LOCUS       sequence-227225-        6507 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry clone containing CAS9p and 35S terminator. Used to make CRISPR
            construct . For use in plants and compatible with the MultiSite 
            Gateway system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6507)
  AUTHORS   Wang X, Ye L, Lyu M, Ursache R, Loytynoja A, Mahonen AP
  TITLE     An inducible genome editing system for plants.
  JOURNAL   Nat Plants. 2020 Jun 29. pii: 10.1038/s41477-020-0695-2. doi: 
            10.1038/s41477-020-0695-2.
  PUBMED    32601420
REFERENCE   2  (bases 1 to 6507)
  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: "Nat Plants.
            2020 Jun 29. pii: 10.1038/s41477-020-0695-2. doi: 
            10.1038/s41477-020-0695-2."
COMMENT     Sequence Label: p221z-CAS9p-t35s
FEATURES             Location/Qualifiers
     source          1..6507
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(11..97)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     146..168
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     160..177
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     161..177
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    193..292
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             301..321
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             346..4446
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             346..4446
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     RBS             1532..1540
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             4447..4494
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     protein_bind    complement(4727..4826)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4843..4862)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4844..4862)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4867..4883)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4867..4883)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(4951..5325)
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     promoter        complement(5344..5391)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     rep_origin      5481..6069
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5970..5989
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6223..6240
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(6399..6426)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ctcccggcgg atttgtccta ctcaggagag cgttcaccga caaacaacag ataaaacgaa
       61 aggcccagtc ttccgactga gcctttcgtt ttatttgatg cctggcagtt ccctactctc
      121 gcgttaacgc tagcatggat gttttcccag tcacgacgtt gtaaaacgac ggccagtctt
      181 aagctcgggc cccaaataat gattttattt tgactgatag tgacctgttc gttgcaacaa
      241 attgatgagc aatgcttttt tataatgcca actttgtaca aaaaagcagg ctcgatggct
      301 cctaagaaga agcggaaggt tggtattcac ggggtgcctg cggctgacaa gaagtactcc
      361 atcggcctcg acatcggcac caacagcgtc ggctgggcgg tgatcaccga cgagtacaag
      421 gtcccgtcca agaagttcaa ggtcctgggc aacaccgacc gccactccat caagaagaac
      481 ctcatcggcg ccctcctctt cgactccggc gagacggcgg aggcgacccg cctcaagcgc
      541 accgcccgcc gccgctacac ccgccgcaag aaccgcatct gctacctcca ggagatcttc
      601 tccaacgaga tggcgaaggt cgacgactcc ttcttccacc gcctcgagga gtccttcctc
      661 gtggaggagg acaagaagca cgagcgccac cccatcttcg gcaacatcgt cgacgaggtc
      721 gcctaccacg agaagtaccc cactatctac caccttcgta agaagcttgt tgactctact
      781 gataaggctg atcttcgtct catctacctt gctctcgctc acatgatcaa gttccgtggt
      841 cacttcctta tcgagggtga ccttaaccct gataactccg acgtggacaa gctcttcatc
      901 cagctcgtcc agacctacaa ccagctcttc gaggagaacc ctatcaacgc ttccggtgtc
      961 gacgctaagg cgatcctttc cgctaggctc tccaagtcca ggcgtctcga gaacctcatc
     1021 gcccagctcc ctggtgagaa gaagaacggt cttttcggta acctcatcgc tctctccctc
     1081 ggtctgaccc ctaacttcaa gtccaacttc gacctcgctg aggacgctaa gcttcagctc
     1141 tccaaggata cctacgacga tgatctcgac aacctcctcg ctcagattgg agatcagtac
     1201 gctgatctct tccttgctgc taagaacctc tccgatgcta tcctcctttc ggatatcctt
     1261 agggttaaca ctgagatcac taaggctcct ctttctgctt ccatgatcaa gcgctacgac
     1321 gagcaccacc aggacctcac cctcctcaag gctcttgttc gtcagcagct ccccgagaag
     1381 tacaaggaga tcttcttcga ccagtccaag aacggctacg ccggttacat tgacggtgga
     1441 gctagccagg aggagttcta caagttcatc aagccaatcc ttgagaagat ggatggtact
     1501 gaggagcttc tcgttaagct taaccgtgag gacctcctta ggaagcagag gactttcgat
     1561 aacggctcta tccctcacca gatccacctt ggtgagcttc acgccatcct tcgtaggcag
     1621 gaggacttct accctttcct caaggacaac cgtgagaaga tcgagaagat ccttactttc
     1681 cgtattcctt actacgttgg tcctcttgct cgtggtaact cccgtttcgc ttggatgact
     1741 aggaagtccg aggagactat caccccttgg aacttcgagg aggttgttga caagggtgct
     1801 tccgcccagt ccttcatcga gcgcatgacc aacttcgaca agaacctccc caacgagaag
     1861 gtcctcccca agcactccct cctctacgag tacttcacgg tctacaacga gctcaccaag
     1921 gtcaagtacg tcaccgaggg tatgcgcaag cctgccttcc tctccggcga gcagaagaag
     1981 gctatcgttg acctcctctt caagaccaac cgcaaggtca ccgtcaagca gctcaaggag
     2041 gactacttca agaagatcga gtgcttcgac tccgtcgaga tcagcggcgt tgaggaccgt
     2101 ttcaacgctt ctctcggtac ctaccacgat ctcctcaaga tcatcaagga caaggacttc
     2161 ctcgacaacg aggagaacga ggacatcctc gaggacatcg tcctcactct tactctcttc
     2221 gaggataggg agatgatcga ggagaggctc aagacttacg ctcatctctt cgatgacaag
     2281 gttatgaagc agctcaagcg tcgccgttac accggttggg gtaggctctc ccgcaagctc
     2341 atcaacggta tcagggataa gcagagcggc aagactatcc tcgacttcct caagtctgat
     2401 ggtttcgcta acaggaactt catgcagctc atccacgatg actctcttac cttcaaggag
     2461 gatattcaga aggctcaggt gtccggtcag ggcgactctc tccacgagca cattgctaac
     2521 cttgctggtt cccctgctat caagaagggc atccttcaga ctgttaaggt tgtcgatgag
     2581 cttgtcaagg ttatgggtcg tcacaagcct gagaacatcg tcatcgagat ggctcgtgag
     2641 aaccagacta cccagaaggg tcagaagaac tcgagggagc gcatgaagag gattgaggag
     2701 ggtatcaagg agcttggttc tcagatcctt aaggagcacc ctgtcgagaa cacccagctc
     2761 cagaacgaga agctctacct ctactacctc cagaacggta gggatatgta cgttgaccag
     2821 gagctcgaca tcaacaggct ttctgactac gacgtcgacc acattgttcc tcagtctttc
     2881 cttaaggatg actccatcga caacaaggtc ctcacgaggt ccgacaagaa caggggtaag
     2941 tcggacaacg tcccttccga ggaggttgtc aagaagatga agaactactg gaggcagctt
     3001 ctcaacgcta agctcattac ccagaggaag ttcgacaacc tcacgaaggc tgagaggggt
     3061 ggcctttccg agcttgacaa ggctggtttc atcaagaggc agcttgttga gacgaggcag
     3121 attaccaagc acgttgctca gatcctcgat tctaggatga acaccaagta cgacgagaac
     3181 gacaagctca tccgcgaggt caaggtgatc accctcaagt ccaagctcgt ctccgacttc
     3241 cgcaaggact tccagttcta caaggtccgc gagatcaaca actaccacca cgctcacgat
     3301 gcttacctta acgctgtcgt tggtaccgct cttatcaaga agtaccctaa gcttgagtcc
     3361 gagttcgtct acggtgacta caaggtctac gacgttcgta agatgatcgc caagtccgag
     3421 caggagatcg gcaaggccac cgccaagtac ttcttctact ccaacatcat gaacttcttc
     3481 aagaccgaga tcaccctcgc caacggcgag atccgcaagc gccctcttat cgagacgaac
     3541 ggtgagactg gtgagatcgt ttgggacaag ggtcgcgact tcgctactgt tcgcaaggtc
     3601 ctttctatgc ctcaggttaa catcgtcaag aagaccgagg tccagaccgg tggcttctcc
     3661 aaggagtcta tccttccaaa gagaaactcg gacaagctca tcgctaggaa gaaggattgg
     3721 gaccctaaga agtacggtgg tttcgactcc cctactgtcg cctactccgt cctcgtggtc
     3781 gccaaggtgg agaagggtaa gtcgaagaag ctcaagtccg tcaaggagct cctcggcatc
     3841 accatcatgg agcgctcctc cttcgagaag aacccgatcg acttcctcga ggccaagggc
     3901 tacaaggagg tcaagaagga cctcatcatc aagctcccca agtactctct tttcgagctc
     3961 gagaacggtc gtaagaggat gctggcttcc gctggtgagc tccagaaggg taacgagctt
     4021 gctcttcctt ccaagtacgt gaacttcctc tacctcgcct cccactacga gaagctcaag
     4081 ggttcccctg aggataacga gcagaagcag ctcttcgtgg agcagcacaa gcactacctc
     4141 gacgagatca tcgagcagat ctccgagttc tccaagcgcg tcatcctcgc tgacgctaac
     4201 ctcgacaagg tcctctccgc ctacaacaag caccgcgaca agcccatccg cgagcaggcc
     4261 gagaacatca tccacctctt cacgctcacg aacctcggcg cccctgctgc tttcaagtac
     4321 ttcgacacca ccatcgacag gaagcgttac acgtccacca aggaggttct cgacgctact
     4381 ctcatccacc agtccatcac cggtctttac gagactcgta tcgacctttc ccagcttggt
     4441 ggtgataagc gtcctgctgc caccaaaaag gccggacagg ctaagaaaaa gaagtaggat
     4501 ccacctgatc tagagtccgc aaaaatcacc agtctctctc tacaaatcta tctctctcta
     4561 tttttctcca gaataatgtg tgagtagttc ccagataagg gaattagggt tcttataggg
     4621 tttcgctcat gtgttgagca tataagaaac ccttagtatg tatttgtatt tgtaaaatac
     4681 ttctatcaat aaaatttcta attcctaaaa ccaaaatcca gtgaccaccc agctttcttg
     4741 tacaaagttg gcattataag aaagcattgc ttatcaattt gttgcaacga acaggtcact
     4801 atcagtcaaa ataaaatcat tatttgccat ccagctgata tcccctatag tgagtcgtat
     4861 tacatggtca tagctgtttc ctggcagctc tggcccgtgt ctcaaaatct ctgatgttac
     4921 attgcacaag ataaaaatat atcatcatga tcagtcctgc tcctcggcca cgaagtgcac
     4981 gcagttgccg gccgggtcgc gcagggcgaa ctcccgcccc cacggctgct cgccgatctc
     5041 ggtcatggcc ggcccggagg cgtcccggaa gttcgtggac acgacctccg accactcggc
     5101 gtacagctcg tccaggccgc gcacccacac ccaggccagg gtgttgtccg gcaccacctg
     5161 gtcctggacc gcgctgatga acagggtcac gtcgtcccgg accacaccgg cgaagtcgtc
     5221 ctccacgaag tcccgggaga acccgagccg gtcggtccag aactcgaccg ctccggcgac
     5281 gtcgcgcgcg gtgagcaccg gaacggcact ggtcaacttg gccatggttt agttcctcac
     5341 cttgtcgtat tatactatgc cgatatacta tgccgatgat taattgtcaa cacgtgctga
     5401 tcatgaccaa aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc agaccccgta
     5461 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     5521 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     5581 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     5641 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     5701 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     5761 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     5821 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     5881 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     5941 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     6001 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     6061 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     6121 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     6181 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     6241 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     6301 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     6361 acgcgtaccg ctagccagga agagtttgta gaaacgcaaa aaggccatcc gtcaggatgg
     6421 ccttctgctt agtttgatgc ctggcagttt atggcgggcg tcctgcccgc caccctccgg
     6481 gccgttgctt cacaacgttc aaatccg
//
