LOCUS       sequence-255116-        6579 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 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: pYPQ291-E827A
FEATURES             Location/Qualifiers
     source          1..6579
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     49..71
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     63..80
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     64..80
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    96..195
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             220..240
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3586..3633
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      3664..3916
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    3961..4084
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4101..4120)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4102..4120)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4125..4141)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4125..4141)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4572..5363
                     /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      5456..6044
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5945..5964
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6198..6215
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(6374..6401)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(6493..6579)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 atgcctggca gttccctact ctcgcgttaa cgcttgcatg gatgttttcc cagtcacgac
       61 gttgtaaaac gacggccagt cttaagctcg ggccccaaat aatgatttta ttttgactga
      121 tagtgacctg ttcgttgcaa caaattgatg agcaatgctt ttttataatg ccaactttgt
      181 acaaaaaagc aggctccgaa ttcgcccttc accatggctc ctaagaagaa gcggaaggtt
      241 ggtattcacg gggtgcctgc ggctgccaca aggtctttca tacttaagat agagccaaac
      301 gaagaggtca aaaagggatt gtggaaaacc catgaagtcc tgaaccatgg cattgcctac
      361 tacatgaaca tcctgaaact tatacggcag gaggctattt atgagcacca cgagcaggat
      421 ccaaaaaacc ccaaaaaggt ttcgaaggct gaaatccagg ccgaactgtg ggacttcgtt
      481 ctcaaaatgc agaaatgtaa ttcgttcact catgaagttg acaaagacgt cgtgtttaac
      541 attttgaggg agctttacga ggagttggtt ccgagctccg tcgaaaagaa gggtgaagca
      601 aatcagctgt cgaataagtt cttgtaccct ttggtggacc cgaacagcca atctggaaaa
      661 gggacagcat catcagggcg gaagcctcgg tggtataact tgaagattgc tggagaccct
      721 tcgtgggaag aggaaaagaa aaagtgggag gaagataaga agaaggaccc acttgccaaa
      781 attctcggca aacttgccga atatggattg ataccgctgt tcatcccctt tacggattct
      841 aacgaaccca tcgttaaaga aatcaagtgg atggaaaaat ctcgcaatca gtccgtccgg
      901 aggctggaca aagatatgtt tatacaagct ttggaacgct ttctctcgtg ggagtcgtgg
      961 aatcttaagg tcaaagaaga gtatgaaaag gtcgagaagg aacacaagac actggaggag
     1021 aggattaagg aagacattca agcattcaag tcactggagc aatacgaaaa ggaacggcag
     1081 gagcaattgc ttcgcgacac gctcaatacc aatgaatata ggctttccaa gaggggcctg
     1141 agaggatggc gggaaataat ccagaaatgg ctcaagatgg acgagaatga accttcagaa
     1201 aaatatctcg aggtttttaa agattaccaa aggaaacatc cacgcgaggc aggggattac
     1261 agcgtgtacg agtttctctc caagaaggaa aaccatttta tctggcgcaa tcatcccgaa
     1321 tacccgtacc tctatgcgac gttctgcgaa atagacaaaa agaaaaaaga tgctaagcaa
     1381 caagcgactt tcacacttgc agatcccata aatcacccat tgtgggtgcg gtttgaagaa
     1441 aggtcgggct ctaacctcaa taagtacaga attttgacgg agcagttgca cacagaaaag
     1501 ctgaagaaga agttgacggt tcagctggat cgccttatct acccaaccga gtctggtggc
     1561 tgggaagaga aggggaaagt cgacatagtg ttgctgccat ctaggcagtt ctataaccag
     1621 atttttctcg atatagaaga aaagggtaaa catgcattta cgtataaaga cgagtccata
     1681 aagtttccac tgaaaggaac acttggcggc gcaagggtgc agtttgatcg ggaccacctt
     1741 cgcaggtacc cccacaaggt tgaaagtgga aacgttggac ggatctattt taatatgacc
     1801 gtcaacatag aacccacaga atcccctgtt tccaaatccc tgaaaataca ccgggacgat
     1861 tttcctaaat ttgtgaactt taaaccgaag gagttgaccg agtggataaa ggacagtaaa
     1921 gggaaaaagc tgaagtccgg tatcgaaagc ctggagattg ggctcagagt tatgtcgata
     1981 gatctgggtc aaaggcaggc agcagccgcc tctatatttg aggtcgtgga ccagaagccc
     2041 gacattgaag gtaaactgtt ctttccgatt aaggggacgg aactctacgc agtccatcgc
     2101 gcctccttca atataaagct gccgggcgaa acactggtta aatcacgcga ggttttgcgc
     2161 aaagcgcggg aagacaacct gaaactcatg aatcaaaagc tcaatttcct gcgcaatgtg
     2221 ttgcacttcc agcagtttga ggatattacc gaaagagaga aaagggttac aaaatggata
     2281 tcccggcaag aaaactctga tgttccgctg gtttaccagg atgagcttat acagattagg
     2341 gaacttatgt ataaacctta caaagattgg gttgcattcc tcaagcagct gcataagaga
     2401 cttgaagtcg agatcggcaa agaagtcaaa cactggcgca agagcctgag cgatggtcgg
     2461 aaagggttgt acggaatcag tttgaaaaat atcgacgaaa tagatagaac caggaaattt
     2521 ttgttgcgct ggtcactgag accaacggaa ccgggagaag tcagaaggtt ggagccaggc
     2581 cagagatttg caattgacca gctgaaccat ctgaatgcac tgaaagagga cagattgaag
     2641 aagatggcga atacgattat tatgcatgct ttgggttatt gttacgacgt taggaagaag
     2701 aaatggcagg ccaagaaccc tgcgtgccaa atcatcctgt tcgccgatct gagtaactac
     2761 aatccgtatg aagaaaggag tcgcttcgag aacagtaaac tgatgaaatg gtcccggcgc
     2821 gagataccac gccaagttgc gcttcaaggg gaaatatacg ggcttcaagt tggggaagtt
     2881 ggagcgcagt tttctagccg gttccacgcc aagacagggt ccccgggtat aaggtgcagt
     2941 gtggtgacga aagaaaagtt gcaggataat agattcttta aaaatcttca acgggaaggg
     3001 cgcctgacgc ttgacaagat tgcagtgttg aaagaggggg atttgtaccc cgataaaggc
     3061 ggggagaagt tcatttcttt gtcgaaggac cgcaagttgg ttacgacgca tgcagacatt
     3121 aacgcagcac aaaatctgca aaaaagattc tggactcgga cgcatggttt ttacaaggtt
     3181 tactgtaaag catatcaagt cgatggtcag acggtttaca ttcccgaatc taaagatcag
     3241 aaacagaaaa tcattgagga gttcggtgaa ggttacttta tactcaagga cggtgtttac
     3301 gaatggggta atgctggtaa actgaaaatt aagaaggggt cctccaagca atcatcttct
     3361 gagctcgtcg acagcgacat ccttaaggat agcttcgatc ttgcctctga gctcaaggga
     3421 gaaaagttga tgctgtatcg cgatcctagt ggaaatgtct ttccctcaga taaatggatg
     3481 gcagcaggtg tgttcttcgg gaaattggaa cgcatactga tatcaaaact gaccaatcaa
     3541 tactctatat ctactattga agacgattca agtaagcaat cgatgaagcg tcctgctgcc
     3601 accaaaaagg ccggacaggc taagaaaaag aagtgagacg actagtggcg gccgccgacg
     3661 tccgatcgtt caaacatttg gcaataaagt ttcttaagat tgaatcctgt tgccggtctt
     3721 gcgatgatta tcatataatt tctgttgaat tacgttaagc atgtaataat taacatgtaa
     3781 tgcatgacgt tatttatgag atgggttttt atgattagag tcccgcaatt atacatttaa
     3841 tacgcgatag aaaacaaaat atagcgcgca aactaggata aattatcgcg cgcggtgtca
     3901 tctatgttac tagatcggga attgatcccc cctcgacagc ttccggaaag ggcgaattcg
     3961 caactttgta tacaaaagtt gaacgagaaa cgtaaaatga tataaatatc aatatattaa
     4021 attagatttt gcataaaaaa cagactacat aatactgtaa aacacaacat atccagtcac
     4081 tatgccatcc agctgatatc ccctatagtg agtcgtatta catggtcata gctgtttcct
     4141 ggcagctctg gcccgtgtct caaaatctct gatgttacat tgcacaagat aaaaatatat
     4201 catcatgcct cctctggacc agccaggaca gaaatgcctc gacttcgctg ctacccaagg
     4261 ttgccgggtg acgcacaccg tggaaacgga tgaaggcacg aacccagtgg acataagcct
     4321 gttcggttcg taagctgtaa tgcaagtagc gtatgcgctc acgcaactgg tccagaacct
     4381 tgaccgaacg cagcggtggt aacggcgcag tggcggtttt catggcttgt tatgactgtt
     4441 tttttggggt acagtctatg cctcgggcat ccaagcagca agcgcgttac gccgtgggtc
     4501 gatgtttgat gttatggagc agcaacgatg ttacgcagca gggcagtcgc cctaaaacaa
     4561 agttaaacat tatgagggaa gcggtgatcg ccgaagtatc gactcaacta tcagaggtag
     4621 ttggcgtcat cgagcgccat ctcgaaccga cgttgctggc cgtacatttg tacggctccg
     4681 cagtggatgg cggcctgaag ccacacagtg atattgattt gctggttacg gtgaccgtaa
     4741 ggcttgatga aacaacgcgg cgagctttga tcaacgacct tttggaaact tcggcttccc
     4801 ctggagagag cgagattctc cgcgctgtag aagtcaccat tgttgtgcac gacgacatca
     4861 ttccgtggcg ttatccagct aagcgcgaac tgcaatttgg agaatggcag cgcaatgaca
     4921 ttcttgcagg tatcttcgag ccagccacga tcgacattga tctggctatc ttgctgacaa
     4981 aagcaagaga acatagcgtt gccttggtag gtccagcggc ggaggaactc tttgatccgg
     5041 ttcctgaaca ggatctattt gaggcgctaa atgaaacctt aacgctatgg aactcgccgc
     5101 ccgactgggc tggcgatgag cgaaatgtag tgcttacgtt gtcccgcatt tggtacagcg
     5161 cagtaaccgg caaaatcgcg ccgaaggatg tcgctgccga ctgggcaatg gagcgcctgc
     5221 cggcccagta tcagcccgtc atacttgaag ctagacaggc ttatcttgga caagaagaag
     5281 atcgcttggc ctcgcgcgca gatcagttgg aagaatttgt ccactacgtg aaaggcgaga
     5341 tcaccaaggt agtcggcaaa taaccctcga gccacccatg accaaaatcc cttaacgtga
     5401 gttacgcgtc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     5461 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     5521 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     5581 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     5641 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     5701 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     5761 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     5821 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     5881 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     5941 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     6001 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     6061 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     6121 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     6181 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     6241 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     6301 actggaaagc gggcagtgag cgcaacgcaa ttaatacgcg taccgcgagc caggaagagt
     6361 ttgtagaaac gcaaaaaggc catccgtcag gatggccttc tgcttagttt gatgcctggc
     6421 agtttatggc gggcgtcctg cccgccaccc tccgggccgt tgcttcacaa cgttcaaatc
     6481 cgctcccggc ggatttgtcc tactcaggag agcgttcacc gacaaacaac agataaaacg
     6541 aaaggcccag tcttccgact gagcctttcg ttttatttg
//
