LOCUS       sequence-223557-        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"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             677..697
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             722..4822
                     /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             722..4822
                     /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             1908..1916
                     /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             4823..4870
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     protein_bind    complement(5103..5202)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5219..5238)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5220..5238)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5243..5259)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5243..5259)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(5327..5701)
                     /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(5720..5767)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     rep_origin      5857..6445
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6346..6365
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacacattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctcg atggctccta agaagaagcg gaaggttggt attcacgggg tgcctgcggc
      721 tgacaagaag tactccatcg gcctcgacat cggcaccaac agcgtcggct gggcggtgat
      781 caccgacgag tacaaggtcc cgtccaagaa gttcaaggtc ctgggcaaca ccgaccgcca
      841 ctccatcaag aagaacctca tcggcgccct cctcttcgac tccggcgaga cggcggaggc
      901 gacccgcctc aagcgcaccg cccgccgccg ctacacccgc cgcaagaacc gcatctgcta
      961 cctccaggag atcttctcca acgagatggc gaaggtcgac gactccttct tccaccgcct
     1021 cgaggagtcc ttcctcgtgg aggaggacaa gaagcacgag cgccacccca tcttcggcaa
     1081 catcgtcgac gaggtcgcct accacgagaa gtaccccact atctaccacc ttcgtaagaa
     1141 gcttgttgac tctactgata aggctgatct tcgtctcatc taccttgctc tcgctcacat
     1201 gatcaagttc cgtggtcact tccttatcga gggtgacctt aaccctgata actccgacgt
     1261 ggacaagctc ttcatccagc tcgtccagac ctacaaccag ctcttcgagg agaaccctat
     1321 caacgcttcc ggtgtcgacg ctaaggcgat cctttccgct aggctctcca agtccaggcg
     1381 tctcgagaac ctcatcgccc agctccctgg tgagaagaag aacggtcttt tcggtaacct
     1441 catcgctctc tccctcggtc tgacccctaa cttcaagtcc aacttcgacc tcgctgagga
     1501 cgctaagctt cagctctcca aggataccta cgacgatgat ctcgacaacc tcctcgctca
     1561 gattggagat cagtacgctg atctcttcct tgctgctaag aacctctccg atgctatcct
     1621 cctttcggat atccttaggg ttaacactga gatcactaag gctcctcttt ctgcttccat
     1681 gatcaagcgc tacgacgagc accaccagga cctcaccctc ctcaaggctc ttgttcgtca
     1741 gcagctcccc gagaagtaca aggagatctt cttcgaccag tccaagaacg gctacgccgg
     1801 ttacattgac ggtggagcta gccaggagga gttctacaag ttcatcaagc caatccttga
     1861 gaagatggat ggtactgagg agcttctcgt taagcttaac cgtgaggacc tccttaggaa
     1921 gcagaggact ttcgataacg gctctatccc tcaccagatc caccttggtg agcttcacgc
     1981 catccttcgt aggcaggagg acttctaccc tttcctcaag gacaaccgtg agaagatcga
     2041 gaagatcctt actttccgta ttccttacta cgttggtcct cttgctcgtg gtaactcccg
     2101 tttcgcttgg atgactagga agtccgagga gactatcacc ccttggaact tcgaggaggt
     2161 tgttgacaag ggtgcttccg cccagtcctt catcgagcgc atgaccaact tcgacaagaa
     2221 cctccccaac gagaaggtcc tccccaagca ctccctcctc tacgagtact tcacggtcta
     2281 caacgagctc accaaggtca agtacgtcac cgagggtatg cgcaagcctg ccttcctctc
     2341 cggcgagcag aagaaggcta tcgttgacct cctcttcaag accaaccgca aggtcaccgt
     2401 caagcagctc aaggaggact acttcaagaa gatcgagtgc ttcgactccg tcgagatcag
     2461 cggcgttgag gaccgtttca acgcttctct cggtacctac cacgatctcc tcaagatcat
     2521 caaggacaag gacttcctcg acaacgagga gaacgaggac atcctcgagg acatcgtcct
     2581 cactcttact ctcttcgagg atagggagat gatcgaggag aggctcaaga cttacgctca
     2641 tctcttcgat gacaaggtta tgaagcagct caagcgtcgc cgttacaccg gttggggtag
     2701 gctctcccgc aagctcatca acggtatcag ggataagcag agcggcaaga ctatcctcga
     2761 cttcctcaag tctgatggtt tcgctaacag gaacttcatg cagctcatcc acgatgactc
     2821 tcttaccttc aaggaggata ttcagaaggc tcaggtgtcc ggtcagggcg actctctcca
     2881 cgagcacatt gctaaccttg ctggttcccc tgctatcaag aagggcatcc ttcagactgt
     2941 taaggttgtc gatgagcttg tcaaggttat gggtcgtcac aagcctgaga acatcgtcat
     3001 cgagatggct cgtgagaacc agactaccca gaagggtcag aagaactcga gggagcgcat
     3061 gaagaggatt gaggagggta tcaaggagct tggttctcag atccttaagg agcaccctgt
     3121 cgagaacacc cagctccaga acgagaagct ctacctctac tacctccaga acggtaggga
     3181 tatgtacgtt gaccaggagc tcgacatcaa caggctttct gactacgacg tcgaccacat
     3241 tgttcctcag tctttcctta aggatgactc catcgacaac aaggtcctca cgaggtccga
     3301 caagaacagg ggtaagtcgg acaacgtccc ttccgaggag gttgtcaaga agatgaagaa
     3361 ctactggagg cagcttctca acgctaagct cattacccag aggaagttcg acaacctcac
     3421 gaaggctgag aggggtggcc tttccgagct tgacaaggct ggtttcatca agaggcagct
     3481 tgttgagacg aggcagatta ccaagcacgt tgctcagatc ctcgattcta ggatgaacac
     3541 caagtacgac gagaacgaca agctcatccg cgaggtcaag gtgatcaccc tcaagtccaa
     3601 gctcgtctcc gacttccgca aggacttcca gttctacaag gtccgcgaga tcaacaacta
     3661 ccaccacgct cacgatgctt accttaacgc tgtcgttggt accgctctta tcaagaagta
     3721 ccctaagctt gagtccgagt tcgtctacgg tgactacaag gtctacgacg ttcgtaagat
     3781 gatcgccaag tccgagcagg agatcggcaa ggccaccgcc aagtacttct tctactccaa
     3841 catcatgaac ttcttcaaga ccgagatcac cctcgccaac ggcgagatcc gcaagcgccc
     3901 tcttatcgag acgaacggtg agactggtga gatcgtttgg gacaagggtc gcgacttcgc
     3961 tactgttcgc aaggtccttt ctatgcctca ggttaacatc gtcaagaaga ccgaggtcca
     4021 gaccggtggc ttctccaagg agtctatcct tccaaagaga aactcggaca agctcatcgc
     4081 taggaagaag gattgggacc ctaagaagta cggtggtttc gactccccta ctgtcgccta
     4141 ctccgtcctc gtggtcgcca aggtggagaa gggtaagtcg aagaagctca agtccgtcaa
     4201 ggagctcctc ggcatcacca tcatggagcg ctcctccttc gagaagaacc cgatcgactt
     4261 cctcgaggcc aagggctaca aggaggtcaa gaaggacctc atcatcaagc tccccaagta
     4321 ctctcttttc gagctcgaga acggtcgtaa gaggatgctg gcttccgctg gtgagctcca
     4381 gaagggtaac gagcttgctc ttccttccaa gtacgtgaac ttcctctacc tcgcctccca
     4441 ctacgagaag ctcaagggtt cccctgagga taacgagcag aagcagctct tcgtggagca
     4501 gcacaagcac tacctcgacg agatcatcga gcagatctcc gagttctcca agcgcgtcat
     4561 cctcgctgac gctaacctcg acaaggtcct ctccgcctac aacaagcacc gcgacaagcc
     4621 catccgcgag caggccgaga acatcatcca cctcttcacg ctcacgaacc tcggcgcccc
     4681 tgctgctttc aagtacttcg acaccaccat cgacaggaag cgttacacgt ccaccaagga
     4741 ggttctcgac gctactctca tccaccagtc catcaccggt ctttacgaga ctcgtatcga
     4801 cctttcccag cttggtggtg ataagcgtcc tgctgccacc aaaaaggccg gacaggctaa
     4861 gaaaaagaag taggatccac ctgatctaga gtccgcaaaa atcaccagtc tctctctaca
     4921 aatctatctc tctctatttt tctccagaat aatgtgtgag tagttcccag ataagggaat
     4981 tagggttctt atagggtttc gctcatgtgt tgagcatata agaaaccctt agtatgtatt
     5041 tgtatttgta aaatacttct atcaataaaa tttctaattc ctaaaaccaa aatccagtga
     5101 ccacccagct ttcttgtaca aagttggcat tataagaaag cattgcttat caatttgttg
     5161 caacgaacag gtcactatca gtcaaaataa aatcattatt tgccatccag ctgatatccc
     5221 ctatagtgag tcgtattaca tggtcatagc tgtttcctgg cagctctggc ccgtgtctca
     5281 aaatctctga tgttacattg cacaagataa aataatatca tcatgatcag tcctgctcct
     5341 cggccacgaa gtgcacgcag ttgccggccg ggtcgcgcag ggcgaactcc cgcccccacg
     5401 gctgctcgcc gatctcggtc atggccggcc cggaggcgtc ccggaagttc gtggacacga
     5461 cctccgacca ctcggcgtac agctcgtcca ggccgcgcac ccacacccag gccagggtgt
     5521 tgtccggcac cacctggtcc tggaccgcgc tgatgaacag ggtcacgtcg tcccggacca
     5581 caccggcgaa gtcgtcctcc acgaagtccc gggagaaccc gagccggtcg gtccagaact
     5641 cgaccgctcc ggcgacgtcg cgcgcggtga gcaccggaac ggcactggtc aacttggcca
     5701 tggtttagtt cctcaccttg tcgtattata ctatgccgat atactatgcc gatgattaat
     5761 tgtcaacacg tgctgatcat gaccaaaatc ccttaacgtg agttacgcgt cgttccactg
     5821 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     5881 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     5941 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6001 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6061 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6121 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6181 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6241 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     6301 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     6361 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     6421 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     6481 cttttgctgg ccttttgctc acatgtt
//
