LOCUS       sequence-267579-        8567 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Components for genome editing in mammalian cells with 
            pCAG-All-in-one-hCascade and pBS-U6-crRNA-targeted..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8567)
  AUTHORS   Morisaka H, Yoshimi K, Okuzaki Y, Gee P, Kunihiro Y, Sonpho E, Xu H,
            Sasakawa N, Naito Y, Nakada S, Yamamoto T, Sano S, Hotta A, Takeda 
            J, Mashimo T
  TITLE     CRISPR-Cas3 induces broad and unidirectional genome editing in human
            cells.
  JOURNAL   Nat Commun. 2019 Dec 6;10(1):5302. doi: 10.1038/s41467-019-13226-x.
  PUBMED    31811138
REFERENCE   2  (bases 1 to 8567)
  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 
            Commun."; date: "2019-12-6"; volume: "10(1):5302. doi"; pages: " 
            10.1038/s41467-019-13226-x"
COMMENT     Sequence Label: pPB-CAG-hCas3
FEATURES             Location/Qualifiers
     source          1..8567
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   674..708
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        1011..1390
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1392..1668
                     /label=chicken beta-actin promoter
     intron          1669..2685
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2609..2631
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2693..2712
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        2752..2770
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2778..2787
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2823..2843
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5541..5561
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5609..5626)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5615..5839
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5886..5902)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5910..5926
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5934..5940,5941..5958,5959..5964))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5979..6000
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   6266..6328
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6379..6399)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6379..6397)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6418..6434)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6442..6458
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(6466..6472,6473..6490,6491..6496))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6511..6532
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6649..6666)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6820..7408)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6900..6919)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7579..8370,8371..8439))
                     /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     8202..8221
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8440..8544)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggggcgcgc
      661 cattctagat taaccctaga aagatagtct gcgtaaaatt gacgcatgca ttcttgaaat
      721 attgctctct ctttctaaat agcgcgaatc cgtcgctgtg catttaggac atctcagtcg
      781 ccgcttggag ctcccgtgag gcgtgcttgt caatgcggta agtgtcactg attttgaact
      841 ataacgaccg cgtgagtcaa aatgacgcat gattatcttt tacgtgactt ttaagattta
      901 actcatacga taattatatt gttatttcat gttctactta cgtgataact tattatatat
      961 atattttctt gttatagata tcaactagaa tgctagcatg ggcccatctc gacattgatt
     1021 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
     1081 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     1141 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
     1201 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
     1261 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
     1321 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
     1381 tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc
     1441 cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg
     1501 gggggggggg gncgcgcgcc aggcggggcg gggcggggcg aggggcgggg cggggcgagg
     1561 cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg
     1621 aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg
     1681 cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga
     1741 ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat
     1801 tagcgcttgg tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag ccttgagggg
     1861 ctccgggagg gccctttgtg cggggggagc ggctcggggg gtgcgtgcgt gtgtgtgtgc
     1921 gtggggagcg ccgcgtgcgg ctccgcgctg cccggcggct gtgagcgctg cgggcgcggc
     1981 gcggggcttt gtgcgctccg cagtgtgcgc gaggggagcg cggccggggg cggtgccccg
     2041 cggtgcgggg ggggctgcga ggggaacaaa ggctgcgtgc ggggtgtgtg cgtggggggg
     2101 tgagcagggg gtgtgggcgc gtcggtcggg ctgcaacccc ccctgcaccc ccctccccga
     2161 gttgctgagc acggcccggc ttcgggtgcg gggctccgta cggggcgtgg cgcggggctc
     2221 gccgtgccgg gcggggggtg gcggcaggtg ggggtgccgg gcggggcggg gccgcctcgg
     2281 gccggggagg gctcggggga ggggcgcggc ggcccccgga gcgccggcgg ctgtcgaggc
     2341 gcggcgagcc gcagccattg ccttttatgg taatcgtgcg agagggcgca gggacttcct
     2401 ttgtcccaaa tctgtgcgga gccgaaatct gggaggcgcc gccgcacccc ctctagcggg
     2461 cgcggggcga agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg ccttcgtgcg
     2521 tcgccgcgcc gccgtcccct tctccctctc cagcctcggg gctgtccgcg gggggacggc
     2581 tgccttcggg ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac cggcggctct
     2641 agagcctctg ctaaccatgt tcatgccttc ttctttttcc tacagctcct gggcaacgtg
     2701 ctggttattg tgctgtctca tcattttggc aaagaattca gatctgaatt ctaatacgac
     2761 tcactatagg gagagccgcc accatggcca agagaacagc cgatggcagc gagttcgaga
     2821 gccccaagaa gaagcggaag gtcgaggaac ccttcaagta catctgccac tactggggca
     2881 agagcagcaa gagcctgacc aagggcaacg acatccatct gctgatctac cactgcctgg
     2941 atgtggccgc tgtggctgac tgttggtggg atcagtctgt ggtgctgcag aacaccttct
     3001 gccggaacga gatgctgagc aagcagagag tgaaggcctg gctgctgttc tttatcgccc
     3061 tgcacgacat cggcaagttc gacatccggt tccagtacaa gagcgccgag agctggctga
     3121 agctgaaccc tgctacaccc agcctgaacg gccctagcac acagatgtgc cggaagttca
     3181 atcacggcgc tgccggcctg tactggttca accaggatag cctgagcgag cagtccctgg
     3241 gcgacttctt cagcttcttc gacgccgctc ctcatcctta cgagtcctgg tttccttggg
     3301 tcgaagccgt gacaggccac cacggcttta ttctgcacag ccaggaccag gacaagagca
     3361 gatgggagat gcctgccagc ctggcctctt atgccgctca ggataagcag gcccgggaag
     3421 agtggatctc tgtgctggaa gccctgtttc tgacaccagc cggcctgagc atcaacgata
     3481 tccctccaga ttgcagcagc ctgctggccg gcttttgttc tctggccgat tggctcggca
     3541 gctggacaac caccaacacc tttctgttca acgaggacgc ccctagcgac atcaacgccc
     3601 tgagaaccta cttccaggac agacagcagg acgccagcag agtgctggaa ctgtctggcc
     3661 tggtgtccaa caagagatgc tacgaagggg tgcacgccct gctggacaat ggctatcagc
     3721 ctagacagct gcaggtcctg gtggatgctc tgcctgttgc tcctggcctg acagtgattg
     3781 aagcccctac aggcagcggc aagacagaaa cagccctggc ctatgcctgg aagctgatcg
     3841 accagcagat cgccgacagc gtgatcttcg ctctgccaac acaggccaca gccaacgcca
     3901 tgctgaccag aatggaagcc tctgccagcc acctgttcag cagccccaat ctgattctgg
     3961 cccacggcaa cagcagattc aatcacctgt tccagagcat caagtcccgg gccatcacag
     4021 agcagggaca agaggaagct tgggtgcagt gctgtcagtg gctgagccag agcaacaaga
     4081 aagtgttcct gggccagatc ggcgtgtgca ccattgacca ggtgctgatc tcagtgctgc
     4141 ccgtgaagca ccggttcatc agaggactcg gcatcggcag atccgtgctg atcgtggatg
     4201 aagtgcacgc ctacgacacc tacatgaacg gcctgcttga ggccgtgctg aaagcccagg
     4261 ctgatgttgg cggaagcgtg atcctgctga gcgccacact gcctatgaag cagaagcaga
     4321 aactgctgga tacctacggc ctgcacaccg atcctgtgga aaacaacagc gcctatcctc
     4381 tgatcaattg gagaggcgtg aacggcgccc agagattcga tctgcttgcc catcctgagc
     4441 agctgcctcc aagattcagc atccagcctg agcctatctg cctggccgac atgctgcctg
     4501 acctgaccat gctggaacgg atgatcgccg ctgctaatgc cggtgctcaa gtgtgcctga
     4561 tctgcaacct ggtggacgtg gcccaagtgt gctaccagag actgaaagag ctgaacaaca
     4621 cccaggtgga catcgacctg tttcacgccc ggttcaccct gaacgaccgg cgcgagaaag
     4681 aaaacagagt gatcagcaac ttcggcaaga acggcaagcg gaatgtgggc agaatcctgg
     4741 tggctacaca ggtggtggaa cagagcctgg acgtggactt cgactggctg atcacccagc
     4801 actgtcccgc cgacctgctg tttcaaagac tgggcagact gcaccggcac caccggaagt
     4861 atagacctgc cggctttgag atccccgtgg ccacaatttt gctgcccgat ggcgaaggct
     4921 acggcagaca cgagcacatc tacagcaacg tgcgagtgat gtggcggacc cagcagcaca
     4981 tcgaggaact gaatggcgcc agcctgttct tccctgatgc ctatcgacag tggctggaca
     5041 gcatctacga cgacgccgag atggatgagc ctgagtgggt cggaaacggc atggacaagt
     5101 ttgagtccgc cgagtgcgag aagcggttca aggccagaaa ggttctgcag tgggccgaag
     5161 agtacagcct gcaggacaac gacgagacaa tcctggccgt gaccagagat ggcgagatgt
     5221 ctctgcctct gctgccctac gtgcagacca gctctggaaa acagctgctg gacggccagg
     5281 tgtacgagga tctgtctcac gagcagcagt acgaggccct ggctctgaat agagtgaacg
     5341 tgcccttcac ctggaagcgg agctttagcg aagtggtgga cgaggatgga ctgctgtggc
     5401 tcgaagggaa gcagaatctg gacggctggg tctggcaggg caactccatc gtgatcacct
     5461 acacaggcga cgagggcatg acaagagtga tccccgccaa tcctaagaag aggaccgccg
     5521 acggctccga gttcgaatct cctaagaaaa aacgcaaggt cgagtgatat cctcgagatg
     5581 catcgatgat ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     5641 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     5701 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     5761 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     5821 gatgcggtgg gctctatggc ttctgaggcg gaaagaacct gcagcccaag cttggcgtaa
     5881 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     5941 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     6001 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gcggatccat
     6061 tcatgaatga attcatgtcg acatactagt taaaagtttt gttactttat agaagaaatt
     6121 ttgagttttt gttttttttt aataaataaa taaacataaa taaattgttt gttgaattta
     6181 ttattagtat gtaagtgtaa atataataaa acttaatatc tattcaaatt aataaataaa
     6241 cctcgatata cagaccgata aaacacatgc gtcaatttta cgcatgatta tctttaacgt
     6301 acgtcacaat atgattatct ttctagggtt aatctagtat acgcgtatgc ggccgcttaa
     6361 ttaatccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc
     6421 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     6481 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     6541 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     6601 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     6661 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     6721 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     6781 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     6841 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     6901 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     6961 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     7021 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     7081 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     7141 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     7201 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     7261 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     7321 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     7381 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     7441 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     7501 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     7561 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     7621 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     7681 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     7741 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     7801 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     7861 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     7921 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     7981 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     8041 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     8101 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     8161 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     8221 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     8281 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     8341 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     8401 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     8461 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     8521 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccac
//
