LOCUS       sequence-485642-        8128 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ttLbCas12a (LbCas12a-D156R)-intron Gateway entry plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8128)
  AUTHORS   Luo W, Ors S, Fang H, Zeng M, Cheng Y, Sidawy AE, Qi Y
  TITLE     Efficient genome editing by CRISPR-Cas12a in common wheat via shoot 
            apical meristem delivery.
  JOURNAL   Plant Physiol. 2025 Nov 26;199(4):kiaf611. doi: 
            10.1093/plphys/kiaf611.
  PUBMED    41335006
REFERENCE   2  (bases 1 to 8128)
  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"; journalName: "Plant 
            Physiol."; date: "2025-11-26"; volume: "199(4):kiaf611. doi"; pages:
            " 10.1093/plphys/kiaf611"
COMMENT     Sequence Label: ttLbCas12a-intron
FEATURES             Location/Qualifiers
     source          1..8128
                     /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             7500..7547
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      7578..7830
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    7875..7998
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(8015..8034)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8016..8034)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8039..8055)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8039..8055)
                     /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 aaacattatg
      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 aatactgttc 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 cctgcggcta gcaagctcga
     2641 gaagtttacc aactgctaca gcctgtctaa gaccctgagg ttcaaggcta ttcctgtggg
     2701 taagacccaa gagaatatcg acaacaagcg gctgctggtt gaggatgaga agagagctga
     2761 ggattacaag ggcgtgaaga agctgctgga tcggtactac ctgagcttca tcaacgatgt
     2821 gctgcacagc atcaagctga agaacctgaa caactacatc agcctgttcc ggaagaaaac
     2881 ccggaccgag aaagagaaca aagagcttga gaacctcgag atcaacctgc ggaaagagat
     2941 cgctaaggct ttcaagggta acgaaggtaa ggatttttat gatatactat gcttatgtat
     3001 tttgtactga aagcatatcc tgcttcattg ggatattact gaaagcattt aactacatgt
     3061 aaactcactt gatgatcaat aaacttgatt ttgcaggtta caagagcctg ttcaagaagg
     3121 atattatcga gactatcctg cctgagttcc tggacgataa ggatgagatt gccctggtga
     3181 acagcttcaa cggtttcact actgccttca ccggtttctt cagaaaccgg gaaaacatgt
     3241 tcagcgaaga ggccaagtct acctctatcg ctttccggtg cattaacgag aacttgaccc
     3301 ggtacatcag caacatggac atcttcgaga aggtggacgc catcttcgat aagcacgagg
     3361 tgcaagaaat caaagagaag atcctgaact ccgactacga cgtcgaggat ttttttgagg
     3421 gcgagttctt caacttcgtg ctcacccaag aaggtaacat tccttagtta cctttctttt
     3481 ctttttccat cataagttta tagattgtac atgctttgag atttttcttt gcaaacaatc
     3541 tcaggtatcg atgtgtacaa cgctatcatc ggtggtttcg tgactgagag cggtgagaag
     3601 attaagggcc tgaacgagta cattaacctg tacaatcaaa agaccaagca gaagctgccg
     3661 aagttcaagc cgctttacaa gcaggttctg agcgatcgtg agagcctgtc tttttacgga
     3721 gagggataca cctctgatga agaggttttg gaggtaatct tgaaattgga actcttcttt
     3781 tgttgtctaa acctatcaat ttctttgcgg aaatttattt gaagctgtag agttaaaatt
     3841 gagtctttta aacttttgta ggtgttccgt aacaccctga acaagaacag cgagatcttc
     3901 agctccatca agaagctgga aaagctgttt aagaacttcg acgagtacag cagcgctggc
     3961 atcttcgtta agaacggtcc tgctatcagc accatcagca aggatatttt cggcgagtgg
     4021 aacgtgatcc gggataagtg gaatgctgag tacgatgaca tccacctgaa gaaaaaggct
     4081 gtggtgaccg agaagtacga ggatgatagg cggaagtcct tcaagaagat aggtaagttg
     4141 ttacttatga ttgttttcct ctctgctaca tgtattttgt tgttcatttc tgtaagatat
     4201 aagaattgag ttttcctctg atgatattat taggttcctt tagcctcgag cagcttcaag
     4261 agtatgctga cgctgatctg tccgtggtcg agaagcttaa agagatcatc atccagaagg
     4321 tcgacgagat ctacaaggtg tacggcagct ctgagaagct tttcgatgct gacttcgtgt
     4381 tggagaagtc tctgaagaag aacgacgccg ttgtcgctat catgaaggat ctgctggaca
     4441 gcgtgaagtc tttcgagaac tatatcaagg ccttcttcgg cgaaggttag tatcatatga
     4501 agaaatacct agtttcagtt gatgaatgct attttctgac ctcagttgtt ctcttttgag
     4561 aattatttct tttctaattt gcctgatttt tctattaatt cattaggtaa agagactaat
     4621 agggacgagt cattctacgg cgatttcgtg ctggcttacg acatccttct taaggtggac
     4681 cacatctacg acgccatcag aaattacgtg acccagaagc cgtacagcaa ggacaagttc
     4741 aagttgtact tccagaatcc gcagttcatg ggcggctggg acaaagacaa agagacagat
     4801 tacagggcta ccatcctgcg gtacggctct aagtactacc ttgccatcat ggacaagaaa
     4861 tacgccaagt gcctgcaaaa gatcgacaag gatgatgtga acggcaacta cgagaagatc
     4921 aactacaagc tcctgccagg taaatcctgg tccacacttt tacgataaaa acacaagatt
     4981 ttaaactatg aactgatcaa taatcattcc taaaagacca cacttttgtt ttgtttctaa
     5041 agtaattttt actgttataa caggtcctaa caagatgctt cctaaggtgt tcttctcaaa
     5101 gaaatggatg gcctactaca acccgagcga ggacatccag aaaatctaca agaacggcac
     5161 cttcaaaaag ggcgacatgt tcaacctgaa cgactgccac aagctgatcg atttcttcaa
     5221 ggacagcatc agccggtatc cgaagtggtc taacgcttac gatttcaact tcagcgagac
     5281 tgagaagtat aaggatatcg ccggcttcta ccgtgaggtt gaggaacagg gttacaaggt
     5341 tagcttcgag agcgccagca agaaagaggt ggacaagttg gttgaagaag gtaagaggtc
     5401 aaaaggtttc cgcaatgatc cctctttttt tgtttctcta gtttcaagaa tttgggtata
     5461 tgactaactt ctgagtgttc cttgatgcat atttgtgatg agacaaatgt ttgttctatg
     5521 ttttaggtaa gctgtacatg ttccaaatct ataacaagga cttctccgac aagtctcacg
     5581 gcactcctaa tctgcataca atgtacttca agctgctgtt cgacgagaac aaccacggtc
     5641 agattaggct ttctggtggt gctgagctgt tcatgagaag ggcctcactg aagaaagaag
     5701 agttggtcgt tcaccctgcc aactctccaa tcgctaacaa gaaccctgac aacccgaaaa
     5761 agaccaccac cttgtcttac gacgtgtaca aggataagcg gttcagcgag gatcagtacg
     5821 agcttcacat tccgatcgcc atcaacaagt gcccgaagaa catcttcaag atcaataccg
     5881 aggtgcgggt gctgctgaag cacgatgata atccttacgt gatcggcatc gataggggcg
     5941 agagaaacct tctttacatc gtggtggtgg acggcaaggg caatatcgtt gagcagtact
     6001 ctctgaacga gattatcaac aatttcaacg gcatccggat caagaccgac taccactctc
     6061 tgctggataa gaaagaaaaa gagcggttcg aggccaggca gaactggact tctatcgaaa
     6121 acatcaaaga gctgaaggcc ggctacatct ctcaggtggt gcataagatt tgcgagctgg
     6181 tggaaaagta cgacgctgtg attgctctcg aggatctgaa cagcggcttc aagaactcac
     6241 gtgtgaaggt aaagcaactg tgttttaatc aatttcttgt caggatatat ggattataac
     6301 ttaatttttg agaaatctgt agtatttggc gtgaaatgag tttgcttttt ggtttctccc
     6361 gtgttatagg ttgagaagca ggtctaccaa aagttcgaga agatgctcat cgacaagctg
     6421 aactacatgg tggacaaaaa gagcaaccct tgcgctaccg gtggtgctct taagggttac
     6481 cagatcacta acaagttcga gtctttcaag agcatgagca cccagaacgg cttcatcttc
     6541 tacatccctg cttggctgac cagcaagatc gatccttcta ctggcttcgt caacctgctc
     6601 aagaccaagt acaccagcat tgccgacagc aagaagttca tcagctcatt cgaccggatc
     6661 atgtacgtgc cagaagagga tcttttcgag ttcgccctcg attacaagaa cttctctagg
     6721 accgacgccg actacattaa gaagtggaag ctgtactcct acggcaaccg gattcggatc
     6781 tttcggaacc cgaagaaaaa caacgtgttc gactgggaag aggtaaagtt tccaactttc
     6841 ctttaccata tcaaactata gttcgaaact ttttatttga tcaacttcaa ggccacccga
     6901 tctttctatt cctgattaat ttgtgatgaa tccatattga cttttgatgg ttacgcaggt
     6961 gtgcctgacc tctgcctaca aagaactgtt caacaagtac ggcatcaact accagcaggg
     7021 tgatattagg gctctgcttt gcgagcagtc tgacaaggct ttctacagct ctttcatggc
     7081 cctgatgtct ctgatgctgc aaatgaggaa ctctatcacc ggtaggaccg atgtggactt
     7141 ccttatctct ccggtgaaga acagtgacgg gatcttctac gacagccgga attatgaggc
     7201 tcaagagaac gcaatcctgc cgaagaatgc tgatgctaac ggcgcttaca acattgccag
     7261 aaaggtgctg tgggctatcg gccagtttaa gaaagccgaa gatgagaagt tggacaaggt
     7321 ctgtctttcc tatttcatat gtttaatcct aggaatttga tcaattgatt gtatgtatgt
     7381 cgatcccaag actttcttgt tcacttatat cttaactctc tctttgctgt ttcttgcagg
     7441 tgaagatcgc tatctccaac aaagagtggc tcgagtacgc tcagactagc gttaagcata
     7501 agcgtcctgc tgccaccaaa aaggccggac aggctaagaa aaagaagtga gacgactagt
     7561 ggcggccgcc gacgtccgat cgttcaaaca tttggcaata aagtttctta agattgaatc
     7621 ctgttgccgg tcttgcgatg attatcatat aatttctgtt gaattacgtt aagcatgtaa
     7681 taattaacat gtaatgcatg acgttattta tgagatgggt ttttatgatt agagtcccgc
     7741 aattatacat ttaatacgcg atagaaaaca aaatatagcg cgcaaactag gataaattat
     7801 cgcgcgcggt gtcatctatg ttactagatc gggaattgat cccccctcga cagcttccgg
     7861 aaagggcgaa ttcgcaactt tgtatacaaa agttgaacga gaaacgtaaa atgatataaa
     7921 tatcaatata ttaaattaga ttttgcataa aaaacagact acataatact gtaaaacaca
     7981 acatatccag tcactatgcc atccagctga tatcccctat agtgagtcgt attacatggt
     8041 catagctgtt tcctggcagc tctggcccgt gtctcaaaat ctctgatgtt acattgcaca
     8101 agataaaaat atatcatcat gcctcctc
//
