LOCUS       sequence-274412-        5600 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid of VP64-SadCas9 sgRNA 1, 2 and 3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5600)
  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 5600)
  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: mLama1 AAV sgRNA 1, 2, 5
FEATURES             Location/Qualifiers
     source          1..5600
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        154..533
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        534..737
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     688..708
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      757..766
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(763..765,766..768,769..1479)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(763..765,766..768,769..1479)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(808..829)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1069..1088)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1416..1437
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1533..1550)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1539..1746
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1754..2002
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        2122..2370
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        2491..2739
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     repeat_region   2863..3003
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2863..2992
                     /label=AAV2 ITR
     rep_origin      3078..3533
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3165..3184)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3375..3396
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3550..3569)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3669..3691
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3729..3747)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3815..3919
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3920..3988,3989..4780)
                     /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(4138..4157)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4951..5539
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5440..5459
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact agttattaat
      181 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
      241 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
      301 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      361 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      421 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
      481 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
      541 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
      601 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
      661 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
      721 tctatataag cagagctctc tggctaacta ccggtcgcca ccatggtgag caagggcgag
      781 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
      841 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
      901 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc
      961 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
     1021 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
     1081 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     1141 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
     1201 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc
     1261 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     1321 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc
     1381 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     1441 gccgccggga tcactctcgg catggacgag ctgtacaagt ccggactcag atctcgagct
     1501 caagcttcga attcctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
     1561 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     1621 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     1681 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagagaata gcaggcatgc
     1741 tggggaggta cctgagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     1801 ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa
     1861 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     1921 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     1981 cttgtggaaa ggacgaaaca ccgaagggcc tgcgcaccag gcgtttaagt actctgtgct
     2041 ggaaacagca cagaatctac ttaaacaagg caaaatgccg tgtttatctc gtcaacttgt
     2101 tggcgagatt tttttccgcg ggagggccta tttcccatga ttccttcata tttgcatata
     2161 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta
     2221 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
     2281 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
     2341 ttatatatct tgtggaaagg acgaaacacc gcggggcgcg gccaggcagg cgtttaagta
     2401 ctctgtgctg gaaacagcac agaatctact taaacaaggc aaaatgccgt gtttatctcg
     2461 tcaacttgtt ggcgagattt ttttccgcgg gagggcctat ttcccatgat tccttcatat
     2521 ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca
     2581 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt
     2641 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat
     2701 ttcttggctt tatatatctt gtggaaagga cgaaacaccg accccaactg gagtggaggg
     2761 tgtttaagta ctctgtgctg gaaacagcac agaatctact taaacaaggc aaaatgccgt
     2821 gtttatctcg tcaacttgtt ggcgagattt ttttgcggcc gcaggaaccc ctagtgatgg
     2881 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg
     2941 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc
     3001 aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     3061 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     3121 tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt
     3181 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3241 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     3301 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     3361 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg
     3421 ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct
     3481 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
     3541 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     3601 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     3661 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     3721 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     3781 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     3841 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     3901 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     3961 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     4021 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     4081 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     4141 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     4201 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     4261 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     4321 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     4381 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     4441 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     4501 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     4561 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     4621 agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     4681 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     4741 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     4801 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     4861 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     4921 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     4981 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     5041 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     5101 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     5161 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     5221 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     5281 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     5341 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     5401 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     5461 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     5521 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     5581 ctggcctttt gctcacatgt
//
