LOCUS       sequence-271504-        8029 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPRa Gateway entry clone for catalytically dead AaCas12b (D570A) 
            fused with TV activation system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8029)
  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 8029)
  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 (AaCas12b)-D570A-TV
FEATURES             Location/Qualifiers
     source          1..8029
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(1..87)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     136..158
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     150..167
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     151..167
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    183..282
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             307..327
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3736..3783
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4867..5016
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             5029..5178
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     terminator      5201..5453
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    5498..5621
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5638..5657)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5639..5657)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5662..5678)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5662..5678)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             6109..6900
                     /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      6993..7581
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7482..7501
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7735..7752
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(7911..7938)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 atttgtccta ctcaggagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc
       61 ttccgactga gcctttcgtt ttatttgatg cctggcagtt ccctactctc gcgttaacgc
      121 ttgcatggat gttttcccag tcacgacgtt gtaaaacgac ggccagtctt aagctcgggc
      181 cccaaataat gattttattt tgactgatag tgacctgttc gttgcaacaa attgatgagc
      241 aatgcttttt tataatgcca actttgtaca aaaaagcagg ctccgaattc gcccttcacc
      301 atggctccta agaagaagcg gaaggttggt attcacgggg tgcctgcggc tgccgtcaag
      361 tccatgaagg tcaagttgcg cctggataac atgccagaga tcagagccgg actttggaaa
      421 cttcacaccg aggttaatgc gggtgtgcgg tactatacgg aatggcttag ccttttgagg
      481 caagaaaatc tttatcggag gagtcccaat ggcgatggag aacaagaatg ctataaaact
      541 gctgaggaat gcaaggctga actccttgag agactcagag cccgccaagt tgagaatggg
      601 cactgcggcc ctgctgggag tgatgacgaa ctgctgcaat tggcacggca actttatgaa
      661 cttctggtcc cacaagcaat cggggctaaa ggtgatgcgc agcaaatcgc aaggaagttt
      721 cttagtcccc ttgccgacaa ggatgccgtg ggtggtttgg gaatagcaaa agcaggaaat
      781 aagcctaggt gggttcggat gagggaggct ggagagccag gttgggaaga ggaaaaggct
      841 aaagccgagg cgagaaagag tacggataga accgccgatg ttcttcgcgc tcttgcagac
      901 ttcggtctta aacctcttat gagagtctac acagactcag acatgtccag cgtgcagtgg
      961 aaaccacttc gcaaaggaca agcggtcaga acctgggata gagacatgtt ccaacaagcg
     1021 atcgaaagaa tgatgagttg ggaatcgtgg aatcagcgcg ttggagaagc gtacgcaaag
     1081 ctcgtggaac aaaagtcgag gtttgaacag aaaaattttg tgggacaaga acatcttgtc
     1141 caacttgtca atcaacttca acaagacatg aaggaagcat cacacggcct ggagtcgaaa
     1201 gaacaaactg cgcattactt gactgggaga gcgctgagag ggagcgacaa agtttttgag
     1261 aagtgggaaa aactcgatcc tgatgcccca tttgacctct atgataccga aatcaagaat
     1321 gttcaacgga ggaatactcg caggttcgga tctcatgatc tgtttgcgaa gctcgcggaa
     1381 cctaaatatc aggcgctctg gagagaggac gcttctttcc tcacgaggta tgcggtttac
     1441 aatagcattg tcagaaaact gaatcacgct aaaatgtttg cgacttttac tcttccggat
     1501 gctaccgccc acccgatctg gacgcggttt gacaaactcg gcggcaacct gcaccagtac
     1561 actttcttgt ttaacgaatt tggcgagggc aggcacgcca ttcggtttca gaagctgttg
     1621 acggttgagg atggcgttgc taaagaggtc gacgacgtca cggttccgat ttctatgtcc
     1681 gcgcagctgg atgacctctt gcctcgggac ccacacgagc tcgttgcact ctacttccag
     1741 gactacggtg cagaacaaca tctggctgga gagtttggcg gcgcgaaaat tcaataccgc
     1801 cgcgatcaat tgaaccacct gcacgccaga agaggcgcca gagatgtcta ccttaatctg
     1861 agcgtccgcg ttcagtcaca atccgaagcc aggggagaaa ggcgccctcc gtatgcagcg
     1921 gtcttcaggc ttgttggcga taaccaccgc gcgtttgttc actttgataa attgtcagat
     1981 tacctcgcag aacacccaga cgatggtaag ctggggtcgg aaggtttgct ctctgggctc
     2041 agagtcatgt cagttgcctt gggtcttagg acttccgcga gcatatctgt cttccgcgtc
     2101 gcaagaaagg acgaattgaa gccgaacagt gaaggccggg tccctttttg cttcccgatc
     2161 gaagggaacg aaaacctcgt tgctgtccac gagcggagcc aactgttgaa gcttcccggt
     2221 gaaacggaat cgaaagatct gagagcgatc agagaagagc gccaaaggac gcttagacag
     2281 ctccggacgc aacttgcata cttgcgcctt ctggttcgct gcggtagtga agacgttgga
     2341 agaagagaga ggtcatgggc taaactcata gagcaaccta tggatgctaa tcaaatgacg
     2401 cctgattgga gagaagcatt cgaagacgaa cttcagaaac tgaaatccct ttacgggata
     2461 tgcggcgatc gcgagtggac agaagcagtg tatgagtctg tgaggcgcgt gtggcggcat
     2521 atgggtaaac aggtgcgcga ttggagaaaa gacgttagga gcggggaaag acctaagata
     2581 cggggatatc agaaagacgt tgtcggggga aatagcattg aacagattga atatttggag
     2641 cgccaatata agttcctcaa atcctggtct ttcttcggca aagtgtcagg ccaggtgata
     2701 cgcgcggaaa agggatcgcg ctttgcaata actctgagag aacatattga tcatgccaaa
     2761 gaagatcggt tgaagaaact cgccgataga atcatcatgg aggcgcttgg ttatgtctac
     2821 gccttggacg atgaacgggg aaagggaaag tgggtcgcca agtatccacc ttgccaactc
     2881 attctcctcg aagaactttc cgaataccag tttaacaacg atcggccgcc atcagagaat
     2941 aatcaactga tgcagtggtc ccatcgcggt gtgtttcaag agttgctcaa tcaggcccaa
     3001 gtccatgatc tgcttgttgg cacaatgtat gcagcctttt cctcccggtt tgatgcaaga
     3061 acaggggctc ctggcatacg ctgtagacgg gtcccggcga ggtgcgcccg cgaacaaaac
     3121 cctgaaccgt tcccctggtg gttgaacaag ttcgttgcgg agcacaagct ggacgggtgt
     3181 cctctgcggg ccgacgatct tattcccacc ggggaagggg aattctttgt gagccctttc
     3241 tcggcggagg aaggggattt tcaccaaata catgcagatc ttaatgccgc acaaaatttg
     3301 cagaggagac tgtggtcaga ctttgatatt agtcagatac gcctccgctg tgactgggga
     3361 gaggtcgatg gcgagcctgt gttgatacca agaacgaccg gaaagaggac agccgattcg
     3421 tatggaaaca aggtttttta cacgaagacg ggcgttactt actacgaaag agaaagaggg
     3481 aagaagagaa ggaaagtctt tgcccaagaa gaattgagcg aggaagaagc cgagctcttg
     3541 gtcgaagcgg acgaggcacg ggaaaagtct gtcgtcctca tgagggaccc ttccggaatt
     3601 attaaccggg gagattggac gcggcagaaa gagttttggt ccatggttaa tcaacgcata
     3661 gaaggctacc ttgtcaagca aataagaagt cgcgtgagat tgcaggagag tgcatgtgag
     3721 aacactgggg acataaagcg tcctgctgcc accaaaaagg ccggacaggc taagaaaaag
     3781 aagggagacg gctctggatc ggggtcgggt tctggctcag tcgacttgct tgatccgggg
     3841 acaccaatgg acgcggacct ggtggcttca tcgaccgtgg tttgggaaca ggacgccgat
     3901 ccattcgccg ggaccgccga tgactttcct gcttttaatg aggaagagtt ggcttggctg
     3961 atggaactcc tgccgcaggg cggctcaggg ggtctccttg accccggcac ccccatggac
     4021 gctgacctcg ttgcaagttc gacggttgtt tgggagcaag atgcagatcc gtttgcgggt
     4081 acagctgatg actttccagc cttcaacgaa gaggagctgg catggcttat ggagcttctg
     4141 cctcaggcca gaggagggtc gggtgggctc ttggatcctg ggacgccgat ggatgccgac
     4201 cttgtcgcgt cctctactgt ggtttgggag caggacgccg acccgttcgc tggtactgct
     4261 gatgactttc cggcctttaa tgaggaggaa ctcgcttggc tcatggagtt gttgccacaa
     4321 gggggtagtg gcggtcttct ggacccggga acgcctatgg atgctgactt ggtggcatcg
     4381 agcaccgtcg tctgggaaca ggatgcggac ccgtttgctg gtaccgctga tgattttccg
     4441 gcatttaacg aggaagaatt ggcgtggctc atggaattgc ttccccaggc gagagggggt
     4501 tccggtggct tgctggatcc gggaacgcct atggatgccg atctcgtggc gtcgtcaacc
     4561 gtggtctggg agcaagacgc tgacccgttt gccggcacag cagatgattt tccagctttc
     4621 aatgaggaag aactggcatg gttgatggaa ttgcttccac agggagggag cggaggcctc
     4681 ctcgatcctg gaacacctat ggacgcagac ctggttgcct cttcaactgt cgtgtgggaa
     4741 caagatgcag acccgttcgc aggtacagct gatgacttcc ctgcctttaa tgaggaggaa
     4801 ctcgcgtggc tcatggaact tttgccccaa gcacgcggtg gctctggggg tggcggatcg
     4861 ggaggagatg cgctggacga tttcgacttg gacatgttgg gctcagacgc tttggacgac
     4921 tttgatctcg acatgcttgg gtccgacgca ttggatgatt tcgaccttga catgcttggt
     4981 tccgacgcac tcgatgactt cgatcttgat atgctcgccc gcggatctga cgctctcgac
     5041 gacttcgatc ttgacatgtt gggctcagat gcgctcgacg actttgatct cgatatgttg
     5101 ggtagcgacg cactcgatga ctttgacttg gatatgctgg ggagcgacgc cttggacgat
     5161 ttcgatctgg acatgctgta aaagctttga taggacgtcc gatcgttcaa acatttggca
     5221 ataaagtttc ttaagattga atcctgttgc cggtcttgcg atgattatca tataatttct
     5281 gttgaattac gttaagcatg taataattaa catgtaatgc atgacgttat ttatgagatg
     5341 ggtttttatg attagagtcc cgcaattata catttaatac gcgatagaaa acaaaatata
     5401 gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct atgttactag atcgggaatt
     5461 gatcccccct cgacagcttc cggaaagggc gaattcgcaa ctttgtatac aaaagttgaa
     5521 cgagaaacgt aaaatgatat aaatatcaat atattaaatt agattttgca taaaaaacag
     5581 actacataat actgtaaaac acaacatatc cagtcactat gccatccagc tgatatcccc
     5641 tatagtgagt cgtattacat ggtcatagct gtttcctggc agctctggcc cgtgtctcaa
     5701 aatctctgat gttacattgc acaagataaa aatatatcat catgcctcct ctggaccagc
     5761 caggacagaa atgcctcgac ttcgctgcta cccaaggttg ccgggtgacg cacaccgtgg
     5821 aaacggatga aggcacgaac ccagtggaca taagcctgtt cggttcgtaa gctgtaatgc
     5881 aagtagcgta tgcgctcacg caactggtcc agaaccttga ccgaacgcag cggtggtaac
     5941 ggcgcagtgg cggttttcat ggcttgttat gactgttttt ttggggtaca gtctatgcct
     6001 cgggcatcca agcagcaagc gcgttacgcc gtgggtcgat gtttgatgtt atggagcagc
     6061 aacgatgtta cgcagcaggg cagtcgccct aaaacaaagt taaacattat gagggaagcg
     6121 gtgatcgccg aagtatcgac tcaactatca gaggtagttg gcgtcatcga gcgccatctc
     6181 gaaccgacgt tgctggccgt acatttgtac ggctccgcag tggatggcgg cctgaagcca
     6241 cacagtgata ttgatttgct ggttacggtg accgtaaggc ttgatgaaac aacgcggcga
     6301 gctttgatca acgacctttt ggaaacttcg gcttcccctg gagagagcga gattctccgc
     6361 gctgtagaag tcaccattgt tgtgcacgac gacatcattc cgtggcgtta tccagctaag
     6421 cgcgaactgc aatttggaga atggcagcgc aatgacattc ttgcaggtat cttcgagcca
     6481 gccacgatcg acattgatct ggctatcttg ctgacaaaag caagagaaca tagcgttgcc
     6541 ttggtaggtc cagcggcgga ggaactcttt gatccggttc ctgaacagga tctatttgag
     6601 gcgctaaatg aaaccttaac gctatggaac tcgccgcccg actgggctgg cgatgagcga
     6661 aatgtagtgc ttacgttgtc ccgcatttgg tacagcgcag taaccggcaa aatcgcgccg
     6721 aaggatgtcg ctgccgactg ggcaatggag cgcctgccgg cccagtatca gcccgtcata
     6781 cttgaagcta gacaggctta tcttggacaa gaagaagatc gcttggcctc gcgcgcagat
     6841 cagttggaag aatttgtcca ctacgtgaaa ggcgagatca ccaaggtagt cggcaaataa
     6901 ccctcgagcc acccatgacc aaaatccctt aacgtgagtt acgcgtcgtt ccactgagcg
     6961 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     7021 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     7081 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     7141 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     7201 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     7261 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     7321 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     7381 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     7441 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     7501 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     7561 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     7621 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     7681 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     7741 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg
     7801 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     7861 aacgcaatta atacgcgtac cgcgagccag gaagagtttg tagaaacgca aaaaggccat
     7921 ccgtcaggat ggccttctgc ttagtttgat gcctggcagt ttatggcggg cgtcctgccc
     7981 gccaccctcc gggccgttgc ttcacaacgt tcaaatccgc tcccggcgg
//
