LOCUS       sequence-274405-        7666 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3xFLAG 2xVP64 SadCas9 for single guide cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7666)
  AUTHORS   Kemaladewi DU, Bassi PS, Erwood S, Al-Basha D, Gawlik KI, Lindsay K,
            Hyatt E, Kember R, Place KM, Marks RM, Durbeej M, Prescott SA, 
            Ivakine EA, Cohn RD
  TITLE     A mutation-independent approach for muscular dystrophy via 
            upregulation of a modifier gene.
  JOURNAL   Nature. 2019 Aug;572(7767):125-130. doi: 10.1038/s41586-019-1430-x. 
            Epub 2019 Jul 24.
  PUBMED    31341277
REFERENCE   2  (bases 1 to 7666)
  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"; doi: 
            "10.1038/s41586-019-1430-x"; journalName: "Nature"; date: "2019-08";
            volume: "572"; issue: "7767"; pages: "125-130"
COMMENT     Sequence Label: 3XFLAG-VP64-SadCas9-NLS-VP64
FEATURES             Location/Qualifiers
     source          1..7666
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   154..283
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        307..686
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        687..890
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     841..861
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      909..918
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             918..983
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             999..1145
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDM"
     CDS             1152..1172
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4353..4400
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4407..4556
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     polyA_signal    4585..4706
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4622..4641)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4676..4695
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        4724..4972
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4724..4744
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4895..4914
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4993..5068
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   5082..5222
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5082..5211
                     /label=AAV2 ITR
     rep_origin      5297..5752
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5384..5403)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5594..5615
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5769..5788)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5888..5910
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5948..5966)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6034..6138
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6139..6207,6208..6999)
                     /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(6357..6376)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7170..7666,1..92)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     join(7659..7666,1..12)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
       61 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
      121 tcctggcctt ttgctggcct tttgctcaca tgtcctgcag gcagctgcgc gctcgctcgc
      181 tcactgaggc cgcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga
      241 gcgcgcagag agggagtggc caactccatc actaggggtt cctgcggcct ctagactcga
      301 ggcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
      361 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      421 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      481 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      541 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      601 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      661 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      721 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      781 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      841 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa
      901 ctaccggtgc caccatggac tacaaagacc atgacggtga ttataaagat catgacatcg
      961 attacaagga tgacgatgac aagcacggtc cgcgggctga cgcattggac gattttgatc
     1021 tggatatgct gggaagtgac gccctcgatg attttgacct tgacatgctt ggttcggatg
     1081 cccttgatga ctttgacctc gacatgctcg gcagtgacgc ccttgatgat ttcgacctgg
     1141 acatggttaa cccaaagaag aagcggaagg tcggtatcca cggagtccca gcagccaagc
     1201 ggaactacat cctgggcctg gccatcggca tcaccagcgt gggctacggc atcatcgact
     1261 acgagacacg ggacgtgatc gatgccggcg tgcggctgtt caaagaggcc aacgtggaaa
     1321 acaacgaggg caggcggagc aagagaggcg ccagaaggct gaagcggcgg aggcggcata
     1381 gaatccagag agtgaagaag ctgctgttcg actacaacct gctgaccgac cacagcgagc
     1441 tgagcggcat caacccctac gaggccagag tgaagggcct gagccagaag ctgagcgagg
     1501 aagagttctc tgccgccctg ctgcacctgg ccaagagaag aggcgtgcac aacgtgaacg
     1561 aggtggaaga ggacaccggc aacgagctgt ccaccaaaga gcagatcagc cggaacagca
     1621 aggccctgga agagaaatac gtggccgaac tgcagctgga acggctgaag aaagacggcg
     1681 aagtgcgggg cagcatcaac agattcaaga ccagcgacta cgtgaaagaa gccaaacagc
     1741 tgctgaaggt gcagaaggcc taccaccagc tggaccagag cttcatcgac acctacatcg
     1801 acctgctgga aacccggcgg acctactatg agggacctgg cgagggcagc cccttcggct
     1861 ggaaggacat caaagaatgg tacgagatgc tgatgggcca ctgcacctac ttccccgagg
     1921 aactgcggag cgtgaagtac gcctacaacg ccgacctgta caacgccctg aacgacctga
     1981 acaatctcgt gatcaccagg gacgagaacg agaagctgga atattacgag aagttccaga
     2041 tcatcgagaa cgtgttcaag cagaagaaga agcccaccct gaagcagatc gccaaagaaa
     2101 tcctcgtgaa cgaagaggat attaagggct acagagtgac cagcaccggc aagcccgagt
     2161 tcaccaacct gaaggtgtac cacgacatca aggacattac cgcccggaaa gagattattg
     2221 agaacgccga gctgctggat cagattgcca agatcctgac catctaccag agcagcgagg
     2281 acatccagga agaactgacc aatctgaact ccgagctgac ccaggaagag atcgagcaga
     2341 tctctaatct gaagggctat accggcaccc acaacctgag cctgaaggcc atcaacctga
     2401 tcctggacga gctgtggcac accaacgaca accagatcgc tatcttcaac cggctgaagc
     2461 tggtgcccaa gaaggtggac ctgtcccagc agaaagagat ccccaccacc ctggtggacg
     2521 acttcatcct gagccccgtc gtgaagagaa gcttcatcca gagcatcaaa gtgatcaacg
     2581 ccatcatcaa gaagtacggc ctgcccaacg acatcattat cgagctggcc cgcgagaaga
     2641 actccaagga cgcccagaaa atgatcaacg agatgcagaa gcggaaccgg cagaccaacg
     2701 agcggatcga ggaaatcatc cggaccaccg gcaaagagaa cgccaagtac ctgatcgaga
     2761 agatcaagct gcacgacatg caggaaggca agtgcctgta cagcctggaa gccatccctc
     2821 tggaagatct gctgaacaac cccttcaact atgaggtgga ccacatcatc cccagaagcg
     2881 tgtccttcga caacagcttc aacaacaagg tgctcgtgaa gcaggaagaa gccagcaaga
     2941 agggcaaccg gaccccattc cagtacctga gcagcagcga cagcaagatc agctacgaaa
     3001 ccttcaagaa gcacatcctg aatctggcca agggcaaggg cagaatcagc aagaccaaga
     3061 aagagtatct gctggaagaa cgggacatca acaggttctc cgtgcagaaa gacttcatca
     3121 accggaacct ggtggatacc agatacgcca ccagaggcct gatgaacctg ctgcggagct
     3181 acttcagagt gaacaacctg gacgtgaaag tgaagtccat caatggcggc ttcaccagct
     3241 ttctgcggcg gaagtggaag tttaagaaag agcggaacaa ggggtacaag caccacgccg
     3301 aggacgccct gatcattgcc aacgccgatt tcatcttcaa agagtggaag aaactggaca
     3361 aggccaaaaa agtgatggaa aaccagatgt tcgaggaaaa gcaggccgag agcatgcccg
     3421 agatcgaaac cgagcaggag tacaaagaga tcttcatcac cccccaccag atcaagcaca
     3481 ttaaggactt caaggactac aagtacagcc accgggtgga caagaagcct aatagagagc
     3541 tgattaacga caccctgtac tccacccgga aggacgacaa gggcaacacc ctgatcgtga
     3601 acaatctgaa cggcctgtac gacaaggaca atgacaagct gaaaaagctg atcaacaaga
     3661 gccccgaaaa gctgctgatg taccaccacg acccccagac ctaccagaaa ctgaagctga
     3721 ttatggaaca gtacggcgac gagaagaatc ccctgtacaa gtactacgag gaaaccggga
     3781 actacctgac caagtactcc aaaaaggaca acggccccgt gatcaagaag attaagtatt
     3841 acggcaacaa actgaacgcc catctggaca tcaccgacga ctaccccaac agcagaaaca
     3901 aggtcgtgaa gctgtccctg aagccctaca gattcgacgt gtacctggac aatggcgtgt
     3961 acaagttcgt gaccgtgaag aatctggatg tgatcaaaaa agaaaactac tacgaagtga
     4021 atagcaagtg ctatgaggaa gctaagaagc tgaagaagat cagcaaccag gccgagttta
     4081 tcgcctcctt ctacaacaac gatctgatca agatcaacgg cgagctgtat agagtgatcg
     4141 gcgtgaacaa cgacctgctg aaccggatcg aagtgaacat gatcgacatc acctaccgcg
     4201 agtacctgga aaacatgaac gacaagaggc cccccaggat cattaagaca atcgcctcca
     4261 agacccagag cattaagaag tacagcacag acattctggg caacctgtat gaagtgaaat
     4321 ctaagaagca ccctcagatc atcaaaaagg gcaaaaggcc ggcggccacg aaaaaggccg
     4381 gccaggcaaa aaagaaaaag ggatccgacg cgctggacga tttcgatctc gacatgctgg
     4441 gttctgatgc cctcgatgac tttgacctgg atatgttggg aagcgacgca ttggatgact
     4501 ttgatctgga catgctcggc tccgatgctc tggacgattt cgatctcgat atgttataag
     4561 aattcatcga taccgtcgag atctaacttg tttattgcag cttataatgg ttacaaataa
     4621 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     4681 ttgtccaaac tcatcaatgt atcttatcat gtctggaggt accgagggcc tatttcccat
     4741 gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt
     4801 gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg
     4861 gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg
     4921 aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccggagacca
     4981 cggcaggtct cagttttagt actctggaaa cagaatctac taaaacaagg caaaatgccg
     5041 tgtttatctc gtcaacttgt tggcgagatt tttgcggccg caggaacccc tagtgatgga
     5101 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc
     5161 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca
     5221 ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg
     5281 tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     5341 acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc
     5401 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     5461 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat
     5521 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     5581 acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc
     5641 tattcttttg atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg
     5701 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc
     5761 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     5821 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     5881 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     5941 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     6001 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     6061 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     6121 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     6181 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     6241 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     6301 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     6361 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     6421 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     6481 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     6541 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     6601 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     6661 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     6721 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     6781 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     6841 gccggtgagc gtggaagccg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     6901 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     6961 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     7021 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     7081 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     7141 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     7201 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     7261 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     7321 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     7381 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     7441 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     7501 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     7561 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     7621 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacg
//
