LOCUS       sequence-232543-        3172 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient transfection; Expressed DR-RG6-SA-DR CasRx gRNA with 
            preprocessed DR 3'; Gateway Donor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3172)
  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 3172)
  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: pAC1820_pCR8-DR-RG6-SA-DR
FEATURES             Location/Qualifiers
     source          1..3172
                     /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)"
     protein_bind    complement(940..1039)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1056..1075)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1057..1075)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1080..1096)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1080..1096)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1527..2318
                     /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      2411..2999
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2900..2919
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3153..3170
                     /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 catatcgcct ggatcctgag ccacaagtaa acccctacca actggtcggg
      901 gtttgaaact ttttttggcg cgccttaatt aagaattcga cccagctttc ttgtacaaag
      961 ttggcattat aaaaaataat tgctcatcaa tttgttgcaa cgaacaggtc actatcagtc
     1021 aaaataaaat cattatttgc catccagctg atatccccta tagtgagtcg tattacatgg
     1081 tcatagctgt ttcctggcag ctctggcccg tgtctcaaaa tctctgatgt tacattgcac
     1141 aagataaaaa tatatcatca tgcctcctct agaccagcca ggacagaaat gcctcgactt
     1201 cgctgctacc caaggttgcc gggtgacgca caccgtggaa acggatgaag gcacgaaccc
     1261 agtggacata agcctgttcg gttcgtaagc tgtaatgcaa gtagcgtatg cgctcacgca
     1321 actggtccag aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg
     1381 cttgttatga ctgttttttt ggggtacagt ctatgcctcg ggcatccaag cagcaagcgc
     1441 gttacgccgt gggtcgatgt ttgatgttat ggagcagcaa cgatgttacg cagcagggca
     1501 gtcgccctaa aacaaagtta aacattatga gggaagcggt gatcgccgaa gtatcgactc
     1561 aactatcaga ggtagttggc gtcatcgagc gccatctcga accgacgttg ctggccgtac
     1621 atttgtacgg ctccgcagtg gatggcggcc tgaagccaca cagtgatatt gatttgctgg
     1681 ttacggtgac cgtaaggctt gatgaaacaa cgcggcgagc tttgatcaac gaccttttgg
     1741 aaacttcggc ttcccctgga gagagcgaga ttctccgcgc tgtagaagtc accattgttg
     1801 tgcacgacga catcattccg tggcgttatc cagctaagcg cgaactgcaa tttggagaat
     1861 ggcagcgcaa tgacattctt gcaggtatct tcgagccagc cacgatcgac attgatctgg
     1921 ctatcttgct gacaaaagca agagaacata gcgttgcctt ggtaggtcca gcggcggagg
     1981 aactctttga tccggttcct gaacaggatc tatttgaggc gctaaatgaa accttaacgc
     2041 tatggaactc gccgcccgac tgggctggcg atgagcgaaa tgtagtgctt acgttgtccc
     2101 gcatttggta cagcgcagta accggcaaaa tcgcgccgaa ggatgtcgct gccgactggg
     2161 caatggagcg cctgccggcc cagtatcagc ccgtcatact tgaagctaga caggcttatc
     2221 ttggacaaga agaagatcgc ttggcctcgc gcgcagatca gttggaagaa tttgtccact
     2281 acgtgaaagg cgagatcacc aaggtagtcg gcaaataacc ctcgagccac ccatgaccaa
     2341 aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc agaccccgta gaaaagatca
     2401 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     2461 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2521 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     2581 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     2641 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     2701 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     2761 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     2821 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     2881 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     2941 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     3001 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     3061 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     3121 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag ga
//
