LOCUS       sequence-97157-e        7434 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Truncated form of SpCas9 without C' domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7434)
  AUTHORS   Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, 
            Ishitani R, Zhang F, Nureki O
  TITLE     Crystal structure of Cas9 in complex with guide RNA and target DNA.
  JOURNAL   Cell. 2014 Feb 27;156(5):935-49. doi: 10.1016/j.cell.2014.02.001. 
            Epub 2014 Feb 13.
  PUBMED    24529477
REFERENCE   2  (bases 1 to 7434)
  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: 
            "10.1016/j.cell.2014.02.001"; journalName: "Cell"; date: 
            "2014-02-27- 27"; volume: "156"; issue: "5"; pages: "935-49"
COMMENT     Sequence Label: PX544 SpCas9(del1098-1368)
FEATURES             Location/Qualifiers
     source          1..7434
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     enhancer        160..463
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        465..742
                     /label=chicken beta-actin promoter
     intron          743..971
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      983..992
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             992..1057
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1064..1084
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1109..2824
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     CDS             4400..4447
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(4475..4492)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4481..4688
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4697..4837
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4697..4826
                     /label=AAV2 ITR
     rep_origin      4912..5367
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4999..5018)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5209..5230
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5384..5403)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5503..5525
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5563..5581)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5649..5753
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5754..5822,5823..6614)
                     /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     complement(5972..5991)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6785..7373
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7274..7293
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggcctct agaggtaccc gttacataac ttacggtaaa
      181 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
      241 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
      301 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
      361 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
      421 tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac
      481 gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat
      541 tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg
      601 ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca
      661 gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa
      721 aaagcgaagc gcgcggcggg cgggagtcgc tgcgacgctg ccttcgcccc gtgccccgct
      781 ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga
      841 gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg taagggttta
      901 agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg cctgaaatca
      961 ctttttttca ggttggaccg gtgccaccat ggactataag gaccacgacg gagactacaa
     1021 ggatcatgat attgattaca aagacgatga cgataagatg gccccaaaga agaagcggaa
     1081 ggtcggtatc cacggagtcc cagcagccga caagaagtac agcatcggcc tggacatcgg
     1141 caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt
     1201 caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct
     1261 gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata
     1321 caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa
     1381 ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa
     1441 gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta
     1501 ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg
     1561 gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg
     1621 cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta
     1681 caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct
     1741 gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga
     1801 gaagaagaat ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt
     1861 caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga
     1921 cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc
     1981 cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat
     2041 caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct
     2101 gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt
     2161 cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt
     2221 ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa
     2281 gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca
     2341 ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt
     2401 cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt
     2461 gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac
     2521 catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat
     2581 cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag
     2641 cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga
     2701 gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct
     2761 gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat
     2821 cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg
     2881 cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa
     2941 cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat
     3001 cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa
     3061 gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga
     3121 caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa
     3181 cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca
     3241 ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc
     3301 cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg
     3361 ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa
     3421 gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg
     3481 cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta
     3541 cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg
     3601 gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat
     3661 cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc
     3721 cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat
     3781 tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga
     3841 taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc
     3901 acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga
     3961 agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt
     4021 ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt
     4081 cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga
     4141 ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc
     4201 taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct
     4261 ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat
     4321 cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt
     4381 gaatatcgtg aaaaagacca aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa
     4441 gaaaaagtaa gaattcctag agctcgctga tcagcctcga ctgtgccttc tagttgccag
     4501 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     4561 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     4621 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagagaa tagcaggcat
     4681 gctggggagc ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     4741 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
     4801 gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc
     4861 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
     4921 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     4981 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     5041 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     5101 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
     5161 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     5221 tccaaactgg aacaacactc aaccctatct cgggctattc ttttgattta taagggattt
     5281 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     5341 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
     5401 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     5461 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     5521 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     5581 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     5641 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     5701 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     5761 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     5821 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     5881 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     5941 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     6001 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     6061 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     6121 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     6181 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     6241 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     6301 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     6361 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     6421 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta
     6481 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     6541 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     6601 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     6661 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     6721 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     6781 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     6841 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     6901 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     6961 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     7021 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     7081 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     7141 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     7201 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     7261 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     7321 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     7381 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgt
//
