LOCUS       sequence-228013-        5594 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level0 Golden Gate part: CDS, eSaCas9 (no stop codon).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5594)
  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 5594)
  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: pEPOR0SP0012
FEATURES             Location/Qualifiers
     source          1..5594
                     /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             2241..2261
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5442..5489
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5496..5576
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
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 tcaaatggcc ccaaagaaga agcggaaggt cggtatccac ggagtcccag
     2281 cagccaagcg gaactacatc ctgggcctgg acatcggcat caccagcgtg ggctacggca
     2341 tcatcgacta cgagacacgg gacgtgatcg atgccggcgt gcggctgttc aaagaggcca
     2401 acgtggaaaa caacgagggc aggcggagca agagaggcgc cagaaggctg aagcggcgga
     2461 ggcggcatag aatccagaga gtgaagaagc tgctgttcga ctacaacctg ctgaccgacc
     2521 acagcgagct gagcggcatc aacccctacg aggccagagt gaagggcctg agccagaagc
     2581 tgagcgagga agagttctct gccgccctgc tgcacctggc caagagaaga ggcgtgcaca
     2641 acgtgaacga ggtggaagag gacaccggca acgagctgtc caccaaagag cagatcagcc
     2701 ggaacagcaa ggccctggaa gagaaatacg tggccgaact gcagctggaa cggctgaaga
     2761 aagacggcga agtgcggggc agcatcaaca gattcaagac cagcgactac gtgaaagaag
     2821 ccaaacagct gctgaaggtg cagaaggcct accaccagct ggaccagagc ttcatcgaca
     2881 cctacatcga cctgctggaa acccggcgga cctactatga gggacctggc gagggcagcc
     2941 ccttcggctg gaaggacatc aaagaatggt acgagatgct gatgggccac tgcacctact
     3001 tccccgagga actgcggagc gtgaagtacg cctacaacgc cgacctgtac aacgccctga
     3061 acgacctgaa caatctcgtg atcaccaggg acgagaacga gaagctggaa tattacgaga
     3121 agttccagat catcgagaac gtgttcaagc agaagaagaa gcccaccctg aagcagatcg
     3181 ccaaagaaat cctcgtgaac gaagaggata ttaagggcta cagagtgacc agcaccggca
     3241 agcccgagtt caccaacctg aaggtgtacc acgacatcaa ggacattacc gcccggaaag
     3301 agattattga gaacgccgag ctgctggatc agattgccaa gatcctgacc atctaccaga
     3361 gcagcgagga catccaggaa gaactgacca atctgaactc cgagctgacc caggaagaga
     3421 tcgagcagat ctctaatctg aagggctata ccggcaccca caacctgagc ctgaaggcca
     3481 tcaacctgat cctggacgag ctgtggcaca ccaacgacaa ccagatcgct atcttcaacc
     3541 ggctgaagct ggtgcccaag aaggtggacc tgtcccagca gaaagagatc cccaccaccc
     3601 tggtggacga cttcatcctg agccccgtcg tgaagagaag cttcatccag agcatcaaag
     3661 tgatcaacgc catcatcaag aagtacggcc tgcccaacga catcattatc gagctggccc
     3721 gcgagaagaa ctccaaggac gcccagaaaa tgatcaacga gatgcagaag cggaacgcga
     3781 aaaccaacga gcggatcgag gaaatcatcc ggaccaccgg caaagagaac gccaagtacc
     3841 tgatcgagaa gatcaagctg cacgacatgc aggaaggcaa gtgcctgtac agcctggaag
     3901 ccatccctct ggaagatctg ctgaacaacc ccttcaacta tgaggtggac cacatcatcc
     3961 ccagaagcgt gtccttcgac aacagcttca acaacaaggt gctcgtgaag caggaagaaa
     4021 acagcaagaa gggcaaccgg accccattcc agtacctgag cagcagcgac agcaagatca
     4081 gctacgaaac cttcaagaag cacatcctga atctggccaa gggcaagggc agaatcagca
     4141 agaccaagaa agagtatctg ctggaagaac gggacatcaa caggttctcc gtgcagaaag
     4201 acttcatcaa ccggaacctg gtggatacca gatacgccac cgcacgtctg atgaacctgc
     4261 tgcggagcta cttcagagtg aacaacctgg acgtgaaagt gaagtccatc aatggcggct
     4321 tcaccagctt tctgcggcgg aagtggaagt ttaagaaaga gcggaacaag gggtacaagc
     4381 accacgccga ggacgccctg atcattgcca acgccgattt catcttcaaa gagtggaaga
     4441 aactggacaa ggccaaaaaa gtgatggaaa accagatgtt cgaggaaaag caggccgaga
     4501 gcatgcccga gatcgaaacc gagcaggagt acaaagagat cttcatcacc ccccaccaga
     4561 tcaagcacat taaggacttc aaggactaca agtacagcca ccgggtggac aagaagccta
     4621 atagagagct gattaacgac accctgtact ccacccggaa ggacgacaag ggcaacaccc
     4681 tgatcgtgaa caatctgaac ggcctgtacg acaaggacaa tgacaagctg aaaaagctga
     4741 tcaacaagag ccccgaaaag ctgctgatgt accaccacga cccccagacc taccagaaac
     4801 tgaagctgat tatggaacag tacggcgacg agaagaatcc cctgtacaag tactacgagg
     4861 aaaccgggaa ctacctgacc aagtactcca aaaaggacaa cggccccgtg atcaagaaga
     4921 ttaagtatta cggcaacaaa ctgaacgccc atctggacat caccgacgac taccccaaca
     4981 gcagaaacaa ggtcgtgaag ctgtccctga agccctacag attcgacgtg tacctggaca
     5041 atggcgtgta caagttcgtg accgtgaaga atctggatgt gatcaaaaaa gaaaactact
     5101 acgaagtgaa tagcaagtgc tatgaggaag ctaagaagct gaagaagatc agcaaccagg
     5161 ccgagtttat cgcctccttc tacaacaacg atctgatcaa gatcaacggc gagctgtata
     5221 gagtgatcgg cgtgaacaac gacctgctga accggatcga agtgaacatg atcgacatca
     5281 cctaccgcga gtacctggaa aacatgaacg acaagaggcc ccccaggatc attaagacaa
     5341 tcgcctccaa gacccagagc attaagaagt acagcacaga cattctgggc aacctgtatg
     5401 aagtgaaatc taagaagcac cctcagatca tcaaaaaggg caaaaggccg gcggccacga
     5461 aaaaggccgg ccaggcaaaa aagaaaaagg gatcctaccc atacgatgtt ccagattacg
     5521 cttacccata cgatgttcca gattacgctt acccatacga tgttccagat tacgctggtt
     5581 cgtgagacca cgaa
//
