LOCUS       sequence-332887-        7145 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses nSpCas9-NG in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7145)
  AUTHORS   Wang L, Xue W, Zhang H, Gao R, Qiu H, Wei J, Zhou L, Lei YN, Wu X, 
            Li X, Liu C, Wu J, Chen Q, Ma H, Huang X, Cai C, Zhang Y, Yang B, 
            Yin H, Yang L, Chen J
  TITLE     Eliminating base-editor-induced genome-wide and transcriptome-wide 
            off-target mutations.
  JOURNAL   Nat Cell Biol. 2021 May 10. pii: 10.1038/s41556-021-00671-4. doi: 
            10.1038/s41556-021-00671-4.
  PUBMED    33972728
REFERENCE   2  (bases 1 to 7145)
  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: "Nat Cell 
            Biol. 2021 May 10. pii: 10.1038/s41556-021-00671-4. doi: 
            10.1038/s41556-021-00671-4."
COMMENT     Sequence Label: pEF1a-nSpCas9-NG
FEATURES             Location/Qualifiers
     source          1..7145
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..212
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      258..267
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             264..4367
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESIRPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARFLQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFDTTIDRKVYRSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4380..4400
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4409..4426
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4449..4466)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4455..4679
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4750..4766)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4750..4766)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4763..4785)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4774..4790
                     /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(4798..4804,4805..4822,4823..4828))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4843..4864
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4981..4998)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5152..5740)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5232..5251)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5911..6702,6703..6771))
                     /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     6534..6553
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6772..6876)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6955..6974)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa
       61 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
      121 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
      181 tttttcgcaa cgggtttgcc gccagaacac aggtgtcgtg acgcggttcc ggttaaggtg
      241 aacgcgtgca ggctggcgcc accatggaca agaagtacag catcggcctg gccatcggca
      301 ccaactctgt gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc aagaaattca
      361 aggtgctggg caacaccgac cggcacagca tcaagaagaa cctgatcgga gccctgctgt
      421 tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag aaccgccaga agaagataca
      481 ccagacggaa gaaccggatc tgctatctgc aagagatctt cagcaacgag atggccaagg
      541 tggacgacag cttcttccac agactggaag agtccttcct ggtggaagag gataagaagc
      601 acgagcggca ccccatcttc ggcaacatcg tggacgaggt ggcctaccac gagaagtacc
      661 ccaccatcta ccacctgaga aagaaactgg tggacagcac cgacaaggcc gacctgcggc
      721 tgatctatct ggccctggcc cacatgatca agttccgggg ccacttcctg atcgagggcg
      781 acctgaaccc cgacaacagc gacgtggaca agctgttcat ccagctggtg cagacctaca
      841 accagctgtt cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag gccatcctgt
      901 ctgccagact gagcaagagc agacggctgg aaaatctgat cgcccagctg cccggcgaga
      961 agaagaatgg cctgttcgga aacctgattg ccctgagcct gggcctgacc cccaacttca
     1021 agagcaactt cgacctggcc gaggatgcca aactgcagct gagcaaggac acctacgacg
     1081 acgacctgga caacctgctg gcccagatcg gcgaccagta cgccgacctg tttctggccg
     1141 ccaagaacct gtccgacgcc atcctgctga gcgacatcct gagagtgaac accgagatca
     1201 ccaaggcccc cctgagcgcc tctatgatca agagatacga cgagcaccac caggacctga
     1261 ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa gtacaaagag attttcttcg
     1321 accagagcaa gaacggctac gccggctaca ttgacggcgg agccagccag gaagagttct
     1381 acaagttcat caagcccatc ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc
     1441 tgaacagaga ggacctgctg cggaagcagc ggaccttcga caacggcagc atcccccacc
     1501 agatccacct gggagagctg cacgccattc tgcggcggca ggaagatttt tacccattcc
     1561 tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc tactacgtgg
     1621 gccctctggc caggggaaac agcagattcg cctggatgac cagaaagagc gaggaaacca
     1681 tcaccccctg gaacttcgag gaagtggtgg acaagggcgc ttccgcccag agcttcatcg
     1741 agcggatgac caacttcgat aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc
     1801 tgctgtacga gtacttcacc gtgtataacg agctgaccaa agtgaaatac gtgaccgagg
     1861 gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt
     1921 tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga ggactacttc aagaaaatcg
     1981 agtgcttcga ctccgtggaa atctccggcg tggaagatcg gttcaacgcc tccctgggca
     2041 cataccacga tctgctgaaa attatcaagg acaaggactt cctggacaat gaggaaaacg
     2101 aggacattct ggaagatatc gtgctgaccc tgacactgtt tgaggacaga gagatgatcg
     2161 aggaacggct gaaaacctat gcccacctgt tcgacgacaa agtgatgaag cagctgaagc
     2221 ggcggagata caccggctgg ggcaggctga gccggaagct gatcaacggc atccgggaca
     2281 agcagtccgg caagacaatc ctggatttcc tgaagtccga cggcttcgcc aacagaaact
     2341 tcatgcagct gatccacgac gacagcctga cctttaaaga ggacatccag aaagcccagg
     2401 tgtccggcca gggcgatagc ctgcacgagc acattgccaa tctggccggc agccccgcca
     2461 ttaagaaggg catcctgcag acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc
     2521 ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga gaaccagacc acccagaagg
     2581 gacagaagaa cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa gagctgggca
     2641 gccagatcct gaaagaacac cccgtggaaa acacccagct gcagaacgag aagctgtacc
     2701 tgtactacct gcagaatggg cgggatatgt acgtggacca ggaactggac atcaaccggc
     2761 tgtccgacta cgatgtggac catatcgtgc ctcagagctt tctgaaggac gactccatcg
     2821 acaacaaggt gctgaccaga agcgacaaga accggggcaa gagcgacaac gtgccctccg
     2881 aagaggtcgt gaagaagatg aagaactact ggcggcagct gctgaacgcc aagctgatta
     2941 cccagagaaa gttcgacaat ctgaccaagg ccgagagagg cggcctgagc gaactggata
     3001 aggccggctt catcaagaga cagctggtgg aaacccggca gatcacaaag cacgtggcac
     3061 agatcctgga ctcccggatg aacactaagt acgacgagaa tgacaagctg atccgggaag
     3121 tgaaagtgat caccctgaag tccaagctgg tgtccgattt ccggaaggat ttccagtttt
     3181 acaaagtgcg cgagatcaac aactaccacc acgcccacga cgcctaccta aacgccgtcg
     3241 tgggaaccgc cctgatcaaa aagtacccta agctggaaag cgagttcgtg tacggcgact
     3301 acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta
     3361 ccgccaagta cttcttctac agcaacatca tgaacttttt caagaccgag attaccctgg
     3421 ccaacggcga gatccggaag cggcctctga tcgagacaaa cggcgaaacc ggggagatcg
     3481 tgtgggataa gggccgggat tttgccaccg tgcggaaagt gctgagcatg ccccaagtga
     3541 atatcgtgaa aaagaccgag gtgcagacag gcggcttcag caaagagtct atcaggccca
     3601 agaggaacag cgataagctg atcgccagaa agaaggactg ggaccctaag aagtacggcg
     3661 gcttcgtgag ccccaccgtg gcctattctg tgctggtggt ggccaaagtg gaaaagggca
     3721 agtccaagaa actgaagagt gtgaaagagc tgctggggat caccatcatg gaaagaagca
     3781 gcttcgagaa gaatcccatc gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg
     3841 acctgatcat caagctgcct aagtactccc tgttcgagct ggaaaacggc cggaagagaa
     3901 tgctggcctc tgccaggttc ctgcagaagg gaaacgaact ggccctgccc tccaaatatg
     3961 tgaacttcct gtacctggcc agccactatg agaagctgaa gggctccccc gaggataatg
     4021 agcagaaaca gctgtttgtg gaacagcaca agcactacct ggacgagatc atcgagcaga
     4081 tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg
     4141 cctacaacaa gcaccgggat aagcccatca gagagcaggc cgagaatatc atccacctgt
     4201 ttaccctgac caatctggga gcccctaggg ccttcaagta ctttgacacc accatcgacc
     4261 ggaaggtgta caggagcacc aaagaggtgc tggacgccac cctgatccac cagagcatca
     4321 ccggcctgta cgagacacgg atcgacctgt ctcagctggg aggcgacagc ggaggatccc
     4381 ccaagaagaa gaggaaagtc taaccggtca tcatcaccat caccattgag tttaaacccg
     4441 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     4501 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     4561 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     4621 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     4681 ttctgaggcg gaaagaacca gctggggctc gataccgtcg acctctagct agagcttggc
     4741 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     4801 catacgagcc ggaagcataa agtgtaaagc ctagggtgcc taatgagtga gctaactcac
     4861 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4921 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     4981 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     5041 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     5101 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5161 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5221 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5281 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5341 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5401 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5461 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5521 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5581 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5641 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5701 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5761 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     5821 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5881 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     5941 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     6001 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     6061 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6121 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6181 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     6241 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     6301 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     6361 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     6421 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6481 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6541 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6601 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6661 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6721 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     6781 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     6841 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     6901 tgacgtcgac ggatcgggag atcgatctcc cgatccccta gggtcgactc tcagtacaat
     6961 ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc
     7021 tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat
     7081 gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac
     7141 gcgtt
//
