LOCUS       sequence-361473-        5985 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gives high editing efficiency in barley..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5985)
  AUTHORS   Lawrenson T, Hinchliffe A, Forner M, Harwood W
  TITLE     Highly efficient genome editing in barley using novel LbCas12a 
            variants and impact of sgRNA architecture
  JOURNAL   bioRxiv 2022.04.28.489853
REFERENCE   2  (bases 1 to 5985)
  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: "bioRxiv 
            2022.04.28.489853"
COMMENT     Sequence Label: ttHsCas12a
FEATURES             Location/Qualifiers
     source          1..5985
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(24..42)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        110..214
                     /gene="bla"
                     /label=AmpR promoter
     CDS             436..1227
                     /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      1398..1986
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1887..1906
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2140..2157
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             2235..5918
                     /codon_start=1
                     /product="CRISPR-associated protein from Lachnospiraceae 
                     bacterium ND2006 (Zetsche et al., 2015)"
                     /label=LbCpf1
                     /translation="MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAE
                     DYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKE
                     IAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFRNRENMFS
                     EEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEG
                     EFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVL
                     SDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKN
                     GPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQ
                     LQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKD
                     LLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYS
                     KDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNG
                     NYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKL
                     IDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLV
                     EEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKK
                     EELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKI
                     NTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTD
                     YHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSG
                     FKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQ
                     NGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALD
                     YKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQ
                     QGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRN
                     YEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTS
                     VKH"
     CDS             5919..5966
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
ORIGIN
        1 gtggctcttc agtggacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
       61 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
      121 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
      181 ataaatgctt caataatatt gaaaaaggaa gagtatgcgc tcacgcaact ggtccagaac
      241 cttgaccgaa cgcagcggtg gtaacggcgc agtggcggtt ttcatggctt gttatgactg
      301 tttttttttg gggtacagtc tatgcctcgg gcatccaagc agcaagcgcg ttacgccgtg
      361 ggtcgatgtt tgatgttatg gagcagcaac gatgttacgc agcagggcag tcgccctaaa
      421 acaaagttaa acatcatgag ggaagcggtg atcgccgaag tatcgactca actatcagag
      481 gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc tggccgtaca tttgtacggc
      541 tccgcagtgg atggcggcct gaagccacac agcgatattg atttgctggt tacggtgacc
      601 gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg accttttgga aacttcggct
      661 tcccctggag agagcgagat tctccgcgct gtagaagtca ccattgttgt gcacgacgac
      721 atcattccgt ggcgttatcc agctaagcgc gaactgcaat ttggagaatg gcagcgcaat
      781 gacattcttg caggtatctt cgagccagcc acgatcgaca ttgatctggc tatcttgctg
      841 acaaaagcaa gagaacatag cgttgccttg gtaggtccag cggcggagga actctttgat
      901 ccggttcctg aacaggatct atttgaggcg ctaaatgaaa ccttaacgct atggaactcg
      961 ccgcccgact gggctggcga tgagcgaaat gtagtgctta cgttgtcccg catttggtac
     1021 agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg ccgactgggc aatggagcgc
     1081 ctgccggccc agtatcagcc cgtcatactt gaagctagac aggcttatct tggacaagaa
     1141 gaagatcgct tggcctcgcg cgcagatcag ttggaagaat ttgtccatta cgtaaaaggc
     1201 gagatcacca aggtagtcgg caaataactg tcagaccaag tttactcata tatactttag
     1261 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     1321 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     1381 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     1441 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     1501 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     1561 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     1621 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     1681 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     1741 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     1801 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     1861 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     1921 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     1981 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     2041 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     2101 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     2161 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatca
     2221 ctctgtggtc tcaaatgagc aagctggaga agtttacaaa ctgctactcc ctgtctaaga
     2281 ccctgaggtt caaggccatc cctgtgggca agacccagga gaacatcgac aataagcggc
     2341 tgctggtgga ggacgagaag agagccgagg attataaggg cgtgaagaag ctgctggatc
     2401 gctactatct gtcttttatc aacgacgtgc tgcacagcat caagctgaag aatctgaaca
     2461 attacatcag cctgttccgg aagaaaacca gaaccgagaa ggagaataag gagctggaga
     2521 acctggagat caatctgcgg aaggagatcg ccaaggcctt caagggcaac gagggctaca
     2581 agtccctgtt taagaaggat atcatcgaga caatcctgcc agagttcctg gacgataagg
     2641 acgagatcgc cctggtgaac agcttcaatg gctttaccac agccttcacc ggcttctttc
     2701 gcaacagaga gaatatgttt tccgaggagg ccaagagcac atccatcgcc ttcaggtgta
     2761 tcaacgagaa tctgacccgc tacatctcta atatggacat cttcgagaag gtggacgcca
     2821 tctttgataa gcacgaggtg caggagatca aggagaagat cctgaacagc gactatgatg
     2881 tggaggattt ctttgagggc gagttcttta actttgtgct gacacaggag ggcatcgacg
     2941 tgtataacgc catcatcggc ggcttcgtga ccgagagcgg cgagaagatc aagggcctga
     3001 acgagtacat caacctgtat aatcagaaaa ccaagcagaa gctgcctaag tttaagccac
     3061 tgtataagca ggtgctgagc gatcgggagt ctctgagctt ctacggcgag ggctatacat
     3121 ccgatgagga ggtgctggag gtgtttagaa acaccctgaa caagaacagc gagatcttca
     3181 gctccatcaa gaagctggag aagctgttca agaattttga cgagtactct agcgccggca
     3241 tctttgtgaa gaacggcccc gccatcagca caatctccaa ggatatcttc ggcgagtgga
     3301 acgtgatccg ggacaagtgg aatgccgagt atgacgatat ccacctgaag aagaaggccg
     3361 tggtgaccga gaagtacgag gacgatcgga gaaagtcctt caagaagatc ggctcctttt
     3421 ctctggagca gctgcaggag tacgccgacg ccgatctgtc tgtggtggag aagctgaagg
     3481 agatcatcat ccagaaggtg gatgagatct acaaggtgta tggctcctct gagaagctgt
     3541 tcgacgccga ttttgtgctg gagaagagcc tgaagaagaa cgacgccgtg gtggccatca
     3601 tgaaggacct gctggattct gtgaagagct tcgagaatta catcaaggcc ttctttggcg
     3661 agggcaagga gacaaacagg gacgagtcct tctatggcga ttttgtgctg gcctacgaca
     3721 tcctgctgaa ggtggaccac atctacgatg ccatccgcaa ttatgtgacc cagaagccct
     3781 actctaagga taagttcaag ctgtattttc agaaccctca gttcatgggc ggctgggaca
     3841 aggataagga gacagactat cgggccacca tcctgagata cggctccaag tactatctgg
     3901 ccatcatgga taagaagtac gccaagtgcc tgcagaagat cgacaaggac gatgtgaacg
     3961 gcaattacga gaagatcaac tataagctgc tgcccggccc taataagatg ctgccaaagg
     4021 tgttcttttc taagaagtgg atggcctact ataaccccag cgaggacatc cagaagatct
     4081 acaagaatgg cacattcaag aagggcgata tgtttaacct gaatgactgt cacaagctga
     4141 tcgacttctt taaggatagc atctcccggt atccaaagtg gtccaatgcc tacgatttca
     4201 acttttctga gacagagaag tataaggaca tcgccggctt ttacagagag gtggaggagc
     4261 agggctataa ggtgagcttc gagtctgcca gcaagaagga ggtggataag ctggtggagg
     4321 agggcaagct gtatatgttc cagatctata acaaggactt ttccgataag tctcacggca
     4381 cacccaatct gcacaccatg tacttcaagc tgctgtttga cgagaacaat cacggacaga
     4441 tcaggctgag cggaggagca gagctgttca tgaggcgcgc ctccctgaag aaggaggagc
     4501 tggtggtgca cccagccaac tcccctatcg ccaacaagaa tccagataat cccaagaaaa
     4561 ccacaaccct gtcctacgac gtgtataagg ataagaggtt ttctgaggac cagtacgagc
     4621 tgcacatccc aatcgccatc aataagtgcc ccaagaacat cttcaagatc aatacagagg
     4681 tgcgcgtgct gctgaagcac gacgataacc cctatgtgat cggcatcgat aggggcgagc
     4741 gcaatctgct gtatatcgtg gtggtggacg gcaagggcaa catcgtggag cagtattccc
     4801 tgaacgagat catcaacaac ttcaacggca tcaggatcaa gacagattac cactctctgc
     4861 tggacaagaa ggagaaggag aggttcgagg cccgccagaa ctggacctcc atcgagaata
     4921 tcaaggagct gaaggccggc tatatctctc aggtggtgca caagatctgc gagctggtgg
     4981 agaagtacga tgccgtgatc gccctggagg acctgaactc tggctttaag aatagccgcg
     5041 tgaaggtgga gaagcaggtg tatcagaagt tcgagaagat gctgatcgat aagctgaact
     5101 acatggtgga caagaagtct aatccttgtg caacaggcgg cgccctgaag ggctatcaga
     5161 tcaccaataa gttcgagagc tttaagtcca tgtctaccca gaacggcttc atcttttaca
     5221 tccctgcctg gctgacatcc aagatcgatc catctaccgg ctttgtgaac ctgctgaaaa
     5281 ccaagtatac cagcatcgcc gattccaaga agttcatcag ctcctttgac aggatcatgt
     5341 acgtgcccga ggaggatctg ttcgagtttg ccctggacta taagaacttc tctcgcacag
     5401 acgccgatta catcaagaag tggaagctgt actcctacgg caaccggatc agaatcttcc
     5461 ggaatcctaa gaagaacaac gtgttcgact gggaggaggt gtgcctgacc agcgcctata
     5521 aggagctgtt caacaagtac ggcatcaatt atcagcaggg cgatatcaga gccctgctgt
     5581 gcgagcagtc cgacaaggcc ttctactcta gctttatggc cctgatgagc ctgatgctgc
     5641 agatgcggaa cagcatcaca ggccgcaccg acgtggattt tctgatcagc cctgtgaaga
     5701 actccgacgg catcttctac gatagccgga actatgaggc ccaggagaat gccatcctgc
     5761 caaagaacgc cgacgccaat ggcgcctata acatcgccag aaaggtgctg tgggccatcg
     5821 gccagttcaa gaaggccgag gacgagaagc tggataaggt gaagatcgcc atctctaaca
     5881 aggagtggct ggagtacgcc cagaccagcg tgaagcacaa aaggccggcg gccacgaaaa
     5941 aggccggcca ggcaaaaaag aaaaagtagg ctttgagacc acgaa
//
