LOCUS       sequence-271506-        8542 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPRa Gateway entry clone for catalytically dead FnCas12a fused 
            with TV activation system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8542)
  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 8542)
  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: pYPQ239A (dFnCas12a)-TV
FEATURES             Location/Qualifiers
     source          1..8542
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(88..174)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     223..245
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     237..254
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     238..254
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    270..369
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             394..414
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4336..4383
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5467..5616
                     /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             5629..5778
                     /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      5801..6053
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    6098..6221
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(6238..6257)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6239..6257)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6262..6278)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6262..6278)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             6709..7500
                     /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      7593..8181
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8082..8101
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8335..8352
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(8511..8538)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 agtttgatgc ctggcagttt atggcgggcg tcctgcccgc caccctccgg gccgttgctt
       61 cacaacgttc aaatccgctc ccggcggatt tgtcctactc aggagagcgt tcaccgacaa
      121 acaacagata aaacgaaagg cccagtcttc cgactgagcc tttcgtttta tttgatgcct
      181 ggcagttccc tactctcgcg ttaacgcttg catggatgtt ttcccagtca cgacgttgta
      241 aaacgacggc cagtcttaag ctcgggcccc aaataatgat tttattttga ctgatagtga
      301 cctgttcgtt gcaacaaatt gatgagcaat gcttttttat aatgccaact ttgtacaaaa
      361 aagcaggctc cgaattcgcc cttcaccatg gctcctaaga agaagcggaa ggttggtatt
      421 cacggggtgc ctgcggcttc gatctatcaa gaattcgtta ataagtattc gctttctaaa
      481 acactgagat tcgaactcat acctcaagga aagacactcg agaacataaa ggctaggggc
      541 ctcatattgg atgatgaaaa gagagccaag gactataaaa aagccaaaca gataatcgac
      601 aagtaccacc aattcttcat agaagaaatc ctttccagcg tctgcattag cgaggatttg
      661 ttgcaaaatt acagcgatgt ctacttcaag cttaagaaga gcgatgacga caatctccag
      721 aaagacttca aatcagcgaa ggacacaatt aagaagcaga tcagcgagta tatcaaagat
      781 agtgaaaaat tcaagaacct tttcaatcag aacctgatag acgcaaaaaa aggacaagaa
      841 agcgacctca ttctttggtt gaagcagtct aaggacaacg ggattgagct ttttaaggcg
      901 aatagcgata taaccgacat cgacgaggcg cttgagatca taaagtcgtt taagggatgg
      961 acaacatact tcaaaggctt ccatgagaat cgcaaaaacg tctactccag caacgacatt
     1021 ccaacgtcga ttatatacag aattgttgat gataacctcc ctaaattcct cgaaaataag
     1081 gcaaaatatg aaagccttaa agataaagcg cctgaagcaa tcaattatga acaaatcaaa
     1141 aaggatcttg ctgaagaatt gacgtttgat atagactaca agacgtcaga agttaaccag
     1201 agggtgtttt cactcgacga ggtgtttgag attgctaatt tcaacaacta cctcaatcag
     1261 agtgggatca cgaagttcaa cactattata ggtggtaagt tcgtgaatgg tgagaacact
     1321 aaaagaaaag ggattaacga atatataaac ctttatagtc aacagatcaa cgacaagact
     1381 ttgaagaaat ataagatgag cgtcctcttc aagcagatac tcagtgacac ggaatccaag
     1441 agctttgtga tcgacaagct cgaagatgac tcggatgtgg tcactaccat gcaatccttc
     1501 tacgagcaaa ttgccgcttt caaaactgtg gaggagaaga gtataaagga gacactgtct
     1561 ctgctttttg acgatcttaa ggcccagaaa ttggatcttt ccaaaatata tttcaagaat
     1621 gataagtcac ttacggacct ttcccaacaa gtttttgacg attattcagt tattggtacg
     1681 gcggttcttg agtacattac gcagcagata gcccccaaga atctggacaa cccctctaag
     1741 aaagaacagg agttgatagc gaagaagaca gagaaggcga aatacctctc gctggagacc
     1801 ataaaattgg cactggaaga atttaacaag catcgcgaca tagataaaca gtgccgcttc
     1861 gaggaaattt tggcaaattt tgcagccatt ccaatgattt tcgacgagat agcgcaaaac
     1921 aaggataatt tggcacaaat ttcaataaaa tatcaaaacc agggcaagaa ggacctcttg
     1981 caggcttcgg cagaggatga tgttaaggct attaaagact tgttggacca aacgaacaat
     2041 ctgttgcaca agttgaaaat tttccacatt agtcaaagcg aggataaagc aaacatattg
     2101 gacaaagacg agcactttta tctggtgttt gaggagtgct acttcgagct tgctaatatt
     2161 gtcccacttt ataataaaat aagaaactac attacgcaaa agccatattc agatgaaaag
     2221 tttaagctca atttcgaaaa tagtactctt gccaacggct gggacaagaa taaggagcca
     2281 gataataccg ccatactttt tatcaaagat gataaatatt atcttggggt gatgaataag
     2341 aagaataata agatcttcga tgataaagcg ataaaggaaa ataagggtga aggctataaa
     2401 aaaattgttt acaaactgtt gccgggagca aataaaatgc tccccaaggt ttttttttcg
     2461 gcaaagagca ttaaatttta caatccttca gaagacattc tgcgcataag aaatcattcg
     2521 acacacacta aaaatggttc gccacaaaag ggctacgaga aatttgaatt caacattgag
     2581 gactgtcgga agttcattga tttctacaag cagtccatct ccaagcaccc ggagtggaaa
     2641 gattttgggt ttcggttttc cgacacgcag agatacaaca gcattgatga attttataga
     2701 gaggtcgaga atcaaggtta taagcttacc tttgaaaaca tttctgaatc atacattgat
     2761 tcagtggtca atcagggcaa actctatctt tttcaaatat acaacaagga ctttagtgct
     2821 tatagtaaag ggcggcccaa tttgcatact ctctattgga aagcgctgtt tgatgagcgg
     2881 aaccttcaag acgtcgtgta taagctcaac ggggaagccg agctctttta ccgcaagcag
     2941 tccataccga aaaaaataac acaccctgcc aaagaagcca tcgccaacaa gaataaagac
     3001 aatcctaaaa aagagtccgt cttcgaatat gatcttatta aggacaagag gtttacagaa
     3061 gataaatttt tcttccattg tcccataact atcaatttca aaagctctgg cgcgaacaaa
     3121 tttaacgacg aaatcaatct cttgttgaaa gaaaaagcca acgatgtgca cattctgtcg
     3181 atcgccaggg gagagcgcca cttggcatac tacacccttg ttgatgggaa aggaaatatt
     3241 attaaacagg acacatttaa tatcatcggc aacgatcgca tgaagaccaa ctatcatgac
     3301 aaactggcag caattgaaaa ggaccgcgac tcagcgagaa aagactggaa gaagatcaat
     3361 aatatcaaag aaatgaaaga gggttatttg tctcaagtgg tccatgagat cgcgaagttg
     3421 gtcattgaat ataatgccat agtggtcttc gaagatctga attttggatt taagcgcggc
     3481 aggttcaaag tcgaaaaaca ggtctaccaa aagttggaaa agatgctcat cgaaaagctg
     3541 aattaccttg tcttcaaaga taacgaattc gataaaaccg ggggggtctt gagggcctac
     3601 caactgactg caccctttga gacttttaaa aagatgggta aacagacagg aataatttac
     3661 tatgttcctg ccggtttcac tagcaagatt tgccccgtta ccggattcgt gaatcaactc
     3721 tatcccaaat acgaatccgt gagcaagagt caggaattct tctccaaatt tgataaaata
     3781 tgctataatc tcgacaaagg ttatttcgag ttctcgttcg actataagaa cttcggggat
     3841 aaggctgcca agggaaagtg gactatagca agctttggta gtcgccttat aaattttagg
     3901 aacagcgaca agaatcacaa ctgggacact cgggaagtct acccaacaaa agaactggag
     3961 aaactcttga aggattatag tatcgagtat gggcatgggg agtgtatcaa ggcagcgatt
     4021 tgtggagagt ccgacaaaaa gttttttgct aaactcacct cggtgctcaa cactatcctc
     4081 cagatgagaa attcaaaaac agggacagag ctcgattacc tcattagccc cgttgccgac
     4141 gtcaatggaa actttttcga ctcaagacag gctccaaaaa acatgccgca agatgcggac
     4201 gcgaatgggg cctatcacat aggcctgaaa gggcttatgc tccttgggag aattaaaaat
     4261 aaccaagaag gcaaaaaact caacctcgtc attaagaacg aagaatactt cgaatttgtt
     4321 cagaacagga ataacaagcg tcctgctgcc accaaaaagg ccggacaggc taagaaaaag
     4381 aagggagacg gctctggatc ggggtcgggt tctggctcag tcgacttgct tgatccgggg
     4441 acaccaatgg acgcggacct ggtggcttca tcgaccgtgg tttgggaaca ggacgccgat
     4501 ccattcgccg ggaccgccga tgactttcct gcttttaatg aggaagagtt ggcttggctg
     4561 atggaactcc tgccgcaggg cggctcaggg ggtctccttg accccggcac ccccatggac
     4621 gctgacctcg ttgcaagttc gacggttgtt tgggagcaag atgcagatcc gtttgcgggt
     4681 acagctgatg actttccagc cttcaacgaa gaggagctgg catggcttat ggagcttctg
     4741 cctcaggcca gaggagggtc gggtgggctc ttggatcctg ggacgccgat ggatgccgac
     4801 cttgtcgcgt cctctactgt ggtttgggag caggacgccg acccgttcgc tggtactgct
     4861 gatgactttc cggcctttaa tgaggaggaa ctcgcttggc tcatggagtt gttgccacaa
     4921 gggggtagtg gcggtcttct ggacccggga acgcctatgg atgctgactt ggtggcatcg
     4981 agcaccgtcg tctgggaaca ggatgcggac ccgtttgctg gtaccgctga tgattttccg
     5041 gcatttaacg aggaagaatt ggcgtggctc atggaattgc ttccccaggc gagagggggt
     5101 tccggtggct tgctggatcc gggaacgcct atggatgccg atctcgtggc gtcgtcaacc
     5161 gtggtctggg agcaagacgc tgacccgttt gccggcacag cagatgattt tccagctttc
     5221 aatgaggaag aactggcatg gttgatggaa ttgcttccac agggagggag cggaggcctc
     5281 ctcgatcctg gaacacctat ggacgcagac ctggttgcct cttcaactgt cgtgtgggaa
     5341 caagatgcag acccgttcgc aggtacagct gatgacttcc ctgcctttaa tgaggaggaa
     5401 ctcgcgtggc tcatggaact tttgccccaa gcacgcggtg gctctggggg tggcggatcg
     5461 ggaggagatg cgctggacga tttcgacttg gacatgttgg gctcagacgc tttggacgac
     5521 tttgatctcg acatgcttgg gtccgacgca ttggatgatt tcgaccttga catgcttggt
     5581 tccgacgcac tcgatgactt cgatcttgat atgctcgccc gcggatctga cgctctcgac
     5641 gacttcgatc ttgacatgtt gggctcagat gcgctcgacg actttgatct cgatatgttg
     5701 ggtagcgacg cactcgatga ctttgacttg gatatgctgg ggagcgacgc cttggacgat
     5761 ttcgatctgg acatgctgta aaagctttga taggacgtcc gatcgttcaa acatttggca
     5821 ataaagtttc ttaagattga atcctgttgc cggtcttgcg atgattatca tataatttct
     5881 gttgaattac gttaagcatg taataattaa catgtaatgc atgacgttat ttatgagatg
     5941 ggtttttatg attagagtcc cgcaattata catttaatac gcgatagaaa acaaaatata
     6001 gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct atgttactag atcgggaatt
     6061 gatcccccct cgacagcttc cggaaagggc gaattcgcaa ctttgtatac aaaagttgaa
     6121 cgagaaacgt aaaatgatat aaatatcaat atattaaatt agattttgca taaaaaacag
     6181 actacataat actgtaaaac acaacatatc cagtcactat gccatccagc tgatatcccc
     6241 tatagtgagt cgtattacat ggtcatagct gtttcctggc agctctggcc cgtgtctcaa
     6301 aatctctgat gttacattgc acaagataaa aatatatcat catgcctcct ctggaccagc
     6361 caggacagaa atgcctcgac ttcgctgcta cccaaggttg ccgggtgacg cacaccgtgg
     6421 aaacggatga aggcacgaac ccagtggaca taagcctgtt cggttcgtaa gctgtaatgc
     6481 aagtagcgta tgcgctcacg caactggtcc agaaccttga ccgaacgcag cggtggtaac
     6541 ggcgcagtgg cggttttcat ggcttgttat gactgttttt ttggggtaca gtctatgcct
     6601 cgggcatcca agcagcaagc gcgttacgcc gtgggtcgat gtttgatgtt atggagcagc
     6661 aacgatgtta cgcagcaggg cagtcgccct aaaacaaagt taaacattat gagggaagcg
     6721 gtgatcgccg aagtatcgac tcaactatca gaggtagttg gcgtcatcga gcgccatctc
     6781 gaaccgacgt tgctggccgt acatttgtac ggctccgcag tggatggcgg cctgaagcca
     6841 cacagtgata ttgatttgct ggttacggtg accgtaaggc ttgatgaaac aacgcggcga
     6901 gctttgatca acgacctttt ggaaacttcg gcttcccctg gagagagcga gattctccgc
     6961 gctgtagaag tcaccattgt tgtgcacgac gacatcattc cgtggcgtta tccagctaag
     7021 cgcgaactgc aatttggaga atggcagcgc aatgacattc ttgcaggtat cttcgagcca
     7081 gccacgatcg acattgatct ggctatcttg ctgacaaaag caagagaaca tagcgttgcc
     7141 ttggtaggtc cagcggcgga ggaactcttt gatccggttc ctgaacagga tctatttgag
     7201 gcgctaaatg aaaccttaac gctatggaac tcgccgcccg actgggctgg cgatgagcga
     7261 aatgtagtgc ttacgttgtc ccgcatttgg tacagcgcag taaccggcaa aatcgcgccg
     7321 aaggatgtcg ctgccgactg ggcaatggag cgcctgccgg cccagtatca gcccgtcata
     7381 cttgaagcta gacaggctta tcttggacaa gaagaagatc gcttggcctc gcgcgcagat
     7441 cagttggaag aatttgtcca ctacgtgaaa ggcgagatca ccaaggtagt cggcaaataa
     7501 ccctcgagcc acccatgacc aaaatccctt aacgtgagtt acgcgtcgtt ccactgagcg
     7561 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     7621 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     7681 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     7741 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     7801 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     7861 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     7921 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     7981 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     8041 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     8101 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     8161 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     8221 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     8281 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     8341 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg
     8401 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     8461 aacgcaatta atacgcgtac cgcgagccag gaagagtttg tagaaacgca aaaaggccat
     8521 ccgtcaggat ggccttctgc tt
//
