LOCUS       sequence-255057-        6579 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 6579)
  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 6579)
  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: pYPQ291
FEATURES             Location/Qualifiers
     source          1..6579
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             145..936
                     /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      1029..1617
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1518..1537
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1771..1788
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(1947..1974)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2066..2152)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2201..2223
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2215..2232
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2216..2232
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2248..2347
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2372..2392
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5738..5785
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      5816..6068
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    6113..6236
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(6253..6272)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6254..6272)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6277..6293)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6277..6293)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 tgttatgact gtttttttgg ggtacagtct atgcctcggg catccaagca gcaagcgcgt
       61 tacgccgtgg gtcgatgttt gatgttatgg agcagcaacg atgttacgca gcagggcagt
      121 cgccctaaaa caaagttaaa cattatgagg gaagcggtga tcgccgaagt atcgactcaa
      181 ctatcagagg tagttggcgt catcgagcgc catctcgaac cgacgttgct ggccgtacat
      241 ttgtacggct ccgcagtgga tggcggcctg aagccacaca gtgatattga tttgctggtt
      301 acggtgaccg taaggcttga tgaaacaacg cggcgagctt tgatcaacga ccttttggaa
      361 acttcggctt cccctggaga gagcgagatt ctccgcgctg tagaagtcac cattgttgtg
      421 cacgacgaca tcattccgtg gcgttatcca gctaagcgcg aactgcaatt tggagaatgg
      481 cagcgcaatg acattcttgc aggtatcttc gagccagcca cgatcgacat tgatctggct
      541 atcttgctga caaaagcaag agaacatagc gttgccttgg taggtccagc ggcggaggaa
      601 ctctttgatc cggttcctga acaggatcta tttgaggcgc taaatgaaac cttaacgcta
      661 tggaactcgc cgcccgactg ggctggcgat gagcgaaatg tagtgcttac gttgtcccgc
      721 atttggtaca gcgcagtaac cggcaaaatc gcgccgaagg atgtcgctgc cgactgggca
      781 atggagcgcc tgccggccca gtatcagccc gtcatacttg aagctagaca ggcttatctt
      841 ggacaagaag aagatcgctt ggcctcgcgc gcagatcagt tggaagaatt tgtccactac
      901 gtgaaaggcg agatcaccaa ggtagtcggc aaataaccct cgagccaccc atgaccaaaa
      961 tcccttaacg tgagttacgc gtcgttccac tgagcgtcag accccgtaga aaagatcaaa
     1021 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     1081 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     1141 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     1201 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     1261 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     1321 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     1381 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     1441 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     1501 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     1561 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     1621 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     1681 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     1741 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     1801 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     1861 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatac gcgtaccgcg
     1921 agccaggaag agtttgtaga aacgcaaaaa ggccatccgt caggatggcc ttctgcttag
     1981 tttgatgcct ggcagtttat ggcgggcgtc ctgcccgcca ccctccgggc cgttgcttca
     2041 caacgttcaa atccgctccc ggcggatttg tcctactcag gagagcgttc accgacaaac
     2101 aacagataaa acgaaaggcc cagtcttccg actgagcctt tcgttttatt tgatgcctgg
     2161 cagttcccta ctctcgcgtt aacgcttgca tggatgtttt cccagtcacg acgttgtaaa
     2221 acgacggcca gtcttaagct cgggccccaa ataatgattt tattttgact gatagtgacc
     2281 tgttcgttgc aacaaattga tgagcaatgc ttttttataa tgccaacttt gtacaaaaaa
     2341 gcaggctccg aattcgccct tcaccatggc tcctaagaag aagcggaagg ttggtattca
     2401 cggggtgcct gcggctgcca caaggtcttt catacttaag atagagccaa acgaagaggt
     2461 caaaaaggga ttgtggaaaa cccatgaagt cctgaaccat ggcattgcct actacatgaa
     2521 catcctgaaa cttatacggc aggaggctat ttatgagcac cacgagcagg atccaaaaaa
     2581 ccccaaaaag gtttcgaagg ctgaaatcca ggccgaactg tgggacttcg ttctcaaaat
     2641 gcagaaatgt aattcgttca ctcatgaagt tgacaaagac gtcgtgttta acattttgag
     2701 ggagctttac gaggagttgg ttccgagctc cgtcgaaaag aagggtgaag caaatcagct
     2761 gtcgaataag ttcttgtacc ctttggtgga cccgaacagc caatctggaa aagggacagc
     2821 atcatcaggg cggaagcctc ggtggtataa cttgaagatt gctggagacc cttcgtggga
     2881 agaggaaaag aaaaagtggg aggaagataa gaagaaggac ccacttgcca aaattctcgg
     2941 caaacttgcc gaatatggat tgataccgct gttcatcccc tttacggatt ctaacgaacc
     3001 catcgttaaa gaaatcaagt ggatggaaaa atctcgcaat cagtccgtcc ggaggctgga
     3061 caaagatatg tttatacaag ctttggaacg ctttctctcg tgggagtcgt ggaatcttaa
     3121 ggtcaaagaa gagtatgaaa aggtcgagaa ggaacacaag acactggagg agaggattaa
     3181 ggaagacatt caagcattca agtcactgga gcaatacgaa aaggaacggc aggagcaatt
     3241 gcttcgcgac acgctcaata ccaatgaata taggctttcc aagaggggcc tgagaggatg
     3301 gcgggaaata atccagaaat ggctcaagat ggacgagaat gaaccttcag aaaaatatct
     3361 cgaggttttt aaagattacc aaaggaaaca tccacgcgag gcaggggatt acagcgtgta
     3421 cgagtttctc tccaagaagg aaaaccattt tatctggcgc aatcatcccg aatacccgta
     3481 cctctatgcg acgttctgcg aaatagacaa aaagaaaaaa gatgctaagc aacaagcgac
     3541 tttcacactt gcagatccca taaatcaccc attgtgggtg cggtttgaag aaaggtcggg
     3601 ctctaacctc aataagtaca gaattttgac ggagcagttg cacacagaaa agctgaagaa
     3661 gaagttgacg gttcagctgg atcgccttat ctacccaacc gagtctggtg gctgggaaga
     3721 gaaggggaaa gtcgacatag tgttgctgcc atctaggcag ttctataacc agatttttct
     3781 cgatatagaa gaaaagggta aacatgcatt tacgtataaa gacgagtcca taaagtttcc
     3841 actgaaagga acacttggcg gcgcaagggt gcagtttgat cgggaccacc ttcgcaggta
     3901 cccccacaag gttgaaagtg gaaacgttgg acggatctat tttaatatga ccgtcaacat
     3961 agaacccaca gaatcccctg tttccaaatc cctgaaaata caccgggacg attttcctaa
     4021 atttgtgaac tttaaaccga aggagttgac cgagtggata aaggacagta aagggaaaaa
     4081 gctgaagtcc ggtatcgaaa gcctggagat tgggctcaga gttatgtcga tagatctggg
     4141 tcaaaggcag gcagcagccg cctctatatt tgaggtcgtg gaccagaagc ccgacattga
     4201 aggtaaactg ttctttccga ttaaggggac ggaactctac gcagtccatc gcgcctcctt
     4261 caatataaag ctgccgggcg aaacactggt taaatcacgc gaggttttgc gcaaagcgcg
     4321 ggaagacaac ctgaaactca tgaatcaaaa gctcaatttc ctgcgcaatg tgttgcactt
     4381 ccagcagttt gaggatatta ccgaaagaga gaaaagggtt acaaaatgga tatcccggca
     4441 agaaaactct gatgttccgc tggtttacca ggatgagctt atacagatta gggaacttat
     4501 gtataaacct tacaaagatt gggttgcatt cctcaagcag ctgcataaga gacttgaagt
     4561 cgagatcggc aaagaagtca aacactggcg caagagcctg agcgatggtc ggaaagggtt
     4621 gtacggaatc agtttgaaaa atatcgacga aatagataga accaggaaat ttttgttgcg
     4681 ctggtcactg agaccaacgg aaccgggaga agtcagaagg ttggagccag gccagagatt
     4741 tgcaattgac cagctgaacc atctgaatgc actgaaagag gacagattga agaagatggc
     4801 gaatacgatt attatgcatg ctttgggtta ttgttacgac gttaggaaga agaaatggca
     4861 ggccaagaac cctgcgtgcc aaatcatcct gttcgaagat ctgagtaact acaatccgta
     4921 tgaagaaagg agtcgcttcg agaacagtaa actgatgaaa tggtcccggc gcgagatacc
     4981 acgccaagtt gcgcttcaag gggaaatata cgggcttcaa gttggggaag ttggagcgca
     5041 gttttctagc cggttccacg ccaagacagg gtccccgggt ataaggtgca gtgtggtgac
     5101 gaaagaaaag ttgcaggata atagattctt taaaaatctt caacgggaag ggcgcctgac
     5161 gcttgacaag attgcagtgt tgaaagaggg ggatttgtac cccgataaag gcggggagaa
     5221 gttcatttct ttgtcgaagg accgcaagtt ggttacgacg catgcagaca ttaacgcagc
     5281 acaaaatctg caaaaaagat tctggactcg gacgcatggt ttttacaagg tttactgtaa
     5341 agcatatcaa gtcgatggtc agacggttta cattcccgaa tctaaagatc agaaacagaa
     5401 aatcattgag gagttcggtg aaggttactt tatactcaag gacggtgttt acgaatgggg
     5461 taatgctggt aaactgaaaa ttaagaaggg gtcctccaag caatcatctt ctgagctcgt
     5521 cgacagcgac atccttaagg atagcttcga tcttgcctct gagctcaagg gagaaaagtt
     5581 gatgctgtat cgcgatccta gtggaaatgt ctttccctca gataaatgga tggcagcagg
     5641 tgtgttcttc gggaaattgg aacgcatact gatatcaaaa ctgaccaatc aatactctat
     5701 atctactatt gaagacgatt caagtaagca atcgatgaag cgtcctgctg ccaccaaaaa
     5761 ggccggacag gctaagaaaa agaagtgaga cgactagtgg cggccgccga cgtccgatcg
     5821 ttcaaacatt tggcaataaa gtttcttaag attgaatcct gttgccggtc ttgcgatgat
     5881 tatcatataa tttctgttga attacgttaa gcatgtaata attaacatgt aatgcatgac
     5941 gttatttatg agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat
     6001 agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt
     6061 actagatcgg gaattgatcc cccctcgaca gcttccggaa agggcgaatt cgcaactttg
     6121 tatacaaaag ttgaacgaga aacgtaaaat gatataaata tcaatatatt aaattagatt
     6181 ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc actatgccat
     6241 ccagctgata tcccctatag tgagtcgtat tacatggtca tagctgtttc ctggcagctc
     6301 tggcccgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatgc
     6361 ctcctctgga ccagccagga cagaaatgcc tcgacttcgc tgctacccaa ggttgccggg
     6421 tgacgcacac cgtggaaacg gatgaaggca cgaacccagt ggacataagc ctgttcggtt
     6481 cgtaagctgt aatgcaagta gcgtatgcgc tcacgcaact ggtccagaac cttgaccgaa
     6541 cgcagcggtg gtaacggcgc agtggcggtt ttcatggct
//
