LOCUS       sequence-255110-        6642 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry clone for CRISPR-Cas12b systems.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6642)
  AUTHORS   Ming M, Ren Q, Pan C, He Y, Zhang Y, Liu S, Zhong Z, Wang J, Malzahn
            AA, Wu J, Zheng X, Zhang Y, Qi Y
  TITLE     CRISPR-Cas12b enables efficient plant genome engineering.
  JOURNAL   Nat Plants. 2020 Mar;6(3):202-208. doi: 10.1038/s41477-020-0614-6. 
            Epub 2020 Mar 9.
  PUBMED    32170285
REFERENCE   2  (bases 1 to 6642)
  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"; doi: 
            "10.1038/s41477-020-0614-6"; journalName: "Nat Plants"; date: 
            "2020-03"; volume: "6"; issue: "3"; pages: "202-208"
COMMENT     Sequence Label: pYPQ292-D570A
FEATURES             Location/Qualifiers
     source          1..6642
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     133..152
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     386..403
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(562..589)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(681..767)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     816..838
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     830..847
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     831..847
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    863..962
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             987..1007
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4416..4463
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      4494..4746
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4791..4914
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4931..4950)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4932..4950)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4955..4971)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4955..4971)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5402..6193
                     /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      join(6286..6642,1..232)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
       61 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
      121 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
      181 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
      241 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
      301 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
      361 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
      421 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
      481 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatacgcgta ccgcgagcca
      541 ggaagagttt gtagaaacgc aaaaaggcca tccgtcagga tggccttctg cttagtttga
      601 tgcctggcag tttatggcgg gcgtcctgcc cgccaccctc cgggccgttg cttcacaacg
      661 ttcaaatccg ctcccggcgg atttgtccta ctcaggagag cgttcaccga caaacaacag
      721 ataaaacgaa aggcccagtc ttccgactga gcctttcgtt ttatttgatg cctggcagtt
      781 ccctactctc gcgttaacgc ttgcatggat gttttcccag tcacgacgtt gtaaaacgac
      841 ggccagtctt aagctcgggc cccaaataat gattttattt tgactgatag tgacctgttc
      901 gttgcaacaa attgatgagc aatgcttttt tataatgcca actttgtaca aaaaagcagg
      961 ctccgaattc gcccttcacc atggctccta agaagaagcg gaaggttggt attcacgggg
     1021 tgcctgcggc tgccgtcaag tccatgaagg tcaagttgcg cctggataac atgccagaga
     1081 tcagagccgg actttggaaa cttcacaccg aggttaatgc gggtgtgcgg tactatacgg
     1141 aatggcttag ccttttgagg caagaaaatc tttatcggag gagtcccaat ggcgatggag
     1201 aacaagaatg ctataaaact gctgaggaat gcaaggctga actccttgag agactcagag
     1261 cccgccaagt tgagaatggg cactgcggcc ctgctgggag tgatgacgaa ctgctgcaat
     1321 tggcacggca actttatgaa cttctggtcc cacaagcaat cggggctaaa ggtgatgcgc
     1381 agcaaatcgc aaggaagttt cttagtcccc ttgccgacaa ggatgccgtg ggtggtttgg
     1441 gaatagcaaa agcaggaaat aagcctaggt gggttcggat gagggaggct ggagagccag
     1501 gttgggaaga ggaaaaggct aaagccgagg cgagaaagag tacggataga accgccgatg
     1561 ttcttcgcgc tcttgcagac ttcggtctta aacctcttat gagagtctac acagactcag
     1621 acatgtccag cgtgcagtgg aaaccacttc gcaaaggaca agcggtcaga acctgggata
     1681 gagacatgtt ccaacaagcg atcgaaagaa tgatgagttg ggaatcgtgg aatcagcgcg
     1741 ttggagaagc gtacgcaaag ctcgtggaac aaaagtcgag gtttgaacag aaaaattttg
     1801 tgggacaaga acatcttgtc caacttgtca atcaacttca acaagacatg aaggaagcat
     1861 cacacggcct ggagtcgaaa gaacaaactg cgcattactt gactgggaga gcgctgagag
     1921 ggagcgacaa agtttttgag aagtgggaaa aactcgatcc tgatgcccca tttgacctct
     1981 atgataccga aatcaagaat gttcaacgga ggaatactcg caggttcgga tctcatgatc
     2041 tgtttgcgaa gctcgcggaa cctaaatatc aggcgctctg gagagaggac gcttctttcc
     2101 tcacgaggta tgcggtttac aatagcattg tcagaaaact gaatcacgct aaaatgtttg
     2161 cgacttttac tcttccggat gctaccgccc acccgatctg gacgcggttt gacaaactcg
     2221 gcggcaacct gcaccagtac actttcttgt ttaacgaatt tggcgagggc aggcacgcca
     2281 ttcggtttca gaagctgttg acggttgagg atggcgttgc taaagaggtc gacgacgtca
     2341 cggttccgat ttctatgtcc gcgcagctgg atgacctctt gcctcgggac ccacacgagc
     2401 tcgttgcact ctacttccag gactacggtg cagaacaaca tctggctgga gagtttggcg
     2461 gcgcgaaaat tcaataccgc cgcgatcaat tgaaccacct gcacgccaga agaggcgcca
     2521 gagatgtcta ccttaatctg agcgtccgcg ttcagtcaca atccgaagcc aggggagaaa
     2581 ggcgccctcc gtatgcagcg gtcttcaggc ttgttggcga taaccaccgc gcgtttgttc
     2641 actttgataa attgtcagat tacctcgcag aacacccaga cgatggtaag ctggggtcgg
     2701 aaggtttgct ctctgggctc agagtcatgt cagttgcctt gggtcttagg acttccgcga
     2761 gcatatctgt cttccgcgtc gcaagaaagg acgaattgaa gccgaacagt gaaggccggg
     2821 tccctttttg cttcccgatc gaagggaacg aaaacctcgt tgctgtccac gagcggagcc
     2881 aactgttgaa gcttcccggt gaaacggaat cgaaagatct gagagcgatc agagaagagc
     2941 gccaaaggac gcttagacag ctccggacgc aacttgcata cttgcgcctt ctggttcgct
     3001 gcggtagtga agacgttgga agaagagaga ggtcatgggc taaactcata gagcaaccta
     3061 tggatgctaa tcaaatgacg cctgattgga gagaagcatt cgaagacgaa cttcagaaac
     3121 tgaaatccct ttacgggata tgcggcgatc gcgagtggac agaagcagtg tatgagtctg
     3181 tgaggcgcgt gtggcggcat atgggtaaac aggtgcgcga ttggagaaaa gacgttagga
     3241 gcggggaaag acctaagata cggggatatc agaaagacgt tgtcggggga aatagcattg
     3301 aacagattga atatttggag cgccaatata agttcctcaa atcctggtct ttcttcggca
     3361 aagtgtcagg ccaggtgata cgcgcggaaa agggatcgcg ctttgcaata actctgagag
     3421 aacatattga tcatgccaaa gaagatcggt tgaagaaact cgccgataga atcatcatgg
     3481 aggcgcttgg ttatgtctac gccttggacg atgaacgggg aaagggaaag tgggtcgcca
     3541 agtatccacc ttgccaactc attctcctcg aagaactttc cgaataccag tttaacaacg
     3601 atcggccgcc atcagagaat aatcaactga tgcagtggtc ccatcgcggt gtgtttcaag
     3661 agttgctcaa tcaggcccaa gtccatgatc tgcttgttgg cacaatgtat gcagcctttt
     3721 cctcccggtt tgatgcaaga acaggggctc ctggcatacg ctgtagacgg gtcccggcga
     3781 ggtgcgcccg cgaacaaaac cctgaaccgt tcccctggtg gttgaacaag ttcgttgcgg
     3841 agcacaagct ggacgggtgt cctctgcggg ccgacgatct tattcccacc ggggaagggg
     3901 aattctttgt gagccctttc tcggcggagg aaggggattt tcaccaaata catgcagatc
     3961 ttaatgccgc acaaaatttg cagaggagac tgtggtcaga ctttgatatt agtcagatac
     4021 gcctccgctg tgactgggga gaggtcgatg gcgagcctgt gttgatacca agaacgaccg
     4081 gaaagaggac agccgattcg tatggaaaca aggtttttta cacgaagacg ggcgttactt
     4141 actacgaaag agaaagaggg aagaagagaa ggaaagtctt tgcccaagaa gaattgagcg
     4201 aggaagaagc cgagctcttg gtcgaagcgg acgaggcacg ggaaaagtct gtcgtcctca
     4261 tgagggaccc ttccggaatt attaaccggg gagattggac gcggcagaaa gagttttggt
     4321 ccatggttaa tcaacgcata gaaggctacc ttgtcaagca aataagaagt cgcgtgagat
     4381 tgcaggagag tgcatgtgag aacactgggg acataaagcg tcctgctgcc accaaaaagg
     4441 ccggacaggc taagaaaaag aagtgagacg actagtggcg gccgccgacg tccgatcgtt
     4501 caaacatttg gcaataaagt ttcttaagat tgaatcctgt tgccggtctt gcgatgatta
     4561 tcatataatt tctgttgaat tacgttaagc atgtaataat taacatgtaa tgcatgacgt
     4621 tatttatgag atgggttttt atgattagag tcccgcaatt atacatttaa tacgcgatag
     4681 aaaacaaaat atagcgcgca aactaggata aattatcgcg cgcggtgtca tctatgttac
     4741 tagatcggga attgatcccc cctcgacagc ttccggaaag ggcgaattcg caactttgta
     4801 tacaaaagtt gaacgagaaa cgtaaaatga tataaatatc aatatattaa attagatttt
     4861 gcataaaaaa cagactacat aatactgtaa aacacaacat atccagtcac tatgccatcc
     4921 agctgatatc ccctatagtg agtcgtatta catggtcata gctgtttcct ggcagctctg
     4981 gcccgtgtct caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgcct
     5041 cctctggacc agccaggaca gaaatgcctc gacttcgctg ctacccaagg ttgccgggtg
     5101 acgcacaccg tggaaacgga tgaaggcacg aacccagtgg acataagcct gttcggttcg
     5161 taagctgtaa tgcaagtagc gtatgcgctc acgcaactgg tccagaacct tgaccgaacg
     5221 cagcggtggt aacggcgcag tggcggtttt catggcttgt tatgactgtt tttttggggt
     5281 acagtctatg cctcgggcat ccaagcagca agcgcgttac gccgtgggtc gatgtttgat
     5341 gttatggagc agcaacgatg ttacgcagca gggcagtcgc cctaaaacaa agttaaacat
     5401 tatgagggaa gcggtgatcg ccgaagtatc gactcaacta tcagaggtag ttggcgtcat
     5461 cgagcgccat ctcgaaccga cgttgctggc cgtacatttg tacggctccg cagtggatgg
     5521 cggcctgaag ccacacagtg atattgattt gctggttacg gtgaccgtaa ggcttgatga
     5581 aacaacgcgg cgagctttga tcaacgacct tttggaaact tcggcttccc ctggagagag
     5641 cgagattctc cgcgctgtag aagtcaccat tgttgtgcac gacgacatca ttccgtggcg
     5701 ttatccagct aagcgcgaac tgcaatttgg agaatggcag cgcaatgaca ttcttgcagg
     5761 tatcttcgag ccagccacga tcgacattga tctggctatc ttgctgacaa aagcaagaga
     5821 acatagcgtt gccttggtag gtccagcggc ggaggaactc tttgatccgg ttcctgaaca
     5881 ggatctattt gaggcgctaa atgaaacctt aacgctatgg aactcgccgc ccgactgggc
     5941 tggcgatgag cgaaatgtag tgcttacgtt gtcccgcatt tggtacagcg cagtaaccgg
     6001 caaaatcgcg ccgaaggatg tcgctgccga ctgggcaatg gagcgcctgc cggcccagta
     6061 tcagcccgtc atacttgaag ctagacaggc ttatcttgga caagaagaag atcgcttggc
     6121 ctcgcgcgca gatcagttgg aagaatttgt ccactacgtg aaaggcgaga tcaccaaggt
     6181 agtcggcaaa taaccctcga gccacccatg accaaaatcc cttaacgtga gttacgcgtc
     6241 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     6301 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     6361 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     6421 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     6481 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     6541 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     6601 ctgaacgggg ggttcgtgca cacagcccag cttggagcga ac
//
