LOCUS       sequence-232471-        5997 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient transfection; Expresses dCasRx; CAGGS promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5997)
  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 5997)
  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: pAC1801_pmax-dCasRx
FEATURES             Location/Qualifiers
     source          1..5997
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        140..519
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        520..723
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     674..694
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          815..947
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     protein_bind    962..986
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1016..1036
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1046..3943
                     /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             3953..3973
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3983..4009
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    complement(4028..4052)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4105..4146
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(4172..4191)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    4201..4282
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     4226..4245
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(4480..5068)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4560..4579)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5149..5943)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(5149..5943)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5260..5279)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5870..5889
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 tgtcaatatt ggccattagc catattattc attggttata tagcataaat caatattggc
       61 tattggccat tgcatacgtt gtatctatat cataatatgt acatttatat tggctcatgt
      121 ccaatatgac cgccatgttg gcattgatta ttgactagtt attaatagta atcaattacg
      181 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
      241 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
      301 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
      361 gcccacttgg cagtacatca agtgtatcat atgccaagtc cgccccctat tgacgtcaat
      421 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttacggga ctttcctact
      481 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
      541 accaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
      601 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaataac
      661 cccgccccgt tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
      721 ggtcgtttag tgaaccgtca gatcactagt agctttattg cggtagttta tcacagttaa
      781 attgctaacg cagtcagtgc tcgactgatc acaggtaagt atcaaggtta caagacaggt
      841 ttaaggaggc caatagaaac tgggcttgtc gagacagaga agattcttgc gtttctgata
      901 ggcacctatt ggtcttactg acatccactt tgcctttctc tccacaggga aaaaacaatt
      961 gacaagtttg tacaaaaaag caggctccga attcaccggt gccgccacca tgagccccaa
     1021 gaagaagaga aaggtggagg ccagcatcga aaaaaaaaag tccttcgcca agggcatggg
     1081 cgtgaagtcc acactcgtgt ccggctccaa agtgtacatg acaaccttcg ccgaaggcag
     1141 cgacgccagg ctggaaaaga tcgtggaggg cgacagcatc aggagcgtga atgagggcga
     1201 ggccttcagc gctgaaatgg ccgataaaaa cgccggctat aagatcggca acgccaaatt
     1261 cagccatcct aagggctacg ccgtggtggc taacaaccct ctgtatacag gacccgtcca
     1321 gcaggatatg ctcggcctga aggaaactct ggaaaagagg tacttcggcg agagcgctga
     1381 tggcaatgac aatatttgta tccaggtgat ccataacatc ctggacattg aaaaaatcct
     1441 cgccgaatac attaccaacg ccgcctacgc cgtcaacaat atctccggcc tggataagga
     1501 cattattgga ttcggcaagt tctccacagt gtatacctac gacgaattca aagaccccga
     1561 gcaccatagg gccgctttca acaataacga taagctcatc aacgccatca aggcccagta
     1621 tgacgagttc gacaacttcc tcgataaccc cagactcggc tatttcggcc aggccttttt
     1681 cagcaaggag ggcagaaatt acatcatcaa ttacggcaac gaatgctatg acattctggc
     1741 cctcctgagc ggactggcgc actgggtggt cgctaacaac gaagaagagt ccaggatctc
     1801 caggacctgg ctctacaacc tcgataagaa cctcgacaac gaatacatct ccaccctcaa
     1861 ctacctctac gacaggatca ccaatgagct gaccaactcc ttctccaaga actccgccgc
     1921 caacgtgaac tatattgccg aaactctggg aatcaaccct gccgaattcg ccgaacaata
     1981 tttcagattc agcattatga aagagcagaa aaacctcgga ttcaatatca ccaagctcag
     2041 ggaagtgatg ctggacagga aggatatgtc cgagatcagg aaaaatcata aggtgttcga
     2101 ctccatcagg accaaggtct acaccatgat ggactttgtg atttataggt attacatcga
     2161 agaggatgcc aaggtggctg ccgccaataa gtccctcccc gataatgaga agtccctgag
     2221 cgagaaggat atctttgtga ttaacctgag gggctccttc aacgacgacc agaaggatgc
     2281 cctctactac gatgaagcta atagaatttg gagaaagctc gaaaatatca tgcacaacat
     2341 caaggaattt aggggaaaca agacaagaga gtataagaag aaggacgccc ctagactgcc
     2401 cagaatcctg cccgctggcc gtgatgtttc cgccttcagc aaactcatgt atgccctgac
     2461 catgttcctg gatggcaagg agatcaacga cctcctgacc accctgatta ataaattcga
     2521 taacatccag agcttcctga aggtgatgcc tctcatcgga gtcaacgcta agttcgtgga
     2581 ggaatacgcc tttttcaaag actccgccaa gatcgccgat gagctgaggc tgatcaagtc
     2641 cttcgctaga atgggagaac ctattgccga tgccaggagg gccatgtata tcgacgccat
     2701 ccgtatttta ggaaccaacc tgtcctatga tgagctcaag gccctcgccg acaccttttc
     2761 cctggacgag aacggaaaca agctcaagaa aggcaagcac ggcatgagaa atttcattat
     2821 taataacgtg atcagcaata aaaggttcca ctacctgatc agatacggtg atcctgccca
     2881 cctccatgag atcgccaaaa acgaggccgt ggtgaagttc gtgctcggca ggatcgctga
     2941 catccagaaa aaacagggcc agaacggcaa gaaccagatc gacaggtact acgaaacttg
     3001 tatcggaaag gataagggca agagcgtgag cgaaaaggtg gacgctctca caaagatcat
     3061 caccggaatg aactacgacc aattcgacaa gaaaaggagc gtcattgagg acaccggcag
     3121 ggaaaacgcc gagagggaga agtttaaaaa gatcatcagc ctgtacctca ccgtgatcta
     3181 ccacatcctc aagaatattg tcaatatcaa cgccaggtac gtcatcggat tccattgcgt
     3241 cgagcgtgat gctcaactgt acaaggagaa aggctacgac atcaatctca agaaactgga
     3301 agagaaggga ttcagctccg tcaccaagct ctgcgctggc attgatgaaa ctgcccccga
     3361 taagagaaag gacgtggaaa aggagatggc tgaaagagcc aaggagagca ttgacagcct
     3421 cgagagcgcc aaccccaagc tgtatgccaa ttacatcaaa tacagcgacg agaagaaagc
     3481 cgaggagttc accaggcaga ttaacaggga gaaggccaaa accgccctga acgcctacct
     3541 gaggaacacc aagtggaatg tgatcatcag ggaggacctc ctgagaattg acaacaagac
     3601 atgtaccctg ttcgcaaaca aggccgtcgc cctggaagtg gccaggtatg tccacgccta
     3661 tatcaacgac attgccgagg tcaattccta cttccaactg taccattaca tcatgcagag
     3721 aattatcatg aatgagaggt acgagaaaag cagcggaaag gtgtccgagt acttcgacgc
     3781 tgtgaatgac gagaagaagt acaacgatag gctcctgaaa ctgctgtgtg tgcctttcgg
     3841 ctactgtatc cccaggttta agaacctgag catcgaggcc ctgttcgata ggaacgaggc
     3901 cgccaagttc gacaaggaga aaaagaaggt gtccggcaat tccggatccg gacctaagaa
     3961 aaagaggaag gtggcggccg cttacccata cgatgttcca gattacgctt gattaattaa
     4021 gaattcgacc cagctttctt gtacaaagtg gttgatatcc agcacagtgg cggccgctcg
     4081 agtctagagg gcccgcggtt cgaaggtaag cctatcccta accctctcct cggtctcgat
     4141 tctacgcgta ccggttagca attgtttttt cgatgagttt ggacaaacca caactagaat
     4201 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     4261 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     4321 gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtacttaaga
     4381 gggggagacc aaagggcgag acgttaaggc ctcacgtgac atgtgagcaa aaggccagca
     4441 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     4501 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     4561 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     4621 gcttacggga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     4681 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     4741 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     4801 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     4861 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     4921 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     4981 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     5041 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     5101 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgccgtctc agaagaactc
     5161 gtcaagaagg cgatagaagg cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac
     5221 gaggaagcgg tcagcccatt cgccgccaag ctcttcagca atatcacggg tagccaacgc
     5281 tatgtcctga tagcggtccg ccacacccag ccggccacag tcgatgaatc cagaaaagcg
     5341 gccattttcc accatgatat tcggcaagca ggcatcgcca tgggtcacga cgagatcctc
     5401 gccgtcgggc atgctcgcct tgagcctggc gaacagttcg gctggcgcga gcccctgatg
     5461 ctcttcgtcc agatcatcct gatcgacaag accggcttcc atccgagtac gtgctctctc
     5521 gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg
     5581 ccgcattgca tcagccatga tggatacttt ctcggcagga gcaaggtgag atgacaggag
     5641 atcctgcccc ggcacttcgc ccaatagcag ccagtccctt cccgcttcag tgacaacgtc
     5701 gagtacagct gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc
     5761 ttgcagttca ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg
     5821 cgctgacagc cggaacacgg cggcatcaga gcagccgatt gtctgttgtg cccagtcata
     5881 gccgaatagc ctctccaccc aagcggccgg agaacctgcg tgcaatccat cttgttcaat
     5941 cataatatta ttgaagcatt tatcagggtt cgtctcgtcc cggtctcctc ccatgca
//
