LOCUS       sequence-227231-        6507 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry clone containing dCAS9p 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-dCAS9p-t35s
FEATURES             Location/Qualifiers
     source          1..6507
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(144..171)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(263..349)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     398..420
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     412..429
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     413..429
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    445..544
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             553..573
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(598..621,622..624,625..3111,3112..3114,3115..4698)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /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"
     RBS             1784..1792
                     /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             4699..4746
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     protein_bind    complement(4979..5078)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5095..5114)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5096..5114)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5119..5135)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5119..5135)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(5203..5577)
                     /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(5596..5643)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     rep_origin      5733..6321
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6222..6241
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6475..6492
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
       61 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatacgcg taccgctagc
      121 caggaagagt ttgtagaaac gcaaaaaggc catccgtcag gatggccttc tgcttagttt
      181 gatgcctggc agtttatggc gggcgtcctg cccgccaccc tccgggccgt tgcttcacaa
      241 cgttcaaatc cgctcccggc ggatttgtcc tactcaggag agcgttcacc gacaaacaac
      301 agataaaacg aaaggcccag tcttccgact gagcctttcg ttttatttga tgcctggcag
      361 ttccctactc tcgcgttaac gctagcatgg atgttttccc agtcacgacg ttgtaaaacg
      421 acggccagtc ttaagctcgg gccccaaata atgattttat tttgactgat agtgacctgt
      481 tcgttgcaac aaattgatga gcaatgcttt tttataatgc caactttgta caaaaaagca
      541 ggctcgatgg ctcctaagaa gaagcggaag gttggtattc acggggtgcc tgcggctgac
      601 aagaagtact ccatcggcct cgcgatcggc accaacagcg tcggctgggc ggtgatcacc
      661 gacgagtaca aggtcccgtc caagaagttc aaggtcctgg gcaacaccga ccgccactcc
      721 atcaagaaga acctcatcgg cgccctcctc ttcgactccg gcgagacggc ggaggcgacc
      781 cgcctcaagc gcaccgcccg ccgccgctac acccgccgca agaaccgcat ctgctacctc
      841 caggagatct tctccaacga gatggcgaag gtcgacgact ccttcttcca ccgcctcgag
      901 gagtccttcc tcgtggagga ggacaagaag cacgagcgcc accccatctt cggcaacatc
      961 gtcgacgagg tcgcctacca cgagaagtac cccactatct accaccttcg taagaagctt
     1021 gttgactcta ctgataaggc tgatcttcgt ctcatctacc ttgctctcgc tcacatgatc
     1081 aagttccgtg gtcacttcct tatcgagggt gaccttaacc ctgataactc cgacgtggac
     1141 aagctcttca tccagctcgt ccagacctac aaccagctct tcgaggagaa ccctatcaac
     1201 gcttccggtg tcgacgctaa ggcgatcctt tccgctaggc tctccaagtc caggcgtctc
     1261 gagaacctca tcgcccagct ccctggtgag aagaagaacg gtcttttcgg taacctcatc
     1321 gctctctccc tcggtctgac ccctaacttc aagtccaact tcgacctcgc tgaggacgct
     1381 aagcttcagc tctccaagga tacctacgac gatgatctcg acaacctcct cgctcagatt
     1441 ggagatcagt acgctgatct cttccttgct gctaagaacc tctccgatgc tatcctcctt
     1501 tcggatatcc ttagggttaa cactgagatc actaaggctc ctctttctgc ttccatgatc
     1561 aagcgctacg acgagcacca ccaggacctc accctcctca aggctcttgt tcgtcagcag
     1621 ctccccgaga agtacaagga gatcttcttc gaccagtcca agaacggcta cgccggttac
     1681 attgacggtg gagctagcca ggaggagttc tacaagttca tcaagccaat ccttgagaag
     1741 atggatggta ctgaggagct tctcgttaag cttaaccgtg aggacctcct taggaagcag
     1801 aggactttcg ataacggctc tatccctcac cagatccacc ttggtgagct tcacgccatc
     1861 cttcgtaggc aggaggactt ctaccctttc ctcaaggaca accgtgagaa gatcgagaag
     1921 atccttactt tccgtattcc ttactacgtt ggtcctcttg ctcgtggtaa ctcccgtttc
     1981 gcttggatga ctaggaagtc cgaggagact atcacccctt ggaacttcga ggaggttgtt
     2041 gacaagggtg cttccgccca gtccttcatc gagcgcatga ccaacttcga caagaacctc
     2101 cccaacgaga aggtcctccc caagcactcc ctcctctacg agtacttcac ggtctacaac
     2161 gagctcacca aggtcaagta cgtcaccgag ggtatgcgca agcctgcctt cctctccggc
     2221 gagcagaaga aggctatcgt tgacctcctc ttcaagacca accgcaaggt caccgtcaag
     2281 cagctcaagg aggactactt caagaagatc gagtgcttcg actccgtcga gatcagcggc
     2341 gttgaggacc gtttcaacgc ttctctcggt acctaccacg atctcctcaa gatcatcaag
     2401 gacaaggact tcctcgacaa cgaggagaac gaggacatcc tcgaggacat cgtcctcact
     2461 cttactctct tcgaggatag ggagatgatc gaggagaggc tcaagactta cgctcatctc
     2521 ttcgatgaca aggttatgaa gcagctcaag cgtcgccgtt acaccggttg gggtaggctc
     2581 tcccgcaagc tcatcaacgg tatcagggat aagcagagcg gcaagactat cctcgacttc
     2641 ctcaagtctg atggtttcgc taacaggaac ttcatgcagc tcatccacga tgactctctt
     2701 accttcaagg aggatattca gaaggctcag gtgtccggtc agggcgactc tctccacgag
     2761 cacattgcta accttgctgg ttcccctgct atcaagaagg gcatccttca gactgttaag
     2821 gttgtcgatg agcttgtcaa ggttatgggt cgtcacaagc ctgagaacat cgtcatcgag
     2881 atggctcgtg agaaccagac tacccagaag ggtcagaaga actcgaggga gcgcatgaag
     2941 aggattgagg agggtatcaa ggagcttggt tctcagatcc ttaaggagca ccctgtcgag
     3001 aacacccagc tccagaacga gaagctctac ctctactacc tccagaacgg tagggatatg
     3061 tacgttgacc aggagctcga catcaacagg ctttctgact acgacgtcga cgcgattgtt
     3121 cctcagtctt tccttaagga tgactccatc gacaacaagg tcctcacgag gtccgacaag
     3181 aacaggggta agtcggacaa cgtcccttcc gaggaggttg tcaagaagat gaagaactac
     3241 tggaggcagc ttctcaacgc taagctcatt acccagagga agttcgacaa cctcacgaag
     3301 gctgagaggg gtggcctttc cgagcttgac aaggctggtt tcatcaagag gcagcttgtt
     3361 gagacgaggc agattaccaa gcacgttgct cagatcctcg attctaggat gaacaccaag
     3421 tacgacgaga acgacaagct catccgcgag gtcaaggtga tcaccctcaa gtccaagctc
     3481 gtctccgact tccgcaagga cttccagttc tacaaggtcc gcgagatcaa caactaccac
     3541 cacgctcacg atgcttacct taacgctgtc gttggtaccg ctcttatcaa gaagtaccct
     3601 aagcttgagt ccgagttcgt ctacggtgac tacaaggtct acgacgttcg taagatgatc
     3661 gccaagtccg agcaggagat cggcaaggcc accgccaagt acttcttcta ctccaacatc
     3721 atgaacttct tcaagaccga gatcaccctc gccaacggcg agatccgcaa gcgccctctt
     3781 atcgagacga acggtgagac tggtgagatc gtttgggaca agggtcgcga cttcgctact
     3841 gttcgcaagg tcctttctat gcctcaggtt aacatcgtca agaagaccga ggtccagacc
     3901 ggtggcttct ccaaggagtc tatccttcca aagagaaact cggacaagct catcgctagg
     3961 aagaaggatt gggaccctaa gaagtacggt ggtttcgact cccctactgt cgcctactcc
     4021 gtcctcgtgg tcgccaaggt ggagaagggt aagtcgaaga agctcaagtc cgtcaaggag
     4081 ctcctcggca tcaccatcat ggagcgctcc tccttcgaga agaacccgat cgacttcctc
     4141 gaggccaagg gctacaagga ggtcaagaag gacctcatca tcaagctccc caagtactct
     4201 cttttcgagc tcgagaacgg tcgtaagagg atgctggctt ccgctggtga gctccagaag
     4261 ggtaacgagc ttgctcttcc ttccaagtac gtgaacttcc tctacctcgc ctcccactac
     4321 gagaagctca agggttcccc tgaggataac gagcagaagc agctcttcgt ggagcagcac
     4381 aagcactacc tcgacgagat catcgagcag atctccgagt tctccaagcg cgtcatcctc
     4441 gctgacgcta acctcgacaa ggtcctctcc gcctacaaca agcaccgcga caagcccatc
     4501 cgcgagcagg ccgagaacat catccacctc ttcacgctca cgaacctcgg cgcccctgct
     4561 gctttcaagt acttcgacac caccatcgac aggaagcgtt acacgtccac caaggaggtt
     4621 ctcgacgcta ctctcatcca ccagtccatc accggtcttt acgagactcg tatcgacctt
     4681 tcccagcttg gtggtgataa gcgtcctgct gccaccaaaa aggccggaca ggctaagaaa
     4741 aagaagtagg atccacctga tctagagtcc gcaaaaatca ccagtctctc tctacaaatc
     4801 tatctctctc tatttttctc cagaataatg tgtgagtagt tcccagataa gggaattagg
     4861 gttcttatag ggtttcgctc atgtgttgag catataagaa acccttagta tgtatttgta
     4921 tttgtaaaat acttctatca ataaaatttc taattcctaa aaccaaaatc cagtgaccac
     4981 ccagctttct tgtacaaagt tggcattata agaaagcatt gcttatcaat ttgttgcaac
     5041 gaacaggtca ctatcagtca aaataaaatc attatttgcc atccagctga tatcccctat
     5101 agtgagtcgt attacatggt catagctgtt tcctggcagc tctggcccgt gtctcaaaat
     5161 ctctgatgtt acattgcaca agataaaaat atatcatcat gatcagtcct gctcctcggc
     5221 cacgaagtgc acgcagttgc cggccgggtc gcgcagggcg aactcccgcc cccacggctg
     5281 ctcgccgatc tcggtcatgg ccggcccgga ggcgtcccgg aagttcgtgg acacgacctc
     5341 cgaccactcg gcgtacagct cgtccaggcc gcgcacccac acccaggcca gggtgttgtc
     5401 cggcaccacc tggtcctgga ccgcgctgat gaacagggtc acgtcgtccc ggaccacacc
     5461 ggcgaagtcg tcctccacga agtcccggga gaacccgagc cggtcggtcc agaactcgac
     5521 cgctccggcg acgtcgcgcg cggtgagcac cggaacggca ctggtcaact tggccatggt
     5581 ttagttcctc accttgtcgt attatactat gccgatatac tatgccgatg attaattgtc
     5641 aacacgtgct gatcatgacc aaaatccctt aacgtgagtt acgcgtcgtt ccactgagcg
     5701 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     5761 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     5821 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     5881 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     5941 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     6001 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     6061 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     6121 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     6181 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     6241 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     6301 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     6361 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     6421 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     6481 tcagtgagcg aggaagcgga agagcgc
//
