LOCUS       sequence-174891-        8202 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses the ATP1A1 G3 sgRNA in combination with FLAGless 
            eSpCas9(1.1) to target ATP1A1 intron 4. Px330-like plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 8202)
  AUTHORS   Agudelo D, Duringer A, Bozoyan L, Huard CC, Carter S, Loehr J, 
            Synodinou D, Drouin M, Salsman J, Dellaire G, Laganiere J, Doyon Y
  TITLE     Marker-free coselection for CRISPR-driven genome editing in human 
            cells.
  JOURNAL   Nat Methods. 2017 Apr 17. doi: 10.1038/nmeth.4265.
  PUBMED    28417998
REFERENCE   2  (bases 1 to 8202)
  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 
            Methods. 2017 Apr 17. doi: 10.1038/nmeth.4265."
COMMENT     Sequence Label: eSpCas9(1.1)_No_FLAG_ATP1A1_G3
FEATURES             Location/Qualifiers
     source          1..8202
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     regulatory      224..233
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             236..256
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(281..2818,2819..2821,2822..3283,3284..3286,3287..3454,
                     3455..3457,3458..4381)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Slaymaker et al., 2016)"
                     /label=eSpCas9(1.1)
                     /note="carries the mutations K848A, K1003A, and R1060A"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4382..4429
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(4457..4474)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4463..4670
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4679..4819
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4679..4808
                     /label=AAV2 ITR
     rep_origin      4894..5349
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4981..5000)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5191..5212
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5366..5385)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5485..5507
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5545..5563)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5631..5735
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5736..5804,5805..6596)
                     /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(5954..5973)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6767..7355
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7256..7275
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        7417..7665
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7417..7437
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7588..7607
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        7686..7761
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        7858..8143
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga
       61 ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc
      121 tgagcaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg tttaattacc
      181 tggagcacct gcctgaaatc actttttttc aggttggacc ggtgccacca tggccccaaa
      241 gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt acagcatcgg
      301 cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc
      361 cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat
      421 cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc
      481 cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa
      541 cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga
      601 agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta
      661 ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa
      721 ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt
      781 cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct
      841 ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc
      901 caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca
      961 gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct
     1021 gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa
     1081 ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga
     1141 cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt
     1201 gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca
     1261 ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa
     1321 agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag
     1381 ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga
     1441 actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg
     1501 cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga
     1561 tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat
     1621 cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa
     1681 gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc
     1741 ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct
     1801 gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa
     1861 atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat
     1921 cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta
     1981 cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa
     2041 cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga
     2101 caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga
     2161 cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat
     2221 gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa
     2281 cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt
     2341 cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat
     2401 ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc
     2461 cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt
     2521 gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca
     2581 gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat
     2641 caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa
     2701 cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact
     2761 ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctggc
     2821 cgacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga
     2881 caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa
     2941 cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct
     3001 gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac
     3061 aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa
     3121 gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa
     3181 ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta
     3241 cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctgcgctgg aaagcgagtt
     3301 cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga
     3361 aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac
     3421 cgagattacc ctggccaacg gcgagatccg gaaggcgcct ctgatcgaga caaacggcga
     3481 aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag
     3541 catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga
     3601 gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc
     3661 taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa
     3721 agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat
     3781 catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa
     3841 agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa
     3901 cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct
     3961 gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc
     4021 ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga
     4081 gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga
     4141 caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa
     4201 tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga
     4261 caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat
     4321 ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga
     4381 caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagt aagaattcct
     4441 agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     4501 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     4561 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     4621 caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga gcggccgcag
     4681 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     4741 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
     4801 gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     4861 atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg
     4921 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg
     4981 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     5041 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     5101 aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     5161 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     5221 tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt
     5281 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     5341 ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     5401 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     5461 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     5521 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     5581 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     5641 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     5701 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     5761 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     5821 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     5881 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     5941 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     6001 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     6061 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     6121 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     6181 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     6241 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     6301 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     6361 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     6421 ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc
     6481 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     6541 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     6601 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     6661 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     6721 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     6781 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     6841 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     6901 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     6961 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     7021 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     7081 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     7141 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     7201 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     7261 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     7321 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     7381 ggttcctggc cttttgctgg ccttttgctc acatgtgagg gcctatttcc catgattcct
     7441 tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta
     7501 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     7561 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     7621 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgagtt ctgtaattca
     7681 gcatagtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa
     7741 aaagtggcac cgagtcggtg cttttttgtt ttagagctag aaatagcaag ttaaaataag
     7801 gctagtccgt ttttagcgcg tgcgccaatt ctgcagacaa atggctctag aggtacccgt
     7861 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
     7921 gtcaatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     7981 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
     8041 caatgacggt aaatggcccg cctggcattg tgcccagtac atgaccttat gggactttcc
     8101 tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac
     8161 gttctgcttc actctcccca tctccccccc ctccccaccc cc
//
