LOCUS       sequence-255064-        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: pYPQ292
FEATURES             Location/Qualifiers
     source          1..6642
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     108..127
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     361..378
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(537..564)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(656..742)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     791..813
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     805..822
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     806..822
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    838..937
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             962..982
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4391..4438
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      4469..4721
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4766..4889
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4906..4925)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4907..4925)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4930..4946)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4930..4946)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5377..6168
                     /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(6261..6642,1..207)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
       61 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
      121 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
      181 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
      241 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
      301 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
      361 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
      421 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
      481 gagcgcaacg caattaatac gcgtaccgcg agccaggaag agtttgtaga aacgcaaaaa
      541 ggccatccgt caggatggcc ttctgcttag tttgatgcct ggcagtttat ggcgggcgtc
      601 ctgcccgcca ccctccgggc cgttgcttca caacgttcaa atccgctccc ggcggatttg
      661 tcctactcag gagagcgttc accgacaaac aacagataaa acgaaaggcc cagtcttccg
      721 actgagcctt tcgttttatt tgatgcctgg cagttcccta ctctcgcgtt aacgcttgca
      781 tggatgtttt cccagtcacg acgttgtaaa acgacggcca gtcttaagct cgggccccaa
      841 ataatgattt tattttgact gatagtgacc tgttcgttgc aacaaattga tgagcaatgc
      901 ttttttataa tgccaacttt gtacaaaaaa gcaggctccg aattcgccct tcaccatggc
      961 tcctaagaag aagcggaagg ttggtattca cggggtgcct gcggctgccg tcaagtccat
     1021 gaaggtcaag ttgcgcctgg ataacatgcc agagatcaga gccggacttt ggaaacttca
     1081 caccgaggtt aatgcgggtg tgcggtacta tacggaatgg cttagccttt tgaggcaaga
     1141 aaatctttat cggaggagtc ccaatggcga tggagaacaa gaatgctata aaactgctga
     1201 ggaatgcaag gctgaactcc ttgagagact cagagcccgc caagttgaga atgggcactg
     1261 cggccctgct gggagtgatg acgaactgct gcaattggca cggcaacttt atgaacttct
     1321 ggtcccacaa gcaatcgggg ctaaaggtga tgcgcagcaa atcgcaagga agtttcttag
     1381 tccccttgcc gacaaggatg ccgtgggtgg tttgggaata gcaaaagcag gaaataagcc
     1441 taggtgggtt cggatgaggg aggctggaga gccaggttgg gaagaggaaa aggctaaagc
     1501 cgaggcgaga aagagtacgg atagaaccgc cgatgttctt cgcgctcttg cagacttcgg
     1561 tcttaaacct cttatgagag tctacacaga ctcagacatg tccagcgtgc agtggaaacc
     1621 acttcgcaaa ggacaagcgg tcagaacctg ggatagagac atgttccaac aagcgatcga
     1681 aagaatgatg agttgggaat cgtggaatca gcgcgttgga gaagcgtacg caaagctcgt
     1741 ggaacaaaag tcgaggtttg aacagaaaaa ttttgtggga caagaacatc ttgtccaact
     1801 tgtcaatcaa cttcaacaag acatgaagga agcatcacac ggcctggagt cgaaagaaca
     1861 aactgcgcat tacttgactg ggagagcgct gagagggagc gacaaagttt ttgagaagtg
     1921 ggaaaaactc gatcctgatg ccccatttga cctctatgat accgaaatca agaatgttca
     1981 acggaggaat actcgcaggt tcggatctca tgatctgttt gcgaagctcg cggaacctaa
     2041 atatcaggcg ctctggagag aggacgcttc tttcctcacg aggtatgcgg tttacaatag
     2101 cattgtcaga aaactgaatc acgctaaaat gtttgcgact tttactcttc cggatgctac
     2161 cgcccacccg atctggacgc ggtttgacaa actcggcggc aacctgcacc agtacacttt
     2221 cttgtttaac gaatttggcg agggcaggca cgccattcgg tttcagaagc tgttgacggt
     2281 tgaggatggc gttgctaaag aggtcgacga cgtcacggtt ccgatttcta tgtccgcgca
     2341 gctggatgac ctcttgcctc gggacccaca cgagctcgtt gcactctact tccaggacta
     2401 cggtgcagaa caacatctgg ctggagagtt tggcggcgcg aaaattcaat accgccgcga
     2461 tcaattgaac cacctgcacg ccagaagagg cgccagagat gtctacctta atctgagcgt
     2521 ccgcgttcag tcacaatccg aagccagggg agaaaggcgc cctccgtatg cagcggtctt
     2581 caggcttgtt ggcgataacc accgcgcgtt tgttcacttt gataaattgt cagattacct
     2641 cgcagaacac ccagacgatg gtaagctggg gtcggaaggt ttgctctctg ggctcagagt
     2701 catgtcagtt gacttgggtc ttaggacttc cgcgagcata tctgtcttcc gcgtcgcaag
     2761 aaaggacgaa ttgaagccga acagtgaagg ccgggtccct ttttgcttcc cgatcgaagg
     2821 gaacgaaaac ctcgttgctg tccacgagcg gagccaactg ttgaagcttc ccggtgaaac
     2881 ggaatcgaaa gatctgagag cgatcagaga agagcgccaa aggacgctta gacagctccg
     2941 gacgcaactt gcatacttgc gccttctggt tcgctgcggt agtgaagacg ttggaagaag
     3001 agagaggtca tgggctaaac tcatagagca acctatggat gctaatcaaa tgacgcctga
     3061 ttggagagaa gcattcgaag acgaacttca gaaactgaaa tccctttacg ggatatgcgg
     3121 cgatcgcgag tggacagaag cagtgtatga gtctgtgagg cgcgtgtggc ggcatatggg
     3181 taaacaggtg cgcgattgga gaaaagacgt taggagcggg gaaagaccta agatacgggg
     3241 atatcagaaa gacgttgtcg ggggaaatag cattgaacag attgaatatt tggagcgcca
     3301 atataagttc ctcaaatcct ggtctttctt cggcaaagtg tcaggccagg tgatacgcgc
     3361 ggaaaaggga tcgcgctttg caataactct gagagaacat attgatcatg ccaaagaaga
     3421 tcggttgaag aaactcgccg atagaatcat catggaggcg cttggttatg tctacgcctt
     3481 ggacgatgaa cggggaaagg gaaagtgggt cgccaagtat ccaccttgcc aactcattct
     3541 cctcgaagaa ctttccgaat accagtttaa caacgatcgg ccgccatcag agaataatca
     3601 actgatgcag tggtcccatc gcggtgtgtt tcaagagttg ctcaatcagg cccaagtcca
     3661 tgatctgctt gttggcacaa tgtatgcagc cttttcctcc cggtttgatg caagaacagg
     3721 ggctcctggc atacgctgta gacgggtccc ggcgaggtgc gcccgcgaac aaaaccctga
     3781 accgttcccc tggtggttga acaagttcgt tgcggagcac aagctggacg ggtgtcctct
     3841 gcgggccgac gatcttattc ccaccgggga aggggaattc tttgtgagcc ctttctcggc
     3901 ggaggaaggg gattttcacc aaatacatgc agatcttaat gccgcacaaa atttgcagag
     3961 gagactgtgg tcagactttg atattagtca gatacgcctc cgctgtgact ggggagaggt
     4021 cgatggcgag cctgtgttga taccaagaac gaccggaaag aggacagccg attcgtatgg
     4081 aaacaaggtt ttttacacga agacgggcgt tacttactac gaaagagaaa gagggaagaa
     4141 gagaaggaaa gtctttgccc aagaagaatt gagcgaggaa gaagccgagc tcttggtcga
     4201 agcggacgag gcacgggaaa agtctgtcgt cctcatgagg gacccttccg gaattattaa
     4261 ccggggagat tggacgcggc agaaagagtt ttggtccatg gttaatcaac gcatagaagg
     4321 ctaccttgtc aagcaaataa gaagtcgcgt gagattgcag gagagtgcat gtgagaacac
     4381 tggggacata aagcgtcctg ctgccaccaa aaaggccgga caggctaaga aaaagaagtg
     4441 agacgactag tggcggccgc cgacgtccga tcgttcaaac atttggcaat aaagtttctt
     4501 aagattgaat cctgttgccg gtcttgcgat gattatcata taatttctgt tgaattacgt
     4561 taagcatgta ataattaaca tgtaatgcat gacgttattt atgagatggg tttttatgat
     4621 tagagtcccg caattataca tttaatacgc gatagaaaac aaaatatagc gcgcaaacta
     4681 ggataaatta tcgcgcgcgg tgtcatctat gttactagat cgggaattga tcccccctcg
     4741 acagcttccg gaaagggcga attcgcaact ttgtatacaa aagttgaacg agaaacgtaa
     4801 aatgatataa atatcaatat attaaattag attttgcata aaaaacagac tacataatac
     4861 tgtaaaacac aacatatcca gtcactatgc catccagctg atatccccta tagtgagtcg
     4921 tattacatgg tcatagctgt ttcctggcag ctctggcccg tgtctcaaaa tctctgatgt
     4981 tacattgcac aagataaaaa tatatcatca tgcctcctct ggaccagcca ggacagaaat
     5041 gcctcgactt cgctgctacc caaggttgcc gggtgacgca caccgtggaa acggatgaag
     5101 gcacgaaccc agtggacata agcctgttcg gttcgtaagc tgtaatgcaa gtagcgtatg
     5161 cgctcacgca actggtccag aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg
     5221 gttttcatgg cttgttatga ctgttttttt ggggtacagt ctatgcctcg ggcatccaag
     5281 cagcaagcgc gttacgccgt gggtcgatgt ttgatgttat ggagcagcaa cgatgttacg
     5341 cagcagggca gtcgccctaa aacaaagtta aacattatga gggaagcggt gatcgccgaa
     5401 gtatcgactc aactatcaga ggtagttggc gtcatcgagc gccatctcga accgacgttg
     5461 ctggccgtac atttgtacgg ctccgcagtg gatggcggcc tgaagccaca cagtgatatt
     5521 gatttgctgg ttacggtgac cgtaaggctt gatgaaacaa cgcggcgagc tttgatcaac
     5581 gaccttttgg aaacttcggc ttcccctgga gagagcgaga ttctccgcgc tgtagaagtc
     5641 accattgttg tgcacgacga catcattccg tggcgttatc cagctaagcg cgaactgcaa
     5701 tttggagaat ggcagcgcaa tgacattctt gcaggtatct tcgagccagc cacgatcgac
     5761 attgatctgg ctatcttgct gacaaaagca agagaacata gcgttgcctt ggtaggtcca
     5821 gcggcggagg aactctttga tccggttcct gaacaggatc tatttgaggc gctaaatgaa
     5881 accttaacgc tatggaactc gccgcccgac tgggctggcg atgagcgaaa tgtagtgctt
     5941 acgttgtccc gcatttggta cagcgcagta accggcaaaa tcgcgccgaa ggatgtcgct
     6001 gccgactggg caatggagcg cctgccggcc cagtatcagc ccgtcatact tgaagctaga
     6061 caggcttatc ttggacaaga agaagatcgc ttggcctcgc gcgcagatca gttggaagaa
     6121 tttgtccact acgtgaaagg cgagatcacc aaggtagtcg gcaaataacc ctcgagccac
     6181 ccatgaccaa aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc agaccccgta
     6241 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     6301 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     6361 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     6421 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     6481 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     6541 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     6601 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc ta
//
