LOCUS       sequence-232488-        3136 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient transfection; Expressed SMN2-DN4 CasRx gRNA; Gateway 
            Donor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3136)
  AUTHORS   Du M, Jillette N, Zhu JJ, Li S, Cheng AW
  TITLE     CRISPR artificial splicing factors.
  JOURNAL   Nat Commun. 2020 Jun 12;11(1):2973. doi: 10.1038/s41467-020-16806-4.
  PUBMED    32532987
REFERENCE   2  (bases 1 to 3136)
  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: "2020-06-12"; volume: "11(1):2973. doi"; pages: " 
            10.1038/s41467-020-16806-4"
COMMENT     Sequence Label: pAC1816_pCR8-gSMN2-DN4
FEATURES             Location/Qualifiers
     source          1..3136
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        683..931
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     683..703
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     854..873
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        933..962
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     protein_bind    complement(1015..1114)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1131..1150)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1132..1150)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1155..1171)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1155..1171)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1602..2393
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      2486..3074
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2975..2994
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgatca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtattcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctcc gaattcaccg gtgagggcct atttcccatg attccttcat atttgcatat
      721 acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt
      781 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
      841 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
      901 tttatatatc ttgtggaaag gacgaaacac cgaaccccta ccaactggtc ggggtttgaa
      961 acgctggtct gcctactagt gatatttttt tggcgcgcct taattaagaa ttcgacccag
     1021 ctttcttgta caaagttggc attataaaaa ataattgctc atcaatttgt tgcaacgaac
     1081 aggtcactat cagtcaaaat aaaatcatta tttgccatcc agctgatatc ccctatagtg
     1141 agtcgtatta catggtcata gctgtttcct ggcagctctg gcccgtgtct caaaatctct
     1201 gatgttacat tgcacaagat aaaaatatat catcatgcct cctctagacc agccaggaca
     1261 gaaatgcctc gacttcgctg ctgcccaagg ttgccgggtg acgcacaccg tggaaacgga
     1321 tgaaggcacg aacccagtgg acataagcct gttcggttcg taagctgtaa tgcaagtagc
     1381 gtatgcgctc acgcaactgg tccagaacct tgaccgaacg cagcggtggt aacggcgcag
     1441 tggcggtttt catggcttgt tatgactgtt tttttggggt acagtctatg cctcgggcat
     1501 ccaagcagca agcgcgttac gccgtgggtc gatgtttgat gttatggagc agcaacgatg
     1561 ttacgcagca gggcagtcgc cctaaaacaa agttaaacat catgagggaa gcggtgatcg
     1621 ccgaagtatc gactcaacta tcagaggtag ttggcgtcat cgagcgccat ctcgaaccga
     1681 cgttgctggc cgtacatttg tacggctccg cagtggatgg cggcctgaag ccacacagtg
     1741 atattgattt gctggttacg gtgaccgtaa ggcttgatga aacaacgcgg cgagctttga
     1801 tcaacgacct tttggaaact tcggcttccc ctggagagag cgagattctc cgcgctgtag
     1861 aagtcaccat tgttgtgcac gacgacatca ttccgtggcg ttatccagct aagcgcgaac
     1921 tgcaatttgg agaatggcag cgcaatgaca ttcttgcagg tatcttcgag ccagccacga
     1981 tcgacattga tctggctatc ttgctgacaa aagcaagaga acatagcgtt gccttggtag
     2041 gtccagcggc ggaggaactc tttgatccgg ttcctgaaca ggatctattt gaggcgctaa
     2101 atgaaacctt aacgctatgg aactcgccgc ccgactgggc tggcgatgag cgaaatgtag
     2161 tgcttacgtt gtcccgcatt tggtacagcg cagtaaccgg caaaatcgcg ccgaaggatg
     2221 tcgctgccga ctgggcaatg gagcgcctgc cggcccagta tcagcccgtc atacttgaag
     2281 ctagacaggc ttatcttgga caagaagaag atcgcttggc ctcgcgcgca gatcagttgg
     2341 aagaatttgt ccactacgtg aaaggcgaga tcaccaaggt agtcggcaaa taaccctcga
     2401 gccacccatg accaaaatcc cttaacgtga gttacgcgtc gttccactga gcgtcagacc
     2461 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2521 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2581 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     2641 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     2701 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     2761 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     2821 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagcatt
     2881 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     2941 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     3001 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3061 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3121 cttttgctca catgtt
//
