LOCUS       sequence-255062-        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-D570A
FEATURES             Location/Qualifiers
     source          1..6642
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..100
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             125..145
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3554..3601
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      3632..3884
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    3929..4052
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4069..4088)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4070..4088)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4093..4109)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4093..4109)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4540..5331
                     /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      5424..6012
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5913..5932
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6166..6183
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(6342..6369)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(6461..6547)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     6596..6618
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6610..6627
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6611..6627
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 caaataatga ttttattttg actgatagtg acctgttcgt tgcaacaaat tgatgagcaa
       61 tgctttttta taatgccaac tttgtacaaa aaagcaggct ccgaattcgc ccttcaccat
      121 ggctcctaag aagaagcgga aggttggtat tcacggggtg cctgcggctg cggtgaagtc
      181 aataaaagtt aaactccgcc tggacgatat gccagaaatt cgggctggcc tctggaagct
      241 tcacaaagag gttaacgctg gcgtcagata ttacacggaa tggttgtcgc tgctccggca
      301 agaaaatctc tacagaaggt cgcccaatgg tgatggggaa caagagtgcg acaaaacggc
      361 ggaggaatgc aaagcggaac tccttgaaag acttcgcgcg agacaagtcg aaaacggcca
      421 tagaggcccg gccggttccg atgatgaatt gcttcagctt gcgcggcagc tttacgaatt
      481 gctcgtgccg caagccatag gtgcaaaagg agatgcacaa caaattgcaa gaaagttcct
      541 ctccccgctc gcagacaagg atgccgtggg aggtcttgga atcgctaaag cagggaataa
      601 gccaagatgg gtgcggatgc gggaagcagg tgagccaggc tgggaagagg agaaggagaa
      661 agccgaaacg aggaaatcag cggatcgcac tgcagacgtg ttgagagccc tcgcagactt
      721 tggacttaag ccactgatgc gggtttacac ggattcagag atgtcctcgg tggaatggaa
      781 gccgctcaga aagggtcaag ccgtgagaac gtgggaccgc gacatgttcc agcaggcaat
      841 tgagcggatg atgtcctggg agtcttggaa ccaaagggtc gggcaagaat atgcgaaact
      901 ggtggagcaa aaaaataggt ttgaacaaaa aaatttcgtt ggtcaagagc atctggttca
      961 tttggttaat caacttcaac aagatatgaa agaagcatca cctggcttgg aatctaaaga
     1021 acaaacagca cactacgtta cgggtagggc gttgagggga tcggataaag ttttcgagaa
     1081 gtggggtaag ttggcccccg acgccccttt cgatctgtat gacgccgaga taaagaacgt
     1141 tcagcggagg aacactcgcc gctttggttc gcacgatctg tttgcaaaac tggccgagcc
     1201 tgagtaccag gccctttggc gggaggatgc gtcgttcctt acacgctacg cggtttataa
     1261 ttcaattctc agaaagctca atcacgcgaa gatgtttgcg actttcactc ttccagatgc
     1321 gacggcacac cctatatgga ctagatttga taagttgggg ggcaacttgc accagtatac
     1381 atttctgttc aacgaattcg gcgaacgcag gcatgcaatc aggttccata aacttttgaa
     1441 agtcgagaat ggtgttgcca gggaggttga cgatgtcaca gtgcctatct cgatgtccga
     1501 acaattggat aacttgctgc ccagagatcc gaacgaaccg attgcacttt atttcaggga
     1561 ttatggtgcc gaacaacact ttacgggtga gttcggaggg gccaagattc agtgcagacg
     1621 ggaccagctt gctcacatgc accgcaggag aggggctagg gatgtgtatt tgaacgtttc
     1681 agttcgcgtg cagtcccaat ccgaggcgcg gggggagcgc agaccaccat acgcggctgt
     1741 cttccggctg gttggcgata accatagagc gttcgtgcat ttcgataagc tgagcgatta
     1801 cctcgccgaa catcctgatg acggaaagtt ggggtcagag gggcttctgt cgggcctgag
     1861 ggtgatgtcc gtggccctgg gattgcgcac cagtgcctcg atcagcgttt ttagggtggc
     1921 caggaaagat gagttgaaac ccaactcgaa ggggagggtt ccgttctttt tccctataaa
     1981 gggcaacgat aacttggtcg cagtgcatga aaggagccaa ctgctcaaac ttcccgggga
     2041 gacagagtcc aaagatcttc gcgctataag ggaagagaga caaagaactc tccggcagct
     2101 gcgcacgcag ctcgcatacc tgcggttgct tgtccgctgc ggaagtgaag acgttggcag
     2161 gcgcgagagg tcatgggcca aattgattga gcagccggtc gacgccgcaa atcacatgac
     2221 tccggattgg agggaggctt tcgagaacga actgcagaag ttgaagagtc tgcatggcat
     2281 atgctctgac aaagagtgga tggacgcggt ttacgagtcc gtccgccggg tctggcggca
     2341 catggggaaa caagttcgcg attggagaaa ggatgttaga tccggggaaa ggccgaagat
     2401 aagaggttat gccaaagacg tggttggtgg aaattctatc gaacagatcg aatatcttga
     2461 gaggcagtac aagttcctca agagttggtc tttcttcggt aaagtctctg gacaagttat
     2521 aagagcagaa aaggggagcc ggttcgctat caccttgcgg gaacacatag accacgcaaa
     2581 agaagacaga ctgaagaagc tggcggacag aattatcatg gaagcgctgg ggtacgttta
     2641 cgcgctggac gaaaggggga aaggtaaatg ggtggccaaa tacccgccat gccagttgat
     2701 attgctggaa gaattgtccg aatatcaatt taataacgat agaccgccat ccgagaacaa
     2761 ccaacttatg caatggtctc accggggagt tttccaggag ttgatcaacc aagctcaagt
     2821 gcacgatctg cttgttggta caatgtacgc agcgttttcc tcacgcttcg acgctagaac
     2881 aggagcgccg ggaattcggt gccggagggt gcctgcgagg tgtactcagg agcacaaccc
     2941 ggagccattt ccctggtggt tgaataaatt cgttgtggaa catacgttgg atgcttgccc
     3001 gcttcgggcg gacgacctca ttccgacggg tgagggcgag attttcgtgt cgccattctc
     3061 ggctgaggaa ggggacttcc atcaaatcca tgctgacctc aatgcggcgc aaaatctgca
     3121 gcagagattg tggagtgatt ttgacatctc tcagatcagg cttcggtgcg attggggaga
     3181 agtcgatggt gaactcgttc tcattccgag actcaccggt aaaaggactg ctgattcata
     3241 ttcgaacaaa gttttttaca ctaacacagg ggtcacttat tatgaaagag aacgcggtaa
     3301 gaagcgccgc aaggtgttcg cgcaagagaa actttccgag gaagaggccg agttgctcgt
     3361 tgaagctgac gaagctcgcg agaagtccgt cgttctgatg cgggatcctt ctggcataat
     3421 aaacaggggg aattggacac ggcagaagga attttggtcc atggtgaatc agcgcataga
     3481 aggttatctg gtcaaacaga tcagaagcag ggttcccctc caggattcag cgtgcgagaa
     3541 cacgggcgat attaagcgtc ctgctgccac caaaaaggcc ggacaggcta agaaaaagaa
     3601 gtgagacgac tagtggcggc cgccgacgtc cgatcgttca aacatttggc aataaagttt
     3661 cttaagattg aatcctgttg ccggtcttgc gatgattatc atataatttc tgttgaatta
     3721 cgttaagcat gtaataatta acatgtaatg catgacgtta tttatgagat gggtttttat
     3781 gattagagtc ccgcaattat acatttaata cgcgatagaa aacaaaatat agcgcgcaaa
     3841 ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta gatcgggaat tgatcccccc
     3901 tcgacagctt ccggaaaggg cgaattcgca actttgtata caaaagttga acgagaaacg
     3961 taaaatgata taaatatcaa tatattaaat tagattttgc ataaaaaaca gactacataa
     4021 tactgtaaaa cacaacatat ccagtcacta tgccatccag ctgatatccc ctatagtgag
     4081 tcgtattaca tggtcatagc tgtttcctgg cagctctggc ccgtgtctca aaatctctga
     4141 tgttacattg cacaagataa aaatatatca tcatgcctcc tctggaccag ccaggacaga
     4201 aatgcctcga cttcgctgct acccaaggtt gccgggtgac gcacaccgtg gaaacggatg
     4261 aaggcacgaa cccagtggac ataagcctgt tcggttcgta agctgtaatg caagtagcgt
     4321 atgcgctcac gcaactggtc cagaaccttg accgaacgca gcggtggtaa cggcgcagtg
     4381 gcggttttca tggcttgtta tgactgtttt tttggggtac agtctatgcc tcgggcatcc
     4441 aagcagcaag cgcgttacgc cgtgggtcga tgtttgatgt tatggagcag caacgatgtt
     4501 acgcagcagg gcagtcgccc taaaacaaag ttaaacatta tgagggaagc ggtgatcgcc
     4561 gaagtatcga ctcaactatc agaggtagtt ggcgtcatcg agcgccatct cgaaccgacg
     4621 ttgctggccg tacatttgta cggctccgca gtggatggcg gcctgaagcc acacagtgat
     4681 attgatttgc tggttacggt gaccgtaagg cttgatgaaa caacgcggcg agctttgatc
     4741 aacgaccttt tggaaacttc ggcttcccct ggagagagcg agattctccg cgctgtagaa
     4801 gtcaccattg ttgtgcacga cgacatcatt ccgtggcgtt atccagctaa gcgcgaactg
     4861 caatttggag aatggcagcg caatgacatt cttgcaggta tcttcgagcc agccacgatc
     4921 gacattgatc tggctatctt gctgacaaaa gcaagagaac atagcgttgc cttggtaggt
     4981 ccagcggcgg aggaactctt tgatccggtt cctgaacagg atctatttga ggcgctaaat
     5041 gaaaccttaa cgctatggaa ctcgccgccc gactgggctg gcgatgagcg aaatgtagtg
     5101 cttacgttgt cccgcatttg gtacagcgca gtaaccggca aaatcgcgcc gaaggatgtc
     5161 gctgccgact gggcaatgga gcgcctgccg gcccagtatc agcccgtcat acttgaagct
     5221 agacaggctt atcttggaca agaagaagat cgcttggcct cgcgcgcaga tcagttggaa
     5281 gaatttgtcc actacgtgaa aggcgagatc accaaggtag tcggcaaata accctcgagc
     5341 cacccatgac caaaatccct taacgtgagt tacgcgtcgt tccactgagc gtcagacccc
     5401 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     5461 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     5521 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     5581 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     5641 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     5701 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     5761 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     5821 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     5881 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     5941 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     6001 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     6061 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     6121 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     6181 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     6241 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     6301 aatacgcgta ccgcgagcca ggaagagttt gtagaaacgc aaaaaggcca tccgtcagga
     6361 tggccttctg cttagtttga tgcctggcag tttatggcgg gcgtcctgcc cgccaccctc
     6421 cgggccgttg cttcacaacg ttcaaatccg ctcccggcgg atttgtccta ctcaggagag
     6481 cgttcaccga caaacaacag ataaaacgaa aggcccagtc ttccgactga gcctttcgtt
     6541 ttatttgatg cctggcagtt ccctactctc gcgttaacgc ttgcatggat gttttcccag
     6601 tcacgacgtt gtaaaacgac ggccagtctt aagctcgggc cc
//
