LOCUS       sequence-240323-        5857 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level0 Golden Gate part: CDS, SaCas9 (no stop codon).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5857)
  AUTHORS   Raitskin O, Schudoma C, West A, Patron NJ
  TITLE     Comparison of efficiency and specificity of CRISPR-associated (Cas) 
            nucleases in plants: An expanded toolkit for precision genome 
            engineering.
  JOURNAL   PLoS One. 2019 Feb 27;14(2):e0211598. doi: 
            10.1371/journal.pone.0211598. eCollection 2019.
  PUBMED    30811422
REFERENCE   2  (bases 1 to 5857)
  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: "PLoS One.";
            date: "2019-02-27"; volume: "14(2):e0211598. doi"; pages: " 
            10.1371/journal.pone.0211598. eCollection 2019"
COMMENT     Sequence Label: pEPOR0SP0021
FEATURES             Location/Qualifiers
     source          1..5857
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     278..300
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     292..309
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     293..309
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     320..339
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        320..338
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             384..404
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             429..3584
                     /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="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     CDS             3585..3632
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3639..3719
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(3847..3864)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3957..4545)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4037..4056)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        complement(5730..5834)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt
      181 gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
      241 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
      301 acggccagtg agcgcgacgt aatacgactc actatagggc gaattgagtg aaggccgtca
      361 aggccgcatg gtctcaaatg gccccaaaga agaagcggaa ggtcggtatc cacggagtcc
      421 cagcagccaa gcggaactac atcctgggcc tggacatcgg catcaccagc gtgggctacg
      481 gcatcatcga ctacgagaca cgggacgtga tcgatgccgg cgtgcggctg ttcaaagagg
      541 ccaacgtgga aaacaacgag ggcaggcgga gcaagagagg cgccagaagg ctgaagcggc
      601 ggaggcggca tagaatccag agagtgaaga agctgctgtt cgactacaac ctgctgaccg
      661 accacagcga gctgagcggc atcaacccct acgaggccag agtgaagggc ctgagccaga
      721 agctgagcga ggaagagttc tctgccgccc tgctgcacct ggccaagaga agaggcgtgc
      781 acaacgtgaa cgaggtggaa gaggacaccg gcaacgagct gtccaccaaa gagcagatca
      841 gccggaacag caaggccctg gaagagaaat acgtggccga actgcagctg gaacggctga
      901 agaaagacgg cgaagtgcgg ggcagcatca acagattcaa gaccagcgac tacgtgaaag
      961 aagccaaaca gctgctgaag gtgcagaagg cctaccacca gctggaccag agcttcatcg
     1021 acacctacat cgacctgctg gaaacccggc ggacctacta tgagggacct ggcgagggca
     1081 gccccttcgg ctggaaggac atcaaagaat ggtacgagat gctgatgggc cactgcacct
     1141 acttccccga ggaactgcgg agcgtgaagt acgcctacaa cgccgacctg tacaacgccc
     1201 tgaacgacct gaacaatctc gtgatcacca gggacgagaa cgagaagctg gaatattacg
     1261 agaagttcca gatcatcgag aacgtgttca agcagaagaa gaagcccacc ctgaagcaga
     1321 tcgccaaaga aatcctcgtg aacgaagagg atattaaggg ctacagagtg accagcaccg
     1381 gcaagcccga gttcaccaac ctgaaggtgt accacgacat caaggacatt accgcccgga
     1441 aagagattat tgagaacgcc gagctgctgg atcagattgc caagatcctg accatctacc
     1501 agagcagcga ggacatccag gaagaactga ccaatctgaa ctccgagctg acccaggaag
     1561 agatcgagca gatctctaat ctgaagggct ataccggcac ccacaacctg agcctgaagg
     1621 ccatcaacct gatcctggac gagctgtggc acaccaacga caaccagatc gctatcttca
     1681 accggctgaa gctggtgccc aagaaggtgg acctgtccca gcagaaagag atccccacca
     1741 ccctggtgga cgacttcatc ctgagccccg tcgtgaagag aagcttcatc cagagcatca
     1801 aagtgatcaa cgccatcatc aagaagtacg gcctgcccaa cgacatcatt atcgagctgg
     1861 cccgcgagaa gaactccaag gacgcccaga aaatgatcaa cgagatgcag aagcggaacc
     1921 ggcagaccaa cgagcggatc gaggaaatca tccggaccac cggcaaagag aacgccaagt
     1981 acctgatcga gaagatcaag ctgcacgaca tgcaggaagg caagtgcctg tacagcctgg
     2041 aagccatccc tctggaagat ctgctgaaca accccttcaa ctatgaggtg gaccacatca
     2101 tccccagaag cgtgtccttc gacaacagct tcaacaacaa ggtgctcgtg aagcaggaag
     2161 aaaacagcaa gaagggcaac cggaccccat tccagtacct gagcagcagc gacagcaaga
     2221 tcagctacga aaccttcaag aagcacatcc tgaatctggc caagggcaag ggcagaatca
     2281 gcaagaccaa gaaagagtat ctgctggaag aacgggacat caacaggttc tccgtgcaga
     2341 aagacttcat caaccggaac ctggtggata ccagatacgc caccagaggc ctgatgaacc
     2401 tgctgcggag ctacttcaga gtgaacaacc tggacgtgaa agtgaagtcc atcaatggcg
     2461 gcttcaccag ctttctgcgg cggaagtgga agtttaagaa agagcggaac aaggggtaca
     2521 agcaccacgc cgaggacgcc ctgatcattg ccaacgccga tttcatcttc aaagagtgga
     2581 agaaactgga caaggccaaa aaagtgatgg aaaaccagat gttcgaggaa aagcaggccg
     2641 agagcatgcc cgagatcgaa accgagcagg agtacaaaga gatcttcatc accccccacc
     2701 agatcaagca cattaaggac ttcaaggact acaagtacag ccaccgggtg gacaagaagc
     2761 ctaatagaga gctgattaac gacaccctgt actccacccg gaaggacgac aagggcaaca
     2821 ccctgatcgt gaacaatctg aacggcctgt acgacaagga caatgacaag ctgaaaaagc
     2881 tgatcaacaa gagccccgaa aagctgctga tgtaccacca cgacccccag acctaccaga
     2941 aactgaagct gattatggaa cagtacggcg acgagaagaa tcccctgtac aagtactacg
     3001 aggaaaccgg gaactacctg accaagtact ccaaaaagga caacggcccc gtgatcaaga
     3061 agattaagta ttacggcaac aaactgaacg cccatctgga catcaccgac gactacccca
     3121 acagcagaaa caaggtcgtg aagctgtccc tgaagcccta cagattcgac gtgtacctgg
     3181 acaatggcgt gtacaagttc gtgaccgtga agaatctgga tgtgatcaaa aaagaaaact
     3241 actacgaagt gaatagcaag tgctatgagg aagctaagaa gctgaagaag atcagcaacc
     3301 aggccgagtt tatcgcctcc ttctacaaca acgatctgat caagatcaac ggcgagctgt
     3361 atagagtgat cggcgtgaac aacgacctgc tgaaccggat cgaagtgaac atgatcgaca
     3421 tcacctaccg cgagtacctg gaaaacatga acgacaagag gccccccagg atcattaaga
     3481 caatcgcctc caagacccag agcattaaga agtacagcac agacattctg ggcaacctgt
     3541 atgaagtgaa atctaagaag caccctcaga tcatcaaaaa gggcaaaagg ccggcggcca
     3601 cgaaaaaggc cggccaggca aaaaagaaaa agggatccta cccatacgat gttccagatt
     3661 acgcttaccc atacgatgtt ccagattacg cttacccata cgatgttcca gattacgctg
     3721 gttcgtgaga ccctgggcct catgggcctt cacttcactg cccgctttcc agtcgggaaa
     3781 cctgtcgtgc cagctgcatt aacatggtca tagctgtttc cttgcgtatt gggcgctctc
     3841 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg gtaaagcctg gggtgcctaa
     3901 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     3961 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4021 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4081 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4141 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4201 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4261 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4321 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4381 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4441 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4501 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4561 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4621 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4681 atctaaagta tatatgagta aacttggtct gacagttatt atttacccac cactttggta
     4741 atttcgcctt tcacgtaatg cacaaattct tccagctgat ccgcacggct ggccagacga
     4801 tcttcttcct ggcccagata cgcctgacgc gcttccagaa tcaccggctg atactgcgcc
     4861 ggcagacgtt ccatggccca atccgccgcc acatctttcg gcgcaatttt gccggtcacc
     4921 gcgctatacc aaatacggct cagggtcagc accacgttgc gttcatcacc cgcccaatcc
     4981 ggcgggctgt tccacagggt cagggtttcg ttcagcgctt caaacaggtc ctgttccggc
     5041 accggatcaa acagttcttc cgctgccgga ccaaccagcg ccacgctatg ttcacgcgct
     5101 ttggtcagca gaatggccag atcgatatca atggtcgccg gttcaaaaat gccggccaga
     5161 atatcgttac gctgccattc gccaaactgc agctcacgtt tcgccggata acgccacgga
     5221 ataatgtcat catgcaccac gatggtcact tccaccgcac gcagaatttc gctttcaccc
     5281 gggctcgcgc tggtttccag cagatcgtta atcagcgcac gacgggtggt ttcatccaga
     5341 cgcacggtca cggtcaccag cagatcaata tcgctatgcg gtttcaggcc gccatccacc
     5401 gcgctgccat acagatgcac ggccagcagg gtcggttcca gatgacgttc aatcacgccc
     5461 accacttcag acagctgggt gctcacttcc gcaatcaccg cttcacgcat aatgttcagt
     5521 ttggttttcg gacggctgcc ctgctgggtc acatcgttgc tgctccacag catcaggcaa
     5581 cggcccaccg cataacgcgc ctgctgtttg ctcgcacgcg gcatgctctg cacgccaaaa
     5641 aagcaatcat agcacgccat aaacaccgcc accgcaccat taccgccgct acgttcggtc
     5701 agggtacggc tccaattacg gctacgcata ctcttccttt ttcaatatta ttgaagcatt
     5761 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     5821 ataggggttc cgcgcacatt tccccgaaaa gtgccac
//
