LOCUS       sequence-255106-        6788 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 6788)
  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 6788)
  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-SRDX
FEATURES             Location/Qualifiers
     source          1..6788
                     /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             569..589
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3998..4045
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      4222..4474
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4519..4642
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4659..4678)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4660..4678)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4683..4699)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4683..4699)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5130..5921
                     /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      6014..6602
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6503..6522
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6756..6773
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
       61 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatacgcg taccgcgagc
      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 gcttgcatgg atgttttccc agtcacgacg ttgtaaaacg
      421 acggccagtc ttaagctcgg gccccaaata atgattttat tttgactgat agtgacctgt
      481 tcgttgcaac aaattgatga gcaatgcttt tttataatgc caactttgta caaaaaagca
      541 ggctccgaat tcgcccttca ccatggctcc taagaagaag cggaaggttg gtattcacgg
      601 ggtgcctgcg gctgccgtca agtccatgaa ggtcaagttg cgcctggata acatgccaga
      661 gatcagagcc ggactttgga aacttcacac cgaggttaat gcgggtgtgc ggtactatac
      721 ggaatggctt agccttttga ggcaagaaaa tctttatcgg aggagtccca atggcgatgg
      781 agaacaagaa tgctataaaa ctgctgagga atgcaaggct gaactccttg agagactcag
      841 agcccgccaa gttgagaatg ggcactgcgg ccctgctggg agtgatgacg aactgctgca
      901 attggcacgg caactttatg aacttctggt cccacaagca atcggggcta aaggtgatgc
      961 gcagcaaatc gcaaggaagt ttcttagtcc ccttgccgac aaggatgccg tgggtggttt
     1021 gggaatagca aaagcaggaa ataagcctag gtgggttcgg atgagggagg ctggagagcc
     1081 aggttgggaa gaggaaaagg ctaaagccga ggcgagaaag agtacggata gaaccgccga
     1141 tgttcttcgc gctcttgcag acttcggtct taaacctctt atgagagtct acacagactc
     1201 agacatgtcc agcgtgcagt ggaaaccact tcgcaaagga caagcggtca gaacctggga
     1261 tagagacatg ttccaacaag cgatcgaaag aatgatgagt tgggaatcgt ggaatcagcg
     1321 cgttggagaa gcgtacgcaa agctcgtgga acaaaagtcg aggtttgaac agaaaaattt
     1381 tgtgggacaa gaacatcttg tccaacttgt caatcaactt caacaagaca tgaaggaagc
     1441 atcacacggc ctggagtcga aagaacaaac tgcgcattac ttgactggga gagcgctgag
     1501 agggagcgac aaagtttttg agaagtggga aaaactcgat cctgatgccc catttgacct
     1561 ctatgatacc gaaatcaaga atgttcaacg gaggaatact cgcaggttcg gatctcatga
     1621 tctgtttgcg aagctcgcgg aacctaaata tcaggcgctc tggagagagg acgcttcttt
     1681 cctcacgagg tatgcggttt acaatagcat tgtcagaaaa ctgaatcacg ctaaaatgtt
     1741 tgcgactttt actcttccgg atgctaccgc ccacccgatc tggacgcggt ttgacaaact
     1801 cggcggcaac ctgcaccagt acactttctt gtttaacgaa tttggcgagg gcaggcacgc
     1861 cattcggttt cagaagctgt tgacggttga ggatggcgtt gctaaagagg tcgacgacgt
     1921 cacggttccg atttctatgt ccgcgcagct ggatgacctc ttgcctcggg acccacacga
     1981 gctcgttgca ctctacttcc aggactacgg tgcagaacaa catctggctg gagagtttgg
     2041 cggcgcgaaa attcaatacc gccgcgatca attgaaccac ctgcacgcca gaagaggcgc
     2101 cagagatgtc taccttaatc tgagcgtccg cgttcagtca caatccgaag ccaggggaga
     2161 aaggcgccct ccgtatgcag cggtcttcag gcttgttggc gataaccacc gcgcgtttgt
     2221 tcactttgat aaattgtcag attacctcgc agaacaccca gacgatggta agctggggtc
     2281 ggaaggtttg ctctctgggc tcagagtcat gtcagttgcc ttgggtctta ggacttccgc
     2341 gagcatatct gtcttccgcg tcgcaagaaa ggacgaattg aagccgaaca gtgaaggccg
     2401 ggtccctttt tgcttcccga tcgaagggaa cgaaaacctc gttgctgtcc acgagcggag
     2461 ccaactgttg aagcttcccg gtgaaacgga atcgaaagat ctgagagcga tcagagaaga
     2521 gcgccaaagg acgcttagac agctccggac gcaacttgca tacttgcgcc ttctggttcg
     2581 ctgcggtagt gaagacgttg gaagaagaga gaggtcatgg gctaaactca tagagcaacc
     2641 tatggatgct aatcaaatga cgcctgattg gagagaagca ttcgaagacg aacttcagaa
     2701 actgaaatcc ctttacggga tatgcggcga tcgcgagtgg acagaagcag tgtatgagtc
     2761 tgtgaggcgc gtgtggcggc atatgggtaa acaggtgcgc gattggagaa aagacgttag
     2821 gagcggggaa agacctaaga tacggggata tcagaaagac gttgtcgggg gaaatagcat
     2881 tgaacagatt gaatatttgg agcgccaata taagttcctc aaatcctggt ctttcttcgg
     2941 caaagtgtca ggccaggtga tacgcgcgga aaagggatcg cgctttgcaa taactctgag
     3001 agaacatatt gatcatgcca aagaagatcg gttgaagaaa ctcgccgata gaatcatcat
     3061 ggaggcgctt ggttatgtct acgccttgga cgatgaacgg ggaaagggaa agtgggtcgc
     3121 caagtatcca ccttgccaac tcattctcct cgaagaactt tccgaatacc agtttaacaa
     3181 cgatcggccg ccatcagaga ataatcaact gatgcagtgg tcccatcgcg gtgtgtttca
     3241 agagttgctc aatcaggccc aagtccatga tctgcttgtt ggcacaatgt atgcagcctt
     3301 ttcctcccgg tttgatgcaa gaacaggggc tcctggcata cgctgtagac gggtcccggc
     3361 gaggtgcgcc cgcgaacaaa accctgaacc gttcccctgg tggttgaaca agttcgttgc
     3421 ggagcacaag ctggacgggt gtcctctgcg ggccgacgat cttattccca ccggggaagg
     3481 ggaattcttt gtgagccctt tctcggcgga ggaaggggat tttcaccaaa tacatgcaga
     3541 tcttaatgcc gcacaaaatt tgcagaggag actgtggtca gactttgata ttagtcagat
     3601 acgcctccgc tgtgactggg gagaggtcga tggcgagcct gtgttgatac caagaacgac
     3661 cggaaagagg acagccgatt cgtatggaaa caaggttttt tacacgaaga cgggcgttac
     3721 ttactacgaa agagaaagag ggaagaagag aaggaaagtc tttgcccaag aagaattgag
     3781 cgaggaagaa gccgagctct tggtcgaagc ggacgaggca cgggaaaagt ctgtcgtcct
     3841 catgagggac ccttccggaa ttattaaccg gggagattgg acgcggcaga aagagttttg
     3901 gtccatggtt aatcaacgca tagaaggcta ccttgtcaag caaataagaa gtcgcgtgag
     3961 attgcaggag agtgcatgtg agaacactgg ggacataaag cgtcctgctg ccaccaaaaa
     4021 ggccggacag gctaagaaaa agaagggaga cggctctgga tcggggtcgg gttctggctc
     4081 agtcgacctt gatcttgacc tcgaactcag acttggattt gctctcgatc tcgaccttga
     4141 acttagactc ggatttgctc ttgacctcga tcttgagctt agactcggat tcgcttagga
     4201 cgtcggcggc cgccgacgtc cgatcgttca aacatttggc aataaagttt cttaagattg
     4261 aatcctgttg ccggtcttgc gatgattatc atataatttc tgttgaatta cgttaagcat
     4321 gtaataatta acatgtaatg catgacgtta tttatgagat gggtttttat gattagagtc
     4381 ccgcaattat acatttaata cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa
     4441 ttatcgcgcg cggtgtcatc tatgttacta gatcgggaat tgatcccccc tcgacagctt
     4501 ccggaaaggg cgaattcgca actttgtata caaaagttga acgagaaacg taaaatgata
     4561 taaatatcaa tatattaaat tagattttgc ataaaaaaca gactacataa tactgtaaaa
     4621 cacaacatat ccagtcacta tgccatccag ctgatatccc ctatagtgag tcgtattaca
     4681 tggtcatagc tgtttcctgg cagctctggc ccgtgtctca aaatctctga tgttacattg
     4741 cacaagataa aaatatatca tcatgcctcc tctggaccag ccaggacaga aatgcctcga
     4801 cttcgctgct acccaaggtt gccgggtgac gcacaccgtg gaaacggatg aaggcacgaa
     4861 cccagtggac ataagcctgt tcggttcgta agctgtaatg caagtagcgt atgcgctcac
     4921 gcaactggtc cagaaccttg accgaacgca gcggtggtaa cggcgcagtg gcggttttca
     4981 tggcttgtta tgactgtttt tttggggtac agtctatgcc tcgggcatcc aagcagcaag
     5041 cgcgttacgc cgtgggtcga tgtttgatgt tatggagcag caacgatgtt acgcagcagg
     5101 gcagtcgccc taaaacaaag ttaaacatta tgagggaagc ggtgatcgcc gaagtatcga
     5161 ctcaactatc agaggtagtt ggcgtcatcg agcgccatct cgaaccgacg ttgctggccg
     5221 tacatttgta cggctccgca gtggatggcg gcctgaagcc acacagtgat attgatttgc
     5281 tggttacggt gaccgtaagg cttgatgaaa caacgcggcg agctttgatc aacgaccttt
     5341 tggaaacttc ggcttcccct ggagagagcg agattctccg cgctgtagaa gtcaccattg
     5401 ttgtgcacga cgacatcatt ccgtggcgtt atccagctaa gcgcgaactg caatttggag
     5461 aatggcagcg caatgacatt cttgcaggta tcttcgagcc agccacgatc gacattgatc
     5521 tggctatctt gctgacaaaa gcaagagaac atagcgttgc cttggtaggt ccagcggcgg
     5581 aggaactctt tgatccggtt cctgaacagg atctatttga ggcgctaaat gaaaccttaa
     5641 cgctatggaa ctcgccgccc gactgggctg gcgatgagcg aaatgtagtg cttacgttgt
     5701 cccgcatttg gtacagcgca gtaaccggca aaatcgcgcc gaaggatgtc gctgccgact
     5761 gggcaatgga gcgcctgccg gcccagtatc agcccgtcat acttgaagct agacaggctt
     5821 atcttggaca agaagaagat cgcttggcct cgcgcgcaga tcagttggaa gaatttgtcc
     5881 actacgtgaa aggcgagatc accaaggtag tcggcaaata accctcgagc cacccatgac
     5941 caaaatccct taacgtgagt tacgcgtcgt tccactgagc gtcagacccc gtagaaaaga
     6001 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     6061 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     6121 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     6181 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     6241 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     6301 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     6361 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     6421 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     6481 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     6541 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     6601 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     6661 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     6721 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     6781 aagagcgc
//
