LOCUS       sequence-232483-        3136 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient transfection; Expressed SMN2-DN3 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: pAC1815_pCR8-gSMN2-DN3
FEATURES             Location/Qualifiers
     source          1..3136
                     /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)"
     protein_bind    complement(904..1003)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1020..1039)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1021..1039)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1044..1060)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1044..1060)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1491..2282
                     /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      2375..2963
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2864..2883
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3117..3134
                     /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 ctttcttcca cacaaccaac cagttttttt ggcgcgcctt aattaagaat
      901 tcgacccagc tttcttgtac aaagttggca ttataaaaaa taattgctca tcaatttgtt
      961 gcaacgaaca ggtcactatc agtcaaaata aaatcattat ttgccatcca gctgatatcc
     1021 cctatagtga gtcgtattac atggtcatag ctgtttcctg gcagctctgg cccgtgtctc
     1081 aaaatctctg atgttacatt gcacaagata aaaatatatc atcatgcctc ctctagacca
     1141 gccaggacag aaatgcctcg acttcgctgc tacccaaggt tgccgggtga cgcacaccgt
     1201 ggaaacggat gaaggcacga acccagtgga cataagcctg ttcggttcgt aagctgtaat
     1261 gcaagtagcg tatgcgctca cgcaactggt ccagaacctt gaccgaacgc agcggtggta
     1321 acggcgcagt ggcggttttc atggcttgtt atgactgttt ttttggggta cagtctatgc
     1381 ctcgggcatc caagcagcaa gcgcgttacg ccgtgggtcg atgtttgatg ttatggagca
     1441 gcaacgatgt tacgcagcag ggcagtcgcc ctaaaacaaa gttaaacatt atgagggaag
     1501 cggtgatcgc cgaagtatcg actcaactat cagaggtagt tggcgtcatc gagcgccatc
     1561 tcgaaccgac gttgctggcc gtacatttgt acggctccgc agtggatggc ggcctgaagc
     1621 cacacagtga tattgatttg ctggttacgg tgaccgtaag gcttgatgaa acaacgcggc
     1681 gagctttgat caacgacctt ttggaaactt cggcttcccc tggagagagc gagattctcc
     1741 gcgctgtaga agtcaccatt gttgtgcacg acgacatcat tccgtggcgt tatccagcta
     1801 agcgcgaact gcaatttgga gaatggcagc gcaatgacat tcttgcaggt atcttcgagc
     1861 cagccacgat cgacattgat ctggctatct tgctgacaaa agcaagagaa catagcgttg
     1921 ccttggtagg tccagcggcg gaggaactct ttgatccggt tcctgaacag gatctatttg
     1981 aggcgctaaa tgaaacctta acgctatgga actcgccgcc cgactgggct ggcgatgagc
     2041 gaaatgtagt gcttacgttg tcccgcattt ggtacagcgc agtaaccggc aaaatcgcgc
     2101 cgaaggatgt cgctgccgac tgggcaatgg agcgcctgcc ggcccagtat cagcccgtca
     2161 tacttgaagc tagacaggct tatcttggac aagaagaaga tcgcttggcc tcgcgcgcag
     2221 atcagttgga agaatttgtc cactacgtga aaggcgagat caccaaggta gtcggcaaat
     2281 aaccctcgag ccacccatga ccaaaatccc ttaacgtgag ttacgcgtcg ttccactgag
     2341 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     2401 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     2461 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     2521 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     2581 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     2641 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     2701 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     2761 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     2821 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     2881 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     2941 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     3001 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     3061 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     3121 agtcagtgag cgagga
//
