LOCUS       sequence-263177-        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"
     CDS             358..1149
                     /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      1242..1830
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1731..1750
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1984..2001
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2160..2187)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2279..2365)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2414..2436
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2428..2445
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2429..2445
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2461..2560
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2585..2605
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6014..6061
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7145..7294
                     /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             7307..7456
                     /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      7479..7731
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    7776..7899
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(7916..7935)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7917..7935)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7940..7956)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7940..7956)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 tggaccagcc aggacagaaa tgcctcgact tcgctgctac ccaaggttgc cgggtgacgc
       61 acaccgtgga aacggatgaa ggcacgaacc cagtggacat aagcctgttc ggttcgtaag
      121 ctgtaatgca agtagcgtat gcgctcacgc aactggtcca gaaccttgac cgaacgcagc
      181 ggtggtaacg gcgcagtggc ggttttcatg gcttgttatg actgtttttt tggggtacag
      241 tctatgcctc gggcatccaa gcagcaagcg cgttacgccg tgggtcgatg tttgatgtta
      301 tggagcagca acgatgttac gcagcagggc agtcgcccta aaacaaagtt aaacatcatg
      361 agggaagcgg tgatcgccga agtatcgact caactatcag aggtagttgg cgtcatcgag
      421 cgccatctcg aaccgacgtt gctggccgta catttgtacg gctccgcagt ggatggcggc
      481 ctgaagccac acagtgatat tgatttgctg gttacggtga ccgtaaggct tgatgaaaca
      541 acgcggcgag ctttgatcaa cgaccttttg gaaacttcgg cttcccctgg agagagcgag
      601 attctccgcg ctgtagaagt caccattgtt gtgcacgacg acatcattcc gtggcgttat
      661 ccagctaagc gcgaactgca atttggagaa tggcagcgca atgacattct tgcaggtatc
      721 ttcgagccag ccacgatcga cattgatctg gctatcttgc tgacaaaagc aagagaacat
      781 agcgttgcct tggtaggtcc agcggcggag gaactctttg atccggttcc tgaacaggat
      841 ctatttgagg cgctaaatga aaccttaacg ctatggaact cgccgcccga ctgggctggc
      901 gatgagcgaa atgtagtgct tacgttgtcc cgcatttggt acagcgcagt aaccggcaaa
      961 atcgcgccga aggatgtcgc tgccgactgg gcaatggagc gcctgccggc ccagtatcag
     1021 cccgtcatac ttgaagctag acaggcttat cttggacaag aagaagatcg cttggcctcg
     1081 cgcgcagatc agttggaaga atttgtccac tacgtgaaag gcgagatcac caaggtagtc
     1141 ggcaaataac cctcgagcca cccatgacca aaatccctta acgtgagtta cgcgtcgttc
     1201 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     1261 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     1321 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     1381 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     1441 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     1501 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     1561 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     1621 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     1681 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     1741 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     1801 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     1861 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     1921 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     1981 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     2041 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     2101 agtgagcgca acgcaattaa tacgcgtacc gcgagccagg aagagtttgt agaaacgcaa
     2161 aaaggccatc cgtcaggatg gccttctgct tagtttgatg cctggcagtt tatggcgggc
     2221 gtcctgcccg ccaccctccg ggccgttgct tcacaacgtt caaatccgct cccggcggat
     2281 ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt
     2341 ccgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc gttaacgctt
     2401 gcatggatgt tttcccagtc acgacgttgt aaaacgacgg ccagtcttaa gctcgggccc
     2461 caaataatga ttttattttg actgatagtg acctgttcgt tgcaacaaat tgatgagcaa
     2521 tgctttttta taatgccaac tttgtacaaa aaagcaggct ccgaattcgc ccttcaccat
     2581 ggctcctaag aagaagcgga aggttggtat tcacggggtg cctgcggctg ccgtcaagtc
     2641 catgaaggtc aagttgcgcc tggataacat gccagagatc agagccggac tttggaaact
     2701 tcacaccgag gttaatgcgg gtgtgcggta ctatacggaa tggcttagcc ttttgaggca
     2761 agaaaatctt tatcggagga gtcccaatgg cgatggagaa caagaatgct ataaaactgc
     2821 tgaggaatgc aaggctgaac tccttgagag actcagagcc cgccaagttg agaatgggca
     2881 ctgcggccct gctgggagtg atgacgaact gctgcaattg gcacggcaac tttatgaact
     2941 tctggtccca caagcaatcg gggctaaagg tgatgcgcag caaatcgcaa ggaagtttct
     3001 tagtcccctt gccgacaagg atgccgtggg tggtttggga atagcaaaag caggaaataa
     3061 gcctaggtgg gttcggatga gggaggctgg agagccaggt tgggaagagg aaaaggctaa
     3121 agccgaggcg agaaagagta cggatagaac cgccgatgtt cttcgcgctc ttgcagactt
     3181 cggtcttaaa cctcttatga gagtctacac agactcagac atgtccagcg tgcagtggaa
     3241 accacttcgc aaaggacaag cggtcagaac ctgggataga gacatgttcc aacaagcgat
     3301 cgaaagaatg atgagttggg aatcgtggaa tcagcgcgtt ggagaagcgt acgcaaagct
     3361 cgtggaacaa aagtcgaggt ttgaacagaa aaattttgtg ggacaagaac atcttgtcca
     3421 acttgtcaat caacttcaac aagacatgaa ggaagcatca cacggcctgg agtcgaaaga
     3481 acaaactgcg cattacttga ctgggagagc gctgagaggg agcgacaaag tttttgagaa
     3541 gtgggaaaaa ctcgatcctg atgccccatt tgacctctat gataccgaaa tcaagaatgt
     3601 tcaacggagg aatactcgca ggttcggatc tcatgatctg tttgcgaagc tcgcggaacc
     3661 taaatatcag gcgctctgga gagaggacgc ttctttcctc acgaggtatg cggtttacaa
     3721 tagcattgtc agaaaactga atcacgctaa aatgtttgcg acttttactc ttccggatgc
     3781 taccgcccac ccgatctgga cgcggtttga caaactcggc ggcaacctgc accagtacac
     3841 tttcttgttt aacgaatttg gcgagggcag gcacgccatt cggtttcaga agctgttgac
     3901 ggttgaggat ggcgttgcta aagaggtcga cgacgtcacg gttccgattt ctatgtccgc
     3961 gcagctggat gacctcttgc ctcgggaccc acacgagctc gttgcactct acttccagga
     4021 ctacggtgca gaacaacatc tggctggaga gtttggcggc gcgaaaattc aataccgccg
     4081 cgatcaattg aaccacctgc acgccagaag aggcgccaga gatgtctacc ttaatctgag
     4141 cgtccgcgtt cagtcacaat ccgaagccag gggagaaagg cgccctccgt atgcagcggt
     4201 cttcaggctt gttggcgata accaccgcgc gtttgttcac tttgataaat tgtcagatta
     4261 cctcgcagaa cacccagacg atggtaagct ggggtcggaa ggtttgctct ctgggctcag
     4321 agtcatgtca gttgccttgg gtcttaggac ttccgcgagc atatctgtct tccgcgtcgc
     4381 aagaaaggac gaattgaagc cgaacagtga aggccgggtc cctttttgct tcccgatcga
     4441 agggaacgaa aacctcgttg ctgtccacga gcggagccaa ctgttgaagc ttcccggtga
     4501 aacggaatcg aaagatctga gagcgatcag agaagagcgc caaaggacgc ttagacagct
     4561 ccggacgcaa cttgcatact tgcgccttct ggttcgctgc ggtagtgaag acgttggaag
     4621 aagagagagg tcatgggcta aactcataga gcaacctatg gatgctaatc aaatgacgcc
     4681 tgattggaga gaagcattcg aagacgaact tcagaaactg aaatcccttt acgggatatg
     4741 cggcgatcgc gagtggacag aagcagtgta tgagtctgtg aggcgcgtgt ggcggcatat
     4801 gggtaaacag gtgcgcgatt ggagaaaaga cgttaggagc ggggaaagac ctaagatacg
     4861 gggatatcag aaagacgttg tcgggggaaa tagcattgaa cagattgaat atttggagcg
     4921 ccaatataag ttcctcaaat cctggtcttt cttcggcaaa gtgtcaggcc aggtgatacg
     4981 cgcggaaaag ggatcgcgct ttgcaataac tctgagagaa catattgatc atgccaaaga
     5041 agatcggttg aagaaactcg ccgatagaat catcatggag gcgcttggtt atgtctacgc
     5101 cttggacgat gaacggggaa agggaaagtg ggtcgccaag tatccacctt gccaactcat
     5161 tctcctcgaa gaactttccg aataccagtt taacaacgat cggccgccat cagagaataa
     5221 tcaactgatg cagtggtccc atcgcggtgt gtttcaagag ttgctcaatc aggcccaagt
     5281 ccatgatctg cttgttggca caatgtatgc agccttttcc tcccggtttg atgcaagaac
     5341 aggggctcct ggcatacgct gtagacgggt cccggcgagg tgcgcccgcg aacaaaaccc
     5401 tgaaccgttc ccctggtggt tgaacaagtt cgttgcggag cacaagctgg acgggtgtcc
     5461 tctgcgggcc gacgatctta ttcccaccgg ggaaggggaa ttctttgtga gccctttctc
     5521 ggcggaggaa ggggattttc accaaataca tgcagatctt aatgccgcac aaaatttgca
     5581 gaggagactg tggtcagact ttgatattag tcagatacgc ctccgctgtg actggggaga
     5641 ggtcgatggc gagcctgtgt tgataccaag aacgaccgga aagaggacag ccgattcgta
     5701 tggaaacaag gttttttaca cgaagacggg cgttacttac tacgaaagag aaagagggaa
     5761 gaagagaagg aaagtctttg cccaagaaga attgagcgag gaagaagccg agctcttggt
     5821 cgaagcggac gaggcacggg aaaagtctgt cgtcctcatg agggaccctt ccggaattat
     5881 taaccgggga gattggacgc ggcagaaaga gttttggtcc atggttaatc aacgcataga
     5941 aggctacctt gtcaagcaaa taagaagtcg cgtgagattg caggagagtg catgtgagaa
     6001 cactggggac ataaagcgtc ctgctgccac caaaaaggcc ggacaggcta agaaaaagaa
     6061 gggagacggc tctggatcgg ggtcgggttc tggctcagtc gacttgcttg atccggggac
     6121 accaatggac gcggacctgg tggcttcatc gaccgtggtt tgggaacagg acgccgatcc
     6181 attcgccggg accgccgatg actttcctgc ttttaatgag gaagagttgg cttggctgat
     6241 ggaactcctg ccgcagggcg gctcaggggg tctccttgac cccggcaccc ccatggacgc
     6301 tgacctcgtt gcaagttcga cggttgtttg ggagcaagat gcagatccgt ttgcgggtac
     6361 agctgatgac tttccagcct tcaacgaaga ggagctggca tggcttatgg agcttctgcc
     6421 tcaggccaga ggagggtcgg gtgggctctt ggatcctggg acgccgatgg atgccgacct
     6481 tgtcgcgtcc tctactgtgg tttgggagca ggacgccgac ccgttcgctg gtactgctga
     6541 tgactttccg gcctttaatg aggaggaact cgcttggctc atggagttgt tgccacaagg
     6601 gggtagtggc ggtcttctgg acccgggaac gcctatggat gctgacttgg tggcatcgag
     6661 caccgtcgtc tgggaacagg atgcggaccc gtttgctggt accgctgatg attttccggc
     6721 atttaacgag gaagaattgg cgtggctcat ggaattgctt ccccaggcga gagggggttc
     6781 cggtggcttg ctggatccgg gaacgcctat ggatgccgat ctcgtggcgt cgtcaaccgt
     6841 ggtctgggag caagacgctg acccgtttgc cggcacagca gatgattttc cagctttcaa
     6901 tgaggaagaa ctggcatggt tgatggaatt gcttccacag ggagggagcg gaggcctcct
     6961 cgatcctgga acacctatgg acgcagacct ggttgcctct tcaactgtcg tgtgggaaca
     7021 agatgcagac ccgttcgcag gtacagctga tgacttccct gcctttaatg aggaggaact
     7081 cgcgtggctc atggaacttt tgccccaagc acgcggtggc tctgggggtg gcggatcggg
     7141 aggagatgcg ctggacgatt tcgacttgga catgttgggc tcagacgctt tggacgactt
     7201 tgatctcgac atgcttgggt ccgacgcatt ggatgatttc gaccttgaca tgcttggttc
     7261 cgacgcactc gatgacttcg atcttgatat gctcgcccgc ggatctgacg ctctcgacga
     7321 cttcgatctt gacatgttgg gctcagatgc gctcgacgac tttgatctcg atatgttggg
     7381 tagcgacgca ctcgatgact ttgacttgga tatgctgggg agcgacgcct tggacgattt
     7441 cgatctggac atgctgtaaa agctttgata ggacgtccga tcgttcaaac atttggcaat
     7501 aaagtttctt aagattgaat cctgttgccg gtcttgcgat gattatcata taatttctgt
     7561 tgaattacgt taagcatgta ataattaaca tgtaatgcat gacgttattt atgagatggg
     7621 tttttatgat tagagtcccg caattataca tttaatacgc gatagaaaac aaaatatagc
     7681 gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat gttactagat cgggaattga
     7741 tcccccctcg acagcttccg gaaagggcga attcgcaact ttgtatacaa aagttgaacg
     7801 agaaacgtaa aatgatataa atatcaatat attaaattag attttgcata aaaaacagac
     7861 tacataatac tgtaaaacac aacatatcca gtcactatgc catccagctg atatccccta
     7921 tagtgagtcg tattacatgg tcatagctgt ttcctggcag ctctggcccg tgtctcaaaa
     7981 tctctgatgt tacattgcac aagataaaaa tatatcatca tgcctcctc
//
