LOCUS       sequence-271510-        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"
     terminator      complement(1..28)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(120..206)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     255..277
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     269..286
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     270..286
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    302..401
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             426..446
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3792..3839
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      3870..4122
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4167..4290
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4307..4326)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4308..4326)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4331..4347)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4331..4347)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4778..5569
                     /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      5662..6250
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6151..6170
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6404..6421
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aaaaggccat ccgtcaggat ggccttctgc ttagtttgat gcctggcagt ttatggcggg
       61 cgtcctgccc gccaccctcc gggccgttgc ttcacaacgt tcaaatccgc tcccggcgga
      121 tttgtcctac tcaggagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct
      181 tccgactgag cctttcgttt tatttgatgc ctggcagttc cctactctcg cgttaacgct
      241 tgcatggatg ttttcccagt cacgacgttg taaaacgacg gccagtctta agctcgggcc
      301 ccaaataatg attttatttt gactgatagt gacctgttcg ttgcaacaaa ttgatgagca
      361 atgctttttt ataatgccaa ctttgtacaa aaaagcaggc tccgaattcg cccttcacca
      421 tggctcctaa gaagaagcgg aaggttggta ttcacggggt gcctgcggct gccaccagat
      481 cgttcatctt gaaaatcgaa cccaatgaag aggttaaaaa gggcctgtgg aaaacccacg
      541 aggtgttgaa tcatggtatc gcgtactata tgaatatact taagctcata agacaggaag
      601 ccatttatga gcatcatgaa caggatccta agaaccccaa aaaagtctct aaggcagaaa
      661 tacaggctga gctttgggac tttgtgctca agatgcaaaa gtgcaattca tttacccacg
      721 aggttgataa agacgaagtc ttcaatattc ttcgggaatt gtatgaagaa ctggtcccat
      781 catcggtgga aaagaaaggt gaagctaacc aacttagcaa taagtttctg tatccgctcg
      841 ttgacccgaa ttctcaatcg ggaaaaggga ctgcctcctc gggacggaaa ccacggtggt
      901 ataacctgaa aatagcaggg gacccaagtt gggaggagga gaagaaaaaa tgggaggagg
      961 acaagaagaa ggaccccctt gctaagatac tcggcaagct tgctgaatat gggttgattc
     1021 ctcttttcat tccctatacg gatagcaatg aaccaattgt caaggagatc aagtggatgg
     1081 agaaatcgcg gaatcagagt gttagaaggt tggacaaaga catgtttata caggcgctgg
     1141 agagattcct gtcgtgggaa tcgtggaact tgaaggttaa ggaggaatat gaaaaagtcg
     1201 aaaaggaata taagacgctt gaagaacgga ttaaagaaga tattcaggct cttaaagccc
     1261 ttgagcagta tgaaaaggaa cggcaagagc aactccttcg ggataccttg aacacaaacg
     1321 agtatcgcct ctcgaagcgg ggtctgcgcg gctggagaga aatcatacaa aagtggctta
     1381 agatggatga gaatgagccg agcgagaagt acctggaggt ttttaaagat tatcaaagga
     1441 aacatccgag agaggcgggg gattattcgg tgtatgagtt tctctccaaa aaagagaatc
     1501 acttcatttg gagaaatcac ccggaatacc cctaccttta tgctacattt tgcgagattg
     1561 ataagaaaaa aaaggatgcc aaacaacaag ccacctttac cctggcggac cccatcaacc
     1621 acccattgtg ggttcgcttt gaggagagaa gcggatctaa tcttaataaa tatagaatcc
     1681 ttacggaaca gttgcatacg gagaaactga aaaaaaagct caccgtccaa ctcgatcgct
     1741 tgatctatcc tacagaatcg ggaggatggg aagagaaggg taaagttgat attgtcctcc
     1801 ttccatctag acaattctat aaccaaatct tcttggacat agaggagaag ggtaaacacg
     1861 cctttactta caaagacgaa tccattaagt ttcccctgaa agggacattg ggaggagccc
     1921 gcgtccaatt tgatcgggac caccttcgcc ggtatcccca caaagtcgaa agcggcaatg
     1981 tcgggcggat ctacttcaac atgacagtta atattgagcc tacagaatcc ccagtctcca
     2041 agtcgctgaa gatacatcgc gacgattttc ctaaagttgt gaattttaaa cctaaggaac
     2101 tgacggaatg gattaaggat tctaaaggca aaaagttgaa atctggaatc gagtctctcg
     2161 aaataggact tagggtgatg agtatagatc ttgggcaaag acaagcggcc gctgcatcaa
     2221 tctttgaagt cgtggaccaa aaacccgata ttgaaggcaa gcttttcttc cccattaagg
     2281 gaacggagct ctacgccgtc catcgcgcct catttaacat aaaactgcca ggcgagaccc
     2341 tggttaagag tcgcgaggtc ttgcgcaagg cgcgcgaaga taatcttaag cttatgaacc
     2401 aaaaacttaa tttcctcagg aacgtgctgc attttcaaca atttgaagat attactgaga
     2461 gagaaaaacg ggtcacaaaa tggatctctc gccaagaaaa cagcgatgtc ccacttgtgt
     2521 atcaggatga acttattcaa attagagagt tgatgtataa accgtacaag gattgggtgg
     2581 cgtttttgaa acagctccac aagcgcctgg aggtggaaat agggaaagaa gttaagcact
     2641 ggcgcaaatc cctgagcgac ggcaggaagg ggctttacgg gattagcctg aagaacattg
     2701 acgaaatcga ccggactaga aaattccttc tcaggtggag tctgaggcct actgagccgg
     2761 gtgaagttcg ccgcttggaa ccaggccagc gctttgcgat tgatcagttg aatcacctta
     2821 acgcccttaa agaggatcgg cttaagaaga tggcgaatac cattattatg cacgcgttgg
     2881 gctattgtta cgacgtgaga aagaagaagt ggcaggctaa aaatcccgcg tgccagatca
     2941 tcctcttcga agatctttcc aactacaacc catatggcga gaggtcaagg ttcgagaata
     3001 gtcggctgat gaaatggagt cgcagggaaa tcccacgcca ggttgccctg caaggagaaa
     3061 tctatggcct tcaagttggt gaggtcgggg cgcaattcag cagccggttt cacgcgaaaa
     3121 ctggtagtcc ggggatacgc tgccgggtgg tcacaaaaga gaaactccag gataaccggt
     3181 tttttaaaaa tctgcagagg gaaggtcgcc tgactcttga taaaatcgca gtgctgaaag
     3241 agggtgacct ctatcccgac aaaggtggcg agaaattcat aagcctctcc aaggatcgca
     3301 aatgtgtcac gactcacgca gacattaacg cggcgcaaaa cctccaaaag cggttttgga
     3361 ccagaaccca cggtttctat aaagtctatt gcaaagccta ccaggttgac ggtcagacgg
     3421 tgtatatccc agaatccaag gatcaaaagc aaaagatcat tgaagaattt ggtgaaggat
     3481 attttattct taaggacggc gtctacgagt gggtcaatgc ggggaagctt aaaattaaaa
     3541 agggctcttc caagcaatcg tcgagcgagc tcgtcgactc agacatcctg aaagactcat
     3601 ttgatctggc cagtgagttg aaaggcgaaa agctcatgtt gtacagggat ccttctggaa
     3661 atgtgttccc ctctgataag tggatggccg caggcgtctt ctttggcaaa ctggagagaa
     3721 tactcatatc aaagttgaca aaccagtatt caataagcac aatagaagat gactcaagca
     3781 agcaaagcat gaagcgtcct gctgccacca aaaaggccgg acaggctaag aaaaagaagt
     3841 gagacgacta gtggcggccg ccgacgtccg atcgttcaaa catttggcaa taaagtttct
     3901 taagattgaa tcctgttgcc ggtcttgcga tgattatcat ataatttctg ttgaattacg
     3961 ttaagcatgt aataattaac atgtaatgca tgacgttatt tatgagatgg gtttttatga
     4021 ttagagtccc gcaattatac atttaatacg cgatagaaaa caaaatatag cgcgcaaact
     4081 aggataaatt atcgcgcgcg gtgtcatcta tgttactaga tcgggaattg atcccccctc
     4141 gacagcttcc ggaaagggcg aattcgcaac tttgtataca aaagttgaac gagaaacgta
     4201 aaatgatata aatatcaata tattaaatta gattttgcat aaaaaacaga ctacataata
     4261 ctgtaaaaca caacatatcc agtcactatg ccatccagct gatatcccct atagtgagtc
     4321 gtattacatg gtcatagctg tttcctggca gctctggccc gtgtctcaaa atctctgatg
     4381 ttacattgca caagataaaa atatatcatc atgcctcctc tggaccagcc aggacagaaa
     4441 tgcctcgact tcgctgctac ccaaggttgc cgggtgacgc acaccgtgga aacggatgaa
     4501 ggcacgaacc cagtggacat aagcctgttc ggttcgtaag ctgtaatgca agtagcgtat
     4561 gcgctcacgc aactggtcca gaaccttgac cgaacgcagc ggtggtaacg gcgcagtggc
     4621 ggttttcatg gcttgttatg actgtttttt tggggtacag tctatgcctc gggcatccaa
     4681 gcagcaagcg cgttacgccg tgggtcgatg tttgatgtta tggagcagca acgatgttac
     4741 gcagcagggc agtcgcccta aaacaaagtt aaacattatg agggaagcgg tgatcgccga
     4801 agtatcgact caactatcag aggtagttgg cgtcatcgag cgccatctcg aaccgacgtt
     4861 gctggccgta catttgtacg gctccgcagt ggatggcggc ctgaagccac acagtgatat
     4921 tgatttgctg gttacggtga ccgtaaggct tgatgaaaca acgcggcgag ctttgatcaa
     4981 cgaccttttg gaaacttcgg cttcccctgg agagagcgag attctccgcg ctgtagaagt
     5041 caccattgtt gtgcacgacg acatcattcc gtggcgttat ccagctaagc gcgaactgca
     5101 atttggagaa tggcagcgca atgacattct tgcaggtatc ttcgagccag ccacgatcga
     5161 cattgatctg gctatcttgc tgacaaaagc aagagaacat agcgttgcct tggtaggtcc
     5221 agcggcggag gaactctttg atccggttcc tgaacaggat ctatttgagg cgctaaatga
     5281 aaccttaacg ctatggaact cgccgcccga ctgggctggc gatgagcgaa atgtagtgct
     5341 tacgttgtcc cgcatttggt acagcgcagt aaccggcaaa atcgcgccga aggatgtcgc
     5401 tgccgactgg gcaatggagc gcctgccggc ccagtatcag cccgtcatac ttgaagctag
     5461 acaggcttat cttggacaag aagaagatcg cttggcctcg cgcgcagatc agttggaaga
     5521 atttgtccac tacgtgaaag gcgagatcac caaggtagtc ggcaaataac cctcgagcca
     5581 cccatgacca aaatccctta acgtgagtta cgcgtcgttc cactgagcgt cagaccccgt
     5641 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     5701 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     5761 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     5821 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     5881 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     5941 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     6001 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     6061 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     6121 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     6181 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     6241 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     6301 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     6361 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     6421 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     6481 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     6541 tacgcgtacc gcgagccagg aagagtttgt agaaacgca
//
