LOCUS       sequence-255109-        6557 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 6557)
  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 6557)
  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-SRDX
FEATURES             Location/Qualifiers
     source          1..6557
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     132..151
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     385..402
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(561..588)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     647..669
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     661..678
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     662..678
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    694..793
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             818..838
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4184..4231
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      4408..4660
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4705..4828
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4845..4864)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4846..4864)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4869..4885)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4869..4885)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5316..6107
                     /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      join(6200..6557,1..231)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
       61 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
      121 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
      181 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
      241 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
      301 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
      361 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
      421 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
      481 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atacgcgtac cgcgagccag
      541 gaagagtttg tagaaacgca aaaaggccat ccgtcaggat ggccttctgc ttagtttgat
      601 gcctggcagt tccctactct cgcgttaacg cttgcatgga tgttttccca gtcacgacgt
      661 tgtaaaacga cggccagtct taagctcggg ccccaaataa tgattttatt ttgactgata
      721 gtgacctgtt cgttgcaaca aattgatgag caatgctttt ttataatgcc aactttgtac
      781 aaaaaagcag gctccgaatt cgcccttcac catggctcct aagaagaagc ggaaggttgg
      841 tattcacggg gtgcctgcgg ctgccacaag gtctttcata cttaagatag agccaaacga
      901 agaggtcaaa aagggattgt ggaaaaccca tgaagtcctg aaccatggca ttgcctacta
      961 catgaacatc ctgaaactta tacggcagga ggctatttat gagcaccacg agcaggatcc
     1021 aaaaaacccc aaaaaggttt cgaaggctga aatccaggcc gaactgtggg acttcgttct
     1081 caaaatgcag aaatgtaatt cgttcactca tgaagttgac aaagacgtcg tgtttaacat
     1141 tttgagggag ctttacgagg agttggttcc gagctccgtc gaaaagaagg gtgaagcaaa
     1201 tcagctgtcg aataagttct tgtacccttt ggtggacccg aacagccaat ctggaaaagg
     1261 gacagcatca tcagggcgga agcctcggtg gtataacttg aagattgctg gagacccttc
     1321 gtgggaagag gaaaagaaaa agtgggagga agataagaag aaggacccac ttgccaaaat
     1381 tctcggcaaa cttgccgaat atggattgat accgctgttc atccccttta cggattctaa
     1441 cgaacccatc gttaaagaaa tcaagtggat ggaaaaatct cgcaatcagt ccgtccggag
     1501 gctggacaaa gatatgttta tacaagcttt ggaacgcttt ctctcgtggg agtcgtggaa
     1561 tcttaaggtc aaagaagagt atgaaaaggt cgagaaggaa cacaagacac tggaggagag
     1621 gattaaggaa gacattcaag cattcaagtc actggagcaa tacgaaaagg aacggcagga
     1681 gcaattgctt cgcgacacgc tcaataccaa tgaatatagg ctttccaaga ggggcctgag
     1741 aggatggcgg gaaataatcc agaaatggct caagatggac gagaatgaac cttcagaaaa
     1801 atatctcgag gtttttaaag attaccaaag gaaacatcca cgcgaggcag gggattacag
     1861 cgtgtacgag tttctctcca agaaggaaaa ccattttatc tggcgcaatc atcccgaata
     1921 cccgtacctc tatgcgacgt tctgcgaaat agacaaaaag aaaaaagatg ctaagcaaca
     1981 agcgactttc acacttgcag atcccataaa tcacccattg tgggtgcggt ttgaagaaag
     2041 gtcgggctct aacctcaata agtacagaat tttgacggag cagttgcaca cagaaaagct
     2101 gaagaagaag ttgacggttc agctggatcg ccttatctac ccaaccgagt ctggtggctg
     2161 ggaagagaag gggaaagtcg acatagtgtt gctgccatct aggcagttct ataaccagat
     2221 ttttctcgat atagaagaaa agggtaaaca tgcatttacg tataaagacg agtccataaa
     2281 gtttccactg aaaggaacac ttggcggcgc aagggtgcag tttgatcggg accaccttcg
     2341 caggtacccc cacaaggttg aaagtggaaa cgttggacgg atctatttta atatgaccgt
     2401 caacatagaa cccacagaat cccctgtttc caaatccctg aaaatacacc gggacgattt
     2461 tcctaaattt gtgaacttta aaccgaagga gttgaccgag tggataaagg acagtaaagg
     2521 gaaaaagctg aagtccggta tcgaaagcct ggagattggg ctcagagtta tgtcgatagc
     2581 gctgggtcaa aggcaggcag cagccgcctc tatatttgag gtcgtggacc agaagcccga
     2641 cattgaaggt aaactgttct ttccgattaa ggggacggaa ctctacgcag tccatcgcgc
     2701 ctccttcaat ataaagctgc cgggcgaaac actggttaaa tcacgcgagg ttttgcgcaa
     2761 agcgcgggaa gacaacctga aactcatgaa tcaaaagctc aatttcctgc gcaatgtgtt
     2821 gcacttccag cagtttgagg atattaccga aagagagaaa agggttacaa aatggatatc
     2881 ccggcaagaa aactctgatg ttccgctggt ttaccaggat gagcttatac agattaggga
     2941 acttatgtat aaaccttaca aagattgggt tgcattcctc aagcagctgc ataagagact
     3001 tgaagtcgag atcggcaaag aagtcaaaca ctggcgcaag agcctgagcg atggtcggaa
     3061 agggttgtac ggaatcagtt tgaaaaatat cgacgaaata gatagaacca ggaaattttt
     3121 gttgcgctgg tcactgagac caacggaacc gggagaagtc agaaggttgg agccaggcca
     3181 gagatttgca attgaccagc tgaaccatct gaatgcactg aaagaggaca gattgaagaa
     3241 gatggcgaat acgattatta tgcatgcttt gggttattgt tacgacgtta ggaagaagaa
     3301 atggcaggcc aagaaccctg cgtgccaaat catcctgttc gaagatctga gtaactacaa
     3361 tccgtatgaa gaaaggagtc gcttcgagaa cagtaaactg atgaaatggt cccggcgcga
     3421 gataccacgc caagttgcgc ttcaagggga aatatacggg cttcaagttg gggaagttgg
     3481 agcgcagttt tctagccggt tccacgccaa gacagggtcc ccgggtataa ggtgcagtgt
     3541 ggtgacgaaa gaaaagttgc aggataatag attctttaaa aatcttcaac gggaagggcg
     3601 cctgacgctt gacaagattg cagtgttgaa agagggggat ttgtaccccg ataaaggcgg
     3661 ggagaagttc atttctttgt cgaaggaccg caagttggtt acgacgcatg cagacattaa
     3721 cgcagcacaa aatctgcaaa aaagattctg gactcggacg catggttttt acaaggttta
     3781 ctgtaaagca tatcaagtcg atggtcagac ggtttacatt cccgaatcta aagatcagaa
     3841 acagaaaatc attgaggagt tcggtgaagg ttactttata ctcaaggacg gtgtttacga
     3901 atggggtaat gctggtaaac tgaaaattaa gaaggggtcc tccaagcaat catcttctga
     3961 gctcgtcgac agcgacatcc ttaaggatag cttcgatctt gcctctgagc tcaagggaga
     4021 aaagttgatg ctgtatcgcg atcctagtgg aaatgtcttt ccctcagata aatggatggc
     4081 agcaggtgtg ttcttcggga aattggaacg catactgata tcaaaactga ccaatcaata
     4141 ctctatatct actattgaag acgattcaag taagcaatcg atgaagcgtc ctgctgccac
     4201 caaaaaggcc ggacaggcta agaaaaagaa gggagacggc tctggatcgg ggtcgggttc
     4261 tggctcagtc gaccttgatc ttgacctcga actcagactt ggatttgctc tcgatctcga
     4321 ccttgaactt agactcggat ttgctcttga cctcgatctt gagcttagac tcggattcgc
     4381 ttaggacgtc ggcggccgcc gacgtccgat cgttcaaaca tttggcaata aagtttctta
     4441 agattgaatc ctgttgccgg tcttgcgatg attatcatat aatttctgtt gaattacgtt
     4501 aagcatgtaa taattaacat gtaatgcatg acgttattta tgagatgggt ttttatgatt
     4561 agagtcccgc aattatacat ttaatacgcg atagaaaaca aaatatagcg cgcaaactag
     4621 gataaattat cgcgcgcggt gtcatctatg ttactagatc gggaattgat cccccctcga
     4681 cagcttccgg aaagggcgaa ttcgcaactt tgtatacaaa agttgaacga gaaacgtaaa
     4741 atgatataaa tatcaatata ttaaattaga ttttgcataa aaaacagact acataatact
     4801 gtaaaacaca acatatccag tcactatgcc atccagctga tatcccctat agtgagtcgt
     4861 attacatggt catagctgtt tcctggcagc tctggcccgt gtctcaaaat ctctgatgtt
     4921 acattgcaca agataaaaat atatcatcat gcctcctctg gaccagccag gacagaaatg
     4981 cctcgacttc gctgctaccc aaggttgccg ggtgacgcac accgtggaaa cggatgaagg
     5041 cacgaaccca gtggacataa gcctgttcgg ttcgtaagct gtaatgcaag tagcgtatgc
     5101 gctcacgcaa ctggtccaga accttgaccg aacgcagcgg tggtaacggc gcagtggcgg
     5161 ttttcatggc ttgttatgac tgtttttttg gggtacagtc tatgcctcgg gcatccaagc
     5221 agcaagcgcg ttacgccgtg ggtcgatgtt tgatgttatg gagcagcaac gatgttacgc
     5281 agcagggcag tcgccctaaa acaaagttaa acattatgag ggaagcggtg atcgccgaag
     5341 tatcgactca actatcagag gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc
     5401 tggccgtaca tttgtacggc tccgcagtgg atggcggcct gaagccacac agtgatattg
     5461 atttgctggt tacggtgacc gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg
     5521 accttttgga aacttcggct tcccctggag agagcgagat tctccgcgct gtagaagtca
     5581 ccattgttgt gcacgacgac atcattccgt ggcgttatcc agctaagcgc gaactgcaat
     5641 ttggagaatg gcagcgcaat gacattcttg caggtatctt cgagccagcc acgatcgaca
     5701 ttgatctggc tatcttgctg acaaaagcaa gagaacatag cgttgccttg gtaggtccag
     5761 cggcggagga actctttgat ccggttcctg aacaggatct atttgaggcg ctaaatgaaa
     5821 ccttaacgct atggaactcg ccgcccgact gggctggcga tgagcgaaat gtagtgctta
     5881 cgttgtcccg catttggtac agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg
     5941 ccgactgggc aatggagcgc ctgccggccc agtatcagcc cgtcatactt gaagctagac
     6001 aggcttatct tggacaagaa gaagatcgct tggcctcgcg cgcagatcag ttggaagaat
     6061 ttgtccacta cgtgaaaggc gagatcacca aggtagtcgg caaataaccc tcgagccacc
     6121 catgaccaaa atcccttaac gtgagttacg cgtcgttcca ctgagcgtca gaccccgtag
     6181 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     6241 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     6301 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     6361 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     6421 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     6481 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     6541 ccagcttgga gcgaacg
//
