LOCUS       sequence-263180-        6579 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry clone for BhCas12b_v4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6579)
  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 6579)
  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: pYPQ293 (BhCas12b_v4)
FEATURES             Location/Qualifiers
     source          1..6579
                     /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             5951..5998
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      6029..6281
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    6326..6449
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(6466..6485)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6467..6485)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6490..6506)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6490..6506)
                     /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 ccaccagatc
     2641 gttcatcttg aaaatcgaac ccaatgaaga ggttaaaaag ggcctgtgga aaacccacga
     2701 ggtgttgaat catggtatcg cgtactatat gaatatactt aagctcataa gacaggaagc
     2761 catttatgag catcatgaac aggatcctaa gaaccccaaa aaagtctcta aggcagaaat
     2821 acaggctgag ctttgggact ttgtgctcaa gatgcaaaag tgcaattcat ttacccacga
     2881 ggttgataaa gacgaagtct tcaatattct tcgggaattg tatgaagaac tggtcccatc
     2941 atcggtggaa aagaaaggtg aagctaacca acttagcaat aagtttctgt atccgctcgt
     3001 tgacccgaat tctcaatcgg gaaaagggac tgcctcctcg ggacggaaac cacggtggta
     3061 taacctgaaa atagcagggg acccaagttg ggaggaggag aagaaaaaat gggaggagga
     3121 caagaagaag gacccccttg ctaagatact cggcaagctt gctgaatatg ggttgattcc
     3181 tcttttcatt ccctatacgg atagcaatga accaattgtc aaggagatca agtggatgga
     3241 gaaatcgcgg aatcagagtg ttagaaggtt ggacaaagac atgtttatac aggcgctgga
     3301 gagattcctg tcgtgggaat cgtggaactt gaaggttaag gaggaatatg aaaaagtcga
     3361 aaaggaatat aagacgcttg aagaacggat taaagaagat attcaggctc ttaaagccct
     3421 tgagcagtat gaaaaggaac ggcaagagca actccttcgg gataccttga acacaaacga
     3481 gtatcgcctc tcgaagcggg gtctgcgcgg ctggagagaa atcatacaaa agtggcttaa
     3541 gatggatgag aatgagccga gcgagaagta cctggaggtt tttaaagatt atcaaaggaa
     3601 acatccgaga gaggcggggg attattcggt gtatgagttt ctctccaaaa aagagaatca
     3661 cttcatttgg agaaatcacc cggaataccc ctacctttat gctacatttt gcgagattga
     3721 taagaaaaaa aaggatgcca aacaacaagc cacctttacc ctggcggacc ccatcaacca
     3781 cccattgtgg gttcgctttg aggagagaag cggatctaat cttaataaat atagaatcct
     3841 tacggaacag ttgcatacgg agaaactgaa aaaaaagctc accgtccaac tcgatcgctt
     3901 gatctatcct acagaatcgg gaggatggga agagaagggt aaagttgata ttgtcctcct
     3961 tccatctaga caattctata accaaatctt cttggacata gaggagaagg gtaaacacgc
     4021 ctttacttac aaagacgaat ccattaagtt tcccctgaaa gggacattgg gaggagcccg
     4081 cgtccaattt gatcgggacc accttcgccg gtatccccac aaagtcgaaa gcggcaatgt
     4141 cgggcggatc tacttcaaca tgacagttaa tattgagcct acagaatccc cagtctccaa
     4201 gtcgctgaag atacatcgcg acgattttcc taaagttgtg aattttaaac ctaaggaact
     4261 gacggaatgg attaaggatt ctaaaggcaa aaagttgaaa tctggaatcg agtctctcga
     4321 aataggactt agggtgatga gtatagatct tgggcaaaga caagcggccg ctgcatcaat
     4381 ctttgaagtc gtggaccaaa aacccgatat tgaaggcaag cttttcttcc ccattaaggg
     4441 aacggagctc tacgccgtcc atcgcgcctc atttaacata aaactgccag gcgagaccct
     4501 ggttaagagt cgcgaggtct tgcgcaaggc gcgcgaagat aatcttaagc ttatgaacca
     4561 aaaacttaat ttcctcagga acgtgctgca ttttcaacaa tttgaagata ttactgagag
     4621 agaaaaacgg gtcacaaaat ggatctctcg ccaagaaaac agcgatgtcc cacttgtgta
     4681 tcaggatgaa cttattcaaa ttagagagtt gatgtataaa ccgtacaagg attgggtggc
     4741 gtttttgaaa cagctccaca agcgcctgga ggtggaaata gggaaagaag ttaagcactg
     4801 gcgcaaatcc ctgagcgacg gcaggaaggg gctttacggg attagcctga agaacattga
     4861 cgaaatcgac cggactagaa aattccttct caggtggagt ctgaggccta ctgagccggg
     4921 tgaagttcgc cgcttggaac caggccagcg ctttgcgatt gatcagttga atcaccttaa
     4981 cgcccttaaa gaggatcggc ttaagaagat ggcgaatacc attattatgc acgcgttggg
     5041 ctattgttac gacgtgagaa agaagaagtg gcaggctaaa aatcccgcgt gccagatcat
     5101 cctcttcgaa gatctttcca actacaaccc atatggcgag aggtcaaggt tcgagaatag
     5161 tcggctgatg aaatggagtc gcagggaaat cccacgccag gttgccctgc aaggagaaat
     5221 ctatggcctt caagttggtg aggtcggggc gcaattcagc agccggtttc acgcgaaaac
     5281 tggtagtccg gggatacgct gccgggtggt cacaaaagag aaactccagg ataaccggtt
     5341 ttttaaaaat ctgcagaggg aaggtcgcct gactcttgat aaaatcgcag tgctgaaaga
     5401 gggtgacctc tatcccgaca aaggtggcga gaaattcata agcctctcca aggatcgcaa
     5461 atgtgtcacg actcacgcag acattaacgc ggcgcaaaac ctccaaaagc ggttttggac
     5521 cagaacccac ggtttctata aagtctattg caaagcctac caggttgacg gtcagacggt
     5581 gtatatccca gaatccaagg atcaaaagca aaagatcatt gaagaatttg gtgaaggata
     5641 ttttattctt aaggacggcg tctacgagtg ggtcaatgcg gggaagctta aaattaaaaa
     5701 gggctcttcc aagcaatcgt cgagcgagct cgtcgactca gacatcctga aagactcatt
     5761 tgatctggcc agtgagttga aaggcgaaaa gctcatgttg tacagggatc cttctggaaa
     5821 tgtgttcccc tctgataagt ggatggccgc aggcgtcttc tttggcaaac tggagagaat
     5881 actcatatca aagttgacaa accagtattc aataagcaca atagaagatg actcaagcaa
     5941 gcaaagcatg aagcgtcctg ctgccaccaa aaaggccgga caggctaaga aaaagaagtg
     6001 agacgactag tggcggccgc cgacgtccga tcgttcaaac atttggcaat aaagtttctt
     6061 aagattgaat cctgttgccg gtcttgcgat gattatcata taatttctgt tgaattacgt
     6121 taagcatgta ataattaaca tgtaatgcat gacgttattt atgagatggg tttttatgat
     6181 tagagtcccg caattataca tttaatacgc gatagaaaac aaaatatagc gcgcaaacta
     6241 ggataaatta tcgcgcgcgg tgtcatctat gttactagat cgggaattga tcccccctcg
     6301 acagcttccg gaaagggcga attcgcaact ttgtatacaa aagttgaacg agaaacgtaa
     6361 aatgatataa atatcaatat attaaattag attttgcata aaaaacagac tacataatac
     6421 tgtaaaacac aacatatcca gtcactatgc catccagctg atatccccta tagtgagtcg
     6481 tattacatgg tcatagctgt ttcctggcag ctctggcccg tgtctcaaaa tctctgatgt
     6541 tacattgcac aagataaaaa tatatcatca tgcctcctc
//
