LOCUS       sequence-330963-        7614 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector for expression of the SpCas9 VRKG variant in human cells: 
            CMV-T7-humanVRKG(SpCas9, 
            D1135V/S1136R/D1332K/R1333G)-1xNLS(SV40)-3xFLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7614)
  AUTHORS   Goldberg GW, Spencer JM, Giganti DO, Camellato BR, Agmon N, Ichikawa
            DM, Boeke JD, Noyes MB
  TITLE     Engineered dual selection for directed evolution of SpCas9 PAM 
            specificity.
  JOURNAL   Nat Commun. 2021 Jan 13;12(1):349. doi: 10.1038/s41467-020-20650-x.
  PUBMED    33441553
REFERENCE   2  (bases 1 to 7614)
  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 
            Commun."; date: "2021-01-13"; volume: "12(1):349. doi"; pages: " 
            10.1038/s41467-020-20650-x"
COMMENT     Sequence Label: pGG438
FEATURES             Location/Qualifiers
     source          1..7614
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      652..661
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(658..4059,4060..4062,4063..4659,4660,4661..4663,
                     4664..4665,4666..4668,4669..4761)
                     /codon_start=1
                     /product="Streptococcus pyogenes Cas9 endonuclease with the
                     D1135V/R1335Q/T1337R mutations"
                     /label=Cas9 VQR
                     /note="has an altered recognition specificity for the 
                     protospacer adjacent motif (PAM) (Kleinstiver et al., 
                     2015)"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFVRPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIKGKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4768..4788
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4795..4860
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4878..4895
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4918..4935)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4924..5148
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5219..5235)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5219..5235)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5232..5254)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5243..5259
                     /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(5267..5273,5274..5291,5292..5297))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5312..5333
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5450..5467)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5621..6209)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5701..5720)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6380..7171,7172..7240))
                     /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     7003..7022
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7241..7345)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7424..7443)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
      601 agatccgcta gagatccgcg gccgctaata cgactcacta tagggagagc cgccaccatg
      661 gataaaaagt attctattgg tttagacatc ggcactaatt ccgttggatg ggctgtcata
      721 accgatgaat acaaagtacc ttcaaagaaa tttaaggtgt tggggaacac agaccgtcat
      781 tcgattaaaa agaatcttat cggtgccctc ctattcgata gtggcgaaac ggcagaggcg
      841 actcgcctga aacgaaccgc tcggagaagg tatacacgtc gcaagaaccg aatatgttac
      901 ttacaagaaa tttttagcaa tgagatggcc aaagttgacg attctttctt tcaccgtttg
      961 gaagagtcct tccttgtcga agaggacaag aaacatgaac ggcaccccat ctttggaaac
     1021 atagtagatg aggtggcata tcatgaaaag tacccaacga tttatcacct cagaaaaaag
     1081 ctagttgact caactgataa agcggacctg aggttaatct acttggctct tgcccatatg
     1141 ataaagttcc gtgggcactt tctcattgag ggtgatctaa atccggacaa ctcggatgtc
     1201 gacaaactgt tcatccagtt agtacaaacc tataatcagt tgtttgaaga gaaccctata
     1261 aatgcaagtg gcgtggatgc gaaggctatt cttagcgccc gcctctctaa atcccgacgg
     1321 ctagaaaacc tgatcgcaca attacccgga gagaagaaaa atgggttgtt cggtaacctt
     1381 atagcgctct cactaggcct gacaccaaat tttaagtcga acttcgactt agctgaagat
     1441 gccaaattgc agcttagtaa ggacacgtac gatgacgatc tcgacaatct actggcacaa
     1501 attggagatc agtatgcgga cttatttttg gctgccaaaa accttagcga tgcaatcctc
     1561 ctatctgaca tactgagagt taatactgag attaccaagg cgccgttatc cgcttcaatg
     1621 atcaaaaggt acgatgaaca tcaccaagac ttgacacttc tcaaggccct agtccgtcag
     1681 caactgcctg agaaatataa ggaaatattc tttgatcagt cgaaaaacgg gtacgcaggt
     1741 tatattgacg gcggagcgag tcaagaggaa ttctacaagt ttatcaaacc catattagag
     1801 aagatggatg ggacggaaga gttgcttgta aaactcaatc gcgaagatct actgcgaaag
     1861 cagcggactt tcgacaacgg tagcattcca catcaaatcc acttaggcga attgcatgct
     1921 atacttagaa ggcaggagga tttttatccg ttcctcaaag acaatcgtga aaagattgag
     1981 aaaatcctaa cctttcgcat accttactat gtgggacccc tggcccgagg gaactctcgg
     2041 ttcgcatgga tgacaagaaa gtccgaagaa acgattactc catggaattt tgaggaagtt
     2101 gtcgataaag gtgcgtcagc tcaatcgttc atcgagagga tgaccaactt tgacaagaat
     2161 ttaccgaacg aaaaagtatt gcctaagcac agtttacttt acgagtattt cacagtgtac
     2221 aatgaactca cgaaagttaa gtatgtcact gagggcatgc gtaaacccgc ctttctaagc
     2281 ggagaacaga agaaagcaat agtagatctg ttattcaaga ccaaccgcaa agtgacagtt
     2341 aagcaattga aagaggacta ctttaagaaa attgaatgct tcgattctgt cgagatctcc
     2401 ggggtagaag atcgatttaa tgcgtcactt ggtacgtatc atgacctcct aaagataatt
     2461 aaagataagg acttcctgga taacgaagag aatgaagata tcttagaaga tatagtgttg
     2521 actcttaccc tctttgaaga tcgggaaatg attgaggaaa gactaaaaac atacgctcac
     2581 ctgttcgacg ataaggttat gaaacagtta aagaggcgtc gctatacggg ctggggacga
     2641 ttgtcgcgga aacttatcaa cgggataaga gacaagcaaa gtggtaaaac tattctcgat
     2701 tttctaaaga gcgacggctt cgccaatagg aactttatgc agctgatcca tgatgactct
     2761 ttaaccttca aagaggatat acaaaaggca caggtttccg gacaagggga ctcattgcac
     2821 gaacatattg cgaatcttgc tggttcgcca gccatcaaaa agggcatact ccagacagtc
     2881 aaagtagtgg atgagctagt taaggtcatg ggacgtcaca aaccggaaaa cattgtaatc
     2941 gagatggcac gcgaaaatca aacgactcag aaggggcaaa aaaacagtcg agagcggatg
     3001 aagagaatag aagagggtat taaagaactg ggcagccaga tcttaaagga gcatcctgtg
     3061 gaaaataccc aattgcagaa cgagaaactt tacctctatt acctacaaaa tggaagggac
     3121 atgtatgttg atcaggaact ggacataaac cgtttatctg attacgacgt cgatcacatt
     3181 gtaccccaat cctttttgaa ggacgattca atcgacaata aagtgcttac acgctcggat
     3241 aagaaccgag ggaaaagtga caatgttcca agcgaggaag tcgtaaagaa aatgaagaac
     3301 tattggcggc agctcctaaa tgcgaaactg ataacgcaaa gaaagttcga taacttaact
     3361 aaagctgaga ggggtggctt gtctgaactt gacaaggccg gatttattaa acgtcagctc
     3421 gtggaaaccc gccaaatcac aaagcatgtt gcacagatac tagattcccg aatgaatacg
     3481 aaatacgacg agaacgataa gctgattcgg gaagtcaaag taatcacttt aaagtcaaaa
     3541 ttggtgtcgg acttcagaaa ggattttcaa ttctataaag ttagggagat aaataactac
     3601 caccatgcgc acgacgctta tcttaatgcc gtcgtaggga ccgcactcat taagaaatac
     3661 ccgaagctag aaagtgagtt tgtgtatggt gattacaaag tttatgacgt ccgtaagatg
     3721 atcgcgaaaa gcgaacagga gataggcaag gctacagcca aatacttctt ttattctaac
     3781 attatgaatt tctttaagac ggaaatcact ctggcaaacg gagagatacg caaacgacct
     3841 ttaattgaaa ccaatgggga gacaggtgaa atcgtatggg ataagggccg ggacttcgcg
     3901 acggtgagaa aagttttgtc catgccccaa gtcaacatag taaagaaaac tgaggtgcag
     3961 accggagggt tttcaaagga atcgattctt ccaaaaagga atagtgataa gctcatcgct
     4021 cgtaaaaagg actgggaccc gaaaaagtac ggtggcttcg tgagacctac agttgcctat
     4081 tctgtcctag tagtggcaaa agttgagaag ggaaaatcca agaaactgaa gtcagtcaaa
     4141 gaattattgg ggataacgat tatggagcgc tcgtcttttg aaaagaaccc catcgacttc
     4201 cttgaggcga aaggttacaa ggaagtaaaa aaggatctca taattaaact accaaagtat
     4261 agtctgtttg agttagaaaa tggccgaaaa cggatgttgg ctagcgccgg agagcttcaa
     4321 aaggggaacg aactcgcact accgtctaaa tacgtgaatt tcctgtattt agcgtcccat
     4381 tacgagaagt tgaaaggttc acctgaagat aacgaacaga agcaactttt tgttgagcag
     4441 cacaaacatt atctcgacga aatcatagag caaatttcgg aattcagtaa gagagtcatc
     4501 ctagctgatg ccaatctgga caaagtatta agcgcataca acaagcacag ggataaaccc
     4561 atacgtgagc aggcggaaaa tattatccat ttgtttactc ttaccaacct cggcgctcca
     4621 gccgcattca agtattttga cacaacgata aagggcaaac gatacacttc taccaaggag
     4681 gtgctagacg cgacactgat tcaccaatcc atcacgggat tatatgaaac tcggatagat
     4741 ttgtcacagc ttgggggtga cggatccccc aagaagaaga ggaaagtctc gagcgactac
     4801 aaagaccatg acggtgatta taaagatcat gacatcgatt acaaggatga cgatgacaag
     4861 gctgcaggat gaccggtcat catcaccatc accattgagt ttaaacccgc tgatcagcct
     4921 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     4981 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     5041 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     5101 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
     5161 aaagaaccag ctggggctcg ataccgtcga cctctagcta gagcttggcg taatcatggt
     5221 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     5281 gaagcataaa gtgtaaagcc tagggtgcct aatgagtgag ctaactcaca ttaattgcgt
     5341 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     5401 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     5461 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     5521 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     5581 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5641 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5701 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5761 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5821 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5881 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5941 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     6001 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     6061 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     6121 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     6181 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     6241 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     6301 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     6361 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     6421 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     6481 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     6541 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     6601 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     6661 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     6721 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6781 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6841 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6901 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6961 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     7021 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     7081 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     7141 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     7201 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     7261 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     7321 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg
     7381 gatcgggaga tcgatctccc gatcccctag ggtcgactct cagtacaatc tgctctgatg
     7441 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
     7501 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
     7561 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgtt
//
