LOCUS       sequence-232550-        3310 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient transfection; Expressed DR-SMN2-DN-RG6-SA-DR CasRx 
            polycistronic gRNA with 3 spacers targeting SMN2 intron and 1 spacer
            targeting RG6 splice acceptor; Gateway Donor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3310)
  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 3310)
  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: pAC1821_pCR8-SMN2-DN-RG6-SA
FEATURES             Location/Qualifiers
     source          1..3310
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(157..184)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(276..362)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     411..433
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     425..442
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     426..442
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    458..557
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        572..820
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     572..592
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     743..762
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        822..851
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     misc_RNA        874..909
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        932..967
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        990..1025
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     protein_bind    complement(1078..1177)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1194..1213)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1195..1213)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1218..1234)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1218..1234)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1665..2456
                     /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      2549..3137
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3038..3057
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3291..3308
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
       61 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatac
      121 gcgtaccgct agccaggaag agtttgtaga aacgcaaaaa ggccatccgt caggatggcc
      181 ttctgcttag tttgatgcct ggcagtttat ggcgggcgtc ctgcccgcca ccctccgggc
      241 cgttgcttca caacgatcaa atccgctccc ggcggatttg tcctactcag gagagcgttc
      301 accgacaaac aacagataaa acgaaaggcc cagtattccg actgagcctt tcgttttatt
      361 tgatgcctgg cagttcccta ctctcgcgtt aacgctagca tggatgtttt cccagtcacg
      421 acgttgtaaa acgacggcca gtcttaagct cgggccccaa ataatgattt tattttgact
      481 gatagtgacc tgttcgttgc aacaaattga tgagcaatgc ttttttataa tgccaacttt
      541 gtacaaaaaa gcaggctccg aattcaccgg tgagggccta tttcccatga ttccttcata
      601 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
      661 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
      721 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
      781 tttcttggct ttatatatct tgtggaaagg acgaaacacc gaacccctac caactggtcg
      841 gggtttgaaa cacaaaagta agattcactt tcacaagtaa acccctacca actggtcggg
      901 gtttgaaacg agaattctag tagggatgta gcaagtaaac ccctaccaac tggtcggggt
      961 ttgaaacttt cttccacaca accaaccagc aagtaaaccc ctaccaactg gtcggggttt
     1021 gaaacatatc gcctggatcc tgagccattt ttttggcgcg ccttaattaa gaattcgacc
     1081 cagctttctt gtacaaagtt ggcattataa aaaataattg ctcatcaatt tgttgcaacg
     1141 aacaggtcac tatcagtcaa aataaaatca ttatttgcca tccagctgat atcccctata
     1201 gtgagtcgta ttacatggtc atagctgttt cctggcagct ctggcccgtg tctcaaaatc
     1261 tctgatgtta cattgcacaa gataaaaata tatcatcatg cctcctctag accagccagg
     1321 acagaaatgc ctcgacttcg ctgctaccca aggttgccgg gtgacgcaca ccgtggaaac
     1381 ggatgaaggc acgaacccag tggacataag cctgttcggt tcgtaagctg taatgcaagt
     1441 agcgtatgcg ctcacgcaac tggtccagaa ccttgaccga acgcagcggt ggtaacggcg
     1501 cagtggcggt tttcatggct tgttatgact gtttttttgg ggtacagtct atgcctcggg
     1561 catccaagca gcaagcgcgt tacgccgtgg gtcgatgttt gatgttatgg agcagcaacg
     1621 atgttacgca gcagggcagt cgccctaaaa caaagttaaa cattatgagg gaagcggtga
     1681 tcgccgaagt atcgactcaa ctatcagagg tagttggcgt catcgagcgc catctcgaac
     1741 cgacgttgct ggccgtacat ttgtacggct ccgcagtgga tggcggcctg aagccacaca
     1801 gtgatattga tttgctggtt acggtgaccg taaggcttga tgaaacaacg cggcgagctt
     1861 tgatcaacga ccttttggaa acttcggctt cccctggaga gagcgagatt ctccgcgctg
     1921 tagaagtcac cattgttgtg cacgacgaca tcattccgtg gcgttatcca gctaagcgcg
     1981 aactgcaatt tggagaatgg cagcgcaatg acattcttgc aggtatcttc gagccagcca
     2041 cgatcgacat tgatctggct atcttgctga caaaagcaag agaacatagc gttgccttgg
     2101 taggtccagc ggcggaggaa ctctttgatc cggttcctga acaggatcta tttgaggcgc
     2161 taaatgaaac cttaacgcta tggaactcgc cgcccgactg ggctggcgat gagcgaaatg
     2221 tagtgcttac gttgtcccgc atttggtaca gcgcagtaac cggcaaaatc gcgccgaagg
     2281 atgtcgctgc cgactgggca atggagcgcc tgccggccca gtatcagccc gtcatacttg
     2341 aagctagaca ggcttatctt ggacaagaag aagatcgctt ggcctcgcgc gcagatcagt
     2401 tggaagaatt tgtccactac gtgaaaggcg agatcaccaa ggtagtcggc aaataaccct
     2461 cgagccaccc atgaccaaaa tcccttaacg tgagttacgc gtcgttccac tgagcgtcag
     2521 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     2581 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     2641 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     2701 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     2761 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     2821 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     2881 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     2941 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     3001 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     3061 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     3121 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     3181 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     3241 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     3301 tgagcgagga
//
