LOCUS       sequence-437014-        7916 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Destination vector for Gateway cloning of ORFs into the C-terminus 
            of dCasRx. CMV promoter. Transient expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7916)
  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 7916)
  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: CMV-dCasRx-ccdB
FEATURES             Location/Qualifiers
     source          1..7916
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        124..503
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        504..707
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     658..678
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          799..931
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     CDS             975..995
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1005..3902
                     /codon_start=1
                     /product="RNA-targeting Type VI CRISPR protein from 
                     Ruminococcus flavefaciens strain XPD3002 (Konermann et al.,
                     2018)"
                     /label=RfxCas13d
                     /translation="IEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDSI
                     RSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEK
                     RYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTVY
                     TYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYIIN
                     YGNECYDILALLSGLAHWVVANNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNE
                     LTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKD
                     MSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVI
                     NLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAG
                     RDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFF
                     KDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDEN
                     GNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQK
                     KQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGREN
                     AEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEE
                     KGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKA
                     EEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFANKAVALEVARYVHA
                     YINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCVP
                     FGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS"
     CDS             3912..3938
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             3948..3974
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    4038..4162
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(4187..4192,4193..4210,4211..4217)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             4271..4930
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(4337..4356)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             5272..5577
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     5456..5475
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(5618..5742)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             5808..5849
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(5875..5894)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    5904..5985
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     5929..5948
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(6141..6729)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6221..6240)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6900..7691,7692..7760))
                     /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     7523..7542
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7761..7865)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tagccatatt attcattggt tatatagcat aaatcaatat tggctattgg ccattgcata
       61 cgttgtatct atatcataat atgtacattt atattggctc atgtccaata tgaccgccat
      121 gttggcattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
      181 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      241 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      301 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      361 atcaagtgta tcatatgcca agtccgcccc ctattgacgt caatgacggt aaatggcccg
      421 cctggcatta tgcccagtac atgaccttac gggactttcc tacttggcag tacatctacg
      481 tattagtcat cgctattacc atggtgatgc ggttttggca gtacaccaat gggcgtggat
      541 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
      601 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa taaccccgcc ccgttgacgc
      661 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagaggtcgt ttagtgaacc
      721 gtcagatcac tagtagcttt attgcggtag tttatcacag ttaaattgct aacgcagtca
      781 gtgctcgact gatcacaggt aagtatcaag gttacaagac aggtttaagg aggccaatag
      841 aaactgggct tgtcgagaca gagaagattc ttgcgtttct gataggcacc tattggtctt
      901 actgacatcc actttgcctt tctctccaca gggaaaaaac aattgccgaa ttcaccggtg
      961 ccgccaccat gagccccaag aagaagagaa aggtggaggc cagcatcgaa aaaaaaaagt
     1021 ccttcgccaa gggcatgggc gtgaagtcca cactcgtgtc cggctccaaa gtgtacatga
     1081 caaccttcgc cgaaggcagc gacgccaggc tggaaaagat cgtggagggc gacagcatca
     1141 ggagcgtgaa tgagggcgag gccttcagcg ctgaaatggc cgataaaaac gccggctata
     1201 agatcggcaa cgccaaattc agccatccta agggctacgc cgtggtggct aacaaccctc
     1261 tgtatacagg acccgtccag caggatatgc tcggcctgaa ggaaactctg gaaaagaggt
     1321 acttcggcga gagcgctgat ggcaatgaca atatttgtat ccaggtgatc cataacatcc
     1381 tggacattga aaaaatcctc gccgaataca ttaccaacgc cgcctacgcc gtcaacaata
     1441 tctccggcct ggataaggac attattggat tcggcaagtt ctccacagtg tatacctacg
     1501 acgaattcaa agaccccgag caccataggg ccgctttcaa caataacgat aagctcatca
     1561 acgccatcaa ggcccagtat gacgagttcg acaacttcct cgataacccc agactcggct
     1621 atttcggcca ggcctttttc agcaaggagg gcagaaatta catcatcaat tacggcaacg
     1681 aatgctatga cattctggcc ctcctgagcg gactggcgca ctgggtggtc gctaacaacg
     1741 aagaagagtc caggatctcc aggacctggc tctacaacct cgataagaac ctcgacaacg
     1801 aatacatctc caccctcaac tacctctacg acaggatcac caatgagctg accaactcct
     1861 tctccaagaa ctccgccgcc aacgtgaact atattgccga aactctggga atcaaccctg
     1921 ccgaattcgc cgaacaatat ttcagattca gcattatgaa agagcagaaa aacctcggat
     1981 tcaatatcac caagctcagg gaagtgatgc tggacaggaa ggatatgtcc gagatcagga
     2041 aaaatcataa ggtgttcgac tccatcagga ccaaggtcta caccatgatg gactttgtga
     2101 tttataggta ttacatcgaa gaggatgcca aggtggctgc cgccaataag tccctccccg
     2161 ataatgagaa gtccctgagc gagaaggata tctttgtgat taacctgagg ggctccttca
     2221 acgacgacca gaaggatgcc ctctactacg atgaagctaa tagaatttgg agaaagctcg
     2281 aaaatatcat gcacaacatc aaggaattta ggggaaacaa gacaagagag tataagaaga
     2341 aggacgcccc tagactgccc agaatcctgc ccgctggccg tgatgtttcc gccttcagca
     2401 aactcatgta tgccctgacc atgttcctgg atggcaagga gatcaacgac ctcctgacca
     2461 ccctgattaa taaattcgat aacatccaga gcttcctgaa ggtgatgcct ctcatcggag
     2521 tcaacgctaa gttcgtggag gaatacgcct ttttcaaaga ctccgccaag atcgccgatg
     2581 agctgaggct gatcaagtcc ttcgctagaa tgggagaacc tattgccgat gccaggaggg
     2641 ccatgtatat cgacgccatc cgtattttag gaaccaacct gtcctatgat gagctcaagg
     2701 ccctcgccga caccttttcc ctggacgaga acggaaacaa gctcaagaaa ggcaagcacg
     2761 gcatgagaaa tttcattatt aataacgtga tcagcaataa aaggttccac tacctgatca
     2821 gatacggtga tcctgcccac ctccatgaga tcgccaaaaa cgaggccgtg gtgaagttcg
     2881 tgctcggcag gatcgctgac atccagaaaa aacagggcca gaacggcaag aaccagatcg
     2941 acaggtacta cgaaacttgt atcggaaagg ataagggcaa gagcgtgagc gaaaaggtgg
     3001 acgctctcac aaagatcatc accggaatga actacgacca attcgacaag aaaaggagcg
     3061 tcattgagga caccggcagg gaaaacgccg agagggagaa gtttaaaaag atcatcagcc
     3121 tgtacctcac cgtgatctac cacatcctca agaatattgt caatatcaac gccaggtacg
     3181 tcatcggatt ccattgcgtc gagcgtgatg ctcaactgta caaggagaaa ggctacgaca
     3241 tcaatctcaa gaaactggaa gagaagggat tcagctccgt caccaagctc tgcgctggca
     3301 ttgatgaaac tgcccccgat aagagaaagg acgtggaaaa ggagatggct gaaagagcca
     3361 aggagagcat tgacagcctc gagagcgcca accccaagct gtatgccaat tacatcaaat
     3421 acagcgacga gaagaaagcc gaggagttca ccaggcagat taacagggag aaggccaaaa
     3481 ccgccctgaa cgcctacctg aggaacacca agtggaatgt gatcatcagg gaggacctcc
     3541 tgagaattga caacaagaca tgtaccctgt tcgcaaacaa ggccgtcgcc ctggaagtgg
     3601 ccaggtatgt ccacgcctat atcaacgaca ttgccgaggt caattcctac ttccaactgt
     3661 accattacat catgcagaga attatcatga atgagaggta cgagaaaagc agcggaaagg
     3721 tgtccgagta cttcgacgct gtgaatgacg agaagaagta caacgatagg ctcctgaaac
     3781 tgctgtgtgt gcctttcggc tactgtatcc ccaggtttaa gaacctgagc atcgaggccc
     3841 tgttcgatag gaacgaggcc gccaagttcg acaaggagaa aaagaaggtg tccggcaatt
     3901 ccggatccgg acctgctgct aagagagtga aactggatgc ggccgcttac ccatacgatg
     3961 ttccagatta cgctggcggc cgcgggggag gcggttccgg tggcggcgga agcggaggtg
     4021 gaggatcagg gccggccaca agtttgtaca aaaaagctga acgagaaacg taaaatgata
     4081 taaatatcaa tatattaaat tagattttgc ataaaaaaca gactacataa tactgtaaaa
     4141 cacaacatat ccagtcacta tggcggccgc attaggcacc ccaggcttta cactttatgc
     4201 ttccggctcg tataatgtgt ggattttgag ttaggatccg tcgagatttt caggagctaa
     4261 ggaagctaaa atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca
     4321 tcgtaaagaa cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt
     4381 tcagctggat attacggcct ttttaaagac cgtaaagaaa aataagcaca agttttatcc
     4441 ggcctttatt cacattcttg cccgcctgat gaatgctcat ccggaattcc gtatggcaat
     4501 gaaagacggt gagctggtga tatgggatag tgttcaccct tgttacaccg ttttccatga
     4561 gcaaactgaa acgttttcat cgctctggag tgaataccac gacgatttcc ggcagtttct
     4621 acacatatat tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg
     4681 gtttattgag aatatgtttt tcgtctcagc caatccctgg gtgagtttca ccagttttga
     4741 tttaaacgtg gccaatatgg acaacttctt cgcccccgtt ttcaccatgg gcaaatatta
     4801 tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga
     4861 tggcttccat gtcggcagaa tgcttaatga attacaacag tactgcgatg agtggcaggg
     4921 cggggcgtaa agatctggat ccggcttact aaaagccaga taacagtatg cgtatttgcg
     4981 cgctgatttt tgcggtataa gaatatatac tgatatgtat acccgaagta tgtcaaaaag
     5041 aggtatgcta tgaagcagcg tattacagtg acagttgaca gcgacagcta tcagttgctc
     5101 aaggcatata tgatgtcaat atctccggtc tggtaagcac aaccatgcag aatgaagccc
     5161 gtcgtctgcg tgccgaacgc tggaaagcgg aaaatcagga agggatggct gaggtcgccc
     5221 ggtttattga aatgaacggc tcttttgctg acgagaacag gggctggtga aatgcagttt
     5281 aaggtttaca cctataaaag agagagccgt tatcgtctgt ttgtggatgt acagagtgat
     5341 attattgaca cgcccgggcg acggatggtg atccccctgg ccagtgcacg tctgctgtca
     5401 gataaagtct cccgtgaact ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg
     5461 atgaccaccg atatggccag tgtgccggtc tccgttatcg gggaagaagt ggctgatctc
     5521 agccaccgcg aaaatgacat caaaaacgcc attaacctga tgttctgggg aatataaatg
     5581 tcaggctccc ttatacacag ccagtctgca ggtcgaccat agtgactgga tatgttgtgt
     5641 tttacagtat tatgtagtct gttttttatg caaaatctaa tttaatatat tgatatttat
     5701 atcattttac gtttctcgtt cagctttctt gtacaaagtg gttgctctat agtaatgata
     5761 tccagcacag tggcggccgc tcgagtctag agggcccgcg gttcgaaggt aagcctatcc
     5821 ctaaccctct cctcggtctc gattctacgc gtaccggtta gcaattgttt tttcgatgag
     5881 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     5941 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     6001 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
     6061 ctctacaaat gtggtacgaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     6121 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     6181 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     6241 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     6301 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     6361 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     6421 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     6481 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     6541 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     6601 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     6661 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     6721 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     6781 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     6841 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     6901 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     6961 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     7021 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     7081 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     7141 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     7201 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     7261 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     7321 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     7381 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     7441 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     7501 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     7561 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     7621 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     7681 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     7741 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     7801 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     7861 ccgcgcacat ttccccgaaa agtgccacct gacgtctgca tgtcaatatt ggccat
//
