LOCUS       sequence-255107-        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-D977A
FEATURES             Location/Qualifiers
     source          1..6642
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(151..178)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(270..356)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     405..427
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     419..436
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     420..436
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    452..551
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             576..596
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4005..4052
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      4083..4335
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4380..4503
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4520..4539)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4521..4539)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4544..4560)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4544..4560)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4991..5782
                     /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      5875..6463
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6364..6383
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6617..6634
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
       61 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atacgcgtac
      121 cgcgagccag gaagagtttg tagaaacgca aaaaggccat ccgtcaggat ggccttctgc
      181 ttagtttgat gcctggcagt ttatggcggg cgtcctgccc gccaccctcc gggccgttgc
      241 ttcacaacgt tcaaatccgc tcccggcgga tttgtcctac tcaggagagc gttcaccgac
      301 aaacaacaga taaaacgaaa ggcccagtct tccgactgag cctttcgttt tatttgatgc
      361 ctggcagttc cctactctcg cgttaacgct tgcatggatg ttttcccagt cacgacgttg
      421 taaaacgacg gccagtctta agctcgggcc ccaaataatg attttatttt gactgatagt
      481 gacctgttcg ttgcaacaaa ttgatgagca atgctttttt ataatgccaa ctttgtacaa
      541 aaaagcaggc tccgaattcg cccttcacca tggctcctaa gaagaagcgg aaggttggta
      601 ttcacggggt gcctgcggct gccgtcaagt ccatgaaggt caagttgcgc ctggataaca
      661 tgccagagat cagagccgga ctttggaaac ttcacaccga ggttaatgcg ggtgtgcggt
      721 actatacgga atggcttagc cttttgaggc aagaaaatct ttatcggagg agtcccaatg
      781 gcgatggaga acaagaatgc tataaaactg ctgaggaatg caaggctgaa ctccttgaga
      841 gactcagagc ccgccaagtt gagaatgggc actgcggccc tgctgggagt gatgacgaac
      901 tgctgcaatt ggcacggcaa ctttatgaac ttctggtccc acaagcaatc ggggctaaag
      961 gtgatgcgca gcaaatcgca aggaagtttc ttagtcccct tgccgacaag gatgccgtgg
     1021 gtggtttggg aatagcaaaa gcaggaaata agcctaggtg ggttcggatg agggaggctg
     1081 gagagccagg ttgggaagag gaaaaggcta aagccgaggc gagaaagagt acggatagaa
     1141 ccgccgatgt tcttcgcgct cttgcagact tcggtcttaa acctcttatg agagtctaca
     1201 cagactcaga catgtccagc gtgcagtgga aaccacttcg caaaggacaa gcggtcagaa
     1261 cctgggatag agacatgttc caacaagcga tcgaaagaat gatgagttgg gaatcgtgga
     1321 atcagcgcgt tggagaagcg tacgcaaagc tcgtggaaca aaagtcgagg tttgaacaga
     1381 aaaattttgt gggacaagaa catcttgtcc aacttgtcaa tcaacttcaa caagacatga
     1441 aggaagcatc acacggcctg gagtcgaaag aacaaactgc gcattacttg actgggagag
     1501 cgctgagagg gagcgacaaa gtttttgaga agtgggaaaa actcgatcct gatgccccat
     1561 ttgacctcta tgataccgaa atcaagaatg ttcaacggag gaatactcgc aggttcggat
     1621 ctcatgatct gtttgcgaag ctcgcggaac ctaaatatca ggcgctctgg agagaggacg
     1681 cttctttcct cacgaggtat gcggtttaca atagcattgt cagaaaactg aatcacgcta
     1741 aaatgtttgc gacttttact cttccggatg ctaccgccca cccgatctgg acgcggtttg
     1801 acaaactcgg cggcaacctg caccagtaca ctttcttgtt taacgaattt ggcgagggca
     1861 ggcacgccat tcggtttcag aagctgttga cggttgagga tggcgttgct aaagaggtcg
     1921 acgacgtcac ggttccgatt tctatgtccg cgcagctgga tgacctcttg cctcgggacc
     1981 cacacgagct cgttgcactc tacttccagg actacggtgc agaacaacat ctggctggag
     2041 agtttggcgg cgcgaaaatt caataccgcc gcgatcaatt gaaccacctg cacgccagaa
     2101 gaggcgccag agatgtctac cttaatctga gcgtccgcgt tcagtcacaa tccgaagcca
     2161 ggggagaaag gcgccctccg tatgcagcgg tcttcaggct tgttggcgat aaccaccgcg
     2221 cgtttgttca ctttgataaa ttgtcagatt acctcgcaga acacccagac gatggtaagc
     2281 tggggtcgga aggtttgctc tctgggctca gagtcatgtc agttgacttg ggtcttagga
     2341 cttccgcgag catatctgtc ttccgcgtcg caagaaagga cgaattgaag ccgaacagtg
     2401 aaggccgggt ccctttttgc ttcccgatcg aagggaacga aaacctcgtt gctgtccacg
     2461 agcggagcca actgttgaag cttcccggtg aaacggaatc gaaagatctg agagcgatca
     2521 gagaagagcg ccaaaggacg cttagacagc tccggacgca acttgcatac ttgcgccttc
     2581 tggttcgctg cggtagtgaa gacgttggaa gaagagagag gtcatgggct aaactcatag
     2641 agcaacctat ggatgctaat caaatgacgc ctgattggag agaagcattc gaagacgaac
     2701 ttcagaaact gaaatccctt tacgggatat gcggcgatcg cgagtggaca gaagcagtgt
     2761 atgagtctgt gaggcgcgtg tggcggcata tgggtaaaca ggtgcgcgat tggagaaaag
     2821 acgttaggag cggggaaaga cctaagatac ggggatatca gaaagacgtt gtcgggggaa
     2881 atagcattga acagattgaa tatttggagc gccaatataa gttcctcaaa tcctggtctt
     2941 tcttcggcaa agtgtcaggc caggtgatac gcgcggaaaa gggatcgcgc tttgcaataa
     3001 ctctgagaga acatattgat catgccaaag aagatcggtt gaagaaactc gccgatagaa
     3061 tcatcatgga ggcgcttggt tatgtctacg ccttggacga tgaacgggga aagggaaagt
     3121 gggtcgccaa gtatccacct tgccaactca ttctcctcga agaactttcc gaataccagt
     3181 ttaacaacga tcggccgcca tcagagaata atcaactgat gcagtggtcc catcgcggtg
     3241 tgtttcaaga gttgctcaat caggcccaag tccatgatct gcttgttggc acaatgtatg
     3301 cagccttttc ctcccggttt gatgcaagaa caggggctcc tggcatacgc tgtagacggg
     3361 tcccggcgag gtgcgcccgc gaacaaaacc ctgaaccgtt cccctggtgg ttgaacaagt
     3421 tcgttgcgga gcacaagctg gacgggtgtc ctctgcgggc cgacgatctt attcccaccg
     3481 gggaagggga attctttgtg agccctttct cggcggagga aggggatttt caccaaatac
     3541 atgcagcgct taatgccgca caaaatttgc agaggagact gtggtcagac tttgatatta
     3601 gtcagatacg cctccgctgt gactggggag aggtcgatgg cgagcctgtg ttgataccaa
     3661 gaacgaccgg aaagaggaca gccgattcgt atggaaacaa ggttttttac acgaagacgg
     3721 gcgttactta ctacgaaaga gaaagaggga agaagagaag gaaagtcttt gcccaagaag
     3781 aattgagcga ggaagaagcc gagctcttgg tcgaagcgga cgaggcacgg gaaaagtctg
     3841 tcgtcctcat gagggaccct tccggaatta ttaaccgggg agattggacg cggcagaaag
     3901 agttttggtc catggttaat caacgcatag aaggctacct tgtcaagcaa ataagaagtc
     3961 gcgtgagatt gcaggagagt gcatgtgaga acactgggga cataaagcgt cctgctgcca
     4021 ccaaaaaggc cggacaggct aagaaaaaga agtgagacga ctagtggcgg ccgccgacgt
     4081 ccgatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg
     4141 cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat
     4201 gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat
     4261 acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat
     4321 ctatgttact agatcgggaa ttgatccccc ctcgacagct tccggaaagg gcgaattcgc
     4381 aactttgtat acaaaagttg aacgagaaac gtaaaatgat ataaatatca atatattaaa
     4441 ttagattttg cataaaaaac agactacata atactgtaaa acacaacata tccagtcact
     4501 atgccatcca gctgatatcc cctatagtga gtcgtattac atggtcatag ctgtttcctg
     4561 gcagctctgg cccgtgtctc aaaatctctg atgttacatt gcacaagata aaaatatatc
     4621 atcatgcctc ctctggacca gccaggacag aaatgcctcg acttcgctgc tacccaaggt
     4681 tgccgggtga cgcacaccgt ggaaacggat gaaggcacga acccagtgga cataagcctg
     4741 ttcggttcgt aagctgtaat gcaagtagcg tatgcgctca cgcaactggt ccagaacctt
     4801 gaccgaacgc agcggtggta acggcgcagt ggcggttttc atggcttgtt atgactgttt
     4861 ttttggggta cagtctatgc ctcgggcatc caagcagcaa gcgcgttacg ccgtgggtcg
     4921 atgtttgatg ttatggagca gcaacgatgt tacgcagcag ggcagtcgcc ctaaaacaaa
     4981 gttaaacatt atgagggaag cggtgatcgc cgaagtatcg actcaactat cagaggtagt
     5041 tggcgtcatc gagcgccatc tcgaaccgac gttgctggcc gtacatttgt acggctccgc
     5101 agtggatggc ggcctgaagc cacacagtga tattgatttg ctggttacgg tgaccgtaag
     5161 gcttgatgaa acaacgcggc gagctttgat caacgacctt ttggaaactt cggcttcccc
     5221 tggagagagc gagattctcc gcgctgtaga agtcaccatt gttgtgcacg acgacatcat
     5281 tccgtggcgt tatccagcta agcgcgaact gcaatttgga gaatggcagc gcaatgacat
     5341 tcttgcaggt atcttcgagc cagccacgat cgacattgat ctggctatct tgctgacaaa
     5401 agcaagagaa catagcgttg ccttggtagg tccagcggcg gaggaactct ttgatccggt
     5461 tcctgaacag gatctatttg aggcgctaaa tgaaacctta acgctatgga actcgccgcc
     5521 cgactgggct ggcgatgagc gaaatgtagt gcttacgttg tcccgcattt ggtacagcgc
     5581 agtaaccggc aaaatcgcgc cgaaggatgt cgctgccgac tgggcaatgg agcgcctgcc
     5641 ggcccagtat cagcccgtca tacttgaagc tagacaggct tatcttggac aagaagaaga
     5701 tcgcttggcc tcgcgcgcag atcagttgga agaatttgtc cactacgtga aaggcgagat
     5761 caccaaggta gtcggcaaat aaccctcgag ccacccatga ccaaaatccc ttaacgtgag
     5821 ttacgcgtcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     5881 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     5941 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     6001 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     6061 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     6121 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     6181 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     6241 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     6301 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     6361 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     6421 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     6481 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     6541 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     6601 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg ga
//
