LOCUS       sequence-171418-        8268 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses the ATP1A1 G5 crRNA in combination with AsCpf1-3xHA to 
            target ATP1A1 intron 4. U6-crRNA(ATP1A1 G5)-CBh-AsCpf1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 8268)
  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 8268)
  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: pY036_ATP1A1_G5
FEATURES             Location/Qualifiers
     source          1..8268
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     intron          5..232
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      264..273
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             276..296
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             321..4238
                     /codon_start=1
                     /product="CRISPR-associated protein from Acidaminococcus 
                     sp. BV3L6 (Zetsche et al., 2015)"
                     /label=AsCpf1
                     /translation="TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDH
                     YKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNA
                     IHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDK
                     FTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFE
                     NVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLN
                     LAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLR
                     NENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGK
                     ITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLK
                     KQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKAR
                     NYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKA
                     LSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPL
                     EITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDL
                     SSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGH
                     HGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKD
                     QKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFH
                     VPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRS
                     LNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVV
                     VLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQF
                     TSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDV
                     KTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENH
                     RFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMR
                     NSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESK
                     DLKLQNGISNQDWLAYIQELRN"
     regulatory      3958..3967
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4239..4286
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4293..4373
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(4401..4418)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4407..4614
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4623..4763
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4623..4752
                     /label=AAV2 ITR
     rep_origin      4838..5293
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4925..4944)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5135..5156
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5310..5329)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5429..5451
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5489..5507)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5575..5679
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5680..5748,5749..6540)
                     /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(5898..5917)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6711..7299
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7200..7219
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7361..7490
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        7503..7751
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7503..7523
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7674..7693
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        7877..8162
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg
       61 cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc
      121 tcctccgggc tgtaattagc tgagcaagag gtaagggttt aagggatggt tggttggtgg
      181 ggtattaatg tttaattacc tggagcacct gcctgaaatc actttttttc aggttggacc
      241 ggctagcgtt taaacttaag cttgccacca tggccccaaa gaagaagcgg aaggtcggta
      301 tccacggagt cccagcagcc acacagttcg agggctttac caacctgtat caggtgagca
      361 agacactgcg gtttgagctg atcccacagg gcaagaccct gaagcacatc caggagcagg
      421 gcttcatcga ggaggacaag gcccgcaatg atcactacaa ggagctgaag cccatcatcg
      481 atcggatcta caagacctat gccgaccagt gcctgcagct ggtgcagctg gattgggaga
      541 acctgagcgc cgccatcgac tcctatagaa aggagaaaac cgaggagaca aggaacgccc
      601 tgatcgagga gcaggccaca tatcgcaatg ccatccacga ctacttcatc ggccggacag
      661 acaacctgac cgatgccatc aataagagac acgccgagat ctacaagggc ctgttcaagg
      721 ccgagctgtt taatggcaag gtgctgaagc agctgggcac cgtgaccaca accgagcacg
      781 agaacgccct gctgcggagc ttcgacaagt ttacaaccta cttctccggc ttttatgaga
      841 acaggaagaa cgtgttcagc gccgaggata tcagcacagc catcccacac cgcatcgtgc
      901 aggacaactt ccccaagttt aaggagaatt gtcacatctt cacacgcctg atcaccgccg
      961 tgcccagcct gcgggagcac tttgagaacg tgaagaaggc catcggcatc ttcgtgagca
     1021 cctccatcga ggaggtgttt tccttccctt tttataacca gctgctgaca cagacccaga
     1081 tcgacctgta taaccagctg ctgggaggaa tctctcggga ggcaggcacc gagaagatca
     1141 agggcctgaa cgaggtgctg aatctggcca tccagaagaa tgatgagaca gcccacatca
     1201 tcgcctccct gccacacaga ttcatccccc tgtttaagca gatcctgtcc gataggaaca
     1261 ccctgtcttt catcctggag gagtttaaga gcgacgagga agtgatccag tccttctgca
     1321 agtacaagac actgctgaga aacgagaacg tgctggagac agccgaggcc ctgtttaacg
     1381 agctgaacag catcgacctg acacacatct tcatcagcca caagaagctg gagacaatca
     1441 gcagcgccct gtgcgaccac tgggatacac tgaggaatgc cctgtatgag cggagaatct
     1501 ccgagctgac aggcaagatc accaagtctg ccaaggagaa ggtgcagcgc agcctgaagc
     1561 acgaggatat caacctgcag gagatcatct ctgccgcagg caaggagctg agcgaggcct
     1621 tcaagcagaa aaccagcgag atcctgtccc acgcacacgc cgccctggat cagccactgc
     1681 ctacaaccct gaagaagcag gaggagaagg agatcctgaa gtctcagctg gacagcctgc
     1741 tgggcctgta ccacctgctg gactggtttg ccgtggatga gtccaacgag gtggaccccg
     1801 agttctctgc ccggctgacc ggcatcaagc tggagatgga gccttctctg agcttctaca
     1861 acaaggccag aaattatgcc accaagaagc cctactccgt ggagaagttc aagctgaact
     1921 ttcagatgcc tacactggcc tctggctggg acgtgaataa ggagaagaac aatggcgcca
     1981 tcctgtttgt gaagaacggc ctgtactatc tgggcatcat gccaaagcag aagggcaggt
     2041 ataaggccct gagcttcgag cccacagaga aaaccagcga gggctttgat aagatgtact
     2101 atgactactt ccctgatgcc gccaagatga tcccaaagtg cagcacccag ctgaaggccg
     2161 tgacagccca ctttcagacc cacacaaccc ccatcctgct gtccaacaat ttcatcgagc
     2221 ctctggagat cacaaaggag atctacgacc tgaacaatcc tgagaaggag ccaaagaagt
     2281 ttcagacagc ctacgccaag aaaaccggcg accagaaggg ctacagagag gccctgtgca
     2341 agtggatcga cttcacaagg gattttctgt ccaagtatac caagacaacc tctatcgatc
     2401 tgtctagcct gcggccatcc tctcagtata aggacctggg cgagtactat gccgagctga
     2461 atcccctgct gtaccacatc agcttccaga gaatcgccga gaaggagatc atggatgccg
     2521 tggagacagg caagctgtac ctgttccaga tctataacaa ggactttgcc aagggccacc
     2581 acggcaagcc taatctgcac acactgtatt ggaccggcct gttttctcca gagaacctgg
     2641 ccaagacaag catcaagctg aatggccagg ccgagctgtt ctaccgccct aagtccagga
     2701 tgaagaggat ggcacaccgg ctgggagaga agatgctgaa caagaagctg aaggatcaga
     2761 aaaccccaat ccccgacacc ctgtaccagg agctgtacga ctatgtgaat cacagactgt
     2821 cccacgacct gtctgatgag gccagggccc tgctgcccaa cgtgatcacc aaggaggtgt
     2881 ctcacgagat catcaaggat aggcgcttta ccagcgacaa gttctttttc cacgtgccta
     2941 tcacactgaa ctatcaggcc gccaattccc catctaagtt caaccagagg gtgaatgcct
     3001 acctgaagga gcaccccgag acacctatca tcggcatcga tcggggcgag agaaacctga
     3061 tctatatcac agtgatcgac tccaccggca agatcctgga gcagcggagc ctgaacacca
     3121 tccagcagtt tgattaccag aagaagctgg acaacaggga gaaggagagg gtggcagcaa
     3181 ggcaggcctg gtctgtggtg ggcacaatca aggatctgaa gcagggctat ctgagccagg
     3241 tcatccacga gatcgtggac ctgatgatcc actaccaggc cgtggtggtg ctggagaacc
     3301 tgaatttcgg ctttaagagc aagaggaccg gcatcgccga gaaggccgtg taccagcagt
     3361 tcgagaagat gctgatcgat aagctgaatt gcctggtgct gaaggactat ccagcagaga
     3421 aagtgggagg cgtgctgaac ccataccagc tgacagacca gttcacctcc tttgccaaga
     3481 tgggcaccca gtctggcttc ctgttttacg tgcctgcccc atatacatct aagatcgatc
     3541 ccctgaccgg cttcgtggac cccttcgtgt ggaaaaccat caagaatcac gagagccgca
     3601 agcacttcct ggagggcttc gactttctgc actacgacgt gaaaaccggc gacttcatcc
     3661 tgcactttaa gatgaacaga aatctgtcct tccagagggg cctgcccggc tttatgcctg
     3721 catgggatat cgtgttcgag aagaacgaga cacagtttga cgccaagggc acccctttca
     3781 tcgccggcaa gagaatcgtg ccagtgatcg agaatcacag attcaccggc agataccggg
     3841 acctgtatcc tgccaacgag ctgatcgccc tgctggagga gaagggcatc gtgttcaggg
     3901 atggctccaa catcctgcca aagctgctgg agaatgacga ttctcacgcc atcgacacca
     3961 tggtggccct gatccgcagc gtgctgcaga tgcggaactc caatgccgcc acaggcgagg
     4021 actatatcaa cagccccgtg cgcgatctga atggcgtgtg cttcgactcc cggtttcaga
     4081 acccagagtg gcccatggac gccgatgcca atggcgccta ccacatcgcc ctgaagggcc
     4141 agctgctgct gaatcacctg aaggagagca aggatctgaa gctgcagaac ggcatctcca
     4201 atcaggactg gctggcctac atccaggagc tgcgcaacaa aaggccggcg gccacgaaaa
     4261 aggccggcca ggcaaaaaag aaaaagggat cctacccata cgatgttcca gattacgctt
     4321 atccctacga cgtgcctgat tatgcatacc catatgatgt ccccgactat gcctaagaat
     4381 tcctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     4441 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     4501 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     4561 ggggcaggac agcaaggggg aggattggga agagaatagc aggcatgctg gggagcggcc
     4621 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     4681 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     4741 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     4801 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     4861 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg
     4921 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     4981 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     5041 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     5101 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     5161 acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc
     5221 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     5281 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     5341 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     5401 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     5461 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     5521 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     5581 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     5641 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     5701 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     5761 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     5821 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     5881 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     5941 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     6001 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     6061 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     6121 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     6181 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     6241 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     6301 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     6361 tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg
     6421 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     6481 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     6541 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     6601 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     6661 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     6721 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     6781 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     6841 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     6901 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     6961 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     7021 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     7081 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     7141 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     7201 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     7261 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     7321 ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca gctgcgcgct
     7381 cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg cctcagtgag
     7441 cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct gcggcctcta
     7501 gagagggcct atttcccatg attccttcat atttgcatat atgatacaag gctgttagag
     7561 agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
     7621 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
     7681 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
     7741 gacgaaacac cgtaatttct actcttgtag attagtacac atcagatatc ttttttttct
     7801 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
     7861 gatatacgcg ggtacccgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
     7921 acgacccccg cccattgacg tcaatagtaa cgccaatagg gactttccat tgacgtcaat
     7981 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
     8041 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattgt gcccagtaca
     8101 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     8161 tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc tccccacccc
     8221 caattttgta tttatttatt ttttaattat tttgtgcagc gatggggg
//
