LOCUS       sequence-255119-        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-E848A
FEATURES             Location/Qualifiers
     source          1..6642
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     123..142
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     376..393
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(552..579)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(671..757)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     806..828
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     820..837
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     821..837
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    853..952
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             977..997
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4406..4453
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      4484..4736
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4781..4904
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4921..4940)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4922..4940)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4945..4961)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4945..4961)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5392..6183
                     /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(6276..6642,1..222)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
       61 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
      121 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
      181 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
      241 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
      301 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
      361 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
      421 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
      481 tggaaagcgg gcagtgagcg caacgcaatt aatacgcgta ccgcgagcca ggaagagttt
      541 gtagaaacgc aaaaaggcca tccgtcagga tggccttctg cttagtttga tgcctggcag
      601 tttatggcgg gcgtcctgcc cgccaccctc cgggccgttg cttcacaacg ttcaaatccg
      661 ctcccggcgg atttgtccta ctcaggagag cgttcaccga caaacaacag ataaaacgaa
      721 aggcccagtc ttccgactga gcctttcgtt ttatttgatg cctggcagtt ccctactctc
      781 gcgttaacgc ttgcatggat gttttcccag tcacgacgtt gtaaaacgac ggccagtctt
      841 aagctcgggc cccaaataat gattttattt tgactgatag tgacctgttc gttgcaacaa
      901 attgatgagc aatgcttttt tataatgcca actttgtaca aaaaagcagg ctccgaattc
      961 gcccttcacc atggctccta agaagaagcg gaaggttggt attcacgggg tgcctgcggc
     1021 tgccgtcaag tccatgaagg tcaagttgcg cctggataac atgccagaga tcagagccgg
     1081 actttggaaa cttcacaccg aggttaatgc gggtgtgcgg tactatacgg aatggcttag
     1141 ccttttgagg caagaaaatc tttatcggag gagtcccaat ggcgatggag aacaagaatg
     1201 ctataaaact gctgaggaat gcaaggctga actccttgag agactcagag cccgccaagt
     1261 tgagaatggg cactgcggcc ctgctgggag tgatgacgaa ctgctgcaat tggcacggca
     1321 actttatgaa cttctggtcc cacaagcaat cggggctaaa ggtgatgcgc agcaaatcgc
     1381 aaggaagttt cttagtcccc ttgccgacaa ggatgccgtg ggtggtttgg gaatagcaaa
     1441 agcaggaaat aagcctaggt gggttcggat gagggaggct ggagagccag gttgggaaga
     1501 ggaaaaggct aaagccgagg cgagaaagag tacggataga accgccgatg ttcttcgcgc
     1561 tcttgcagac ttcggtctta aacctcttat gagagtctac acagactcag acatgtccag
     1621 cgtgcagtgg aaaccacttc gcaaaggaca agcggtcaga acctgggata gagacatgtt
     1681 ccaacaagcg atcgaaagaa tgatgagttg ggaatcgtgg aatcagcgcg ttggagaagc
     1741 gtacgcaaag ctcgtggaac aaaagtcgag gtttgaacag aaaaattttg tgggacaaga
     1801 acatcttgtc caacttgtca atcaacttca acaagacatg aaggaagcat cacacggcct
     1861 ggagtcgaaa gaacaaactg cgcattactt gactgggaga gcgctgagag ggagcgacaa
     1921 agtttttgag aagtgggaaa aactcgatcc tgatgcccca tttgacctct atgataccga
     1981 aatcaagaat gttcaacgga ggaatactcg caggttcgga tctcatgatc tgtttgcgaa
     2041 gctcgcggaa cctaaatatc aggcgctctg gagagaggac gcttctttcc tcacgaggta
     2101 tgcggtttac aatagcattg tcagaaaact gaatcacgct aaaatgtttg cgacttttac
     2161 tcttccggat gctaccgccc acccgatctg gacgcggttt gacaaactcg gcggcaacct
     2221 gcaccagtac actttcttgt ttaacgaatt tggcgagggc aggcacgcca ttcggtttca
     2281 gaagctgttg acggttgagg atggcgttgc taaagaggtc gacgacgtca cggttccgat
     2341 ttctatgtcc gcgcagctgg atgacctctt gcctcgggac ccacacgagc tcgttgcact
     2401 ctacttccag gactacggtg cagaacaaca tctggctgga gagtttggcg gcgcgaaaat
     2461 tcaataccgc cgcgatcaat tgaaccacct gcacgccaga agaggcgcca gagatgtcta
     2521 ccttaatctg agcgtccgcg ttcagtcaca atccgaagcc aggggagaaa ggcgccctcc
     2581 gtatgcagcg gtcttcaggc ttgttggcga taaccaccgc gcgtttgttc actttgataa
     2641 attgtcagat tacctcgcag aacacccaga cgatggtaag ctggggtcgg aaggtttgct
     2701 ctctgggctc agagtcatgt cagttgactt gggtcttagg acttccgcga gcatatctgt
     2761 cttccgcgtc gcaagaaagg acgaattgaa gccgaacagt gaaggccggg tccctttttg
     2821 cttcccgatc gaagggaacg aaaacctcgt tgctgtccac gagcggagcc aactgttgaa
     2881 gcttcccggt gaaacggaat cgaaagatct gagagcgatc agagaagagc gccaaaggac
     2941 gcttagacag ctccggacgc aacttgcata cttgcgcctt ctggttcgct gcggtagtga
     3001 agacgttgga agaagagaga ggtcatgggc taaactcata gagcaaccta tggatgctaa
     3061 tcaaatgacg cctgattgga gagaagcatt cgaagacgaa cttcagaaac tgaaatccct
     3121 ttacgggata tgcggcgatc gcgagtggac agaagcagtg tatgagtctg tgaggcgcgt
     3181 gtggcggcat atgggtaaac aggtgcgcga ttggagaaaa gacgttagga gcggggaaag
     3241 acctaagata cggggatatc agaaagacgt tgtcggggga aatagcattg aacagattga
     3301 atatttggag cgccaatata agttcctcaa atcctggtct ttcttcggca aagtgtcagg
     3361 ccaggtgata cgcgcggaaa agggatcgcg ctttgcaata actctgagag aacatattga
     3421 tcatgccaaa gaagatcggt tgaagaaact cgccgataga atcatcatgg aggcgcttgg
     3481 ttatgtctac gccttggacg atgaacgggg aaagggaaag tgggtcgcca agtatccacc
     3541 ttgccaactc attctcctcg ccgaactttc cgaataccag tttaacaacg atcggccgcc
     3601 atcagagaat aatcaactga tgcagtggtc ccatcgcggt gtgtttcaag agttgctcaa
     3661 tcaggcccaa gtccatgatc tgcttgttgg cacaatgtat gcagcctttt cctcccggtt
     3721 tgatgcaaga acaggggctc ctggcatacg ctgtagacgg gtcccggcga ggtgcgcccg
     3781 cgaacaaaac cctgaaccgt tcccctggtg gttgaacaag ttcgttgcgg agcacaagct
     3841 ggacgggtgt cctctgcggg ccgacgatct tattcccacc ggggaagggg aattctttgt
     3901 gagccctttc tcggcggagg aaggggattt tcaccaaata catgcagatc ttaatgccgc
     3961 acaaaatttg cagaggagac tgtggtcaga ctttgatatt agtcagatac gcctccgctg
     4021 tgactgggga gaggtcgatg gcgagcctgt gttgatacca agaacgaccg gaaagaggac
     4081 agccgattcg tatggaaaca aggtttttta cacgaagacg ggcgttactt actacgaaag
     4141 agaaagaggg aagaagagaa ggaaagtctt tgcccaagaa gaattgagcg aggaagaagc
     4201 cgagctcttg gtcgaagcgg acgaggcacg ggaaaagtct gtcgtcctca tgagggaccc
     4261 ttccggaatt attaaccggg gagattggac gcggcagaaa gagttttggt ccatggttaa
     4321 tcaacgcata gaaggctacc ttgtcaagca aataagaagt cgcgtgagat tgcaggagag
     4381 tgcatgtgag aacactgggg acataaagcg tcctgctgcc accaaaaagg ccggacaggc
     4441 taagaaaaag aagtgagacg actagtggcg gccgccgacg tccgatcgtt caaacatttg
     4501 gcaataaagt ttcttaagat tgaatcctgt tgccggtctt gcgatgatta tcatataatt
     4561 tctgttgaat tacgttaagc atgtaataat taacatgtaa tgcatgacgt tatttatgag
     4621 atgggttttt atgattagag tcccgcaatt atacatttaa tacgcgatag aaaacaaaat
     4681 atagcgcgca aactaggata aattatcgcg cgcggtgtca tctatgttac tagatcggga
     4741 attgatcccc cctcgacagc ttccggaaag ggcgaattcg caactttgta tacaaaagtt
     4801 gaacgagaaa cgtaaaatga tataaatatc aatatattaa attagatttt gcataaaaaa
     4861 cagactacat aatactgtaa aacacaacat atccagtcac tatgccatcc agctgatatc
     4921 ccctatagtg agtcgtatta catggtcata gctgtttcct ggcagctctg gcccgtgtct
     4981 caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgcct cctctggacc
     5041 agccaggaca gaaatgcctc gacttcgctg ctacccaagg ttgccgggtg acgcacaccg
     5101 tggaaacgga tgaaggcacg aacccagtgg acataagcct gttcggttcg taagctgtaa
     5161 tgcaagtagc gtatgcgctc acgcaactgg tccagaacct tgaccgaacg cagcggtggt
     5221 aacggcgcag tggcggtttt catggcttgt tatgactgtt tttttggggt acagtctatg
     5281 cctcgggcat ccaagcagca agcgcgttac gccgtgggtc gatgtttgat gttatggagc
     5341 agcaacgatg ttacgcagca gggcagtcgc cctaaaacaa agttaaacat tatgagggaa
     5401 gcggtgatcg ccgaagtatc gactcaacta tcagaggtag ttggcgtcat cgagcgccat
     5461 ctcgaaccga cgttgctggc cgtacatttg tacggctccg cagtggatgg cggcctgaag
     5521 ccacacagtg atattgattt gctggttacg gtgaccgtaa ggcttgatga aacaacgcgg
     5581 cgagctttga tcaacgacct tttggaaact tcggcttccc ctggagagag cgagattctc
     5641 cgcgctgtag aagtcaccat tgttgtgcac gacgacatca ttccgtggcg ttatccagct
     5701 aagcgcgaac tgcaatttgg agaatggcag cgcaatgaca ttcttgcagg tatcttcgag
     5761 ccagccacga tcgacattga tctggctatc ttgctgacaa aagcaagaga acatagcgtt
     5821 gccttggtag gtccagcggc ggaggaactc tttgatccgg ttcctgaaca ggatctattt
     5881 gaggcgctaa atgaaacctt aacgctatgg aactcgccgc ccgactgggc tggcgatgag
     5941 cgaaatgtag tgcttacgtt gtcccgcatt tggtacagcg cagtaaccgg caaaatcgcg
     6001 ccgaaggatg tcgctgccga ctgggcaatg gagcgcctgc cggcccagta tcagcccgtc
     6061 atacttgaag ctagacaggc ttatcttgga caagaagaag atcgcttggc ctcgcgcgca
     6121 gatcagttgg aagaatttgt ccactacgtg aaaggcgaga tcaccaaggt agtcggcaaa
     6181 taaccctcga gccacccatg accaaaatcc cttaacgtga gttacgcgtc gttccactga
     6241 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     6301 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     6361 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     6421 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     6481 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     6541 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     6601 ggttcgtgca cacagcccag cttggagcga acgacctaca cc
//
