LOCUS       sequence-255108-        6788 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 6788)
  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 6788)
  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-SRDX
FEATURES             Location/Qualifiers
     source          1..6788
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(171..198)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(290..376)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     425..447
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     439..456
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     440..456
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    472..571
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             596..616
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4025..4072
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      4249..4501
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4546..4669
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4686..4705)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4687..4705)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4710..4726)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4710..4726)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5157..5948
                     /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      6041..6629
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6530..6549
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(6783..6788,1..12)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg
       61 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
      121 aacgcaatta atacgcgtac cgcgagccag gaagagtttg tagaaacgca aaaaggccat
      181 ccgtcaggat ggccttctgc ttagtttgat gcctggcagt ttatggcggg cgtcctgccc
      241 gccaccctcc gggccgttgc ttcacaacgt tcaaatccgc tcccggcgga tttgtcctac
      301 tcaggagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct tccgactgag
      361 cctttcgttt tatttgatgc ctggcagttc cctactctcg cgttaacgct tgcatggatg
      421 ttttcccagt cacgacgttg taaaacgacg gccagtctta agctcgggcc ccaaataatg
      481 attttatttt gactgatagt gacctgttcg ttgcaacaaa ttgatgagca atgctttttt
      541 ataatgccaa ctttgtacaa aaaagcaggc tccgaattcg cccttcacca tggctcctaa
      601 gaagaagcgg aaggttggta ttcacggggt gcctgcggct gcggtgaagt caataaaagt
      661 taaactccgc ctggacgata tgccagaaat tcgggctggc ctctggaagc ttcacaaaga
      721 ggttaacgct ggcgtcagat attacacgga atggttgtcg ctgctccggc aagaaaatct
      781 ctacagaagg tcgcccaatg gtgatgggga acaagagtgc gacaaaacgg cggaggaatg
      841 caaagcggaa ctccttgaaa gacttcgcgc gagacaagtc gaaaacggcc atagaggccc
      901 ggccggttcc gatgatgaat tgcttcagct tgcgcggcag ctttacgaat tgctcgtgcc
      961 gcaagccata ggtgcaaaag gagatgcaca acaaattgca agaaagttcc tctccccgct
     1021 cgcagacaag gatgccgtgg gaggtcttgg aatcgctaaa gcagggaata agccaagatg
     1081 ggtgcggatg cgggaagcag gtgagccagg ctgggaagag gagaaggaga aagccgaaac
     1141 gaggaaatca gcggatcgca ctgcagacgt gttgagagcc ctcgcagact ttggacttaa
     1201 gccactgatg cgggtttaca cggattcaga gatgtcctcg gtggaatgga agccgctcag
     1261 aaagggtcaa gccgtgagaa cgtgggaccg cgacatgttc cagcaggcaa ttgagcggat
     1321 gatgtcctgg gagtcttgga accaaagggt cgggcaagaa tatgcgaaac tggtggagca
     1381 aaaaaatagg tttgaacaaa aaaatttcgt tggtcaagag catctggttc atttggttaa
     1441 tcaacttcaa caagatatga aagaagcatc acctggcttg gaatctaaag aacaaacagc
     1501 acactacgtt acgggtaggg cgttgagggg atcggataaa gttttcgaga agtggggtaa
     1561 gttggccccc gacgcccctt tcgatctgta tgacgccgag ataaagaacg ttcagcggag
     1621 gaacactcgc cgctttggtt cgcacgatct gtttgcaaaa ctggccgagc ctgagtacca
     1681 ggccctttgg cgggaggatg cgtcgttcct tacacgctac gcggtttata attcaattct
     1741 cagaaagctc aatcacgcga agatgtttgc gactttcact cttccagatg cgacggcaca
     1801 ccctatatgg actagatttg ataagttggg gggcaacttg caccagtata catttctgtt
     1861 caacgaattc ggcgaacgca ggcatgcaat caggttccat aaacttttga aagtcgagaa
     1921 tggtgttgcc agggaggttg acgatgtcac agtgcctatc tcgatgtccg aacaattgga
     1981 taacttgctg cccagagatc cgaacgaacc gattgcactt tatttcaggg attatggtgc
     2041 cgaacaacac tttacgggtg agttcggagg ggccaagatt cagtgcagac gggaccagct
     2101 tgctcacatg caccgcagga gaggggctag ggatgtgtat ttgaacgttt cagttcgcgt
     2161 gcagtcccaa tccgaggcgc ggggggagcg cagaccacca tacgcggctg tcttccggct
     2221 ggttggcgat aaccatagag cgttcgtgca tttcgataag ctgagcgatt acctcgccga
     2281 acatcctgat gacggaaagt tggggtcaga ggggcttctg tcgggcctga gggtgatgtc
     2341 cgtggccctg ggattgcgca ccagtgcctc gatcagcgtt tttagggtgg ccaggaaaga
     2401 tgagttgaaa cccaactcga aggggagggt tccgttcttt ttccctataa agggcaacga
     2461 taacttggtc gcagtgcatg aaaggagcca actgctcaaa cttcccgggg agacagagtc
     2521 caaagatctt cgcgctataa gggaagagag acaaagaact ctccggcagc tgcgcacgca
     2581 gctcgcatac ctgcggttgc ttgtccgctg cggaagtgaa gacgttggca ggcgcgagag
     2641 gtcatgggcc aaattgattg agcagccggt cgacgccgca aatcacatga ctccggattg
     2701 gagggaggct ttcgagaacg aactgcagaa gttgaagagt ctgcatggca tatgctctga
     2761 caaagagtgg atggacgcgg tttacgagtc cgtccgccgg gtctggcggc acatggggaa
     2821 acaagttcgc gattggagaa aggatgttag atccggggaa aggccgaaga taagaggtta
     2881 tgccaaagac gtggttggtg gaaattctat cgaacagatc gaatatcttg agaggcagta
     2941 caagttcctc aagagttggt ctttcttcgg taaagtctct ggacaagtta taagagcaga
     3001 aaaggggagc cggttcgcta tcaccttgcg ggaacacata gaccacgcaa aagaagacag
     3061 actgaagaag ctggcggaca gaattatcat ggaagcgctg gggtacgttt acgcgctgga
     3121 cgaaaggggg aaaggtaaat gggtggccaa atacccgcca tgccagttga tattgctgga
     3181 agaattgtcc gaatatcaat ttaataacga tagaccgcca tccgagaaca accaacttat
     3241 gcaatggtct caccggggag ttttccagga gttgatcaac caagctcaag tgcacgatct
     3301 gcttgttggt acaatgtacg cagcgttttc ctcacgcttc gacgctagaa caggagcgcc
     3361 gggaattcgg tgccggaggg tgcctgcgag gtgtactcag gagcacaacc cggagccatt
     3421 tccctggtgg ttgaataaat tcgttgtgga acatacgttg gatgcttgcc cgcttcgggc
     3481 ggacgacctc attccgacgg gtgagggcga gattttcgtg tcgccattct cggctgagga
     3541 aggggacttc catcaaatcc atgctgacct caatgcggcg caaaatctgc agcagagatt
     3601 gtggagtgat tttgacatct ctcagatcag gcttcggtgc gattggggag aagtcgatgg
     3661 tgaactcgtt ctcattccga gactcaccgg taaaaggact gctgattcat attcgaacaa
     3721 agttttttac actaacacag gggtcactta ttatgaaaga gaacgcggta agaagcgccg
     3781 caaggtgttc gcgcaagaga aactttccga ggaagaggcc gagttgctcg ttgaagctga
     3841 cgaagctcgc gagaagtccg tcgttctgat gcgggatcct tctggcataa taaacagggg
     3901 gaattggaca cggcagaagg aattttggtc catggtgaat cagcgcatag aaggttatct
     3961 ggtcaaacag atcagaagca gggttcccct ccaggattca gcgtgcgaga acacgggcga
     4021 tattaagcgt cctgctgcca ccaaaaaggc cggacaggct aagaaaaaga agggagacgg
     4081 ctctggatcg gggtcgggtt ctggctcagt cgaccttgat cttgacctcg aactcagact
     4141 tggatttgct ctcgatctcg accttgaact tagactcgga tttgctcttg acctcgatct
     4201 tgagcttaga ctcggattcg cttaggacgt cggcggccgc cgacgtccga tcgttcaaac
     4261 atttggcaat aaagtttctt aagattgaat cctgttgccg gtcttgcgat gattatcata
     4321 taatttctgt tgaattacgt taagcatgta ataattaaca tgtaatgcat gacgttattt
     4381 atgagatggg tttttatgat tagagtcccg caattataca tttaatacgc gatagaaaac
     4441 aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat gttactagat
     4501 cgggaattga tcccccctcg acagcttccg gaaagggcga attcgcaact ttgtatacaa
     4561 aagttgaacg agaaacgtaa aatgatataa atatcaatat attaaattag attttgcata
     4621 aaaaacagac tacataatac tgtaaaacac aacatatcca gtcactatgc catccagctg
     4681 atatccccta tagtgagtcg tattacatgg tcatagctgt ttcctggcag ctctggcccg
     4741 tgtctcaaaa tctctgatgt tacattgcac aagataaaaa tatatcatca tgcctcctct
     4801 ggaccagcca ggacagaaat gcctcgactt cgctgctacc caaggttgcc gggtgacgca
     4861 caccgtggaa acggatgaag gcacgaaccc agtggacata agcctgttcg gttcgtaagc
     4921 tgtaatgcaa gtagcgtatg cgctcacgca actggtccag aaccttgacc gaacgcagcg
     4981 gtggtaacgg cgcagtggcg gttttcatgg cttgttatga ctgttttttt ggggtacagt
     5041 ctatgcctcg ggcatccaag cagcaagcgc gttacgccgt gggtcgatgt ttgatgttat
     5101 ggagcagcaa cgatgttacg cagcagggca gtcgccctaa aacaaagtta aacattatga
     5161 gggaagcggt gatcgccgaa gtatcgactc aactatcaga ggtagttggc gtcatcgagc
     5221 gccatctcga accgacgttg ctggccgtac atttgtacgg ctccgcagtg gatggcggcc
     5281 tgaagccaca cagtgatatt gatttgctgg ttacggtgac cgtaaggctt gatgaaacaa
     5341 cgcggcgagc tttgatcaac gaccttttgg aaacttcggc ttcccctgga gagagcgaga
     5401 ttctccgcgc tgtagaagtc accattgttg tgcacgacga catcattccg tggcgttatc
     5461 cagctaagcg cgaactgcaa tttggagaat ggcagcgcaa tgacattctt gcaggtatct
     5521 tcgagccagc cacgatcgac attgatctgg ctatcttgct gacaaaagca agagaacata
     5581 gcgttgcctt ggtaggtcca gcggcggagg aactctttga tccggttcct gaacaggatc
     5641 tatttgaggc gctaaatgaa accttaacgc tatggaactc gccgcccgac tgggctggcg
     5701 atgagcgaaa tgtagtgctt acgttgtccc gcatttggta cagcgcagta accggcaaaa
     5761 tcgcgccgaa ggatgtcgct gccgactggg caatggagcg cctgccggcc cagtatcagc
     5821 ccgtcatact tgaagctaga caggcttatc ttggacaaga agaagatcgc ttggcctcgc
     5881 gcgcagatca gttggaagaa tttgtccact acgtgaaagg cgagatcacc aaggtagtcg
     5941 gcaaataacc ctcgagccac ccatgaccaa aatcccttaa cgtgagttac gcgtcgttcc
     6001 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     6061 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     6121 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     6181 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     6241 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     6301 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     6361 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     6421 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     6481 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     6541 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     6601 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     6661 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     6721 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     6781 gcagcgag
//
