LOCUS       sequence-252665-        5445 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level0 SaCas9, Arabidopsis codon optimized.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5445)
  AUTHORS   Hahn F, Korolev A, Sanjurjo Loures L, Nekrasov V
  TITLE     A modular cloning toolkit for genome editing in plants.
  JOURNAL   BMC Plant Biol. 2020 Apr 23;20(1):179. doi: 
            10.1186/s12870-020-02388-2.
  PUBMED    32326901
REFERENCE   2  (bases 1 to 5445)
  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: "BMC Plant 
            Biol."; date: "2020-04-23"; volume: "20(1):179. doi"; pages: " 
            10.1186/s12870-020-02388-2"
COMMENT     Sequence Label: pFH14
FEATURES             Location/Qualifiers
     source          1..5445
                     /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..5393
                     /codon_start=1
                     /product="Cas9 endonuclease from the Staphylococcus aureus 
                     Type II CRISPR/Cas system"
                     /label=SaCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNE
                     GRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEE
                     FSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGE
                     VRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFG
                     WKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKF
                     QIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKE
                     IIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKA
                     INLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSI
                     KVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENA
                     KYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVK
                     QEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRF
                     SVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKER
                     NKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEI
                     FITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKD
                     NDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKK
                     DNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKN
                     LDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLL
                     NRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQ
                     IIKKG"
     CDS             5406..5426
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
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 tcaaatgaag aggaactaca tcctcggact cgatatcgga atcacctctg
     2281 tgggatacgg aatcatcgat tacgagacta gggatgtgat cgatgctggt gtgagactct
     2341 tcaaagaggc taacgttgag aacaacgagg gaagaaggtc taagagggga gctagaaggc
     2401 tcaagagaag aagaaggcac agaatccaga gggtgaagaa gctcctcttc gattacaacc
     2461 tcctcaccga tcactctgag ctttctggaa tcaaccctta cgaggcaaga gtgaagggac
     2521 tctctcagaa gttgtctgag gaagagttct ctgctgcttt gctccacctt gctaagagaa
     2581 ggggtgtgca taacgtgaac gaggtggaag aggataccgg aaacgagctt tctaccaaag
     2641 agcagatctc taggaactct aaggctctcg aagagaagta cgtggcagag cttcagcttg
     2701 agagactcaa gaaagatggt gaggtgaggg gatctatcaa caggttcaag acctctgatt
     2761 acgtgaaaga agctaagcag ctcctcaagg tgcagaaggc ttaccatcag ctcgatcagt
     2821 ctttcatcga tacctacatc gatcttctcg agactagaag gacctactac gagggacctg
     2881 gtgaaggatc tccattcgga tggaaggata tcaaagaatg gtacgagatg ctcatgggac
     2941 actgcactta cttccctgaa gaactcagat ctgtgaagta cgcttacaac gctgatctct
     3001 acaacgctct taacgatctc aacaacctcg tgatcaccag ggatgagaac gagaagcttg
     3061 agtactacga gaagttccag atcatcgaga acgtgttcaa gcaaaagaag aagcctaccc
     3121 tcaagcagat cgctaaagag atccttgtta acgaggaaga tatcaaggga tacagggtga
     3181 cctctaccgg aaagcctgag ttcactaacc tcaaggttta ccacgatatt aaggatatta
     3241 ccgctagaaa agagattatt gagaacgctg agcttctcga tcaaatcgct aagatcctta
     3301 ccatctacca gtcatctgag gatatccaag aggaattgac caacctcaac tcagagctta
     3361 cccaagaaga gatcgagcag atcagtaact tgaagggtta caccggaacc cacaacctct
     3421 cactcaaggc tatcaacctc atcctcgatg agctttggca caccaacgat aaccagatcg
     3481 caatcttcaa cagactcaag ctcgtgccta agaaagtgga tctctctcag cagaaagaga
     3541 ttcctaccac cctcgtggat gatttcatcc tctcacctgt tgtgaagaga tctttcatcc
     3601 agtctatcaa ggtgatcaac gctattatca agaaatacgg actccctaac gatatcatca
     3661 tcgagttggc tagggaaaag aactcaaagg atgctcaaaa gatgatcaac gagatgcaga
     3721 agagaaacag gcagaccaac gagaggatcg aagagatcat caggaccacc ggaaaagaga
     3781 acgctaagta cctcattgag aagatcaagc tccacgatat gcaagaggga aagtgcctct
     3841 actctctcga ggctatccct cttgaggatc tccttaacaa ccctttcaac tacgaagtgg
     3901 atcacatcat ccctagatct gtgtctttcg ataactcttt caacaacaag gtgctcgtga
     3961 agcaagagga aaactctaag aagggtaaca ggaccccttt ccagtacctc tcttcatctg
     4021 attctaagat ctcatacgag actttcaaga agcacatcct caacctcgct aagggaaagg
     4081 gtaggatctc taagacaaag aaagagtacc ttctcgagga aagggatatc aacagattct
     4141 ctgttcagaa ggatttcatc aacagaaacc ttgtggatac caggtacgct accaggggtc
     4201 ttatgaacct tctcaggtct tacttcaggg tgaacaacct tgatgtgaag gtgaagtcaa
     4261 tcaacggtgg attcacctca ttccttagaa gaaaatggaa gtttaagaaa gagaggaaca
     4321 agggttataa gcaccacgct gaggatgctc tcattatcgc taacgcagat ttcattttca
     4381 aagagtggaa gaagttggat aaggctaaaa aggtgatgga aaaccagatg ttcgaggaaa
     4441 agcaggctga gtctatgcct gagatcgaga ctgagcaaga gtacaaagaa atcttcatca
     4501 cccctcacca gatcaagcac atcaaagatt tcaaggatta caagtactct cacagagtgg
     4561 ataagaagcc aaacagagag cttatcaacg ataccctcta ctcaaccaga aaggatgata
     4621 agggaaacac cctcatcgtg aacaatctca acggactcta cgataaggat aacgataagc
     4681 tcaagaagct cattaacaag tctccagaga agttgctcat gtaccaccac gatcctcaga
     4741 cctaccaaaa gctcaagctc atcatggaac agtacggtga tgagaagaac cctctctaca
     4801 agtattacga ggaaaccggt aactacctca ccaagtactc aaaaaaggat aatggacctg
     4861 tgatcaagaa gattaagtac tacggaaaca agctcaacgc acacctcgat atcaccgatg
     4921 attaccctaa cagtagaaac aaggtggtga agctttcact caagccttac aggttcgatg
     4981 tgtacctcga taacggtgtg tacaagttcg tgaccgtgaa gaacctcgat gttatcaaaa
     5041 aagaaaacta ctatgaggtt aactctaagt gctacgagga agctaagaag ttgaagaaga
     5101 tctctaacca ggcagagttc attgcttcat tctacaacaa cgatcttatc aagattaacg
     5161 gtgagcttta cagggttatc ggagtgaaca acgatttgct caacaggatc gaggtgaaca
     5221 tgattgatat cacctacagg gaatacctcg agaatatgaa cgataagagg cctcctagaa
     5281 tcatcaagac tatcgcttct aagacccaga gtatcaagaa gtactctacc gatatcctcg
     5341 gaaacctcta cgaggttaag tcaaagaagc accctcagat cattaagaag ggatcaaggg
     5401 ctgatcctaa gaagaagagg aaggtttgag ctttgagacc acgaa
//
