pCMV-nCas9

LOCUS       pCMV-nCas9         7638 bp DNA     circular SYN 07-NOV-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7638)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 20, 2026 from SnapGene 6.0.2
            https://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..7638
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        87..466
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        467..670
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     621..641
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     712..731
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        712..730
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(774..800)
                     /label=OL1013
     primer_bind     complement(778..800)
                     /label=OL1036
     CDS             780..800
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     781..822
                     /label=OL1039
     CDS             801..4901
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(R221K N394K H840A)
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRKLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLKREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     primer_bind     complement(1409..1419)
                     /label=OL900
     CDS             4902..4979
                     /label=SGGSx2-sv40NLS-SGGSx2
                     /note="/created_by=chenp"
     primer_bind     complement(4953..5009)
                     /label=OL1015
     primer_bind     4958..5012
                     /label=OL1014
     CDS             4959..4979
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4964..5007)
                     /label=OL1040
     primer_bind     complement(4979..5012)
                     /label=OL900
     primer_bind     4988..5011
                     /label=OL1035
     CDS             4988..5005
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5028..5045)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5034..5258
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal
                     牛生长激素聚腺苷酸化信号
                     牛生长激素（BGH）多聚腺苷酸化信号能够保证高效的转录终止和mRNA的poly A结构"
     primer_bind     complement(5329..5345)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5329..5345)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5342..5364)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5353..5369
                     /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(5377..5407)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5422..5443
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5560..5577)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5731..6319)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5811..5830)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6490..7350)
                     /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     7113..7132
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7351..7455)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7534..7553)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg
       61 cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc
      121 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      181 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      241 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      301 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      361 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      421 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
      481 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
      541 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
      601 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
      661 aagcagagct ggtttagtga accgtcagat ccgctagaga tccgcggccg ctaatacgac
      721 tcactatagg gagagccgcc accatgaaac ggacagccga cggaagcgag ttcgagtcac
      781 caaagaagaa gcggaaagtc gacaagaagt acagcatcgg cctggacatc ggcaccaact
      841 ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc
      901 tgggcaacac cgaccggcac agcatcaaga agaacctgat cggagccctg ctgttcgaca
      961 gcggcgaaac agccgaggcc acccggctga agagaaccgc cagaagaaga tacaccagac
     1021 ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg
     1081 acagcttctt ccacagactg gaagagtcct tcctggtgga agaggataag aagcacgagc
     1141 ggcaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag taccccacca
     1201 tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg cggctgatct
     1261 atctggccct ggcccacatg atcaagttcc ggggccactt cctgatcgag ggcgacctga
     1321 accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc tacaaccagc
     1381 tgttcgagga aaaccccatc aacgccagcg gcgtggacgc caaggccatc ctgtctgcca
     1441 gactgagcaa gagcagaaag ctggaaaatc tgatcgccca gctgcccggc gagaagaaga
     1501 atggcctgtt cggaaacctg attgccctga gcctgggcct gacccccaac ttcaagagca
     1561 acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac gacgacgacc
     1621 tggacaacct gctggcccag atcggcgacc agtacgccga cctgtttctg gccgccaaga
     1681 acctgtccga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag atcaccaagg
     1741 cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac ctgaccctgc
     1801 tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agagattttc ttcgaccaga
     1861 gcaagaacgg ctacgccggc tacattgacg gcggagccag ccaggaagag ttctacaagt
     1921 tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg aagctgaaga
     1981 gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc caccagatcc
     2041 acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca ttcctgaagg
     2101 acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac gtgggccctc
     2161 tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc
     2221 cctggaactt cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc atcgagcgga
     2281 tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac agcctgctgt
     2341 acgagtactt caccgtgtat aacgagctga ccaaagtgaa atacgtgacc gagggaatga
     2401 gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga
     2461 ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa atcgagtgct
     2521 tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg ggcacatacc
     2581 acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa aacgaggaca
     2641 ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg atcgaggaac
     2701 ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg aagcggcgga
     2761 gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg gacaagcagt
     2821 ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga aacttcatgc
     2881 agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc caggtgtccg
     2941 gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc gccattaaga
     3001 agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca
     3061 agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag aagggacaga
     3121 agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg ggcagccaga
     3181 tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg tacctgtact
     3241 acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac cggctgtccg
     3301 actacgatgt ggacgctatc gtgcctcaga gctttctgaa ggacgactcc atcgacaaca
     3361 aggtgctgac cagaagcgac aagaaccggg gcaagagcga caacgtgccc tccgaagagg
     3421 tcgtgaagaa gatgaagaac tactggcggc agctgctgaa cgccaagctg attacccaga
     3481 gaaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg gataaggccg
     3541 gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc
     3601 tggactcccg gatgaacact aagtacgacg agaatgacaa gctgatccgg gaagtgaaag
     3661 tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag ttttacaaag
     3721 tgcgcgagat caacaactac caccacgccc acgacgccta cctgaacgcc gtcgtgggaa
     3781 ccgccctgat caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc gactacaagg
     3841 tgtacgacgt gcggaagatg atcgccaaga gcgagcagga aatcggcaag gctaccgcca
     3901 agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc ctggccaacg
     3961 gcgagatccg gaagcggcct ctgatcgaga caaacggcga aaccggggag atcgtgtggg
     4021 ataagggccg ggattttgcc accgtgcgga aagtgctgag catgccccaa gtgaatatcg
     4081 tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg cccaagagga
     4141 acagcgataa gctgatcgcc agaaagaagg actgggaccc taagaagtac ggcggcttcg
     4201 acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca
     4261 agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga agcagcttcg
     4321 agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa aaggacctga
     4381 tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag agaatgctgg
     4441 cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa tatgtgaact
     4501 tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat aatgagcaga
     4561 aacagctgtt tgtggaacag cacaagcact acctggacga gatcatcgag cagatcagcg
     4621 agttctccaa gagagtgatc ctggccgacg ctaatctgga caaagtgctg tccgcctaca
     4681 acaagcaccg ggataagccc atcagagagc aggccgagaa tatcatccac ctgtttaccc
     4741 tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc gaccggaaga
     4801 ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc atcaccggcc
     4861 tgtacgagac acggatcgac ctgtctcagc tgggaggtga ctccggcgga agctctggtg
     4921 gcagcaagcg gaccgccgac ggctctgaat tcgagagccc taagaagaaa agaaaggtgt
     4981 aaccggtcat catcaccatc accattgagt ttaaacccgc tgatcagcct cgactgtgcc
     5041 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     5101 tgccactccc actgtccttt cctaataaaa tgagaaaatt gcatcgcatt gtctgagtag
     5161 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     5221 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     5281 ctggggctcg ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     5341 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     5401 gtgtaaagcc tagggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     5461 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5521 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     5581 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     5641 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5701 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5761 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5821 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5881 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5941 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     6001 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     6061 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     6121 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     6181 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     6241 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6301 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acactcagtg
     6361 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6421 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6481 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6541 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6601 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     6661 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6721 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6781 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6841 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     6901 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     6961 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     7021 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     7081 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     7141 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     7201 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     7261 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7321 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7381 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7441 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
     7501 tcgatctccc gatcccctag ggtctactct cagtacaatc tgctctgatg ccgcatagtt
     7561 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
     7621 ttaagctaca acaaggca
//