacctctacc aaggaggttc ttgatgctac
     6721 ccttatccac cagtctatca ccggacttta cgagaccaga atcgatcttt ctcagcttgg
     6781 aggagataag agaccagctg ctaccaagaa ggctggacag gctaagaaga agaagtgaga
     6841 cgtccgatcg ttcaaacatt tggcaataaa gtttcttaag attgaatcct gttgccggtc
     6901 ttgcgatgat tatcatataa tttctgttga attacgttaa gcatgtaata attaacatgt
     6961 aatgcatgac gttatttatg agatgggttt ttatgattag agtcccgcaa ttatacattt
     7021 aatacgcgat agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt
     7081 catctatgtt actagatcgg gaattgatcc cccctcgaca gcttccggaa agggcgaatt
     7141 cgcaactttg tatacaaaag ttgaacgaga aacgtaaaat gatataaata tcaatatatt
     7201 aaattagatt ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc
     7261 actatgccat ccagctgata tcccctatag tgagtcgtat tacatggtca tagctgtttc
     7321 ctggcagctc tggcccgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat
     7381 atcatcatgc
//
