LOCUS       sequence-319794-        9021 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one vector for CRISPR/Cas9-mediated homology-independent 
            knock-in system. The plasmid contains BPNLS-HiFi Cas9-BPNLS and two 
            sgRNA expression cassettes..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9021)
  AUTHORS   Fujisawa S, Qiu H, Nozaki S, Chiba S, Katoh Y, Nakayama K
  TITLE     ARL3 and ARL13B GTPases participate in distinct steps of INPP5E 
            targeting to the ciliary membrane.
  JOURNAL   Biol Open. 2021 Aug 27. pii: 271969. doi: 10.1242/bio.058843.
  PUBMED    34447983
REFERENCE   2  (bases 1 to 9021)
  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: "Biol Open. 
            2021 Aug 27. pii: 271969. doi: 10.1242/bio.058843."
COMMENT     Sequence Label: pHiFi Cas9-2×sgRNA (empty, donor)
FEATURES             Location/Qualifiers
     source          1..9021
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        434..682
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        703..778
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        869..1154
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        1156..1433
                     /label=chicken beta-actin promoter
     intron          1434..1661
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1715..1735
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      1769..1778
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1775..3493
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     CDS             5924..5944
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5951..6016
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(6062..6079)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6068..6275
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6284..6424
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6284..6413
                     /label=AAV2 ITR
     rep_origin      6499..6954
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6586..6605)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6796..6817
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6971..6990)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7090..7112
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7150..7168)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7236..7340
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7341..7409,7410..8201)
                     /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(7559..7578)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8372..8960
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8861..8880
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agagagggcc tatttcccat gattccttca tatttgcata tacgatacaa
      481 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa
      541 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
      601 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
      661 cttgtggaaa ggacgaaaca ccgcatcgta cgcgtacgtg ttgttttaga gctagaaata
      721 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
      781 ttttgtttta gagctagaaa tagcaagtta aaataaggct agtccgtttt tagcgcgtgc
      841 gccaattctg cagacaaatg gcggtacccg ttacataact tacggtaaat ggcccgcctg
      901 gctgaccgcc caacgacccc cgcccattga cgtcaatagt aacgccaata gggactttcc
      961 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     1021 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
     1081 gtgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
     1141 tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc
     1201 cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg
     1261 cggggggggg gggggggsgc gcgccrgggg gggggggggg gggggggggg gggggsgggg
     1321 sgrggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta
     1381 tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg
     1441 ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc
     1501 tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct
     1561 gtaattagct gagcaagagg taagggttta agggatggtt ggttggtggg gtattaatgt
     1621 ttaattacct ggagcacctg cctgaaatca ctttttttca ggttggaccg gtgccaccat
     1681 gaagcggact gctgatggca gtgaatttga gtccccaaag aagaagagaa aggtggaagg
     1741 tatccacgga gtcccagcag ccgaattcgc caccatggac aagaagtatt ctatcggact
     1801 ggacatcggg actaatagcg tcgggtgggc cgtgatcact gacgagtaca aggtgccctc
     1861 taagaagttc aaggtgctcg ggaacaccga ccggcattcc atcaagaaaa atctgatcgg
     1921 agctctcctc tttgattcag gggagaccgc tgaagcaacc cgcctcaagc ggactgctag
     1981 acggcggtac accaggagga agaaccggat ttgttacctt caagagatat tctccaacga
     2041 aatggcaaag gtcgacgaca gcttcttcca taggctggaa gaatcattcc tcgtggaaga
     2101 ggataagaag catgaacggc atcccatctt cggtaatatc gtcgacgagg tggcctatca
     2161 cgagaaatac ccaaccatct accatcttcg caaaaagctg gtggactcaa ccgacaaggc
     2221 agacctccgg cttatctacc tggccctggc ccacatgatc aagttcagag gccacttcct
     2281 gatcgagggc gacctcaatc ctgacaatag cgatgtggat aaactgttca tccagctggt
     2341 gcagacttac aaccagctct ttgaagagaa ccccatcaat gcaagcggag tcgatgccaa
     2401 ggccattctg tcagcccggc tgtcaaagag ccgcagactt gagaatctta tcgctcagct
     2461 gccgggtgaa aagaaaaatg gactgttcgg gaacctgatt gctctttcac ttgggctgac
     2521 tcccaatttc aagtctaatt tcgacctggc agaggatgcc aagctgcaac tgtccaagga
     2581 cacctatgat gacgatctcg acaacctcct ggcccagatc ggtgaccaat acgccgacct
     2641 tttccttgct gctaagaatc tttctgacgc catcctgctg tctgacattc tccgcgtgaa
     2701 cactgaaatc accaaggccc ctctttcagc ttcaatgatt aagcggtatg atgagcacca
     2761 ccaggacctg accctgctta aggcactcgt ccggcagcag cttccggaga agtacaagga
     2821 aatcttcttt gaccagtcaa agaatggata cgccggctac atcgacggag gtgcctccca
     2881 agaggaattt tataagttta tcaaacctat ccttgagaag atggacggca ccgaagagct
     2941 cctcgtgaaa ctgaatcggg aggatctgct gcggaagcag cgcactttcg acaatgggag
     3001 cattccccac cagatccatc ttggggagct tcacgccatc cttcggcgcc aagaggactt
     3061 ctaccccttt cttaaggaca acagggagaa gattgagaaa attctcactt tccgcatccc
     3121 ctactacgtg ggacccctcg ccagaggaaa tagccggttt gcttggatga ccagaaagtc
     3181 agaagaaact atcactccct ggaacttcga agaggtggtg gacaagggag ccagcgctca
     3241 gtcattcatc gaacggatga ctaacttcga taagaacctc cccaatgaga aggtcctgcc
     3301 gaaacattcc ctgctctacg agtactttac cgtgtacaac gagctgacca aggtgaaata
     3361 tgtcaccgaa gggatgagga agcccgcatt cctgtcaggc gaacaaaaga aggcaattgt
     3421 ggaccttctg ttcaagacca atagaaaggt gaccgtgaag cagctgaagg aggactattt
     3481 caagaaaatt gaatgcttcg actctgtgga gattagcggg gtcgaagatc ggttcaacgc
     3541 aagcctgggt acctaccatg atctgcttaa gatcatcaag gacaaggatt ttctggacaa
     3601 tgaggagaac gaggacatcc ttgaggacat tgtcctgact ctcactctgt tcgaggaccg
     3661 ggaaatgatc gaggagaggc ttaagaccta cgcccatctg ttcgacgata aagtgatgaa
     3721 gcaacttaaa cggagaagat ataccggatg gggacgcctt agccgcaaac tcatcaacgg
     3781 aatccgggac aaacagagcg gaaagaccat tcttgatttc cttaagagcg acggattcgc
     3841 taatgccaac ttcatgcaac ttatccatga tgattccctg acctttaagg aggacatcca
     3901 gaaggcccaa gtgtctggac aaggtgactc actgcacgag catatcgcaa atctggctgg
     3961 ttcacccgct attaagaagg gtattctcca gaccgtgaaa gtcgtggacg agctggtcaa
     4021 ggtgatgggt cgccataaac cagagaacat tgtcatcgag atggccaggg aaaaccagac
     4081 tacccagaag ggacagaaga acagcaggga gcggatgaaa agaattgagg aagggattaa
     4141 ggagctcggg tcacagatcc ttaaagagca cccggtggaa aacacccagc ttcagaatga
     4201 gaagctctat ctgtactacc ttcaaaatgg acgcgatatg tatgtggacc aagagcttga
     4261 tatcaacagg ctctcagact acgacgtgga ccacatcgtc cctcagagct tcctcaaaga
     4321 cgactcaatt gacaataagg tgctgactcg ctcagacaag aaccggggaa agtcagataa
     4381 cgtgccctca gaggaagtcg tgaaaaagat gaagaactat tggcgccagc ttctgaacgc
     4441 aaagctgatc actcagcgga agttcgacaa tctcactaag gctgagaggg gcggactgag
     4501 cgaactggac aaagcaggat tcattaaacg gcaacttgtg gagactcggc agattactaa
     4561 acatgtcgcc caaatccttg actcacgcat gaataccaag tacgacgaaa acgacaaact
     4621 tatccgcgag gtgaaggtga ttaccctgaa gtccaagctg gtcagcgatt tcagaaagga
     4681 ctttcaattc tacaaagtgc gggagatcaa taactatcat catgctcatg acgcatatct
     4741 gaatgccgtg gtgggaaccg ccctgatcaa gaagtaccca aagctggaaa gcgagttcgt
     4801 gtacggagac tacaaggtct acgacgtgcg caagatgatt gccaaatctg agcaggagat
     4861 cggaaaggcc accgcaaagt acttcttcta cagcaacatc atgaatttct tcaagaccga
     4921 aatcaccctt gcaaacggtg agatccggaa gaggccgctc atcgagacta atggggagac
     4981 tggcgaaatc gtgtgggaca agggcagaga tttcgctacc gtgcgcaaag tgctttctat
     5041 gcctcaagtg aacatcgtga agaaaaccga ggtgcaaacc ggaggctttt ctaaggaatc
     5101 aatcctcccc aagcgcaact ccgacaagct cattgcaagg aagaaggatt gggaccctaa
     5161 gaagtacggc ggattcgatt caccaactgt ggcttattct gtcctggtcg tggctaaggt
     5221 ggaaaaagga aagtctaaga agctcaagag cgtgaaggaa ctgctgggta tcaccattat
     5281 ggagcgcagc tccttcgaga agaacccaat tgactttctc gaagccaaag gttacaagga
     5341 agtcaagaag gaccttatca tcaagctccc aaagtatagc ctgttcgaac tggagaatgg
     5401 gcggaagcgg atgctcgcct ccgctggcga acttcagaag ggtaatgagc tggctctccc
     5461 ctccaagtac gtgaatttcc tctaccttgc aagccattac gagaagctga aggggagccc
     5521 cgaggacaac gagcaaaagc aactgtttgt ggagcagcat aagcattatc tggacgagat
     5581 cattgagcag atttccgagt tttctaaacg cgtcattctc gctgatgcca acctcgataa
     5641 agtccttagc gcatacaata agcacagaga caaaccaatt cgggagcagg ctgagaatat
     5701 catccacctg ttcaccctca ccaatcttgg tgcccctgcc gcattcaagt acttcgacac
     5761 caccatcgac cggaaacgct atacctccac caaagaagtg ctggacgcca ccctcatcca
     5821 ccagagcatc accggacttt acgaaactcg gattgacctc tcacagctcg gaggggatga
     5881 gggagctgat aagcgcaccg ccgatggttc cgagttcgaa agccccaaaa aaaagcgcaa
     5941 ggtcgaagat gactataagg atcacgacgg tgattataaa gaccacgata tcgactataa
     6001 agacgacgat gacaaagagc tgtataaact gctgtaagaa ttcctagagc tcgctgatca
     6061 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     6121 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     6181 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     6241 gaggattggg aagagaatag caggcatgct ggggagcggc cgcaggaacc cctagtgatg
     6301 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
     6361 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg
     6421 caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     6481 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     6541 ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct
     6601 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     6661 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     6721 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     6781 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg
     6841 gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc
     6901 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     6961 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     7021 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     7081 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     7141 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     7201 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     7261 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     7321 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     7381 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     7441 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     7501 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     7561 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     7621 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     7681 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     7741 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     7801 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     7861 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     7921 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     7981 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     8041 gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct
     8101 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     8161 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     8221 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     8281 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     8341 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     8401 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     8461 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     8521 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     8581 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     8641 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     8701 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     8761 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     8821 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     8881 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     8941 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     9001 gctggccttt tgctcacatg t
//
