LOCUS       sequence-263176-        8542 bp    DNA     circular SYN 15-APR-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 Apr 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"
     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             6527..6574
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7658..7807
                     /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             7820..7969
                     /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      7992..8244
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    8289..8412
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(8429..8448)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8430..8448)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8453..8469)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8453..8469)
                     /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 cctgcggctt cgatctatca
     2641 agaattcgtt aataagtatt cgctttctaa aacactgaga ttcgaactca tacctcaagg
     2701 aaagacactc gagaacataa aggctagggg cctcatattg gatgatgaaa agagagccaa
     2761 ggactataaa aaagccaaac agataatcga caagtaccac caattcttca tagaagaaat
     2821 cctttccagc gtctgcatta gcgaggattt gttgcaaaat tacagcgatg tctacttcaa
     2881 gcttaagaag agcgatgacg acaatctcca gaaagacttc aaatcagcga aggacacaat
     2941 taagaagcag atcagcgagt atatcaaaga tagtgaaaaa ttcaagaacc ttttcaatca
     3001 gaacctgata gacgcaaaaa aaggacaaga aagcgacctc attctttggt tgaagcagtc
     3061 taaggacaac gggattgagc tttttaaggc gaatagcgat ataaccgaca tcgacgaggc
     3121 gcttgagatc ataaagtcgt ttaagggatg gacaacatac ttcaaaggct tccatgagaa
     3181 tcgcaaaaac gtctactcca gcaacgacat tccaacgtcg attatataca gaattgttga
     3241 tgataacctc cctaaattcc tcgaaaataa ggcaaaatat gaaagcctta aagataaagc
     3301 gcctgaagca atcaattatg aacaaatcaa aaaggatctt gctgaagaat tgacgtttga
     3361 tatagactac aagacgtcag aagttaacca gagggtgttt tcactcgacg aggtgtttga
     3421 gattgctaat ttcaacaact acctcaatca gagtgggatc acgaagttca acactattat
     3481 aggtggtaag ttcgtgaatg gtgagaacac taaaagaaaa gggattaacg aatatataaa
     3541 cctttatagt caacagatca acgacaagac tttgaagaaa tataagatga gcgtcctctt
     3601 caagcagata ctcagtgaca cggaatccaa gagctttgtg atcgacaagc tcgaagatga
     3661 ctcggatgtg gtcactacca tgcaatcctt ctacgagcaa attgccgctt tcaaaactgt
     3721 ggaggagaag agtataaagg agacactgtc tctgcttttt gacgatctta aggcccagaa
     3781 attggatctt tccaaaatat atttcaagaa tgataagtca cttacggacc tttcccaaca
     3841 agtttttgac gattattcag ttattggtac ggcggttctt gagtacatta cgcagcagat
     3901 agcccccaag aatctggaca acccctctaa gaaagaacag gagttgatag cgaagaagac
     3961 agagaaggcg aaatacctct cgctggagac cataaaattg gcactggaag aatttaacaa
     4021 gcatcgcgac atagataaac agtgccgctt cgaggaaatt ttggcaaatt ttgcagccat
     4081 tccaatgatt ttcgacgaga tagcgcaaaa caaggataat ttggcacaaa tttcaataaa
     4141 atatcaaaac cagggcaaga aggacctctt gcaggcttcg gcagaggatg atgttaaggc
     4201 tattaaagac ttgttggacc aaacgaacaa tctgttgcac aagttgaaaa ttttccacat
     4261 tagtcaaagc gaggataaag caaacatatt ggacaaagac gagcactttt atctggtgtt
     4321 tgaggagtgc tacttcgagc ttgctaatat tgtcccactt tataataaaa taagaaacta
     4381 cattacgcaa aagccatatt cagatgaaaa gtttaagctc aatttcgaaa atagtactct
     4441 tgccaacggc tgggacaaga ataaggagcc agataatacc gccatacttt ttatcaaaga
     4501 tgataaatat tatcttgggg tgatgaataa gaagaataat aagatcttcg atgataaagc
     4561 gataaaggaa aataagggtg aaggctataa aaaaattgtt tacaaactgt tgccgggagc
     4621 aaataaaatg ctccccaagg tttttttttc ggcaaagagc attaaatttt acaatccttc
     4681 agaagacatt ctgcgcataa gaaatcattc gacacacact aaaaatggtt cgccacaaaa
     4741 gggctacgag aaatttgaat tcaacattga ggactgtcgg aagttcattg atttctacaa
     4801 gcagtccatc tccaagcacc cggagtggaa agattttggg tttcggtttt ccgacacgca
     4861 gagatacaac agcattgatg aattttatag agaggtcgag aatcaaggtt ataagcttac
     4921 ctttgaaaac atttctgaat catacattga ttcagtggtc aatcagggca aactctatct
     4981 ttttcaaata tacaacaagg actttagtgc ttatagtaaa gggcggccca atttgcatac
     5041 tctctattgg aaagcgctgt ttgatgagcg gaaccttcaa gacgtcgtgt ataagctcaa
     5101 cggggaagcc gagctctttt accgcaagca gtccataccg aaaaaaataa cacaccctgc
     5161 caaagaagcc atcgccaaca agaataaaga caatcctaaa aaagagtccg tcttcgaata
     5221 tgatcttatt aaggacaaga ggtttacaga agataaattt ttcttccatt gtcccataac
     5281 tatcaatttc aaaagctctg gcgcgaacaa atttaacgac gaaatcaatc tcttgttgaa
     5341 agaaaaagcc aacgatgtgc acattctgtc gatcgccagg ggagagcgcc acttggcata
     5401 ctacaccctt gttgatggga aaggaaatat tattaaacag gacacattta atatcatcgg
     5461 caacgatcgc atgaagacca actatcatga caaactggca gcaattgaaa aggaccgcga
     5521 ctcagcgaga aaagactgga agaagatcaa taatatcaaa gaaatgaaag agggttattt
     5581 gtctcaagtg gtccatgaga tcgcgaagtt ggtcattgaa tataatgcca tagtggtctt
     5641 cgaagatctg aattttggat ttaagcgcgg caggttcaaa gtcgaaaaac aggtctacca
     5701 aaagttggaa aagatgctca tcgaaaagct gaattacctt gtcttcaaag ataacgaatt
     5761 cgataaaacc gggggggtct tgagggccta ccaactgact gcaccctttg agacttttaa
     5821 aaagatgggt aaacagacag gaataattta ctatgttcct gccggtttca ctagcaagat
     5881 ttgccccgtt accggattcg tgaatcaact ctatcccaaa tacgaatccg tgagcaagag
     5941 tcaggaattc ttctccaaat ttgataaaat atgctataat ctcgacaaag gttatttcga
     6001 gttctcgttc gactataaga acttcgggga taaggctgcc aagggaaagt ggactatagc
     6061 aagctttggt agtcgcctta taaattttag gaacagcgac aagaatcaca actgggacac
     6121 tcgggaagtc tacccaacaa aagaactgga gaaactcttg aaggattata gtatcgagta
     6181 tgggcatggg gagtgtatca aggcagcgat ttgtggagag tccgacaaaa agttttttgc
     6241 taaactcacc tcggtgctca acactatcct ccagatgaga aattcaaaaa cagggacaga
     6301 gctcgattac ctcattagcc ccgttgccga cgtcaatgga aactttttcg actcaagaca
     6361 ggctccaaaa aacatgccgc aagatgcgga cgcgaatggg gcctatcaca taggcctgaa
     6421 agggcttatg ctccttggga gaattaaaaa taaccaagaa ggcaaaaaac tcaacctcgt
     6481 cattaagaac gaagaatact tcgaatttgt tcagaacagg aataacaagc gtcctgctgc
     6541 caccaaaaag gccggacagg ctaagaaaaa gaagggagac ggctctggat cggggtcggg
     6601 ttctggctca gtcgacttgc ttgatccggg gacaccaatg gacgcggacc tggtggcttc
     6661 atcgaccgtg gtttgggaac aggacgccga tccattcgcc gggaccgccg atgactttcc
     6721 tgcttttaat gaggaagagt tggcttggct gatggaactc ctgccgcagg gcggctcagg
     6781 gggtctcctt gaccccggca cccccatgga cgctgacctc gttgcaagtt cgacggttgt
     6841 ttgggagcaa gatgcagatc cgtttgcggg tacagctgat gactttccag ccttcaacga
     6901 agaggagctg gcatggctta tggagcttct gcctcaggcc agaggagggt cgggtgggct
     6961 cttggatcct gggacgccga tggatgccga ccttgtcgcg tcctctactg tggtttggga
     7021 gcaggacgcc gacccgttcg ctggtactgc tgatgacttt ccggccttta atgaggagga
     7081 actcgcttgg ctcatggagt tgttgccaca agggggtagt ggcggtcttc tggacccggg
     7141 aacgcctatg gatgctgact tggtggcatc gagcaccgtc gtctgggaac aggatgcgga
     7201 cccgtttgct ggtaccgctg atgattttcc ggcatttaac gaggaagaat tggcgtggct
     7261 catggaattg cttccccagg cgagaggggg ttccggtggc ttgctggatc cgggaacgcc
     7321 tatggatgcc gatctcgtgg cgtcgtcaac cgtggtctgg gagcaagacg ctgacccgtt
     7381 tgccggcaca gcagatgatt ttccagcttt caatgaggaa gaactggcat ggttgatgga
     7441 attgcttcca cagggaggga gcggaggcct cctcgatcct ggaacaccta tggacgcaga
     7501 cctggttgcc tcttcaactg tcgtgtggga acaagatgca gacccgttcg caggtacagc
     7561 tgatgacttc cctgccttta atgaggagga actcgcgtgg ctcatggaac ttttgcccca
     7621 agcacgcggt ggctctgggg gtggcggatc gggaggagat gcgctggacg atttcgactt
     7681 ggacatgttg ggctcagacg ctttggacga ctttgatctc gacatgcttg ggtccgacgc
     7741 attggatgat ttcgaccttg acatgcttgg ttccgacgca ctcgatgact tcgatcttga
     7801 tatgctcgcc cgcggatctg acgctctcga cgacttcgat cttgacatgt tgggctcaga
     7861 tgcgctcgac gactttgatc tcgatatgtt gggtagcgac gcactcgatg actttgactt
     7921 ggatatgctg gggagcgacg ccttggacga tttcgatctg gacatgctgt aaaagctttg
     7981 ataggacgtc cgatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg
     8041 ccggtcttgc gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta
     8101 acatgtaatg catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat
     8161 acatttaata cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg
     8221 cggtgtcatc tatgttacta gatcgggaat tgatcccccc tcgacagctt ccggaaaggg
     8281 cgaattcgca actttgtata caaaagttga acgagaaacg taaaatgata taaatatcaa
     8341 tatattaaat tagattttgc ataaaaaaca gactacataa tactgtaaaa cacaacatat
     8401 ccagtcacta tgccatccag ctgatatccc ctatagtgag tcgtattaca tggtcatagc
     8461 tgtttcctgg cagctctggc ccgtgtctca aaatctctga tgttacattg cacaagataa
     8521 aaatatatca tcatgcctcc tc
//
