LOCUS       sequence-255066-        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
FEATURES             Location/Qualifiers
     source          1..6642
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     118..137
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     371..388
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(547..574)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(666..752)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     801..823
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     815..832
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     816..832
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    848..947
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             972..992
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4401..4448
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      4479..4731
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4776..4899
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4916..4935)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4917..4935)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4940..4956)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4940..4956)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5387..6178
                     /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(6271..6642,1..217)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
       61 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
      121 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
      181 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
      241 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
      301 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
      361 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
      421 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
      481 agcgggcagt gagcgcaacg caattaatac gcgtaccgcg agccaggaag agtttgtaga
      541 aacgcaaaaa ggccatccgt caggatggcc ttctgcttag tttgatgcct ggcagtttat
      601 ggcgggcgtc ctgcccgcca ccctccgggc cgttgcttca caacgttcaa atccgctccc
      661 ggcggatttg tcctactcag gagagcgttc accgacaaac aacagataaa acgaaaggcc
      721 cagtcttccg actgagcctt tcgttttatt tgatgcctgg cagttcccta ctctcgcgtt
      781 aacgcttgca tggatgtttt cccagtcacg acgttgtaaa acgacggcca gtcttaagct
      841 cgggccccaa ataatgattt tattttgact gatagtgacc tgttcgttgc aacaaattga
      901 tgagcaatgc ttttttataa tgccaacttt gtacaaaaaa gcaggctccg aattcgccct
      961 tcaccatggc tcctaagaag aagcggaagg ttggtattca cggggtgcct gcggctgcgg
     1021 tgaagtcaat aaaagttaaa ctccgcctgg acgatatgcc agaaattcgg gctggcctct
     1081 ggaagcttca caaagaggtt aacgctggcg tcagatatta cacggaatgg ttgtcgctgc
     1141 tccggcaaga aaatctctac agaaggtcgc ccaatggtga tggggaacaa gagtgcgaca
     1201 aaacggcgga ggaatgcaaa gcggaactcc ttgaaagact tcgcgcgaga caagtcgaaa
     1261 acggccatag aggcccggcc ggttccgatg atgaattgct tcagcttgcg cggcagcttt
     1321 acgaattgct cgtgccgcaa gccataggtg caaaaggaga cgcacaacaa attgcaagaa
     1381 agttcctctc cccgctcgca gacaaggatg ccgtgggagg tcttggaatc gctaaagcag
     1441 ggaataagcc aagatgggtg cggatgcggg aagcaggtga gccaggctgg gaagaggaga
     1501 aggagaaagc cgaaacgagg aaatcagcgg atcgcactgc agacgtgttg agagccctcg
     1561 cagactttgg acttaagcca ctgatgcggg tttacacgga ttcagagatg tcctcggtgg
     1621 aatggaagcc gctcagaaag ggtcaagccg tgagaacgtg ggaccgcgac atgttccagc
     1681 aggcaattga gcggatgatg tcctgggagt cttggaacca aagggtcggg caagaatatg
     1741 cgaaactggt ggagcaaaaa aataggtttg aacaaaaaaa tttcgttggt caagagcatc
     1801 tggttcattt ggttaatcaa cttcaacaag atatgaaaga agcatcacct ggcttggaat
     1861 ctaaagaaca aacagcacac tacgttacgg gtagggcgtt gaggggatcg gataaagttt
     1921 tcgagaagtg gggtaagttg gcccccgacg cccctttcga tctgtatgac gccgagataa
     1981 agaacgttca gcggaggaac actcgccgct ttggttcgca cgatctgttt gcaaaactgg
     2041 ccgagcctga gtaccaggcc ctttggcggg aggatgcgtc gttccttaca cgctacgcgg
     2101 tttataattc aattctcaga aagctcaatc acgcgaagat gtttgcgact ttcactcttc
     2161 cagatgcgac ggcacaccct atatggacta gatttgataa gttggggggc aacttgcacc
     2221 agtatacatt tctgttcaac gaattcggcg aacgcaggca tgcaatcagg ttccataaac
     2281 ttttgaaagt cgagaatggt gttgccaggg aggttgacga tgtcacagtg cctatctcga
     2341 tgtccgaaca attggataac ttgctgccca gagatccgaa cgaaccgatt gcactttatt
     2401 tcagggatta tggtgccgaa caacacttta cgggtgagtt cggaggggcc aagattcagt
     2461 gcagacggga ccagcttgct cacatgcacc gcaggagagg ggctagggat gtgtatttga
     2521 acgtttcagt tcgcgtgcag tcccaatccg aggcgcgggg ggagcgcaga ccaccatacg
     2581 cggctgtctt ccggctggtt ggcgataacc atagagcgtt cgtgcatttc gataagctga
     2641 gcgattacct cgccgaacat cctgatgacg gaaagttggg gtcagagggg cttctgtcgg
     2701 gcctgagggt gatgtccgtg gacctgggat tgcgcaccag tgcctcgatc agcgttttta
     2761 gggtggccag gaaagatgag ttgaaaccca actcgaaggg gagggttccg ttctttttcc
     2821 ctataaaggg caacgataac ttggtcgcag tgcatgaaag gagccaactg ctcaaacttc
     2881 ccggggagac agagtccaaa gatcttcgcg ctataaggga agagagacaa agaactctcc
     2941 ggcagctgcg cacgcagctc gcatacctgc ggttgcttgt ccgctgcgga agtgaagacg
     3001 ttggcaggcg cgagaggtca tgggccaaat tgattgagca gccggtcgac gccgcaaatc
     3061 acatgactcc ggattggagg gaggctttcg agaacgaact gcagaagttg aagagtctgc
     3121 atggcatatg ctctgacaaa gagtggatgg acgcggttta cgagtccgtc cgccgggtct
     3181 ggcggcacat ggggaaacaa gttcgcgatt ggagaaagga tgttagatcc ggggaaaggc
     3241 cgaagataag aggttatgcc aaagacgtgg ttggtggaaa ttctatcgaa cagatcgaat
     3301 atcttgagag gcagtacaag ttcctcaaga gttggtcttt cttcggtaaa gtctctggac
     3361 aagttataag agcagaaaag gggagccggt tcgctatcac cttgcgggaa cacatagacc
     3421 acgcaaaaga agacagactg aagaagctgg cggacagaat tatcatggaa gcgctggggt
     3481 acgtttacgc gctggacgaa agggggaaag gtaaatgggt ggccaaatac ccgccatgcc
     3541 agttgatatt gctggaagaa ttgtccgaat atcaatttaa taacgataga ccgccatccg
     3601 agaacaacca acttatgcaa tggtctcacc ggggagtttt ccaggagttg atcaaccaag
     3661 ctcaagtgca cgatctgctt gttggtacaa tgtacgcagc gttttcctca cgcttcgacg
     3721 ctagaacagg agcgccggga attcggtgcc ggagggtgcc tgcgaggtgt actcaggagc
     3781 acaacccgga gccatttccc tggtggttga ataaattcgt tgtggaacat acgttggatg
     3841 cttgcccgct tcgggcggac gacctcattc cgacgggtga gggcgagatt ttcgtgtcgc
     3901 cattctcggc tgaggaaggg gacttccatc aaatccatgc tgacctcaat gcggcgcaaa
     3961 atctgcagca gagattgtgg agtgattttg acatctctca gatcaggctt cggtgcgatt
     4021 ggggagaagt cgatggtgaa ctcgttctca ttccgagact caccggtaaa aggactgctg
     4081 attcatattc gaacaaagtt ttttacacta acacaggggt cacttattat gaaagagaac
     4141 gcggtaagaa gcgccgcaag gtgttcgcgc aagagaaact ttccgaggaa gaggccgagt
     4201 tgctcgttga agctgacgaa gctcgcgaga agtccgtcgt tctgatgcgg gatccttctg
     4261 gcataataaa cagggggaat tggacacggc agaaggaatt ttggtccatg gtgaatcagc
     4321 gcatagaagg ttatctggtc aaacagatca gaagcagggt tcccctccag gattcagcgt
     4381 gcgagaacac gggcgatatt aagcgtcctg ctgccaccaa aaaggccgga caggctaaga
     4441 aaaagaagtg agacgactag tggcggccgc cgacgtccga tcgttcaaac atttggcaat
     4501 aaagtttctt aagattgaat cctgttgccg gtcttgcgat gattatcata taatttctgt
     4561 tgaattacgt taagcatgta ataattaaca tgtaatgcat gacgttattt atgagatggg
     4621 tttttatgat tagagtcccg caattataca tttaatacgc gatagaaaac aaaatatagc
     4681 gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat gttactagat cgggaattga
     4741 tcccccctcg acagcttccg gaaagggcga attcgcaact ttgtatacaa aagttgaacg
     4801 agaaacgtaa aatgatataa atatcaatat attaaattag attttgcata aaaaacagac
     4861 tacataatac tgtaaaacac aacatatcca gtcactatgc catccagctg atatccccta
     4921 tagtgagtcg tattacatgg tcatagctgt ttcctggcag ctctggcccg tgtctcaaaa
     4981 tctctgatgt tacattgcac aagataaaaa tatatcatca tgcctcctct ggaccagcca
     5041 ggacagaaat gcctcgactt cgctgctacc caaggttgcc gggtgacgca caccgtggaa
     5101 acggatgaag gcacgaaccc agtggacata agcctgttcg gttcgtaagc tgtaatgcaa
     5161 gtagcgtatg cgctcacgca actggtccag aaccttgacc gaacgcagcg gtggtaacgg
     5221 cgcagtggcg gttttcatgg cttgttatga ctgttttttt ggggtacagt ctatgcctcg
     5281 ggcatccaag cagcaagcgc gttacgccgt gggtcgatgt ttgatgttat ggagcagcaa
     5341 cgatgttacg cagcagggca gtcgccctaa aacaaagtta aacattatga gggaagcggt
     5401 gatcgccgaa gtatcgactc aactatcaga ggtagttggc gtcatcgagc gccatctcga
     5461 accgacgttg ctggccgtac atttgtacgg ctccgcagtg gatggcggcc tgaagccaca
     5521 cagtgatatt gatttgctgg ttacggtgac cgtaaggctt gatgaaacaa cgcggcgagc
     5581 tttgatcaac gaccttttgg aaacttcggc ttcccctgga gagagcgaga ttctccgcgc
     5641 tgtagaagtc accattgttg tgcacgacga catcattccg tggcgttatc cagctaagcg
     5701 cgaactgcaa tttggagaat ggcagcgcaa tgacattctt gcaggtatct tcgagccagc
     5761 cacgatcgac attgatctgg ctatcttgct gacaaaagca agagaacata gcgttgcctt
     5821 ggtaggtcca gcggcggagg aactctttga tccggttcct gaacaggatc tatttgaggc
     5881 gctaaatgaa accttaacgc tatggaactc gccgcccgac tgggctggcg atgagcgaaa
     5941 tgtagtgctt acgttgtccc gcatttggta cagcgcagta accggcaaaa tcgcgccgaa
     6001 ggatgtcgct gccgactggg caatggagcg cctgccggcc cagtatcagc ccgtcatact
     6061 tgaagctaga caggcttatc ttggacaaga agaagatcgc ttggcctcgc gcgcagatca
     6121 gttggaagaa tttgtccact acgtgaaagg cgagatcacc aaggtagtcg gcaaataacc
     6181 ctcgagccac ccatgaccaa aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc
     6241 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     6301 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     6361 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     6421 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     6481 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     6541 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     6601 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ct
//
