LOCUS       sequence-255061-        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"
     primer_bind     146..165
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     399..416
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(575..602)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(694..780)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     829..851
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     843..860
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     844..860
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    876..975
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             1000..1020
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4429..4476
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      4507..4759
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4804..4927
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4944..4963)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4945..4963)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4968..4984)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4968..4984)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5415..6206
                     /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(6299..6642,1..245)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
       61 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      121 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      181 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      241 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
      301 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
      361 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
      421 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
      481 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatacgc
      541 gtaccgcgag ccaggaagag tttgtagaaa cgcaaaaagg ccatccgtca ggatggcctt
      601 ctgcttagtt tgatgcctgg cagtttatgg cgggcgtcct gcccgccacc ctccgggccg
      661 ttgcttcaca acgttcaaat ccgctcccgg cggatttgtc ctactcagga gagcgttcac
      721 cgacaaacaa cagataaaac gaaaggccca gtcttccgac tgagcctttc gttttatttg
      781 atgcctggca gttccctact ctcgcgttaa cgcttgcatg gatgttttcc cagtcacgac
      841 gttgtaaaac gacggccagt cttaagctcg ggccccaaat aatgatttta ttttgactga
      901 tagtgacctg ttcgttgcaa caaattgatg agcaatgctt ttttataatg ccaactttgt
      961 acaaaaaagc aggctccgaa ttcgcccttc accatggctc ctaagaagaa gcggaaggtt
     1021 ggtattcacg gggtgcctgc ggctgcggtg aagtcaataa aagttaaact ccgcctggac
     1081 gatatgccag aaattcgggc tggcctctgg aagcttcaca aagaggttaa cgctggcgtc
     1141 agatattaca cggaatggtt gtcgctgctc cggcaagaaa atctctacag aaggtcgccc
     1201 aatggtgatg gggaacaaga gtgcgacaaa acggcggagg aatgcaaagc ggaactcctt
     1261 gaaagacttc gcgcgagaca agtcgaaaac ggccatagag gcccggccgg ttccgatgat
     1321 gaattgcttc agcttgcgcg gcagctttac gaattgctcg tgccgcaagc cataggtgca
     1381 aaaggagatg cacaacaaat tgcaagaaag ttcctctccc cgctcgcaga caaggatgcc
     1441 gtgggaggtc ttggaatcgc taaagcaggg aataagccaa gatgggtgcg gatgcgggaa
     1501 gcaggtgagc caggctggga agaggagaag gagaaagccg aaacgaggaa atcagcggat
     1561 cgcactgcag acgtgttgag agccctcgca gactttggac ttaagccact gatgcgggtt
     1621 tacacggatt cagagatgtc ctcggtggaa tggaagccgc tcagaaaggg tcaagccgtg
     1681 agaacgtggg accgcgacat gttccagcag gcaattgagc ggatgatgtc ctgggagtct
     1741 tggaaccaaa gggtcgggca agaatatgcg aaactggtgg agcaaaaaaa taggtttgaa
     1801 caaaaaaatt tcgttggtca agagcatctg gttcatttgg ttaatcaact tcaacaagat
     1861 atgaaagaag catcacctgg cttggaatct aaagaacaaa cagcacacta cgttacgggt
     1921 agggcgttga ggggatcgga taaagttttc gagaagtggg gtaagttggc ccccgacgcc
     1981 cctttcgatc tgtatgacgc cgagataaag aacgttcagc ggaggaacac tcgccgcttt
     2041 ggttcgcacg atctgtttgc aaaactggcc gagcctgagt accaggccct ttggcgggag
     2101 gatgcgtcgt tccttacacg ctacgcggtt tataattcaa ttctcagaaa gctcaatcac
     2161 gcgaagatgt ttgcgacttt cactcttcca gatgcgacgg cacaccctat atggactaga
     2221 tttgataagt tggggggcaa cttgcaccag tatacatttc tgttcaacga attcggcgaa
     2281 cgcaggcatg caatcaggtt ccataaactt ttgaaagtcg agaatggtgt tgccagggag
     2341 gttgacgatg tcacagtgcc tatctcgatg tccgaacaat tggataactt gctgcccaga
     2401 gatccgaacg aaccgattgc actttatttc agggattatg gtgccgaaca acactttacg
     2461 ggtgagttcg gaggggccaa gattcagtgc agacgggacc agcttgctca catgcaccgc
     2521 aggagagggg ctagggatgt gtatttgaac gtttcagttc gcgtgcagtc ccaatccgag
     2581 gcgcgggggg agcgcagacc accatacgcg gctgtcttcc ggctggttgg cgataaccat
     2641 agagcgttcg tgcatttcga taagctgagc gattacctcg ccgaacatcc tgatgacgga
     2701 aagttggggt cagaggggct tctgtcgggc ctgagggtga tgtccgtgga cctgggattg
     2761 cgcaccagtg cctcgatcag cgtttttagg gtggccagga aagatgagtt gaaacccaac
     2821 tcgaagggga gggttccgtt ctttttccct ataaagggca acgataactt ggtcgcagtg
     2881 catgaaagga gccaactgct caaacttccc ggggagacag agtccaaaga tcttcgcgct
     2941 ataagggaag agagacaaag aactctccgg cagctgcgca cgcagctcgc atacctgcgg
     3001 ttgcttgtcc gctgcggaag tgaagacgtt ggcaggcgcg agaggtcatg ggccaaattg
     3061 attgagcagc cggtcgacgc cgcaaatcac atgactccgg attggaggga ggctttcgag
     3121 aacgaactgc agaagttgaa gagtctgcat ggcatatgct ctgacaaaga gtggatggac
     3181 gcggtttacg agtccgtccg ccgggtctgg cggcacatgg ggaaacaagt tcgcgattgg
     3241 agaaaggatg ttagatccgg ggaaaggccg aagataagag gttatgccaa agacgtggtt
     3301 ggtggaaatt ctatcgaaca gatcgaatat cttgagaggc agtacaagtt cctcaagagt
     3361 tggtctttct tcggtaaagt ctctggacaa gttataagag cagaaaaggg gagccggttc
     3421 gctatcacct tgcgggaaca catagaccac gcaaaagaag acagactgaa gaagctggcg
     3481 gacagaatta tcatggaagc gctggggtac gtttacgcgc tggacgaaag ggggaaaggt
     3541 aaatgggtgg ccaaataccc gccatgccag ttgatattgc tggaagaatt gtccgaatat
     3601 caatttaata acgatagacc gccatccgag aacaaccaac ttatgcaatg gtctcaccgg
     3661 ggagttttcc aggagttgat caaccaagct caagtgcacg atctgcttgt tggtacaatg
     3721 tacgcagcgt tttcctcacg cttcgacgct agaacaggag cgccgggaat tcggtgccgg
     3781 agggtgcctg cgaggtgtac tcaggagcac aacccggagc catttccctg gtggttgaat
     3841 aaattcgttg tggaacatac gttggatgct tgcccgcttc gggcggacga cctcattccg
     3901 acgggtgagg gcgagatttt cgtgtcgcca ttctcggctg aggaagggga cttccatcaa
     3961 atccatgctg ccctcaatgc ggcgcaaaat ctgcagcaga gattgtggag tgattttgac
     4021 atctctcaga tcaggcttcg gtgcgattgg ggagaagtcg atggtgaact cgttctcatt
     4081 ccgagactca ccggtaaaag gactgctgat tcatattcga acaaagtttt ttacactaac
     4141 acaggggtca cttattatga aagagaacgc ggtaagaagc gccgcaaggt gttcgcgcaa
     4201 gagaaacttt ccgaggaaga ggccgagttg ctcgttgaag ctgacgaagc tcgcgagaag
     4261 tccgtcgttc tgatgcggga tccttctggc ataataaaca gggggaattg gacacggcag
     4321 aaggaatttt ggtccatggt gaatcagcgc atagaaggtt atctggtcaa acagatcaga
     4381 agcagggttc ccctccagga ttcagcgtgc gagaacacgg gcgatattaa gcgtcctgct
     4441 gccaccaaaa aggccggaca ggctaagaaa aagaagtgag acgactagtg gcggccgccg
     4501 acgtccgatc gttcaaacat ttggcaataa agtttcttaa gattgaatcc tgttgccggt
     4561 cttgcgatga ttatcatata atttctgttg aattacgtta agcatgtaat aattaacatg
     4621 taatgcatga cgttatttat gagatgggtt tttatgatta gagtcccgca attatacatt
     4681 taatacgcga tagaaaacaa aatatagcgc gcaaactagg ataaattatc gcgcgcggtg
     4741 tcatctatgt tactagatcg ggaattgatc ccccctcgac agcttccgga aagggcgaat
     4801 tcgcaacttt gtatacaaaa gttgaacgag aaacgtaaaa tgatataaat atcaatatat
     4861 taaattagat tttgcataaa aaacagacta cataatactg taaaacacaa catatccagt
     4921 cactatgcca tccagctgat atcccctata gtgagtcgta ttacatggtc atagctgttt
     4981 cctggcagct ctggcccgtg tctcaaaatc tctgatgtta cattgcacaa gataaaaata
     5041 tatcatcatg cctcctctgg accagccagg acagaaatgc ctcgacttcg ctgctaccca
     5101 aggttgccgg gtgacgcaca ccgtggaaac ggatgaaggc acgaacccag tggacataag
     5161 cctgttcggt tcgtaagctg taatgcaagt agcgtatgcg ctcacgcaac tggtccagaa
     5221 ccttgaccga acgcagcggt ggtaacggcg cagtggcggt tttcatggct tgttatgact
     5281 gtttttttgg ggtacagtct atgcctcggg catccaagca gcaagcgcgt tacgccgtgg
     5341 gtcgatgttt gatgttatgg agcagcaacg atgttacgca gcagggcagt cgccctaaaa
     5401 caaagttaaa cattatgagg gaagcggtga tcgccgaagt atcgactcaa ctatcagagg
     5461 tagttggcgt catcgagcgc catctcgaac cgacgttgct ggccgtacat ttgtacggct
     5521 ccgcagtgga tggcggcctg aagccacaca gtgatattga tttgctggtt acggtgaccg
     5581 taaggcttga tgaaacaacg cggcgagctt tgatcaacga ccttttggaa acttcggctt
     5641 cccctggaga gagcgagatt ctccgcgctg tagaagtcac cattgttgtg cacgacgaca
     5701 tcattccgtg gcgttatcca gctaagcgcg aactgcaatt tggagaatgg cagcgcaatg
     5761 acattcttgc aggtatcttc gagccagcca cgatcgacat tgatctggct atcttgctga
     5821 caaaagcaag agaacatagc gttgccttgg taggtccagc ggcggaggaa ctctttgatc
     5881 cggttcctga acaggatcta tttgaggcgc taaatgaaac cttaacgcta tggaactcgc
     5941 cgcccgactg ggctggcgat gagcgaaatg tagtgcttac gttgtcccgc atttggtaca
     6001 gcgcagtaac cggcaaaatc gcgccgaagg atgtcgctgc cgactgggca atggagcgcc
     6061 tgccggccca gtatcagccc gtcatacttg aagctagaca ggcttatctt ggacaagaag
     6121 aagatcgctt ggcctcgcgc gcagatcagt tggaagaatt tgtccactac gtgaaaggcg
     6181 agatcaccaa ggtagtcggc aaataaccct cgagccaccc atgaccaaaa tcccttaacg
     6241 tgagttacgc gtcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     6301 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     6361 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     6421 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     6481 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     6541 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     6601 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc ca
//
