LOCUS       sequence-250138-        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-D977A
FEATURES             Location/Qualifiers
     source          1..6642
                     /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             6014..6061
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      6092..6344
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    6389..6512
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(6529..6548)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6530..6548)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6553..6569)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6553..6569)
                     /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 cggtgaagtc
     2641 aataaaagtt aaactccgcc tggacgatat gccagaaatt cgggctggcc tctggaagct
     2701 tcacaaagag gttaacgctg gcgtcagata ttacacggaa tggttgtcgc tgctccggca
     2761 agaaaatctc tacagaaggt cgcccaatgg tgatggggaa caagagtgcg acaaaacggc
     2821 ggaggaatgc aaagcggaac tccttgaaag acttcgcgcg agacaagtcg aaaacggcca
     2881 tagaggcccg gccggttccg atgatgaatt gcttcagctt gcgcggcagc tttacgaatt
     2941 gctcgtgccg caagccatag gtgcaaaagg agatgcacaa caaattgcaa gaaagttcct
     3001 ctccccgctc gcagacaagg atgccgtggg aggtcttgga atcgctaaag cagggaataa
     3061 gccaagatgg gtgcggatgc gggaagcagg tgagccaggc tgggaagagg agaaggagaa
     3121 agccgaaacg aggaaatcag cggatcgcac tgcagacgtg ttgagagccc tcgcagactt
     3181 tggacttaag ccactgatgc gggtttacac ggattcagag atgtcctcgg tggaatggaa
     3241 gccgctcaga aagggtcaag ccgtgagaac gtgggaccgc gacatgttcc agcaggcaat
     3301 tgagcggatg atgtcctggg agtcttggaa ccaaagggtc gggcaagaat atgcgaaact
     3361 ggtggagcaa aaaaataggt ttgaacaaaa aaatttcgtt ggtcaagagc atctggttca
     3421 tttggttaat caacttcaac aagatatgaa agaagcatca cctggcttgg aatctaaaga
     3481 acaaacagca cactacgtta cgggtagggc gttgagggga tcggataaag ttttcgagaa
     3541 gtggggtaag ttggcccccg acgccccttt cgatctgtat gacgccgaga taaagaacgt
     3601 tcagcggagg aacactcgcc gctttggttc gcacgatctg tttgcaaaac tggccgagcc
     3661 tgagtaccag gccctttggc gggaggatgc gtcgttcctt acacgctacg cggtttataa
     3721 ttcaattctc agaaagctca atcacgcgaa gatgtttgcg actttcactc ttccagatgc
     3781 gacggcacac cctatatgga ctagatttga taagttgggg ggcaacttgc accagtatac
     3841 atttctgttc aacgaattcg gcgaacgcag gcatgcaatc aggttccata aacttttgaa
     3901 agtcgagaat ggtgttgcca gggaggttga cgatgtcaca gtgcctatct cgatgtccga
     3961 acaattggat aacttgctgc ccagagatcc gaacgaaccg attgcacttt atttcaggga
     4021 ttatggtgcc gaacaacact ttacgggtga gttcggaggg gccaagattc agtgcagacg
     4081 ggaccagctt gctcacatgc accgcaggag aggggctagg gatgtgtatt tgaacgtttc
     4141 agttcgcgtg cagtcccaat ccgaggcgcg gggggagcgc agaccaccat acgcggctgt
     4201 cttccggctg gttggcgata accatagagc gttcgtgcat ttcgataagc tgagcgatta
     4261 cctcgccgaa catcctgatg acggaaagtt ggggtcagag gggcttctgt cgggcctgag
     4321 ggtgatgtcc gtggacctgg gattgcgcac cagtgcctcg atcagcgttt ttagggtggc
     4381 caggaaagat gagttgaaac ccaactcgaa ggggagggtt ccgttctttt tccctataaa
     4441 gggcaacgat aacttggtcg cagtgcatga aaggagccaa ctgctcaaac ttcccgggga
     4501 gacagagtcc aaagatcttc gcgctataag ggaagagaga caaagaactc tccggcagct
     4561 gcgcacgcag ctcgcatacc tgcggttgct tgtccgctgc ggaagtgaag acgttggcag
     4621 gcgcgagagg tcatgggcca aattgattga gcagccggtc gacgccgcaa atcacatgac
     4681 tccggattgg agggaggctt tcgagaacga actgcagaag ttgaagagtc tgcatggcat
     4741 atgctctgac aaagagtgga tggacgcggt ttacgagtcc gtccgccggg tctggcggca
     4801 catggggaaa caagttcgcg attggagaaa ggatgttaga tccggggaaa ggccgaagat
     4861 aagaggttat gccaaagacg tggttggtgg aaattctatc gaacagatcg aatatcttga
     4921 gaggcagtac aagttcctca agagttggtc tttcttcggt aaagtctctg gacaagttat
     4981 aagagcagaa aaggggagcc ggttcgctat caccttgcgg gaacacatag accacgcaaa
     5041 agaagacaga ctgaagaagc tggcggacag aattatcatg gaagcgctgg ggtacgttta
     5101 cgcgctggac gaaaggggga aaggtaaatg ggtggccaaa tacccgccat gccagttgat
     5161 attgctggaa gaattgtccg aatatcaatt taataacgat agaccgccat ccgagaacaa
     5221 ccaacttatg caatggtctc accggggagt tttccaggag ttgatcaacc aagctcaagt
     5281 gcacgatctg cttgttggta caatgtacgc agcgttttcc tcacgcttcg acgctagaac
     5341 aggagcgccg ggaattcggt gccggagggt gcctgcgagg tgtactcagg agcacaaccc
     5401 ggagccattt ccctggtggt tgaataaatt cgttgtggaa catacgttgg atgcttgccc
     5461 gcttcgggcg gacgacctca ttccgacggg tgagggcgag attttcgtgt cgccattctc
     5521 ggctgaggaa ggggacttcc atcaaatcca tgctgccctc aatgcggcgc aaaatctgca
     5581 gcagagattg tggagtgatt ttgacatctc tcagatcagg cttcggtgcg attggggaga
     5641 agtcgatggt gaactcgttc tcattccgag actcaccggt aaaaggactg ctgattcata
     5701 ttcgaacaaa gttttttaca ctaacacagg ggtcacttat tatgaaagag aacgcggtaa
     5761 gaagcgccgc aaggtgttcg cgcaagagaa actttccgag gaagaggccg agttgctcgt
     5821 tgaagctgac gaagctcgcg agaagtccgt cgttctgatg cgggatcctt ctggcataat
     5881 aaacaggggg aattggacac ggcagaagga attttggtcc atggtgaatc agcgcataga
     5941 aggttatctg gtcaaacaga tcagaagcag ggttcccctc caggattcag cgtgcgagaa
     6001 cacgggcgat attaagcgtc ctgctgccac caaaaaggcc ggacaggcta agaaaaagaa
     6061 gtgagacgac tagtggcggc cgccgacgtc cgatcgttca aacatttggc aataaagttt
     6121 cttaagattg aatcctgttg ccggtcttgc gatgattatc atataatttc tgttgaatta
     6181 cgttaagcat gtaataatta acatgtaatg catgacgtta tttatgagat gggtttttat
     6241 gattagagtc ccgcaattat acatttaata cgcgatagaa aacaaaatat agcgcgcaaa
     6301 ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta gatcgggaat tgatcccccc
     6361 tcgacagctt ccggaaaggg cgaattcgca actttgtata caaaagttga acgagaaacg
     6421 taaaatgata taaatatcaa tatattaaat tagattttgc ataaaaaaca gactacataa
     6481 tactgtaaaa cacaacatat ccagtcacta tgccatccag ctgatatccc ctatagtgag
     6541 tcgtattaca tggtcatagc tgtttcctgg cagctctggc ccgtgtctca aaatctctga
     6601 tgttacattg cacaagataa aaatatatca tcatgcctcc tc
//
