LOCUS       sequence-255113-        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-D573A
FEATURES             Location/Qualifiers
     source          1..6579
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(96..123)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(215..301)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     350..372
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     364..381
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     365..381
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    397..496
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             521..541
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3887..3934
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      3965..4217
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4262..4385
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4402..4421)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4403..4421)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4426..4442)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4426..4442)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4873..5664
                     /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      5757..6345
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6246..6265
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6499..6516
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatacg
       61 cgtaccgcga gccaggaaga gtttgtagaa acgcaaaaag gccatccgtc aggatggcct
      121 tctgcttagt ttgatgcctg gcagtttatg gcgggcgtcc tgcccgccac cctccgggcc
      181 gttgcttcac aacgttcaaa tccgctcccg gcggatttgt cctactcagg agagcgttca
      241 ccgacaaaca acagataaaa cgaaaggccc agtcttccga ctgagccttt cgttttattt
      301 gatgcctggc agttccctac tctcgcgtta acgcttgcat ggatgttttc ccagtcacga
      361 cgttgtaaaa cgacggccag tcttaagctc gggccccaaa taatgatttt attttgactg
      421 atagtgacct gttcgttgca acaaattgat gagcaatgct tttttataat gccaactttg
      481 tacaaaaaag caggctccga attcgccctt caccatggct cctaagaaga agcggaaggt
      541 tggtattcac ggggtgcctg cggctgccac aaggtctttc atacttaaga tagagccaaa
      601 cgaagaggtc aaaaagggat tgtggaaaac ccatgaagtc ctgaaccatg gcattgccta
      661 ctacatgaac atcctgaaac ttatacggca ggaggctatt tatgagcacc acgagcagga
      721 tccaaaaaac cccaaaaagg tttcgaaggc tgaaatccag gccgaactgt gggacttcgt
      781 tctcaaaatg cagaaatgta attcgttcac tcatgaagtt gacaaagacg tcgtgtttaa
      841 cattttgagg gagctttacg aggagttggt tccgagctcc gtcgaaaaga agggtgaagc
      901 aaatcagctg tcgaataagt tcttgtaccc tttggtggac ccgaacagcc aatctggaaa
      961 agggacagca tcatcagggc ggaagcctcg gtggtataac ttgaagattg ctggagaccc
     1021 ttcgtgggaa gaggaaaaga aaaagtggga ggaagataag aagaaggacc cacttgccaa
     1081 aattctcggc aaacttgccg aatatggatt gataccgctg ttcatcccct ttacggattc
     1141 taacgaaccc atcgttaaag aaatcaagtg gatggaaaaa tctcgcaatc agtccgtccg
     1201 gaggctggac aaagatatgt ttatacaagc tttggaacgc tttctctcgt gggagtcgtg
     1261 gaatcttaag gtcaaagaag agtatgaaaa ggtcgagaag gaacacaaga cactggagga
     1321 gaggattaag gaagacattc aagcattcaa gtcactggag caatacgaaa aggaacggca
     1381 ggagcaattg cttcgcgaca cgctcaatac caatgaatat aggctttcca agaggggcct
     1441 gagaggatgg cgggaaataa tccagaaatg gctcaagatg gacgagaatg aaccttcaga
     1501 aaaatatctc gaggttttta aagattacca aaggaaacat ccacgcgagg caggggatta
     1561 cagcgtgtac gagtttctct ccaagaagga aaaccatttt atctggcgca atcatcccga
     1621 atacccgtac ctctatgcga cgttctgcga aatagacaaa aagaaaaaag atgctaagca
     1681 acaagcgact ttcacacttg cagatcccat aaatcaccca ttgtgggtgc ggtttgaaga
     1741 aaggtcgggc tctaacctca ataagtacag aattttgacg gagcagttgc acacagaaaa
     1801 gctgaagaag aagttgacgg ttcagctgga tcgccttatc tacccaaccg agtctggtgg
     1861 ctgggaagag aaggggaaag tcgacatagt gttgctgcca tctaggcagt tctataacca
     1921 gatttttctc gatatagaag aaaagggtaa acatgcattt acgtataaag acgagtccat
     1981 aaagtttcca ctgaaaggaa cacttggcgg cgcaagggtg cagtttgatc gggaccacct
     2041 tcgcaggtac ccccacaagg ttgaaagtgg aaacgttgga cggatctatt ttaatatgac
     2101 cgtcaacata gaacccacag aatcccctgt ttccaaatcc ctgaaaatac accgggacga
     2161 ttttcctaaa tttgtgaact ttaaaccgaa ggagttgacc gagtggataa aggacagtaa
     2221 agggaaaaag ctgaagtccg gtatcgaaag cctggagatt gggctcagag ttatgtcgat
     2281 agcgctgggt caaaggcagg cagcagccgc ctctatattt gaggtcgtgg accagaagcc
     2341 cgacattgaa ggtaaactgt tctttccgat taaggggacg gaactctacg cagtccatcg
     2401 cgcctccttc aatataaagc tgccgggcga aacactggtt aaatcacgcg aggttttgcg
     2461 caaagcgcgg gaagacaacc tgaaactcat gaatcaaaag ctcaatttcc tgcgcaatgt
     2521 gttgcacttc cagcagtttg aggatattac cgaaagagag aaaagggtta caaaatggat
     2581 atcccggcaa gaaaactctg atgttccgct ggtttaccag gatgagctta tacagattag
     2641 ggaacttatg tataaacctt acaaagattg ggttgcattc ctcaagcagc tgcataagag
     2701 acttgaagtc gagatcggca aagaagtcaa acactggcgc aagagcctga gcgatggtcg
     2761 gaaagggttg tacggaatca gtttgaaaaa tatcgacgaa atagatagaa ccaggaaatt
     2821 tttgttgcgc tggtcactga gaccaacgga accgggagaa gtcagaaggt tggagccagg
     2881 ccagagattt gcaattgacc agctgaacca tctgaatgca ctgaaagagg acagattgaa
     2941 gaagatggcg aatacgatta ttatgcatgc tttgggttat tgttacgacg ttaggaagaa
     3001 gaaatggcag gccaagaacc ctgcgtgcca aatcatcctg ttcgaagatc tgagtaacta
     3061 caatccgtat gaagaaagga gtcgcttcga gaacagtaaa ctgatgaaat ggtcccggcg
     3121 cgagatacca cgccaagttg cgcttcaagg ggaaatatac gggcttcaag ttggggaagt
     3181 tggagcgcag ttttctagcc ggttccacgc caagacaggg tccccgggta taaggtgcag
     3241 tgtggtgacg aaagaaaagt tgcaggataa tagattcttt aaaaatcttc aacgggaagg
     3301 gcgcctgacg cttgacaaga ttgcagtgtt gaaagagggg gatttgtacc ccgataaagg
     3361 cggggagaag ttcatttctt tgtcgaagga ccgcaagttg gttacgacgc atgcagacat
     3421 taacgcagca caaaatctgc aaaaaagatt ctggactcgg acgcatggtt tttacaaggt
     3481 ttactgtaaa gcatatcaag tcgatggtca gacggtttac attcccgaat ctaaagatca
     3541 gaaacagaaa atcattgagg agttcggtga aggttacttt atactcaagg acggtgttta
     3601 cgaatggggt aatgctggta aactgaaaat taagaagggg tcctccaagc aatcatcttc
     3661 tgagctcgtc gacagcgaca tccttaagga tagcttcgat cttgcctctg agctcaaggg
     3721 agaaaagttg atgctgtatc gcgatcctag tggaaatgtc tttccctcag ataaatggat
     3781 ggcagcaggt gtgttcttcg ggaaattgga acgcatactg atatcaaaac tgaccaatca
     3841 atactctata tctactattg aagacgattc aagtaagcaa tcgatgaagc gtcctgctgc
     3901 caccaaaaag gccggacagg ctaagaaaaa gaagtgagac gactagtggc ggccgccgac
     3961 gtccgatcgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg ttgccggtct
     4021 tgcgatgatt atcatataat ttctgttgaa ttacgttaag catgtaataa ttaacatgta
     4081 atgcatgacg ttatttatga gatgggtttt tatgattaga gtcccgcaat tatacattta
     4141 atacgcgata gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc gcgcggtgtc
     4201 atctatgtta ctagatcggg aattgatccc ccctcgacag cttccggaaa gggcgaattc
     4261 gcaactttgt atacaaaagt tgaacgagaa acgtaaaatg atataaatat caatatatta
     4321 aattagattt tgcataaaaa acagactaca taatactgta aaacacaaca tatccagtca
     4381 ctatgccatc cagctgatat cccctatagt gagtcgtatt acatggtcat agctgtttcc
     4441 tggcagctct ggcccgtgtc tcaaaatctc tgatgttaca ttgcacaaga taaaaatata
     4501 tcatcatgcc tcctctggac cagccaggac agaaatgcct cgacttcgct gctacccaag
     4561 gttgccgggt gacgcacacc gtggaaacgg atgaaggcac gaacccagtg gacataagcc
     4621 tgttcggttc gtaagctgta atgcaagtag cgtatgcgct cacgcaactg gtccagaacc
     4681 ttgaccgaac gcagcggtgg taacggcgca gtggcggttt tcatggcttg ttatgactgt
     4741 ttttttgggg tacagtctat gcctcgggca tccaagcagc aagcgcgtta cgccgtgggt
     4801 cgatgtttga tgttatggag cagcaacgat gttacgcagc agggcagtcg ccctaaaaca
     4861 aagttaaaca ttatgaggga agcggtgatc gccgaagtat cgactcaact atcagaggta
     4921 gttggcgtca tcgagcgcca tctcgaaccg acgttgctgg ccgtacattt gtacggctcc
     4981 gcagtggatg gcggcctgaa gccacacagt gatattgatt tgctggttac ggtgaccgta
     5041 aggcttgatg aaacaacgcg gcgagctttg atcaacgacc ttttggaaac ttcggcttcc
     5101 cctggagaga gcgagattct ccgcgctgta gaagtcacca ttgttgtgca cgacgacatc
     5161 attccgtggc gttatccagc taagcgcgaa ctgcaatttg gagaatggca gcgcaatgac
     5221 attcttgcag gtatcttcga gccagccacg atcgacattg atctggctat cttgctgaca
     5281 aaagcaagag aacatagcgt tgccttggta ggtccagcgg cggaggaact ctttgatccg
     5341 gttcctgaac aggatctatt tgaggcgcta aatgaaacct taacgctatg gaactcgccg
     5401 cccgactggg ctggcgatga gcgaaatgta gtgcttacgt tgtcccgcat ttggtacagc
     5461 gcagtaaccg gcaaaatcgc gccgaaggat gtcgctgccg actgggcaat ggagcgcctg
     5521 ccggcccagt atcagcccgt catacttgaa gctagacagg cttatcttgg acaagaagaa
     5581 gatcgcttgg cctcgcgcgc agatcagttg gaagaatttg tccactacgt gaaaggcgag
     5641 atcaccaagg tagtcggcaa ataaccctcg agccacccat gaccaaaatc ccttaacgtg
     5701 agttacgcgt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     5761 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     5821 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     5881 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     5941 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     6001 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     6061 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     6121 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     6181 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     6241 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     6301 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     6361 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     6421 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     6481 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
     6541 aaaccgcctc tccccgcgcg ttggccgatt cattaatgc
//
