LOCUS       sequence-172841-        8291 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Vector for expression of a CRISPR/AsCpf1 array containing the ATP1A1
            G3 guide in combination with a user-specified guide. Cloning of 
            oligos for the second guide using BbsI sites. PY036-like plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 8291)
  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 8291)
  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_Array
FEATURES             Location/Qualifiers
     source          1..8291
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     regulatory      244..253
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             256..276
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             301..4218
                     /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      3938..3947
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4219..4266
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4273..4353
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(4381..4398)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4387..4594
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4603..4743
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4603..4732
                     /label=AAV2 ITR
     rep_origin      4818..5273
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4905..4924)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5115..5136
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5290..5309)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5409..5431
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5469..5487)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5555..5659
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5660..5728,5729..6520)
                     /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(5878..5897)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6691..7279
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7180..7199
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7341..7470
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        7483..7731
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7483..7503
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7654..7673
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        7897..8182
                     /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 ggctagcgtt taaacttaag
      241 cttgccacca tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc
      301 acacagttcg agggctttac caacctgtat caggtgagca agacactgcg gtttgagctg
      361 atcccacagg gcaagaccct gaagcacatc caggagcagg gcttcatcga ggaggacaag
      421 gcccgcaatg atcactacaa ggagctgaag cccatcatcg atcggatcta caagacctat
      481 gccgaccagt gcctgcagct ggtgcagctg gattgggaga acctgagcgc cgccatcgac
      541 tcctatagaa aggagaaaac cgaggagaca aggaacgccc tgatcgagga gcaggccaca
      601 tatcgcaatg ccatccacga ctacttcatc ggccggacag acaacctgac cgatgccatc
      661 aataagagac acgccgagat ctacaagggc ctgttcaagg ccgagctgtt taatggcaag
      721 gtgctgaagc agctgggcac cgtgaccaca accgagcacg agaacgccct gctgcggagc
      781 ttcgacaagt ttacaaccta cttctccggc ttttatgaga acaggaagaa cgtgttcagc
      841 gccgaggata tcagcacagc catcccacac cgcatcgtgc aggacaactt ccccaagttt
      901 aaggagaatt gtcacatctt cacacgcctg atcaccgccg tgcccagcct gcgggagcac
      961 tttgagaacg tgaagaaggc catcggcatc ttcgtgagca cctccatcga ggaggtgttt
     1021 tccttccctt tttataacca gctgctgaca cagacccaga tcgacctgta taaccagctg
     1081 ctgggaggaa tctctcggga ggcaggcacc gagaagatca agggcctgaa cgaggtgctg
     1141 aatctggcca tccagaagaa tgatgagaca gcccacatca tcgcctccct gccacacaga
     1201 ttcatccccc tgtttaagca gatcctgtcc gataggaaca ccctgtcttt catcctggag
     1261 gagtttaaga gcgacgagga agtgatccag tccttctgca agtacaagac actgctgaga
     1321 aacgagaacg tgctggagac agccgaggcc ctgtttaacg agctgaacag catcgacctg
     1381 acacacatct tcatcagcca caagaagctg gagacaatca gcagcgccct gtgcgaccac
     1441 tgggatacac tgaggaatgc cctgtatgag cggagaatct ccgagctgac aggcaagatc
     1501 accaagtctg ccaaggagaa ggtgcagcgc agcctgaagc acgaggatat caacctgcag
     1561 gagatcatct ctgccgcagg caaggagctg agcgaggcct tcaagcagaa aaccagcgag
     1621 atcctgtccc acgcacacgc cgccctggat cagccactgc ctacaaccct gaagaagcag
     1681 gaggagaagg agatcctgaa gtctcagctg gacagcctgc tgggcctgta ccacctgctg
     1741 gactggtttg ccgtggatga gtccaacgag gtggaccccg agttctctgc ccggctgacc
     1801 ggcatcaagc tggagatgga gccttctctg agcttctaca acaaggccag aaattatgcc
     1861 accaagaagc cctactccgt ggagaagttc aagctgaact ttcagatgcc tacactggcc
     1921 tctggctggg acgtgaataa ggagaagaac aatggcgcca tcctgtttgt gaagaacggc
     1981 ctgtactatc tgggcatcat gccaaagcag aagggcaggt ataaggccct gagcttcgag
     2041 cccacagaga aaaccagcga gggctttgat aagatgtact atgactactt ccctgatgcc
     2101 gccaagatga tcccaaagtg cagcacccag ctgaaggccg tgacagccca ctttcagacc
     2161 cacacaaccc ccatcctgct gtccaacaat ttcatcgagc ctctggagat cacaaaggag
     2221 atctacgacc tgaacaatcc tgagaaggag ccaaagaagt ttcagacagc ctacgccaag
     2281 aaaaccggcg accagaaggg ctacagagag gccctgtgca agtggatcga cttcacaagg
     2341 gattttctgt ccaagtatac caagacaacc tctatcgatc tgtctagcct gcggccatcc
     2401 tctcagtata aggacctggg cgagtactat gccgagctga atcccctgct gtaccacatc
     2461 agcttccaga gaatcgccga gaaggagatc atggatgccg tggagacagg caagctgtac
     2521 ctgttccaga tctataacaa ggactttgcc aagggccacc acggcaagcc taatctgcac
     2581 acactgtatt ggaccggcct gttttctcca gagaacctgg ccaagacaag catcaagctg
     2641 aatggccagg ccgagctgtt ctaccgccct aagtccagga tgaagaggat ggcacaccgg
     2701 ctgggagaga agatgctgaa caagaagctg aaggatcaga aaaccccaat ccccgacacc
     2761 ctgtaccagg agctgtacga ctatgtgaat cacagactgt cccacgacct gtctgatgag
     2821 gccagggccc tgctgcccaa cgtgatcacc aaggaggtgt ctcacgagat catcaaggat
     2881 aggcgcttta ccagcgacaa gttctttttc cacgtgccta tcacactgaa ctatcaggcc
     2941 gccaattccc catctaagtt caaccagagg gtgaatgcct acctgaagga gcaccccgag
     3001 acacctatca tcggcatcga tcggggcgag agaaacctga tctatatcac agtgatcgac
     3061 tccaccggca agatcctgga gcagcggagc ctgaacacca tccagcagtt tgattaccag
     3121 aagaagctgg acaacaggga gaaggagagg gtggcagcaa ggcaggcctg gtctgtggtg
     3181 ggcacaatca aggatctgaa gcagggctat ctgagccagg tcatccacga gatcgtggac
     3241 ctgatgatcc actaccaggc cgtggtggtg ctggagaacc tgaatttcgg ctttaagagc
     3301 aagaggaccg gcatcgccga gaaggccgtg taccagcagt tcgagaagat gctgatcgat
     3361 aagctgaatt gcctggtgct gaaggactat ccagcagaga aagtgggagg cgtgctgaac
     3421 ccataccagc tgacagacca gttcacctcc tttgccaaga tgggcaccca gtctggcttc
     3481 ctgttttacg tgcctgcccc atatacatct aagatcgatc ccctgaccgg cttcgtggac
     3541 cccttcgtgt ggaaaaccat caagaatcac gagagccgca agcacttcct ggagggcttc
     3601 gactttctgc actacgacgt gaaaaccggc gacttcatcc tgcactttaa gatgaacaga
     3661 aatctgtcct tccagagggg cctgcccggc tttatgcctg catgggatat cgtgttcgag
     3721 aagaacgaga cacagtttga cgccaagggc acccctttca tcgccggcaa gagaatcgtg
     3781 ccagtgatcg agaatcacag attcaccggc agataccggg acctgtatcc tgccaacgag
     3841 ctgatcgccc tgctggagga gaagggcatc gtgttcaggg atggctccaa catcctgcca
     3901 aagctgctgg agaatgacga ttctcacgcc atcgacacca tggtggccct gatccgcagc
     3961 gtgctgcaga tgcggaactc caatgccgcc acaggcgagg actatatcaa cagccccgtg
     4021 cgcgatctga atggcgtgtg cttcgactcc cggtttcaga acccagagtg gcccatggac
     4081 gccgatgcca atggcgccta ccacatcgcc ctgaagggcc agctgctgct gaatcacctg
     4141 aaggagagca aggatctgaa gctgcagaac ggcatctcca atcaggactg gctggcctac
     4201 atccaggagc tgcgcaacaa aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag
     4261 aaaaagggat cctacccata cgatgttcca gattacgctt atccctacga cgtgcctgat
     4321 tatgcatacc catatgatgt ccccgactat gcctaagaat tcctagagct cgctgatcag
     4381 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     4441 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     4501 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     4561 aggattggga agagaatagc aggcatgctg gggagcggcc gcaggaaccc ctagtgatgg
     4621 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg
     4681 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc
     4741 aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     4801 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     4861 tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt
     4921 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     4981 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     5041 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     5101 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg
     5161 ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct
     5221 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
     5281 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     5341 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     5401 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     5461 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     5521 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     5581 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     5641 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     5701 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     5761 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     5821 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     5881 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     5941 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     6001 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     6061 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     6121 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     6181 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     6241 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     6301 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     6361 agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     6421 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     6481 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     6541 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     6601 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     6661 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     6721 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     6781 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     6841 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     6901 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     6961 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     7021 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     7081 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     7141 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     7201 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     7261 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     7321 ctggcctttt gctcacatgt cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc
     7381 ccgggcgtcg ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg
     7441 gagtggccaa ctccatcact aggggttcct gcggcctcta gagagggcct atttcccatg
     7501 attccttcat atttgcatat acgatacaag gctgttagag agataattgg aattaatttg
     7561 actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg
     7621 tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga
     7681 aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac cgtaatttct
     7741 actcttgtag atgggtcttc gagaagacct aatttctact cttgtagata ggttcctctt
     7801 ctgtagcagc ttttttttca attgcatgaa gaatctgctt agggttaggc gttttgcgct
     7861 gcttcgcgat gtacgggcca gatatacgcg ggtacccgtt acataactta cggtaaatgg
     7921 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaatagtaa cgccaatagg
     7981 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     8041 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     8101 ctggcattgt gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     8161 attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat
     8221 ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc
     8281 gatgggggcg g
//
