LOCUS       sequence-250146-        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"
     CDS             358..1149
                     /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      1242..1830
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1731..1750
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1984..2001
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2160..2187)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2279..2365)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2414..2436
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2428..2445
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2429..2445
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2461..2560
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2585..2605
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5951..5998
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      6029..6281
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    6326..6449
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(6466..6485)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6467..6485)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6490..6506)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6490..6506)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 tggaccagcc aggacagaaa tgcctcgact tcgctgctac ccaaggttgc cgggtgacgc
       61 acaccgtgga aacggatgaa ggcacgaacc cagtggacat aagcctgttc ggttcgtaag
      121 ctgtaatgca agtagcgtat gcgctcacgc aactggtcca gaaccttgac cgaacgcagc
      181 ggtggtaacg gcgcagtggc ggttttcatg gcttgttatg actgtttttt tggggtacag
      241 tctatgcctc gggcatccaa gcagcaagcg cgttacgccg tgggtcgatg tttgatgtta
      301 tggagcagca acgatgttac gcagcagggc agtcgcccta aaacaaagtt aaacatcatg
      361 agggaagcgg tgatcgccga agtatcgact caactatcag aggtagttgg cgtcatcgag
      421 cgccatctcg aaccgacgtt gctggccgta catttgtacg gctccgcagt ggatggcggc
      481 ctgaagccac acagtgatat tgatttgctg gttacggtga ccgtaaggct tgatgaaaca
      541 acgcggcgag ctttgatcaa cgaccttttg gaaacttcgg cttcccctgg agagagcgag
      601 attctccgcg ctgtagaagt caccattgtt gtgcacgacg acatcattcc gtggcgttat
      661 ccagctaagc gcgaactgca atttggagaa tggcagcgca atgacattct tgcaggtatc
      721 ttcgagccag ccacgatcga cattgatctg gctatcttgc tgacaaaagc aagagaacat
      781 agcgttgcct tggtaggtcc agcggcggag gaactctttg atccggttcc tgaacaggat
      841 ctatttgagg cgctaaatga aaccttaacg ctatggaact cgccgcccga ctgggctggc
      901 gatgagcgaa atgtagtgct tacgttgtcc cgcatttggt acagcgcagt aaccggcaaa
      961 atcgcgccga aggatgtcgc tgccgactgg gcaatggagc gcctgccggc ccagtatcag
     1021 cccgtcatac ttgaagctag acaggcttat cttggacaag aagaagatcg cttggcctcg
     1081 cgcgcagatc agttggaaga atttgtccac tacgtgaaag gcgagatcac caaggtagtc
     1141 ggcaaataac cctcgagcca cccatgacca aaatccctta acgtgagtta cgcgtcgttc
     1201 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     1261 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     1321 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     1381 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     1441 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     1501 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     1561 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     1621 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     1681 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     1741 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     1801 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     1861 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     1921 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     1981 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     2041 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     2101 agtgagcgca acgcaattaa tacgcgtacc gcgagccagg aagagtttgt agaaacgcaa
     2161 aaaggccatc cgtcaggatg gccttctgct tagtttgatg cctggcagtt tatggcgggc
     2221 gtcctgcccg ccaccctccg ggccgttgct tcacaacgtt caaatccgct cccggcggat
     2281 ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt
     2341 ccgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc gttaacgctt
     2401 gcatggatgt tttcccagtc acgacgttgt aaaacgacgg ccagtcttaa gctcgggccc
     2461 caaataatga ttttattttg actgatagtg acctgttcgt tgcaacaaat tgatgagcaa
     2521 tgctttttta taatgccaac tttgtacaaa aaagcaggct ccgaattcgc ccttcaccat
     2581 ggctcctaag aagaagcgga aggttggtat tcacggggtg cctgcggctg ccacaaggtc
     2641 tttcatactt aagatagagc caaacgaaga ggtcaaaaag ggattgtgga aaacccatga
     2701 agtcctgaac catggcattg cctactacat gaacatcctg aaacttatac ggcaggaggc
     2761 tatttatgag caccacgagc aggatccaaa aaaccccaaa aaggtttcga aggctgaaat
     2821 ccaggccgaa ctgtgggact tcgttctcaa aatgcagaaa tgtaattcgt tcactcatga
     2881 agttgacaaa gacgtcgtgt ttaacatttt gagggagctt tacgaggagt tggttccgag
     2941 ctccgtcgaa aagaagggtg aagcaaatca gctgtcgaat aagttcttgt accctttggt
     3001 ggacccgaac agccaatctg gaaaagggac agcatcatca gggcggaagc ctcggtggta
     3061 taacttgaag attgctggag acccttcgtg ggaagaggaa aagaaaaagt gggaggaaga
     3121 taagaagaag gacccacttg ccaaaattct cggcaaactt gccgaatatg gattgatacc
     3181 gctgttcatc ccctttacgg attctaacga acccatcgtt aaagaaatca agtggatgga
     3241 aaaatctcgc aatcagtccg tccggaggct ggacaaagat atgtttatac aagctttgga
     3301 acgctttctc tcgtgggagt cgtggaatct taaggtcaaa gaagagtatg aaaaggtcga
     3361 gaaggaacac aagacactgg aggagaggat taaggaagac attcaagcat tcaagtcact
     3421 ggagcaatac gaaaaggaac ggcaggagca attgcttcgc gacacgctca ataccaatga
     3481 atataggctt tccaagaggg gcctgagagg atggcgggaa ataatccaga aatggctcaa
     3541 gatggacgag aatgaacctt cagaaaaata tctcgaggtt tttaaagatt accaaaggaa
     3601 acatccacgc gaggcagggg attacagcgt gtacgagttt ctctccaaga aggaaaacca
     3661 ttttatctgg cgcaatcatc ccgaataccc gtacctctat gcgacgttct gcgaaataga
     3721 caaaaagaaa aaagatgcta agcaacaagc gactttcaca cttgcagatc ccataaatca
     3781 cccattgtgg gtgcggtttg aagaaaggtc gggctctaac ctcaataagt acagaatttt
     3841 gacggagcag ttgcacacag aaaagctgaa gaagaagttg acggttcagc tggatcgcct
     3901 tatctaccca accgagtctg gtggctggga agagaagggg aaagtcgaca tagtgttgct
     3961 gccatctagg cagttctata accagatttt tctcgatata gaagaaaagg gtaaacatgc
     4021 atttacgtat aaagacgagt ccataaagtt tccactgaaa ggaacacttg gcggcgcaag
     4081 ggtgcagttt gatcgggacc accttcgcag gtacccccac aaggttgaaa gtggaaacgt
     4141 tggacggatc tattttaata tgaccgtcaa catagaaccc acagaatccc ctgtttccaa
     4201 atccctgaaa atacaccggg acgattttcc taaatttgtg aactttaaac cgaaggagtt
     4261 gaccgagtgg ataaaggaca gtaaagggaa aaagctgaag tccggtatcg aaagcctgga
     4321 gattgggctc agagttatgt cgatagcgct gggtcaaagg caggcagcag ccgcctctat
     4381 atttgaggtc gtggaccaga agcccgacat tgaaggtaaa ctgttctttc cgattaaggg
     4441 gacggaactc tacgcagtcc atcgcgcctc cttcaatata aagctgccgg gcgaaacact
     4501 ggttaaatca cgcgaggttt tgcgcaaagc gcgggaagac aacctgaaac tcatgaatca
     4561 aaagctcaat ttcctgcgca atgtgttgca cttccagcag tttgaggata ttaccgaaag
     4621 agagaaaagg gttacaaaat ggatatcccg gcaagaaaac tctgatgttc cgctggttta
     4681 ccaggatgag cttatacaga ttagggaact tatgtataaa ccttacaaag attgggttgc
     4741 attcctcaag cagctgcata agagacttga agtcgagatc ggcaaagaag tcaaacactg
     4801 gcgcaagagc ctgagcgatg gtcggaaagg gttgtacgga atcagtttga aaaatatcga
     4861 cgaaatagat agaaccagga aatttttgtt gcgctggtca ctgagaccaa cggaaccggg
     4921 agaagtcaga aggttggagc caggccagag atttgcaatt gaccagctga accatctgaa
     4981 tgcactgaaa gaggacagat tgaagaagat ggcgaatacg attattatgc atgctttggg
     5041 ttattgttac gacgttagga agaagaaatg gcaggccaag aaccctgcgt gccaaatcat
     5101 cctgttcgaa gatctgagta actacaatcc gtatgaagaa aggagtcgct tcgagaacag
     5161 taaactgatg aaatggtccc ggcgcgagat accacgccaa gttgcgcttc aaggggaaat
     5221 atacgggctt caagttgggg aagttggagc gcagttttct agccggttcc acgccaagac
     5281 agggtccccg ggtataaggt gcagtgtggt gacgaaagaa aagttgcagg ataatagatt
     5341 ctttaaaaat cttcaacggg aagggcgcct gacgcttgac aagattgcag tgttgaaaga
     5401 gggggatttg taccccgata aaggcgggga gaagttcatt tctttgtcga aggaccgcaa
     5461 gttggttacg acgcatgcag acattaacgc agcacaaaat ctgcaaaaaa gattctggac
     5521 tcggacgcat ggtttttaca aggtttactg taaagcatat caagtcgatg gtcagacggt
     5581 ttacattccc gaatctaaag atcagaaaca gaaaatcatt gaggagttcg gtgaaggtta
     5641 ctttatactc aaggacggtg tttacgaatg gggtaatgct ggtaaactga aaattaagaa
     5701 ggggtcctcc aagcaatcat cttctgagct cgtcgacagc gacatcctta aggatagctt
     5761 cgatcttgcc tctgagctca agggagaaaa gttgatgctg tatcgcgatc ctagtggaaa
     5821 tgtctttccc tcagataaat ggatggcagc aggtgtgttc ttcgggaaat tggaacgcat
     5881 actgatatca aaactgacca atcaatactc tatatctact attgaagacg attcaagtaa
     5941 gcaatcgatg aagcgtcctg ctgccaccaa aaaggccgga caggctaaga aaaagaagtg
     6001 agacgactag tggcggccgc cgacgtccga tcgttcaaac atttggcaat aaagtttctt
     6061 aagattgaat cctgttgccg gtcttgcgat gattatcata taatttctgt tgaattacgt
     6121 taagcatgta ataattaaca tgtaatgcat gacgttattt atgagatggg tttttatgat
     6181 tagagtcccg caattataca tttaatacgc gatagaaaac aaaatatagc gcgcaaacta
     6241 ggataaatta tcgcgcgcgg tgtcatctat gttactagat cgggaattga tcccccctcg
     6301 acagcttccg gaaagggcga attcgcaact ttgtatacaa aagttgaacg agaaacgtaa
     6361 aatgatataa atatcaatat attaaattag attttgcata aaaaacagac tacataatac
     6421 tgtaaaacac aacatatcca gtcactatgc catccagctg atatccccta tagtgagtcg
     6481 tattacatgg tcatagctgt ttcctggcag ctctggcccg tgtctcaaaa tctctgatgt
     6541 tacattgcac aagataaaaa tatatcatca tgcctcctc
//
