LOCUS       sequence-436033-        3898 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transiently express CasRx DR30 repeat with gRNA spacer. Contains SD1
            mutation to enhance efficiency. SV40-TagBFP cassette to monitor 
            transfection efficiency. Use BbsI with overhangs Fw-AAAC, Rv-AAAA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3898)
  AUTHORS   Li JD, Taipale M, Blencowe BJ
  TITLE     Efficient, specific, and combinatorial control of endogenous exon 
            splicing with dCasRx-RBM25.
  JOURNAL   Mol Cell. 2024 Jul 11;84(13):2573-2589.e5. doi: 
            10.1016/j.molcel.2024.05.028. Epub 2024 Jun 24.
  PUBMED    38917795
REFERENCE   2  (bases 1 to 3898)
  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"; doi: 
            "10.1016/j.molcel.2024.05.028"; journalName: "Mol Cell"; date: 
            "2024-07-11- 11"; volume: "84"; issue: "13"; pages: "2573-2589.e5"
COMMENT     Sequence Label: gRNA-CasRx_SD1-pSV40-TagBFP
FEATURES             Location/Qualifiers
     source          1..3898
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        32..280
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     32..52
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     203..222
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        283..311
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     promoter        386..715
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      566..701
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     628..647
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             764..1462
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     polyA_signal    1592..1713
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1629..1648)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1683..1702
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1762..1778)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1762..1778)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1775..1797)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1786..1802
                     /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)."
     promoter        complement(join(1810..1816,1817..1834,1835..1840))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1855..1876
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1993..2010)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2164..2752)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2244..2263)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2923..3714,3715..3783))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3546..3565
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3784..3888)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cgaaaagtgc cacctgacgt cgacggctag cgagggccta tttcccatga ttccttcata
       61 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
      121 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
      181 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
      241 tttcttggct ttatatatct tgtggaaagg acgaaacacc gtacccctac caactggtcg
      301 gggtttgaaa cgggtcttcg agaagacctt ttttttgaat tctgatgcgg tattttctcc
      361 ttacgcatct gtgcggtatt tcacagtgtg tcagttaggg tgtggaaagt ccccaggctc
      421 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
      481 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
      541 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
      601 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
      661 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
      721 cccgggagct tgtatatcca ttttcggatc tgatcagcac gtgatggagc tgattaagga
      781 gaacatgcac atgaagctgt acatggaggg caccgtggac aaccatcact tcaagtgcac
      841 atccgagggc gaaggcaagc cctacgaggg cacccagacc atgagaatca aggtggtcga
      901 gggcggccct ctccccttcg ccttcgacat cctggctact agcttcctct acggcagcaa
      961 gaccttcatc aaccacaccc agggcatccc cgacttcttc aagcagtcct tccctgaggg
     1021 cttcacatgg gagagagtca ccacatacga agatgggggc gtgctgaccg ctacccagga
     1081 caccagcctc caggacggct gcctcatcta caacgtcaag atcagagggg tgaacttcac
     1141 atccaacggc cctgtgatgc agaagaaaac actcggctgg gaggccttca ccgagacgct
     1201 gtaccccgct gacggcggcc tggaaggcag aaacgacatg gccctgaagc tcgtgggcgg
     1261 gagccatctg atcgcaaaca tcaagaccac atatagatcc aagaaacccg ctaagaacct
     1321 caagatgcct ggcgtctact atgtggacta cagactggaa agaatcaagg aggccaacaa
     1381 cgagacctac gtcgagcagc acgaggtggc agtggccaga tactgcgacc tccctagcaa
     1441 actggggcac aaacttaatt aacacgtgct acgagatttc gattccaccg ccgccttcta
     1501 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     1561 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     1621 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     1681 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     1741 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     1801 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     1861 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     1921 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     1981 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     2041 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     2101 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     2161 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     2221 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     2281 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     2341 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     2401 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     2461 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     2521 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     2581 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     2641 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     2701 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     2761 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     2821 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     2881 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     2941 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     3001 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     3061 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     3121 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     3181 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     3241 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     3301 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     3361 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     3421 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     3481 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     3541 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     3601 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     3661 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     3721 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     3781 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     3841 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccc
//
