LOCUS       sequence-183602-        3046 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  DMD intron 43-targeting gRNA expression plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3046)
  AUTHORS   Chen X, Janssen JM, Liu J, Maggio I, 't Jong AEJ, Mikkers HMM, 
            Goncalves MAFV
  TITLE     In trans paired nicking triggers seamless genome editing without 
            double-stranded DNA cutting.
  JOURNAL   Nat Commun. 2017 Sep 22;8(1):657. doi: 10.1038/s41467-017-00687-1.
  PUBMED    28939824
REFERENCE   2  (bases 1 to 3046)
  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: "2017-09-22"; volume: "8(1):657. doi"; pages: " 
            10.1038/s41467-017-00687-1"
COMMENT     Sequence Label: AL08_pgRNA.DMD
FEATURES             Location/Qualifiers
     source          1..3046
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    106..127
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(142..147,148..165,166..172)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    180..196
                     /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)."
     primer_bind     185..207
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     204..220
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     204..220
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        292..540
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     292..312
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     463..482
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        561..636
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(650..667)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(650..666)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(659..681)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(875..894)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     994..1016
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1054..1072)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1140..1244
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1245..1313,1314..2105)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGARERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1463..1482)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2276..2864
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2765..2784
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3018..3035
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
       61 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
      121 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
      181 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttcc
      241 atgggatatc gcatgcctgc agagctctag agtccggccg cccccttcac cgagggccta
      301 tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga
      361 attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa
      421 tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc
      481 gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc
      541 gttacataca ggctagggag gttttagagc tagaaatagc aagttaaaat aaggctagtc
      601 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttgaattca ctggccgtcg
      661 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
      721 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
      781 agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt
      841 gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt
      901 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
      961 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     1021 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     1081 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     1141 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     1201 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     1261 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     1321 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     1381 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     1441 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     1501 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     1561 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     1621 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     1681 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     1741 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     1801 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     1861 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     1921 gctggtttat tgctgataaa tctggagccc gtgagcgtgg gtctcgcggt atcattgcag
     1981 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     2041 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     2101 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     2161 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     2221 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     2281 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     2341 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     2401 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     2461 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     2521 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     2581 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     2641 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     2701 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     2761 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2821 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     2881 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     2941 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     3001 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaaga
//
