LOCUS       sequence-437867-        7864 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Module of intronized NLS-SpCas9-NLS for N-terminal and C-terminal 
            tag fusions (AGGT_N-SpCas9i-N_TTCG).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7864)
  AUTHORS   Schreiber T, Prange A, Schafer P, Iwen T, Grutzner R, Marillonnet S,
            Lepage A, Javelle M, Paul W, Tissier A
  TITLE     Efficient scar-free knock-ins of several kilobases in plants by 
            engineered CRISPR/Cas endonucleases.
  JOURNAL   Mol Plant. 2024 Mar 22:S1674-2052(24)00086-8. doi: 
            10.1016/j.molp.2024.03.013.
  PUBMED    38520090
REFERENCE   2  (bases 1 to 7864)
  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: "Mol Plant. 
            2024 Mar 22:S1674-2052(24)00086-8. doi: 10.1016/j.molp.2024.03.013."
COMMENT     Sequence Label: pAGT6471
FEATURES             Location/Qualifiers
     source          1..7864
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             3639..3659
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3962..4753
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      5094..5682
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5583..5602
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             5750..5770
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 gtctcctgta cgaatacttc acggtctaca acgagctgac gaaggtgaaa tatgtgacgg
       61 aggggatgcg caaaccggcc ttcctgtcag gtaaatcctg gtccacactt ttacgataaa
      121 aacacaagat tttaaactat gaactgatca ataatcattc ctaaaagacc acacttttgt
      181 tttgtttcta aagtaatttt tactgttata acaggtgagc agaagaaggc cattgtcgat
      241 ctcttgttca aaaccaatcg gaaggtcact gtgaaacagc ttaaagagga ctactttaag
      301 aagatcgaat gctttgattc tgtggaaatc agcggcgttg aggataggtt caatgcctct
      361 cttggcacat accatgacct gttgaaaatc atcaaggaca aggacttcct tgacaacgag
      421 gagaacgagg acatcctcga ggacatcgtg ctgactctca cgctgtttga ggacagagaa
      481 atgatcgagg agcgccttaa gacttatgcg catctgttcg atgacaaggt catgaagcag
      541 ttgaagagga ggagatatac aggtaagagg tcaaaaggtt tccgcaatga tccctctttt
      601 tttgtttctc tagtttcaag aatttgggta tatgactaac ttctgagtgt tccttgatgc
      661 atatttgtga tgagacaaat gtttgttcta tgttttaggt tggggaaggc tctccaggaa
      721 gctcatcaac ggcatccgcg acaagcaatc cggcaagact atactggact ttctcaaatc
      781 cgacggtttt gcgaatcgga acttcatgca gcttattcac gatgactcac tgaccttcaa
      841 agaagatatc cagaaggccc aagtgtcagg tcagggcgat agccttcacg aacacatagc
      901 caacctggct ggatcgccag ctataaagaa gggcatactg cagacagtga aggttgtgga
      961 tgagctggtg aaggtaagtt ctgcatttgg ttatgctcct tgcattttag gtgttcgtcg
     1021 cacttccatt tccatgaata gctaagattt tttttctctg cattcattct tcttgcctca
     1081 gttctaactg tttgtggtat ttttgtttta attattgcta caggtcatgg gccgccataa
     1141 gccggagaac atcgtcatcg agatggcgag ggaaaaccag acgactcaga aagggcagaa
     1201 gaactcacgg gagcgcatga agcggataga ggaaggcatc aaggagcttg ggagtcagat
     1261 tctgaaagag cacccagtcg aaaatactca actccagaac gagaagctgt acctctatta
     1321 cctccagaat gggagagata tgtacgtcga ccaagagctc gacattaaca gactctccga
     1381 ctatgatgtg gatcacattg tccctcaatc tttcctgaag gacgatagta ttgacaacaa
     1441 ggtaaagcaa ctgtgtttta atcaatttct tgtcaggata tatggattat aacttaattt
     1501 ttgagaaatc tgtagtattt ggcgtgaaat gagtttgctt tttggtttct cccgtgttat
     1561 aggtccttac gcgctcagac aagaaccgcg gaaaatccga caatgtaccc agcgaggagg
     1621 ttgtgaagaa gatgaagaac tattggaggc agcttttgaa tgctaagctc ataacccaac
     1681 ggaaattcga caatctcacg aaggcagaaa ggggcggact gtctgagctc gacaaagccg
     1741 gcttcatcaa gcgccagttg gttgaaactc gtcagattac gaaacatgtg gcccagatac
     1801 tcgattcgcg tatgaatacg aagtatgatg agaatgacaa acttatcagg gaggtaaagg
     1861 taaagtttcc aactttcctt taccatatca aactaaagtt cgaaactttt tatttgatca
     1921 acttcaaggc cacccgatct ttctattcct gattaatttg tgatgaatcc atattgactt
     1981 ttgatggtta cgcaggtgat caccctcaag agcaaactgg ttagtgactt ccggaaggac
     2041 ttccagtttt acaaggttcg cgagatcaac aactaccatc atgcccatga cgcctacctg
     2101 aacgccgttg ttggcactgc tctcatcaag aagtatccga aactggagtc tgagtttgtg
     2161 tacggggatt acaaggtgta cgacgttagg aagatgatcg cgaagtcaga acaagagatc
     2221 ggcaaggcta ccgcgaaata cttcttttac tcgaatatca tgaacttctt caagacagag
     2281 atcactctgg cgaatggtga aatccggaag aggcctctga tcgagacaaa tggcgaaaca
     2341 ggtctgtctt tcctatttca tatgtttaat cctaggaatt tgatcaattg attgtatgta
     2401 tgtcgatccc aagactttct tgttcactta tatcttaact ctctctttgc tgtttcttgc
     2461 aggtgagatt gtctgggata agggcaggga ttttgcgact gtgcgtaagg ttctcagcat
     2521 gccccaagtc aacatagtca agaaaacgga ggttcaaacc ggtggtttct ccaaggagtc
     2581 cattctccct aagcgcaact ccgacaaact gattgcgagg aagaaggatt gggatccgaa
     2641 gaaatacgga ggctttgata gccctaccgt ggcatacagc gtactggtag tggccaaggt
     2701 ggagaagggc aagagcaaga aactgaaaag cgtcaaggaa ctgcttggaa ttaccataat
     2761 ggaaaggtcc tcgttcgaga agaatccgat cgacttcctc gaggctaaag gtaaaatatt
     2821 ggatgccaga cgatattctt tcttttgatt tgtaactttt tcctgtcaag gtcgataaat
     2881 tttatttttt ttggtaaaag gtcgataatt tttttttgga gccattatgt aattttccta
     2941 attaactgaa ccaaaattat actttgcagg ttacaaagag gtgaagaaag acctcattat
     3001 caaactgccc aagtattcgc ttttcgaatt ggaaaatggc agaaaacgca tgctggcatc
     3061 tgccggagaa ctgcagaagg gcaacgagct ggcattgccc agtaagtacg tcaacttcct
     3121 gtacttggcc tcacactatg agaagctgaa ggggtcacca gaggacaacg agcagaagca
     3181 gttgtttgtc gagcagcaca agcactatct tgatgagatc atagagcaga tcagcgaatt
     3241 ttccaagcgg gtcattcttg cagacgctaa cctcgataag gtaaggactt ctcatgaata
     3301 ttagtggcag attagtgttg ttaaagtctt tggttagata atcgatgcct cctaattgtc
     3361 catgttttac tggttttcta caattacagg tgctttccgc gtacaacaag cacagagata
     3421 agccgataag ggaacaagcg gaaaacatca tccacctgtt cacactgacc aatctgggag
     3481 ccccagcagc ctttaagtac ttcgatacca ctatcgacag aaagcgctac acatcaacca
     3541 aggaagtgtt ggacgctacc cttattcacc aatctattac agggctctat gagacaagga
     3601 tagatctgtc gcagttgggt ggtgactcta gggctgaccc aaagaagaag cgtaaagtcg
     3661 gttcgtgaga ccacgaagtg ccgaggcacg aacccagtgg acataagcct gttcggttcg
     3721 taagctgtaa tgcaagtagc gtatgcgctc acgcaactgg tccagaacct tgaccgaacg
     3781 cagcggtggt aacggcgcag tggcggtttt catggcttgt tatgactgtt tttttggggt
     3841 acagtctatg cctcgggcat ccaagcagca agcgcgttac gccgtgggtc gatgtttgat
     3901 gttatggagc agcaacgatg ttacgcagca gggcagtcgc cctaaaacaa agttaaacat
     3961 catgggggaa gcggtgatcg ccgaagtatc gactcaacta tcagaggtag ttggcgtcat
     4021 cgagcgccat ctcgaaccga cgttgctggc cgtacatttg tacggctccg cagtggatgg
     4081 cggcctgaag ccacacagcg atattgattt gctggttacg gtgaccgtaa ggcttgatga
     4141 aacaacgcgg cgagctttga tcaacgacct tttggaaact tcggcttccc ctggagagag
     4201 cgagattctc cgcgctgtag aagtcaccat tgttgtgcac gacgacatca ttccgtggcg
     4261 ttatccagct aagcgcgaac tgcaatttgg agaatggcag cgcaatgaca ttcttgcagg
     4321 tatcttcgag ccagccacga tcgacattga tctggctatc ttgctgacaa aagcaagaga
     4381 acatagcgtt gccttggtag gtccagcggc ggaggaactc tttgatccgg ttcctgaaca
     4441 ggatctattt gaggcgctaa atgaaacctt aacgctatgg aactcgccgc ccgactgggc
     4501 tggcgatgag cgaaatgtag tgcttacgtt gtcccgcatt tggtacagcg cagtaaccgg
     4561 caaaatcgcg ccgaaggatg tcgctgccga ctgggcaatg gagcgcctgc cggcccagta
     4621 tcagcccgtc atacttgaag ctagacaggc ttatcttgga caagaagaag atcgcttggc
     4681 ctcgcgcgca gatcagttgg aagaatttgt ccattacgtg aaaggcgaga tcaccaaggt
     4741 agtcggcaaa taatgtctag ctagaaattc gttcaagccg acgccgcttc gcggcgcggc
     4801 ttaactcaag cgttagatgc actaagcaca taattgctca cagccaaact atcaggtcaa
     4861 gtctgctttt attattttta agcgtgcata ataagcccta cacaaattgg gagatatatc
     4921 atgctgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     4981 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     5041 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     5101 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     5161 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     5221 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     5281 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     5341 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     5401 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     5461 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     5521 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     5581 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     5641 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     5701 tttttacggt tcctgcactc tgtggtctca aggtatggct tctagcccac cgaagaagaa
     5761 gcggaaggtc agctggaaaa tggacaagaa gtacagcatt ggacttgata ttggtacgaa
     5821 ctcagttggg tgggccgtta tcaccgatga atacaaggta ccttcgaaga aatttaaagt
     5881 gctgggcaac acagataggc acagcattaa gaagaacttg atcggagctc tgctctttga
     5941 ctctggagaa accgcggagg cgacaaggct taaacgtact gcgaggagaa ggtacactcg
     6001 caggaagaac agaatctgtt atctccaaga gatctttagc aacgagatgg cgaaggtaag
     6061 gatttttatg atatagtatg cttatgtatt ttgtactgaa agcatatcct gcttcattgg
     6121 gatattactg aaagcattta actacatgta aactcacttg atgatcaata aacttgattt
     6181 tgcaggttga cgactcgttc ttccatcgcc tcgaggaatc cttcctggta gaggaagata
     6241 agaaacacga gcgtcacccc atctttggga atattgttga cgaagtagcc tatcatgaaa
     6301 agtatccgac tatataccac cttcgcaaga agctggtgga ctcaaccgat aaggcagacc
     6361 ttcggctcat atacctggct ctcgcgcaca tgataaagtt tcgtggccat ttcttgatcg
     6421 aaggggacct caacccggat aactccgatg tggataaact gttcattcag ctcgtccaaa
     6481 cctacaatca gctgttcgag gagaacccca tcaatgcatc aggtaacatt ccttagttac
     6541 ctttcttttc tttttccatc ataagtttat agattgtaca tgctttgaga tttttctttg
     6601 caaacaatct caggtgtcga cgccaaggca atactgtctg ccagactttc gaagtccaga
     6661 cggcttgaga atctgatcgc tcaattgcca ggcgagaaga agaacggctt gttcgggaat
     6721 ctgattgcac tgtctctggg cctcacccct aacttcaaaa gcaactttga cctcgccgag
     6781 gacgcgaagc tgcagctgtc aaaggataca tacgatgatg atctggacaa tctgctcgcc
     6841 caaataggta atcttgaaat tggaactctt cttttgttgt ctaaacctat caatttcttt
     6901 gcggaaattt atttgaagct gtagagttaa aattgagtct tttaaacttt tgtaggtgat
     6961 cagtatgccg acctgttctt ggctgccaag aatctgtcag acgctatctt gctcagtgac
     7021 attctgcggg tcaacacgga gataaccaaa gcgccactta gcgcctccat gatcaagagg
     7081 tacgacgagc atcaccagga tctgaccctt ctgaaggctt tggttcgcca gcaactcccc
     7141 gagaagtaca aggagatttt ctttgaccaa tcgaagaatg gctacgcagg gtacattgat
     7201 ggaggtaagt tgttacttat gattgttttc ctctctgcta catgtatttt gttgttcatt
     7261 tctgtaagat ataagaattg agttttcctc tgatgatatt attaggtgca agtcaggagg
     7321 aattctacaa attcatcaag cctattctgg aaaagatgga cggtacagag gagctgctcg
     7381 ttaaattgaa ccgcgaagat ttgcttcgga agcagcgtac cttcgacaat ggcagcatac
     7441 cgcaccagat ccacctcggt gagctgcatg ctatcttgag gaggcaagag gacttctatc
     7501 cgttcctgaa agacaacaga gagaagattg aaaagatcct cacgttccgc attccctact
     7561 atgtaggtta gtatcatatg aagaaatacc tagtttcagt tgatgaatgc tattttctga
     7621 cctcagttgt tctcttttga gaattatttc ttttctaatt tgcctgattt ttctattaat
     7681 tcattaggtc cactcgcacg cgggaactcg cggtttgcgt ggatgacacg caaatccgag
     7741 gagactatca cgccttggaa cttcgaagag gtcgtggaca agggtgcgag tgcacagtcc
     7801 ttcatcgaaa ggatgaccaa cttcgataag aatctcccaa atgagaaagt cctgcccaag
     7861 cata
//
