LOCUS       sequence-255121-        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-D951A
FEATURES             Location/Qualifiers
     source          1..6579
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(1..87)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     136..158
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     150..167
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     151..167
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    183..282
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             307..327
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3673..3720
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      3751..4003
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4048..4171
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4188..4207)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4189..4207)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4212..4228)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4212..4228)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4659..5450
                     /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      5543..6131
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6032..6051
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6285..6302
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(6461..6488)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 atttgtccta ctcaggagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc
       61 ttccgactga gcctttcgtt ttatttgatg cctggcagtt ccctactctc gcgttaacgc
      121 ttgcatggat gttttcccag tcacgacgtt gtaaaacgac ggccagtctt aagctcgggc
      181 cccaaataat gattttattt tgactgatag tgacctgttc gttgcaacaa attgatgagc
      241 aatgcttttt tataatgcca actttgtaca aaaaagcagg ctccgaattc gcccttcacc
      301 atggctccta agaagaagcg gaaggttggt attcacgggg tgcctgcggc tgccacaagg
      361 tctttcatac ttaagataga gccaaacgaa gaggtcaaaa agggattgtg gaaaacccat
      421 gaagtcctga accatggcat tgcctactac atgaacatcc tgaaacttat acggcaggag
      481 gctatttatg agcaccacga gcaggatcca aaaaacccca aaaaggtttc gaaggctgaa
      541 atccaggccg aactgtggga cttcgttctc aaaatgcaga aatgtaattc gttcactcat
      601 gaagttgaca aagacgtcgt gtttaacatt ttgagggagc tttacgagga gttggttccg
      661 agctccgtcg aaaagaaggg tgaagcaaat cagctgtcga ataagttctt gtaccctttg
      721 gtggacccga acagccaatc tggaaaaggg acagcatcat cagggcggaa gcctcggtgg
      781 tataacttga agattgctgg agacccttcg tgggaagagg aaaagaaaaa gtgggaggaa
      841 gataagaaga aggacccact tgccaaaatt ctcggcaaac ttgccgaata tggattgata
      901 ccgctgttca tcccctttac ggattctaac gaacccatcg ttaaagaaat caagtggatg
      961 gaaaaatctc gcaatcagtc cgtccggagg ctggacaaag atatgtttat acaagctttg
     1021 gaacgctttc tctcgtggga gtcgtggaat cttaaggtca aagaagagta tgaaaaggtc
     1081 gagaaggaac acaagacact ggaggagagg attaaggaag acattcaagc attcaagtca
     1141 ctggagcaat acgaaaagga acggcaggag caattgcttc gcgacacgct caataccaat
     1201 gaatataggc tttccaagag gggcctgaga ggatggcggg aaataatcca gaaatggctc
     1261 aagatggacg agaatgaacc ttcagaaaaa tatctcgagg tttttaaaga ttaccaaagg
     1321 aaacatccac gcgaggcagg ggattacagc gtgtacgagt ttctctccaa gaaggaaaac
     1381 cattttatct ggcgcaatca tcccgaatac ccgtacctct atgcgacgtt ctgcgaaata
     1441 gacaaaaaga aaaaagatgc taagcaacaa gcgactttca cacttgcaga tcccataaat
     1501 cacccattgt gggtgcggtt tgaagaaagg tcgggctcta acctcaataa gtacagaatt
     1561 ttgacggagc agttgcacac agaaaagctg aagaagaagt tgacggttca gctggatcgc
     1621 cttatctacc caaccgagtc tggtggctgg gaagagaagg ggaaagtcga catagtgttg
     1681 ctgccatcta ggcagttcta taaccagatt tttctcgata tagaagaaaa gggtaaacat
     1741 gcatttacgt ataaagacga gtccataaag tttccactga aaggaacact tggcggcgca
     1801 agggtgcagt ttgatcggga ccaccttcgc aggtaccccc acaaggttga aagtggaaac
     1861 gttggacgga tctattttaa tatgaccgtc aacatagaac ccacagaatc ccctgtttcc
     1921 aaatccctga aaatacaccg ggacgatttt cctaaatttg tgaactttaa accgaaggag
     1981 ttgaccgagt ggataaagga cagtaaaggg aaaaagctga agtccggtat cgaaagcctg
     2041 gagattgggc tcagagttat gtcgatagat ctgggtcaaa ggcaggcagc agccgcctct
     2101 atatttgagg tcgtggacca gaagcccgac attgaaggta aactgttctt tccgattaag
     2161 gggacggaac tctacgcagt ccatcgcgcc tccttcaata taaagctgcc gggcgaaaca
     2221 ctggttaaat cacgcgaggt tttgcgcaaa gcgcgggaag acaacctgaa actcatgaat
     2281 caaaagctca atttcctgcg caatgtgttg cacttccagc agtttgagga tattaccgaa
     2341 agagagaaaa gggttacaaa atggatatcc cggcaagaaa actctgatgt tccgctggtt
     2401 taccaggatg agcttataca gattagggaa cttatgtata aaccttacaa agattgggtt
     2461 gcattcctca agcagctgca taagagactt gaagtcgaga tcggcaaaga agtcaaacac
     2521 tggcgcaaga gcctgagcga tggtcggaaa gggttgtacg gaatcagttt gaaaaatatc
     2581 gacgaaatag atagaaccag gaaatttttg ttgcgctggt cactgagacc aacggaaccg
     2641 ggagaagtca gaaggttgga gccaggccag agatttgcaa ttgaccagct gaaccatctg
     2701 aatgcactga aagaggacag attgaagaag atggcgaata cgattattat gcatgctttg
     2761 ggttattgtt acgacgttag gaagaagaaa tggcaggcca agaaccctgc gtgccaaatc
     2821 atcctgttcg aagatctgag taactacaat ccgtatgaag aaaggagtcg cttcgagaac
     2881 agtaaactga tgaaatggtc ccggcgcgag ataccacgcc aagttgcgct tcaaggggaa
     2941 atatacgggc ttcaagttgg ggaagttgga gcgcagtttt ctagccggtt ccacgccaag
     3001 acagggtccc cgggtataag gtgcagtgtg gtgacgaaag aaaagttgca ggataataga
     3061 ttctttaaaa atcttcaacg ggaagggcgc ctgacgcttg acaagattgc agtgttgaaa
     3121 gagggggatt tgtaccccga taaaggcggg gagaagttca tttctttgtc gaaggaccgc
     3181 aagttggtta cgacgcatgc agccattaac gcagcacaaa atctgcaaaa aagattctgg
     3241 actcggacgc atggttttta caaggtttac tgtaaagcat atcaagtcga tggtcagacg
     3301 gtttacattc ccgaatctaa agatcagaaa cagaaaatca ttgaggagtt cggtgaaggt
     3361 tactttatac tcaaggacgg tgtttacgaa tggggtaatg ctggtaaact gaaaattaag
     3421 aaggggtcct ccaagcaatc atcttctgag ctcgtcgaca gcgacatcct taaggatagc
     3481 ttcgatcttg cctctgagct caagggagaa aagttgatgc tgtatcgcga tcctagtgga
     3541 aatgtctttc cctcagataa atggatggca gcaggtgtgt tcttcgggaa attggaacgc
     3601 atactgatat caaaactgac caatcaatac tctatatcta ctattgaaga cgattcaagt
     3661 aagcaatcga tgaagcgtcc tgctgccacc aaaaaggccg gacaggctaa gaaaaagaag
     3721 tgagacgact agtggcggcc gccgacgtcc gatcgttcaa acatttggca ataaagtttc
     3781 ttaagattga atcctgttgc cggtcttgcg atgattatca tataatttct gttgaattac
     3841 gttaagcatg taataattaa catgtaatgc atgacgttat ttatgagatg ggtttttatg
     3901 attagagtcc cgcaattata catttaatac gcgatagaaa acaaaatata gcgcgcaaac
     3961 taggataaat tatcgcgcgc ggtgtcatct atgttactag atcgggaatt gatcccccct
     4021 cgacagcttc cggaaagggc gaattcgcaa ctttgtatac aaaagttgaa cgagaaacgt
     4081 aaaatgatat aaatatcaat atattaaatt agattttgca taaaaaacag actacataat
     4141 actgtaaaac acaacatatc cagtcactat gccatccagc tgatatcccc tatagtgagt
     4201 cgtattacat ggtcatagct gtttcctggc agctctggcc cgtgtctcaa aatctctgat
     4261 gttacattgc acaagataaa aatatatcat catgcctcct ctggaccagc caggacagaa
     4321 atgcctcgac ttcgctgcta cccaaggttg ccgggtgacg cacaccgtgg aaacggatga
     4381 aggcacgaac ccagtggaca taagcctgtt cggttcgtaa gctgtaatgc aagtagcgta
     4441 tgcgctcacg caactggtcc agaaccttga ccgaacgcag cggtggtaac ggcgcagtgg
     4501 cggttttcat ggcttgttat gactgttttt ttggggtaca gtctatgcct cgggcatcca
     4561 agcagcaagc gcgttacgcc gtgggtcgat gtttgatgtt atggagcagc aacgatgtta
     4621 cgcagcaggg cagtcgccct aaaacaaagt taaacattat gagggaagcg gtgatcgccg
     4681 aagtatcgac tcaactatca gaggtagttg gcgtcatcga gcgccatctc gaaccgacgt
     4741 tgctggccgt acatttgtac ggctccgcag tggatggcgg cctgaagcca cacagtgata
     4801 ttgatttgct ggttacggtg accgtaaggc ttgatgaaac aacgcggcga gctttgatca
     4861 acgacctttt ggaaacttcg gcttcccctg gagagagcga gattctccgc gctgtagaag
     4921 tcaccattgt tgtgcacgac gacatcattc cgtggcgtta tccagctaag cgcgaactgc
     4981 aatttggaga atggcagcgc aatgacattc ttgcaggtat cttcgagcca gccacgatcg
     5041 acattgatct ggctatcttg ctgacaaaag caagagaaca tagcgttgcc ttggtaggtc
     5101 cagcggcgga ggaactcttt gatccggttc ctgaacagga tctatttgag gcgctaaatg
     5161 aaaccttaac gctatggaac tcgccgcccg actgggctgg cgatgagcga aatgtagtgc
     5221 ttacgttgtc ccgcatttgg tacagcgcag taaccggcaa aatcgcgccg aaggatgtcg
     5281 ctgccgactg ggcaatggag cgcctgccgg cccagtatca gcccgtcata cttgaagcta
     5341 gacaggctta tcttggacaa gaagaagatc gcttggcctc gcgcgcagat cagttggaag
     5401 aatttgtcca ctacgtgaaa ggcgagatca ccaaggtagt cggcaaataa ccctcgagcc
     5461 acccatgacc aaaatccctt aacgtgagtt acgcgtcgtt ccactgagcg tcagaccccg
     5521 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     5581 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     5641 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     5701 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     5761 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     5821 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     5881 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     5941 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     6001 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     6061 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     6121 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     6181 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     6241 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     6301 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     6361 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     6421 atacgcgtac cgcgagccag gaagagtttg tagaaacgca aaaaggccat ccgtcaggat
     6481 ggccttctgc ttagtttgat gcctggcagt ttatggcggg cgtcctgccc gccaccctcc
     6541 gggccgttgc ttcacaacgt tcaaatccgc tcccggcgg
//
