LOCUS       sequence-255080-        6642 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry clone for CRISPR-Cas12b systems.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6642)
  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 6642)
  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: pYPQ290-E848A
FEATURES             Location/Qualifiers
     source          1..6642
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     28..45
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(204..231)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(323..409)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     458..480
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     472..489
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     473..489
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    505..604
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             629..649
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4058..4105
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      4136..4388
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4433..4556
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4573..4592)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4574..4592)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4597..4613)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4597..4613)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5044..5835
                     /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      5928..6516
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6417..6436
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
       61 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
      121 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatacgcg taccgcgagc
      181 caggaagagt ttgtagaaac gcaaaaaggc catccgtcag gatggccttc tgcttagttt
      241 gatgcctggc agtttatggc gggcgtcctg cccgccaccc tccgggccgt tgcttcacaa
      301 cgttcaaatc cgctcccggc ggatttgtcc tactcaggag agcgttcacc gacaaacaac
      361 agataaaacg aaaggcccag tcttccgact gagcctttcg ttttatttga tgcctggcag
      421 ttccctactc tcgcgttaac gcttgcatgg atgttttccc agtcacgacg ttgtaaaacg
      481 acggccagtc ttaagctcgg gccccaaata atgattttat tttgactgat agtgacctgt
      541 tcgttgcaac aaattgatga gcaatgcttt tttataatgc caactttgta caaaaaagca
      601 ggctccgaat tcgcccttca ccatggctcc taagaagaag cggaaggttg gtattcacgg
      661 ggtgcctgcg gctgcggtga agtcaataaa agttaaactc cgcctggacg atatgccaga
      721 aattcgggct ggcctctgga agcttcacaa agaggttaac gctggcgtca gatattacac
      781 ggaatggttg tcgctgctcc ggcaagaaaa tctctacaga aggtcgccca atggtgatgg
      841 ggaacaagag tgcgacaaaa cggcggagga atgcaaagcg gaactccttg aaagacttcg
      901 cgcgagacaa gtcgaaaacg gccatagagg cccggccggt tccgatgatg aattgcttca
      961 gcttgcgcgg cagctttacg aattgctcgt gccgcaagcc ataggtgcaa aaggagatgc
     1021 acaacaaatt gcaagaaagt tcctctcccc gctcgcagac aaggatgccg tgggaggtct
     1081 tggaatcgct aaagcaggga ataagccaag atgggtgcgg atgcgggaag caggtgagcc
     1141 aggctgggaa gaggagaagg agaaagccga aacgaggaaa tcagcggatc gcactgcaga
     1201 cgtgttgaga gccctcgcag actttggact taagccactg atgcgggttt acacggattc
     1261 agagatgtcc tcggtggaat ggaagccgct cagaaagggt caagccgtga gaacgtggga
     1321 ccgcgacatg ttccagcagg caattgagcg gatgatgtcc tgggagtctt ggaaccaaag
     1381 ggtcgggcaa gaatatgcga aactggtgga gcaaaaaaat aggtttgaac aaaaaaattt
     1441 cgttggtcaa gagcatctgg ttcatttggt taatcaactt caacaagata tgaaagaagc
     1501 atcacctggc ttggaatcta aagaacaaac agcacactac gttacgggta gggcgttgag
     1561 gggatcggat aaagttttcg agaagtgggg taagttggcc cccgacgccc ctttcgatct
     1621 gtatgacgcc gagataaaga acgttcagcg gaggaacact cgccgctttg gttcgcacga
     1681 tctgtttgca aaactggccg agcctgagta ccaggccctt tggcgggagg atgcgtcgtt
     1741 ccttacacgc tacgcggttt ataattcaat tctcagaaag ctcaatcacg cgaagatgtt
     1801 tgcgactttc actcttccag atgcgacggc acaccctata tggactagat ttgataagtt
     1861 ggggggcaac ttgcaccagt atacatttct gttcaacgaa ttcggcgaac gcaggcatgc
     1921 aatcaggttc cataaacttt tgaaagtcga gaatggtgtt gccagggagg ttgacgatgt
     1981 cacagtgcct atctcgatgt ccgaacaatt ggataacttg ctgcccagag atccgaacga
     2041 accgattgca ctttatttca gggattatgg tgccgaacaa cactttacgg gtgagttcgg
     2101 aggggccaag attcagtgca gacgggacca gcttgctcac atgcaccgca ggagaggggc
     2161 tagggatgtg tatttgaacg tttcagttcg cgtgcagtcc caatccgagg cgcgggggga
     2221 gcgcagacca ccatacgcgg ctgtcttccg gctggttggc gataaccata gagcgttcgt
     2281 gcatttcgat aagctgagcg attacctcgc cgaacatcct gatgacggaa agttggggtc
     2341 agaggggctt ctgtcgggcc tgagggtgat gtccgtggac ctgggattgc gcaccagtgc
     2401 ctcgatcagc gtttttaggg tggccaggaa agatgagttg aaacccaact cgaaggggag
     2461 ggttccgttc tttttcccta taaagggcaa cgataacttg gtcgcagtgc atgaaaggag
     2521 ccaactgctc aaacttcccg gggagacaga gtccaaagat cttcgcgcta taagggaaga
     2581 gagacaaaga actctccggc agctgcgcac gcagctcgca tacctgcggt tgcttgtccg
     2641 ctgcggaagt gaagacgttg gcaggcgcga gaggtcatgg gccaaattga ttgagcagcc
     2701 ggtcgacgcc gcaaatcaca tgactccgga ttggagggag gctttcgaga acgaactgca
     2761 gaagttgaag agtctgcatg gcatatgctc tgacaaagag tggatggacg cggtttacga
     2821 gtccgtccgc cgggtctggc ggcacatggg gaaacaagtt cgcgattgga gaaaggatgt
     2881 tagatccggg gaaaggccga agataagagg ttatgccaaa gacgtggttg gtggaaattc
     2941 tatcgaacag atcgaatatc ttgagaggca gtacaagttc ctcaagagtt ggtctttctt
     3001 cggtaaagtc tctggacaag ttataagagc agaaaagggg agccggttcg ctatcacctt
     3061 gcgggaacac atagaccacg caaaagaaga cagactgaag aagctggcgg acagaattat
     3121 catggaagcg ctggggtacg tttacgcgct ggacgaaagg gggaaaggta aatgggtggc
     3181 caaatacccg ccatgccagt tgatattgct ggccgaattg tccgaatatc aatttaataa
     3241 cgatagaccg ccatccgaga acaaccaact tatgcaatgg tctcaccggg gagttttcca
     3301 ggagttgatc aaccaagctc aagtgcacga tctgcttgtt ggtacaatgt acgcagcgtt
     3361 ttcctcacgc ttcgacgcta gaacaggagc gccgggaatt cggtgccgga gggtgcctgc
     3421 gaggtgtact caggagcaca acccggagcc atttccctgg tggttgaata aattcgttgt
     3481 ggaacatacg ttggatgctt gcccgcttcg ggcggacgac ctcattccga cgggtgaggg
     3541 cgagattttc gtgtcgccat tctcggctga ggaaggggac ttccatcaaa tccatgctga
     3601 cctcaatgcg gcgcaaaatc tgcagcagag attgtggagt gattttgaca tctctcagat
     3661 caggcttcgg tgcgattggg gagaagtcga tggtgaactc gttctcattc cgagactcac
     3721 cggtaaaagg actgctgatt catattcgaa caaagttttt tacactaaca caggggtcac
     3781 ttattatgaa agagaacgcg gtaagaagcg ccgcaaggtg ttcgcgcaag agaaactttc
     3841 cgaggaagag gccgagttgc tcgttgaagc tgacgaagct cgcgagaagt ccgtcgttct
     3901 gatgcgggat ccttctggca taataaacag ggggaattgg acacggcaga aggaattttg
     3961 gtccatggtg aatcagcgca tagaaggtta tctggtcaaa cagatcagaa gcagggttcc
     4021 cctccaggat tcagcgtgcg agaacacggg cgatattaag cgtcctgctg ccaccaaaaa
     4081 ggccggacag gctaagaaaa agaagtgaga cgactagtgg cggccgccga cgtccgatcg
     4141 ttcaaacatt tggcaataaa gtttcttaag attgaatcct gttgccggtc ttgcgatgat
     4201 tatcatataa tttctgttga attacgttaa gcatgtaata attaacatgt aatgcatgac
     4261 gttatttatg agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat
     4321 agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt
     4381 actagatcgg gaattgatcc cccctcgaca gcttccggaa agggcgaatt cgcaactttg
     4441 tatacaaaag ttgaacgaga aacgtaaaat gatataaata tcaatatatt aaattagatt
     4501 ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc actatgccat
     4561 ccagctgata tcccctatag tgagtcgtat tacatggtca tagctgtttc ctggcagctc
     4621 tggcccgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatgc
     4681 ctcctctgga ccagccagga cagaaatgcc tcgacttcgc tgctacccaa ggttgccggg
     4741 tgacgcacac cgtggaaacg gatgaaggca cgaacccagt ggacataagc ctgttcggtt
     4801 cgtaagctgt aatgcaagta gcgtatgcgc tcacgcaact ggtccagaac cttgaccgaa
     4861 cgcagcggtg gtaacggcgc agtggcggtt ttcatggctt gttatgactg tttttttggg
     4921 gtacagtcta tgcctcgggc atccaagcag caagcgcgtt acgccgtggg tcgatgtttg
     4981 atgttatgga gcagcaacga tgttacgcag cagggcagtc gccctaaaac aaagttaaac
     5041 attatgaggg aagcggtgat cgccgaagta tcgactcaac tatcagaggt agttggcgtc
     5101 atcgagcgcc atctcgaacc gacgttgctg gccgtacatt tgtacggctc cgcagtggat
     5161 ggcggcctga agccacacag tgatattgat ttgctggtta cggtgaccgt aaggcttgat
     5221 gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa cttcggcttc ccctggagag
     5281 agcgagattc tccgcgctgt agaagtcacc attgttgtgc acgacgacat cattccgtgg
     5341 cgttatccag ctaagcgcga actgcaattt ggagaatggc agcgcaatga cattcttgca
     5401 ggtatcttcg agccagccac gatcgacatt gatctggcta tcttgctgac aaaagcaaga
     5461 gaacatagcg ttgccttggt aggtccagcg gcggaggaac tctttgatcc ggttcctgaa
     5521 caggatctat ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg
     5581 gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca tttggtacag cgcagtaacc
     5641 ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct gccggcccag
     5701 tatcagcccg tcatacttga agctagacag gcttatcttg gacaagaaga agatcgcttg
     5761 gcctcgcgcg cagatcagtt ggaagaattt gtccactacg tgaaaggcga gatcaccaag
     5821 gtagtcggca aataaccctc gagccaccca tgaccaaaat cccttaacgt gagttacgcg
     5881 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     5941 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     6001 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     6061 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     6121 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     6181 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     6241 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     6301 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     6361 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     6421 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     6481 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     6541 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     6601 tctgtggata accgtattac cgcctttgag tgagctgata cc
//
