LOCUS       sequence-269635-        7611 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cas9-NRCH in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7611)
  AUTHORS   Miller SM, Wang T, Randolph PB, Arbab M, Shen MW, Huang TP, Matuszek
            Z, Newby GA, Rees HA, Liu DR
  TITLE     Continuous evolution of SpCas9 variants compatible with non-G PAMs.
  JOURNAL   Nat Biotechnol. 2020 Apr;38(4):471-481. doi: 
            10.1038/s41587-020-0412-8. Epub 2020 Feb 10.
  PUBMED    32042170
REFERENCE   2  (bases 1 to 7611)
  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"; doi: 
            "10.1038/s41587-020-0412-8"; journalName: "Nat Biotechnol"; date: 
            "2020-04"; volume: "38"; issue: "4"; pages: "471-481"
COMMENT     Sequence Label: pCMV-Cas9-NRCH
FEATURES             Location/Qualifiers
     source          1..7611
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     246..265
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        246..264
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             314..334
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             335..4435
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMVKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGIIPHQIHLGELHAILRRQGDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRLRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGGH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKGNSDKLIARKKDWDPKKYGGFNSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTINRKQYNTTKEVLD
                     ATLIRQSITGLYETRIDLSQLGGD"
     CDS             4466..4486
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4495..4512
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4535..4552)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4541..4765
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4836..4852)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4836..4852)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4849..4871)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4860..4876
                     /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(4884..4890,4891..4908,4909..4914))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4929..4950
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5067..5084)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5238..5826)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5318..5337)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5997..6788,6789..6857))
                     /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     6620..6639
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6858..6962)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7041..7060)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7232..7611
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gagatccgcg
      241 gccgctaata cgactcacta tagggagagc cgccaccatg aaacggacag ccgacggaag
      301 cgagttcgag tcaccaaaga agaagcggaa agtcgacaag aagtacagca tcggcctgga
      361 tatcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa
      421 gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc
      481 cctgctgttc gacagcggcg aaacagccga ggccacccgg ctgaagagaa ccgccagaag
      541 aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat
      601 ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga
      661 taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga
      721 gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga
      781 cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat
      841 cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca
      901 gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc
      961 catcctgtct gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc
     1021 cggcgagaag aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc
     1081 caacttcaag agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac
     1141 ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt
     1201 tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac
     1261 cgagatcacc aaggcccccc tgagcgcctc tatggtgaag agatacgacg agcaccacca
     1321 ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat
     1381 tttcttcgac cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga
     1441 agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct
     1501 cgtgaagctg aacagagagg acctgctgcg gaagcagcgg accttcgaca acggcattat
     1561 cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg gcgattttta
     1621 cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta
     1681 ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga
     1741 ggaaaccatc accccctgga acttcgagga agtggtggac aagggcgctt ccgcccagag
     1801 cttcatcgag cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa
     1861 gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt
     1921 gaccgaggga atgagaaagc ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga
     1981 cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa
     2041 gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc
     2101 cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggacaatga
     2161 ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg aggacagaga
     2221 gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag tgatgaagca
     2281 gctgaagcgg ctgagataca ccggctgggg caggctgagc cggaagctga tcaacggcat
     2341 ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa
     2401 cagaaacttc atgcagctga tccacgacga cagcctgacc tttaaagagg acatccagaa
     2461 agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc tggccggcag
     2521 ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt
     2581 gatgggcggc cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac
     2641 ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga
     2701 gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa
     2761 gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg aactggacat
     2821 caaccggctg tccgactacg atgtggacca tatcgtgcct cagagctttc tgaaggacga
     2881 ctccatcgac aacaaggtgc tgaccagaag cgacaagaac cggggcaaga gcgacaacgt
     2941 gccctccgaa gaggtcgtga agaagatgaa gaactactgg cggcagctgc tgaacgccaa
     3001 gctgattacc cagagaaagt tcgacaatct gaccaaggcc gagagaggcg gcctgagcga
     3061 actggataag gccggcttca tcaagagaca gctggtggaa acccggcaga tcacaaagca
     3121 cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaatg acaagctgat
     3181 ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt
     3241 ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa
     3301 cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta
     3361 cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg
     3421 caaggctacc gccaagtact tcttctacag caacatcatg aactttttca agaccgagat
     3481 taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg
     3541 ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc
     3601 ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca aagagtctat
     3661 cctgcccaag ggtaacagcg ataagctgat cgccagaaag aaggactggg accctaagaa
     3721 gtacggcggc ttcaacagcc ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga
     3781 aaagggcaag tccaagaaac tgaagagtgt gaaagagctg ctggggatca ccatcatgga
     3841 aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct acaaagaagt
     3901 gaaaaaggac ctgatcatca agctgcctaa gtactccctg ttcgagctgg aaaacggccg
     3961 gaagagaatg ctggcctctg ccggcgtgct gcagaaggga aacgaactgg ccctgccctc
     4021 caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg gctcccccga
     4081 ggataatgag cagaaacagc tgtttgtgga acagcacaag cactacctgg acgagatcat
     4141 cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc tggacaaagt
     4201 gctgtccgcc tacaacaagc accgggataa gcccatcaga gagcaggccg agaatatcat
     4261 ccacctgttt accctgacca atctgggagc ccctgccgcc ttcaagtact ttgacaccac
     4321 catcaaccgg aagcaataca acacgaccaa agaggtgctg gacgccaccc tgatccgtca
     4381 gagcatcacc ggcctgtacg agacacggat cgacctgtct cagctgggag gtgacaaaag
     4441 aaccgccgac ggcagcgaat tcgagcccaa gaagaagagg aaagtctaac cggtcatcat
     4501 caccatcacc attgagttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     4561 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     4621 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     4681 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     4741 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctcgata
     4801 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     4861 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctag
     4921 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     4981 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     5041 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     5101 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     5161 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     5221 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     5281 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     5341 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5401 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     5461 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5521 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5581 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5641 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     5701 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5761 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5821 tctcaagaag atcctttgat cttttctacg gggtctgaca ctcagtggaa cgaaaactca
     5881 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     5941 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     6001 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     6061 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     6121 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     6181 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     6241 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     6301 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6361 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6421 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6481 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6541 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6601 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6661 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     6721 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6781 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6841 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     6901 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     6961 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatcg atctcccgat
     7021 cccctagggt cgactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg
     7081 ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca
     7141 aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg
     7201 cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag
     7261 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
     7321 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
     7381 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
     7441 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
     7501 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
     7561 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat g
//
