LOCUS       sequence-171416-        8263 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses the ATP1A1 G3 crRNA in combination with AsCpf1-3xHA to 
            target ATP1A1 exon 4. U6-crRNA(ATP1A1 G3)-CBh-AsCpf1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 8263)
  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 8263)
  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_G3
FEATURES             Location/Qualifiers
     source          1..8263
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     intron          6..233
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      265..274
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             277..297
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             322..4239
                     /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      3959..3968
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4240..4287
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4294..4374
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(4402..4419)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4408..4615
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4624..4764
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4624..4753
                     /label=AAV2 ITR
     rep_origin      4839..5294
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4926..4945)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5136..5157
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5311..5330)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5430..5452
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5490..5508)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5576..5680
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5681..5749,5750..6541)
                     /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(5899..5918)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6712..7300
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7201..7220
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7362..7491
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        7504..7752
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7504..7524
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7675..7694
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        7878..8163
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc
       61 gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt
      121 ctcctccggg ctgtaattag ctgagcaaga ggtaagggtt taagggatgg ttggttggtg
      181 gggtattaat gtttaattac ctggagcacc tgcctgaaat cacttttttt caggttggac
      241 cggctagcgt ttaaacttaa gcttgccacc atggccccaa agaagaagcg gaaggtcggt
      301 atccacggag tcccagcagc cacacagttc gagggcttta ccaacctgta tcaggtgagc
      361 aagacactgc ggtttgagct gatcccacag ggcaagaccc tgaagcacat ccaggagcag
      421 ggcttcatcg aggaggacaa ggcccgcaat gatcactaca aggagctgaa gcccatcatc
      481 gatcggatct acaagaccta tgccgaccag tgcctgcagc tggtgcagct ggattgggag
      541 aacctgagcg ccgccatcga ctcctataga aaggagaaaa ccgaggagac aaggaacgcc
      601 ctgatcgagg agcaggccac atatcgcaat gccatccacg actacttcat cggccggaca
      661 gacaacctga ccgatgccat caataagaga cacgccgaga tctacaaggg cctgttcaag
      721 gccgagctgt ttaatggcaa ggtgctgaag cagctgggca ccgtgaccac aaccgagcac
      781 gagaacgccc tgctgcggag cttcgacaag tttacaacct acttctccgg cttttatgag
      841 aacaggaaga acgtgttcag cgccgaggat atcagcacag ccatcccaca ccgcatcgtg
      901 caggacaact tccccaagtt taaggagaat tgtcacatct tcacacgcct gatcaccgcc
      961 gtgcccagcc tgcgggagca ctttgagaac gtgaagaagg ccatcggcat cttcgtgagc
     1021 acctccatcg aggaggtgtt ttccttccct ttttataacc agctgctgac acagacccag
     1081 atcgacctgt ataaccagct gctgggagga atctctcggg aggcaggcac cgagaagatc
     1141 aagggcctga acgaggtgct gaatctggcc atccagaaga atgatgagac agcccacatc
     1201 atcgcctccc tgccacacag attcatcccc ctgtttaagc agatcctgtc cgataggaac
     1261 accctgtctt tcatcctgga ggagtttaag agcgacgagg aagtgatcca gtccttctgc
     1321 aagtacaaga cactgctgag aaacgagaac gtgctggaga cagccgaggc cctgtttaac
     1381 gagctgaaca gcatcgacct gacacacatc ttcatcagcc acaagaagct ggagacaatc
     1441 agcagcgccc tgtgcgacca ctgggataca ctgaggaatg ccctgtatga gcggagaatc
     1501 tccgagctga caggcaagat caccaagtct gccaaggaga aggtgcagcg cagcctgaag
     1561 cacgaggata tcaacctgca ggagatcatc tctgccgcag gcaaggagct gagcgaggcc
     1621 ttcaagcaga aaaccagcga gatcctgtcc cacgcacacg ccgccctgga tcagccactg
     1681 cctacaaccc tgaagaagca ggaggagaag gagatcctga agtctcagct ggacagcctg
     1741 ctgggcctgt accacctgct ggactggttt gccgtggatg agtccaacga ggtggacccc
     1801 gagttctctg cccggctgac cggcatcaag ctggagatgg agccttctct gagcttctac
     1861 aacaaggcca gaaattatgc caccaagaag ccctactccg tggagaagtt caagctgaac
     1921 tttcagatgc ctacactggc ctctggctgg gacgtgaata aggagaagaa caatggcgcc
     1981 atcctgtttg tgaagaacgg cctgtactat ctgggcatca tgccaaagca gaagggcagg
     2041 tataaggccc tgagcttcga gcccacagag aaaaccagcg agggctttga taagatgtac
     2101 tatgactact tccctgatgc cgccaagatg atcccaaagt gcagcaccca gctgaaggcc
     2161 gtgacagccc actttcagac ccacacaacc cccatcctgc tgtccaacaa tttcatcgag
     2221 cctctggaga tcacaaagga gatctacgac ctgaacaatc ctgagaagga gccaaagaag
     2281 tttcagacag cctacgccaa gaaaaccggc gaccagaagg gctacagaga ggccctgtgc
     2341 aagtggatcg acttcacaag ggattttctg tccaagtata ccaagacaac ctctatcgat
     2401 ctgtctagcc tgcggccatc ctctcagtat aaggacctgg gcgagtacta tgccgagctg
     2461 aatcccctgc tgtaccacat cagcttccag agaatcgccg agaaggagat catggatgcc
     2521 gtggagacag gcaagctgta cctgttccag atctataaca aggactttgc caagggccac
     2581 cacggcaagc ctaatctgca cacactgtat tggaccggcc tgttttctcc agagaacctg
     2641 gccaagacaa gcatcaagct gaatggccag gccgagctgt tctaccgccc taagtccagg
     2701 atgaagagga tggcacaccg gctgggagag aagatgctga acaagaagct gaaggatcag
     2761 aaaaccccaa tccccgacac cctgtaccag gagctgtacg actatgtgaa tcacagactg
     2821 tcccacgacc tgtctgatga ggccagggcc ctgctgccca acgtgatcac caaggaggtg
     2881 tctcacgaga tcatcaagga taggcgcttt accagcgaca agttcttttt ccacgtgcct
     2941 atcacactga actatcaggc cgccaattcc ccatctaagt tcaaccagag ggtgaatgcc
     3001 tacctgaagg agcaccccga gacacctatc atcggcatcg atcggggcga gagaaacctg
     3061 atctatatca cagtgatcga ctccaccggc aagatcctgg agcagcggag cctgaacacc
     3121 atccagcagt ttgattacca gaagaagctg gacaacaggg agaaggagag ggtggcagca
     3181 aggcaggcct ggtctgtggt gggcacaatc aaggatctga agcagggcta tctgagccag
     3241 gtcatccacg agatcgtgga cctgatgatc cactaccagg ccgtggtggt gctggagaac
     3301 ctgaatttcg gctttaagag caagaggacc ggcatcgccg agaaggccgt gtaccagcag
     3361 ttcgagaaga tgctgatcga taagctgaat tgcctggtgc tgaaggacta tccagcagag
     3421 aaagtgggag gcgtgctgaa cccataccag ctgacagacc agttcacctc ctttgccaag
     3481 atgggcaccc agtctggctt cctgttttac gtgcctgccc catatacatc taagatcgat
     3541 cccctgaccg gcttcgtgga ccccttcgtg tggaaaacca tcaagaatca cgagagccgc
     3601 aagcacttcc tggagggctt cgactttctg cactacgacg tgaaaaccgg cgacttcatc
     3661 ctgcacttta agatgaacag aaatctgtcc ttccagaggg gcctgcccgg ctttatgcct
     3721 gcatgggata tcgtgttcga gaagaacgag acacagtttg acgccaaggg cacccctttc
     3781 atcgccggca agagaatcgt gccagtgatc gagaatcaca gattcaccgg cagataccgg
     3841 gacctgtatc ctgccaacga gctgatcgcc ctgctggagg agaagggcat cgtgttcagg
     3901 gatggctcca acatcctgcc aaagctgctg gagaatgacg attctcacgc catcgacacc
     3961 atggtggccc tgatccgcag cgtgctgcag atgcggaact ccaatgccgc cacaggcgag
     4021 gactatatca acagccccgt gcgcgatctg aatggcgtgt gcttcgactc ccggtttcag
     4081 aacccagagt ggcccatgga cgccgatgcc aatggcgcct accacatcgc cctgaagggc
     4141 cagctgctgc tgaatcacct gaaggagagc aaggatctga agctgcagaa cggcatctcc
     4201 aatcaggact ggctggccta catccaggag ctgcgcaaca aaaggccggc ggccacgaaa
     4261 aaggccggcc aggcaaaaaa gaaaaaggga tcctacccat acgatgttcc agattacgct
     4321 tatccctacg acgtgcctga ttatgcatac ccatatgatg tccccgacta tgcctaagaa
     4381 ttcctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     4441 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     4501 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     4561 tggggcagga cagcaagggg gaggattggg aagagaatag caggcatgct ggggagcggc
     4621 cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg
     4681 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg
     4741 agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt
     4801 gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt
     4861 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc
     4921 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     4981 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     5041 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     5101 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     5161 aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt
     5221 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     5281 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     5341 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     5401 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     5461 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     5521 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     5581 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     5641 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     5701 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     5761 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     5821 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     5881 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     5941 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     6001 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     6061 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     6121 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     6181 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     6241 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     6301 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     6361 gtttattgct gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact
     6421 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     6481 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     6541 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     6601 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     6661 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     6721 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     6781 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     6841 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     6901 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     6961 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     7021 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     7081 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     7141 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     7201 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     7261 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     7321 tttacggttc ctggcctttt gctggccttt tgctcacatg tcctgcaggc agctgcgcgc
     7381 tcgctcgctc actgaggccg cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga
     7441 gcgagcgagc gcgcagagag ggagtggcca actccatcac taggggttcc tgcggcctct
     7501 agagagggcc tatttcccat gattccttca tatttgcata tatgatacaa ggctgttaga
     7561 gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt
     7621 agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat
     7681 catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa
     7741 ggacgaaaca ccgtaatttc tactcttgta gataggttcc tcttctgtag cagttttttc
     7801 tattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
     7861 agatatacgc gggtacccgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
     7921 aacgaccccc gcccattgac gtcaatagta acgccaatag ggactttcca ttgacgtcaa
     7981 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
     8041 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcattg tgcccagtac
     8101 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
     8161 atggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc
     8221 ccaattttgt atttatttat tttttaatta ttttgtgcag cga
//
