LOCUS       sequence-228010-        5858 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level0 Golden Gate part: CDS, SaCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5858)
  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 5858)
  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: pEPOR0SP0020
FEATURES             Location/Qualifiers
     source          1..5858
                     /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(3848..3865)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3958..4546)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4038..4057)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        complement(5731..5835)
                     /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 gattacgctt
     3721 aagctttgag accctgggcc tcatgggcct tcacttcact gcccgctttc cagtcgggaa
     3781 acctgtcgtg ccagctgcat taacatggtc atagctgttt ccttgcgtat tgggcgctct
     3841 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg ggtaaagcct ggggtgccta
     3901 atgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     3961 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     4021 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4081 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4141 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     4201 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     4261 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     4321 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     4381 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     4441 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     4501 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     4561 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     4621 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     4681 aatctaaagt atatatgagt aaacttggtc tgacagttat tatttaccca ccactttggt
     4741 aatttcgcct ttcacgtaat gcacaaattc ttccagctga tccgcacggc tggccagacg
     4801 atcttcttcc tggcccagat acgcctgacg cgcttccaga atcaccggct gatactgcgc
     4861 cggcagacgt tccatggccc aatccgccgc cacatctttc ggcgcaattt tgccggtcac
     4921 cgcgctatac caaatacggc tcagggtcag caccacgttg cgttcatcac ccgcccaatc
     4981 cggcgggctg ttccacaggg tcagggtttc gttcagcgct tcaaacaggt cctgttccgg
     5041 caccggatca aacagttctt ccgctgccgg accaaccagc gccacgctat gttcacgcgc
     5101 tttggtcagc agaatggcca gatcgatatc aatggtcgcc ggttcaaaaa tgccggccag
     5161 aatatcgtta cgctgccatt cgccaaactg cagctcacgt ttcgccggat aacgccacgg
     5221 aataatgtca tcatgcacca cgatggtcac ttccaccgca cgcagaattt cgctttcacc
     5281 cgggctcgcg ctggtttcca gcagatcgtt aatcagcgca cgacgggtgg tttcatccag
     5341 acgcacggtc acggtcacca gcagatcaat atcgctatgc ggtttcaggc cgccatccac
     5401 cgcgctgcca tacagatgca cggccagcag ggtcggttcc agatgacgtt caatcacgcc
     5461 caccacttca gacagctggg tgctcacttc cgcaatcacc gcttcacgca taatgttcag
     5521 tttggttttc ggacggctgc cctgctgggt cacatcgttg ctgctccaca gcatcaggca
     5581 acggcccacc gcataacgcg cctgctgttt gctcgcacgc ggcatgctct gcacgccaaa
     5641 aaagcaatca tagcacgcca taaacaccgc caccgcacca ttaccgccgc tacgttcggt
     5701 cagggtacgg ctccaattac ggctacgcat actcttcctt tttcaatatt attgaagcat
     5761 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     5821 aataggggtt ccgcgcacat ttccccgaaa agtgccac
//
