LOCUS       sequence-392884-        6939 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A temperature-tolerant LbCas12a mutant (D156R), also known as 
            ttLbCas12a.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6939)
  AUTHORS   Cheng Y, Zhang Y, Li G, Fang H, Sretenovic S, Fan A, Li J, Xu J, Que
            Q, Qi Y
  TITLE     CRISPR-Cas12a base editors confer efficient multiplexed genome 
            editing in rice.
  JOURNAL   Plant Commun. 2023 Apr 13:100601. doi: 10.1016/j.xplc.2023.100601.
  PUBMED    37060177
REFERENCE   2  (bases 1 to 6939)
  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 
            Commun. 2023 Apr 13:100601. doi: 10.1016/j.xplc.2023.100601."
COMMENT     Sequence Label: pYPQ230-D156R (ttLbCas12a)
FEATURES             Location/Qualifiers
     source          1..6939
                     /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             6311..6358
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      6389..6641
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    6686..6809
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(6826..6845)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6827..6845)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6850..6866)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6850..6866)
                     /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 cctgcggctt caaagctcga
     2641 gaaattcacc aactgttatt cgttgagcaa aacactgcgg tttaaagcga ttccagtcgg
     2701 caagactcaa gagaatatag acaataagcg gctgttggtg gaagatgaaa agcgcgcgga
     2761 agactacaaa ggggtgaaga agttgttgga cagatactac ctctctttta tcaatgatgt
     2821 cttgcactca atcaaattga agaatctgaa caactacatc tccctcttca gaaagaaaac
     2881 aaggacagaa aaggagaata aggaacttga aaatttggag atcaatctga ggaaagagat
     2941 cgcgaaagcc tttaaaggca acgaaggata caaaagtctg ttcaagaagg atataattga
     3001 gacaattttg ccagagttcc tcgatgacaa ggacgagatt gcgctggtca attcgttcaa
     3061 cggattcaca acagcattca caggcttctt tcgcaatcgg gaaaatatgt tctctgagga
     3121 ggcaaagtcc acttctattg cgttcaggtg tatcaatgag aatctcacta ggtacatttc
     3181 caacatggat atctttgaga aggttgacgc aatttttgac aagcacgaag ttcaggagat
     3241 taaggagaag atcctcaatt ccgattatga cgttgaggac ttcttcgaag gtgagttttt
     3301 taatttcgtg ctcactcaag agggtatcga cgtgtataat gcgatcatcg gtgggttcgt
     3361 gactgagtcc ggtgaaaaga ttaagggatt gaacgagtat atcaaccttt acaaccaaaa
     3421 gacgaaacag aagctgccaa agttcaagcc tctttacaaa caggttcttt cagaccgcga
     3481 gtcactctcg ttctatgggg agggctacac ttcggatgag gaagtcctgg aggtgttcag
     3541 gaatactctc aataagaatt cggagatttt ctcttctata aaaaaactgg aaaagttgtt
     3601 taagaatttt gacgaatact ctagcgccgg catatttgtg aaaaacggcc cggccatatc
     3661 aacgataagt aaagatatct tcggcgaatg gaacgtgatc agagacaaat ggaacgcgga
     3721 gtatgacgat attcacctga agaagaaggc tgtcgtaacg gagaagtacg aggatgatcg
     3781 caggaaaagc ttcaaaaaga tcggaagttt cagcctggaa cagttgcagg agtatgctga
     3841 cgccgatctt agcgtcgtcg agaagttgaa ggagataatc atccaaaagg tcgacgagat
     3901 atataaagtc tatggatcaa gtgaaaaact gttcgacgcc gacttcgttt tggagaagtc
     3961 cctgaagaag aacgacgctg ttgttgccat tatgaaggat ctgctcgaca gcgtgaagag
     4021 tttcgagaac tatattaagg cttttttcgg ggaggggaag gagactaaca gagatgagtc
     4081 cttctacgga gacttcgtcc tcgcgtacga tatactcctt aaggtagacc acatctacga
     4141 cgcaatcaga aattacgtga cacaaaagcc gtacagcaag gacaagttca aactctactt
     4201 ccagaacccc cagttcatgg gcggctggga caaggacaag gaaacggatt acagggctac
     4261 gatcctgagg tatggttcaa aatactactt ggcgattatg gacaagaagt acgccaagtg
     4321 tctccagaag attgacaaag acgatgtcaa tggcaattat gagaagatca actacaagct
     4381 gcttccgggt ccgaacaaga tgctcccaaa ggttttcttc agcaagaaat ggatggccta
     4441 ctataaccca agcgaggaca tccagaagat ttataagaac ggtacgttca agaagggcga
     4501 catgttcaat cttaacgact gtcacaagct gatcgacttc ttcaaagact caattagccg
     4561 gtacccaaag tggtctaacg cctatgactt caacttttcg gaaaccgaga agtacaagga
     4621 tatagccgga ttttatagag aggtggaaga gcagggctac aaggtgtcat tcgagtccgc
     4681 cagcaagaag gaagtggaca agctcgtgga agagggtaag ctctacatgt tccagattta
     4741 taataaagac tttagcgata agagccacgg gacacctaat ctccacacaa tgtatttcaa
     4801 gctgctcttc gacgagaata accacggcca aatcaggttg tcaggagggg ctgaactctt
     4861 catgcggcgc gctagcctta agaaggagga gcttgtagtc caccctgcga atagtccaat
     4921 tgcgaataag aacccggaca atcctaaaaa gactacaaca ttgagctacg acgtgtacaa
     4981 ggataagagg ttttccgagg atcagtacga gctccacatc ccgattgcga tcaacaagtg
     5041 cccaaagaat attttcaaga taaacacaga ggtgcgtgta ctcctgaagc atgacgacaa
     5101 tccttacgtc attgggattg atcggggcga gaggaacctc ctctatattg tggtggtgga
     5161 cgggaagggg aacatagtcg aacagtactc ccttaacgaa ataattaaca atttcaacgg
     5221 catccgtatc aagaccgact accattcgtt gctggacaag aaggagaagg agagatttga
     5281 ggcgcggcaa aattggacaa gtatcgagaa catcaaggaa ctcaaagcag gttatatctc
     5341 tcaagttgtg cataagatat gcgagctggt tgagaagtat gacgcagtga tcgctcttga
     5401 ggacctcaac tcgggcttta agaattctag agttaaagtg gagaagcagg tctatcaaaa
     5461 gttcgagaag atgcttatag ataagctcaa ctacatggtc gataagaaat cgaacccatg
     5521 tgccaccggc ggcgcactca aaggttacca aataacaaac aaattcgagt ccttcaaatc
     5581 gatgagtact cagaatgggt tcatatttta tataccggcg tggcttacgt ctaagatcga
     5641 cccgtcaact ggttttgtca acctgttgaa gacgaaatac acgtccattg ccgattcgaa
     5701 aaagttcata tctagttttg atcgtattat gtacgtccca gaggaagatc ttttcgagtt
     5761 tgctctcgac tacaaaaact tttcgcggac cgatgcggat tacattaaaa aatggaaact
     5821 ctattcgtac ggcaacagaa tcaggatttt tcgcaaccct aagaagaata acgtctttga
     5881 ttgggaggaa gtttgcttga ctagcgcgta caaggagctc tttaataagt atggcattaa
     5941 ctaccaacag ggtgatatca gagcactgct ttgcgaacaa tctgacaagg ctttctactc
     6001 atccttcatg gctttgatga gcctgatgct ccagatgaga aattcaatta caggcagaac
     6061 cgacgtggat ttcttgatct ccccggttaa aaattctgat ggcatctttt acgatagcag
     6121 gaactatgaa gcgcaagaga atgcgattct gccaaaaaat gcagacgcca acggtgccta
     6181 taacatcgcc aggaaagtcc tgtgggcgat cggccagttc aaaaaggccg aagacgaaaa
     6241 attggacaag gtcaaaatcg ctatcagcaa caaagagtgg ctggagtatg ctcagacatc
     6301 cgtaaagcat aagcgtcctg ctgccaccaa aaaggccgga caggctaaga aaaagaagtg
     6361 agacgactag tggcggccgc cgacgtccga tcgttcaaac atttggcaat aaagtttctt
     6421 aagattgaat cctgttgccg gtcttgcgat gattatcata taatttctgt tgaattacgt
     6481 taagcatgta ataattaaca tgtaatgcat gacgttattt atgagatggg tttttatgat
     6541 tagagtcccg caattataca tttaatacgc gatagaaaac aaaatatagc gcgcaaacta
     6601 ggataaatta tcgcgcgcgg tgtcatctat gttactagat cgggaattga tcccccctcg
     6661 acagcttccg gaaagggcga attcgcaact ttgtatacaa aagttgaacg agaaacgtaa
     6721 aatgatataa atatcaatat attaaattag attttgcata aaaaacagac tacataatac
     6781 tgtaaaacac aacatatcca gtcactatgc catccagctg atatccccta tagtgagtcg
     6841 tattacatgg tcatagctgt ttcctggcag ctctggcccg tgtctcaaaa tctctgatgt
     6901 tacattgcac aagataaaaa tatatcatca tgcctcctc
//
