LOCUS       sequence-232065-        8752 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Target-AID-NG 
            (NLS-nSpCas9-NG-NLS-SH3-3xFLAG-PmCDA1-NLS-UGI) in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8752)
  AUTHORS   Nishimasu H, Shi X, Ishiguro S, Gao L, Hirano S, Okazaki S, Noda T, 
            Abudayyeh OO, Gootenberg JS, Mori H, Oura S, Holmes B, Tanaka M, 
            Seki M, Hirano H, Aburatani H, Ishitani R, Ikawa M, Yachie N, Zhang 
            F, Nureki O
  TITLE     Engineered CRISPR-Cas9 nuclease with expanded targeting space.
  JOURNAL   Science. 2018 Aug 30. pii: science.aas9129. doi: 
            10.1126/science.aas9129.
  PUBMED    30166441
REFERENCE   2  (bases 1 to 8752)
  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: "Science. 
            2018 Aug 30. pii: science.aas9129. doi: 10.1126/science.aas9129."
COMMENT     Sequence Label: pSI-Target-AID-NG
FEATURES             Location/Qualifiers
     source          1..8752
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(12..31)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        203..582
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        583..786
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     737..757
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     828..847
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        828..846
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      854..863
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             866..886
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3498..3509
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             5027..5047
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5255..5320
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5954..5974
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5981..6235
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHT
                     AYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     primer_bind     complement(6258..6275)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6264..6488
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6559..6575)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6559..6575)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6572..6594)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6583..6599
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(6607..6613,6614..6631,6632..6637))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6652..6673
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6790..6807)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6961..7549)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7041..7060)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7720..8511,8512..8580))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     8343..8362
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8581..8685)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcgactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
       61 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc
      121 ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat
      181 gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt
      241 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
      301 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
      361 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
      421 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
      481 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
      541 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
      601 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
      661 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
      721 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
      781 agagctggtt tagtgaaccg tcagatccgc tagagatccg cggccgctaa tacgactcac
      841 tatagggaga gccgccacca tggcaccgaa gaagaagcgt aaagtcggaa tccacggagt
      901 tcctgcggca atggacaaga agtactccat tgggctcgct atcggcacaa acagcgtcgg
      961 ttgggccgtc attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa
     1021 taccgatcgc cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga
     1081 aacggccgaa gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa
     1141 tcggatctgc tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt
     1201 cttccatagg ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc
     1261 aatctttggc aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca
     1321 tctgaggaag aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc
     1381 gctggcgcat atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga
     1441 caacagcgat gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga
     1501 agagaacccg atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc
     1561 caaatcccgg cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct
     1621 gtttggtaat cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga
     1681 cctggccgaa gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa
     1741 tctgctggcc cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc
     1801 agacgccatt ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct
     1861 gagcgctagt atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc
     1921 ccttgtcaga cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa
     1981 tggctacgcc ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa
     2041 gcccatcttg gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga
     2101 tctgttgcgc aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg
     2161 cgaactgcac gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag
     2221 ggaaaagatt gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg
     2281 gggaaattcc agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa
     2341 cttcgaggaa gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa
     2401 ctttgataaa aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta
     2461 cttcacagtt tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc
     2521 agcattcctg tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg
     2581 gaaagttacc gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc
     2641 tgttgaaatc agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct
     2701 cctgaaaatc attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga
     2761 ggacattgtc ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa
     2821 aacttacgct catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac
     2881 aggatggggg cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa
     2941 gacaatcctg gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat
     3001 ccatgatgac tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg
     3061 ggacagtctt cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat
     3121 actgcagacc gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga
     3181 gaatatcgtt atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag
     3241 tagggaaagg atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa
     3301 ggaacaccca gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca
     3361 gaacggcagg gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga
     3421 cgtggatcat atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt
     3481 gacaagatcc gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa
     3541 gaaaatgaaa aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt
     3601 cgataatctg actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat
     3661 caaaaggcag cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc
     3721 acgcatgaac accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac
     3781 tctgaagtct aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga
     3841 gatcaacaat taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact
     3901 tatcaaaaaa tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga
     3961 tgttaggaaa atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt
     4021 cttttacagc aatattatga attttttcaa gaccgagatt acactggcca atggagagat
     4081 tcggaagcga ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg
     4141 tagggatttc gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa
     4201 gaccgaagta cagaccggag gcttctccaa ggaaagtatc cggcccaaga ggaacagcga
     4261 taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct tcgttagccc
     4321 caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact
     4381 gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa
     4441 tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa
     4501 gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc
     4561 ccggttcctg cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta
     4621 cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct
     4681 gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc
     4741 caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca
     4801 ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa
     4861 tctgggagcc cctcgggcct tcaagtactt tgacaccacc atcgaccgga aggtctaccg
     4921 gtctacaaag gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga
     4981 aacaagaatc gacctctctc agctcggtgg agacagcagg gctgacccca agaagaagag
     5041 gaaggtgggt ggaggaggta ccggcggtgg aggctcagca gaatacgtac gagctctgtt
     5101 tgacttcaat gggaatgacg aggaggatct cccctttaag aagggcgata ttctccgcat
     5161 cagagataag cccgaagaac aatggtggaa tgccgaggat agcgaaggga aaaggggcat
     5221 gattctggtg ccatatgtgg agaaatattc cggtgactac aaagaccatg atggggatta
     5281 caaagaccac gacatcgact acaaagacga cgacgataaa tcagggatga cagacgccga
     5341 gtacgtgcgc attcatgaga aactggatat ttacaccttc aagaagcagt tcttcaacaa
     5401 caagaaatct gtgtcacacc gctgctacgt gctgtttgag ttgaagcgaa ggggcgaaag
     5461 aagggcttgc ttttggggct atgccgtcaa caagccccaa agtggcaccg agagaggaat
     5521 acacgctgag atattcagta tccgaaaggt ggaagagtat cttcgggata atcctgggca
     5581 gtttacgatc aactggtatt ccagctggag tccttgcgct gattgtgccg agaaaattct
     5641 ggaatggtat aatcaggaac ttcggggaaa cgggcacaca ttgaaaatct gggcctgcaa
     5701 gctgtactac gagaagaatg cccggaacca gataggactc tggaatctga gggacaatgg
     5761 tgtaggcctg aacgtgatgg tttccgagca ctatcagtgt tgtcggaaga ttttcatcca
     5821 aagctctcat aaccagctca atgaaaaccg ctggttggag aaaacactga aacgtgcgga
     5881 gaagtggaga tccgagctga gcatcatgat ccaggtcaag attctgcata ccactaagtc
     5941 tccagccgtt ggtcccaaga agaaaagaaa agtcggtacc atgaccaacc tttccgacat
     6001 catagagaag gaaacaggca aacagttggt catccaagag tcgatactca tgcttcctga
     6061 agaagttgag gaggtcattg ggaataagcc ggaaagtgac attctcgtac acactgcgta
     6121 tgatgagagc accgatgaga acgtgatgct gctcacgtca gatgccccag agtacaaacc
     6181 ctgggctctg gtgattcagg actctaatgg agagaacaag atcaagatgc tataatgagt
     6241 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     6301 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     6361 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     6421 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     6481 ctctatggct tctgaggcgg aaagaaccag ctggggctcg ataccgtcga cctctagcta
     6541 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     6601 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tagggtgcct aatgagtgag
     6661 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     6721 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     6781 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     6841 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     6901 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     6961 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     7021 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     7081 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     7141 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     7201 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     7261 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     7321 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     7381 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     7441 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     7501 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     7561 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     7621 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     7681 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     7741 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     7801 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     7861 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     7921 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     7981 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     8041 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     8101 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     8161 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     8221 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     8281 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     8341 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     8401 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     8461 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     8521 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     8581 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     8641 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     8701 agtgccacct gacgtcgacg gatcgggaga tcgatctccc gatcccctag gg
//
