LOCUS       sequence-454997-        8039 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of CasRx-NLS-HA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8039)
  AUTHORS   Cheng ECK, Lam JKC, Kwon SC
  TITLE     Cytosolic CRISPR RNAs for efficient application of RNA-targeting 
            CRISPR-Cas systems.
  JOURNAL   EMBO Rep. 2025 Feb 26. doi: 10.1038/s44319-025-00399-4.
  PUBMED    40011676
REFERENCE   2  (bases 1 to 8039)
  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: "EMBO Rep. 
            2025 Feb 26. doi: 10.1038/s44319-025-00399-4."
COMMENT     Sequence Label: pCI5-CasRx-NLS-HA (pKW919)
FEATURES             Location/Qualifiers
     source          1..8039
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        2..381
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        382..585
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     536..556
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     582..606
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          721..1544
                     /label=CMV intron A
                     /note="human cytomegalovirus intron A (Chapman et al., 
                     1991)"
     CDS             1586..1606
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1616..4513
                     /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
                     YGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNE
                     LTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKD
                     MSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVI
                     NLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAG
                     RDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFF
                     KDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDEN
                     GNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQK
                     KQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGREN
                     AEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEE
                     KGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKA
                     EEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHA
                     YINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCVP
                     FGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS"
     CDS             4523..4543
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4553..4579
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(4648..4668)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5066..5077)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    5153..5274
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5164..5183)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5218..5237
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      5455..5910
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5542..5561)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5752..5773
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5977..5996)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6096..6118
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6156..6174)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6242..6346
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6347..6415,6416..7207)
                     /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     complement(6565..6584)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7378..7966
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7867..7886
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
       61 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctcg tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt ccggccccct attgacgtca atgacggtaa atggcccgcc
      301 tggcattatg cccagtacat gaccttacgg gactttccta cttggcagta catctacgta
      361 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acaccaatgg gcgtggatag
      421 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
      481 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaata accccgcccc gttgacgcaa
      541 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt
      601 cagatcgcct ggagtcgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga
      661 tccagcctcc gcggccggga acggtgcatt ggaacgcgga ttccccgtgc caagagtgac
      721 gtaagtaccg cctatagact ctataggcac acccctttgg ctcttatgca tgctatactg
      781 tttttggctt ggggcctata cacccccgct ccttatgcta taggtgatgg tatagcttag
      841 cctataggtg tgggttattg accattattg accactcccc tattggtgac gatactttcc
      901 attactaatc cataacatgg ctctttgcca caactatctc tattggctat atgccaatac
      961 tctgtccttc agagactgac acggactctg tatttttaca ggatggggtc ccatttatta
     1021 tttacaaatt cacatataca acaacgccgt cccccgtgcc cgcagttttt attaaacata
     1081 gcgtgggatc tccacgcgaa tctcgggtac gtgttccgga catgggctct tctccggtag
     1141 cggcggagct tccacatccg agccctggtc ccatgcctcc agcggctcat ggtcgctcgg
     1201 cagctccttg ctcctaacag tggaggccag acttaggcac agcacaatgc ccaccaccac
     1261 cagtgtgccg cacaaggccg tggcggtagg gtatgtgtct gaaaatgagc tcggagattg
     1321 ggctcgcacc gtgacgcaga tggaagactt aaggcagcgg cagaagaaga tgcaggcagc
     1381 tgagttgttg tattctgata agagtcagag gtaactcccg ttgcggtgct gttaacggtg
     1441 gagggcagtg tagtctgagc agtactcgtt gctgccgcgc gcgccaccag acataatagc
     1501 tgacagacta acagactgtt cctttccatg ggtcttttct gcagtcaccg tccttgacac
     1561 ggctagcggt accgccacca tgagccccaa gaagaagaga aaggtggagg ccagcatcga
     1621 aaaaaaaaag tccttcgcca agggcatggg cgtgaagtcc acactcgtgt ccggctccaa
     1681 agtgtacatg acaaccttcg ccgaaggcag cgacgccagg ctggaaaaga tcgtggaggg
     1741 cgacagcatc aggagcgtga atgagggcga ggccttcagc gctgaaatgg ccgataaaaa
     1801 cgccggctat aagatcggca acgccaaatt cagccatcct aagggctacg ccgtggtggc
     1861 taacaaccct ctgtatacag gacccgtcca gcaggatatg ctcggcctga aggaaactct
     1921 ggaaaagagg tacttcggcg agagcgctga tggcaatgac aatatttgta tccaggtgat
     1981 ccataacatc ctggacattg aaaaaatcct cgccgaatac attaccaacg ccgcctacgc
     2041 cgtcaacaat atctccggcc tggataagga cattattgga ttcggcaagt tctccacagt
     2101 gtatacctac gacgaattca aagaccccga gcaccatagg gccgctttca acaataacga
     2161 taagctcatc aacgccatca aggcccagta tgacgagttc gacaacttcc tcgataaccc
     2221 cagactcggc tatttcggcc aggccttttt cagcaaggag ggcagaaatt acatcatcaa
     2281 ttacggcaac gaatgctatg acattctggc cctcctgagc ggactgaggc actgggtggt
     2341 ccataacaac gaagaagagt ccaggatctc caggacctgg ctctacaacc tcgataagaa
     2401 cctcgacaac gaatacatct ccaccctcaa ctacctctac gacaggatca ccaatgagct
     2461 gaccaactcc ttctccaaga actccgccgc caacgtgaac tatattgccg aaactctggg
     2521 aatcaaccct gccgaattcg ccgaacaata tttcagattc agcattatga aagagcagaa
     2581 aaacctcgga ttcaatatca ccaagctcag ggaagtgatg ctggacagga aggatatgtc
     2641 cgagatcagg aaaaatcata aggtgttcga ctccatcagg accaaggtct acaccatgat
     2701 ggactttgtg atttataggt attacatcga agaggatgcc aaggtggctg ccgccaataa
     2761 gtccctcccc gataatgaga agtccctgag cgagaaggat atctttgtga ttaacctgag
     2821 gggctccttc aacgacgacc agaaggatgc cctctactac gatgaagcta atagaatttg
     2881 gagaaagctc gaaaatatca tgcacaacat caaggaattt aggggaaaca agacaagaga
     2941 gtataagaag aaggacgccc ctagactgcc cagaatcctg cccgctggcc gtgatgtttc
     3001 cgccttcagc aaactcatgt atgccctgac catgttcctg gatggcaagg agatcaacga
     3061 cctcctgacc accctgatta ataaattcga taacatccag agcttcctga aggtgatgcc
     3121 tctcatcgga gtcaacgcta agttcgtgga ggaatacgcc tttttcaaag actccgccaa
     3181 gatcgccgat gagctgaggc tgatcaagtc cttcgctaga atgggagaac ctattgccga
     3241 tgccaggagg gccatgtata tcgacgccat ccgtatttta ggaaccaacc tgtcctatga
     3301 tgagctcaag gccctcgccg acaccttttc cctggacgag aacggaaaca agctcaagaa
     3361 aggcaagcac ggcatgagaa atttcattat taataacgtg atcagcaata aaaggttcca
     3421 ctacctgatc agatacggtg atcctgccca cctccatgag atcgccaaaa acgaggccgt
     3481 ggtgaagttc gtgctcggca ggatcgctga catccagaaa aaacagggcc agaacggcaa
     3541 gaaccagatc gacaggtact acgaaacttg tatcggaaag gataagggca agagcgtgag
     3601 cgaaaaggtg gacgctctca caaagatcat caccggaatg aactacgacc aattcgacaa
     3661 gaaaaggagc gtcattgagg acaccggcag ggaaaacgcc gagagggaga agtttaaaaa
     3721 gatcatcagc ctgtacctca ccgtgatcta ccacatcctc aagaatattg tcaatatcaa
     3781 cgccaggtac gtcatcggat tccattgcgt cgagcgtgat gctcaactgt acaaggagaa
     3841 aggctacgac atcaatctca agaaactgga agagaaggga ttcagctccg tcaccaagct
     3901 ctgcgctggc attgatgaaa ctgcccccga taagagaaag gacgtggaaa aggagatggc
     3961 tgaaagagcc aaggagagca ttgacagcct cgagagcgcc aaccccaagc tgtatgccaa
     4021 ttacatcaaa tacagcgacg agaagaaagc cgaggagttc accaggcaga ttaacaggga
     4081 gaaggccaaa accgccctga acgcctacct gaggaacacc aagtggaatg tgatcatcag
     4141 ggaggacctc ctgagaattg acaacaagac atgtaccctg ttcagaaaca aggccgtcca
     4201 cctggaagtg gccaggtatg tccacgccta tatcaacgac attgccgagg tcaattccta
     4261 cttccaactg taccattaca tcatgcagag aattatcatg aatgagaggt acgagaaaag
     4321 cagcggaaag gtgtccgagt acttcgacgc tgtgaatgac gagaagaagt acaacgatag
     4381 gctcctgaaa ctgctgtgtg tgcctttcgg ctactgtatc cccaggttta agaacctgag
     4441 catcgaggcc ctgttcgata ggaacgaggc cgccaagttc gacaaggaga aaaagaaggt
     4501 gtccggcaat tccggatccg gacctaagaa aaagaggaag gtggcggccg cttacccata
     4561 cgatgttcca gattacgctt aatctagagg gcccaatcaa cctctggatt acaaaatttg
     4621 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     4681 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta
     4741 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt
     4801 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca
     4861 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc
     4921 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt
     4981 gtcggggaag ctgacgtcct ttccatggct gctcgcctgt gttgctacct ggattctgcg
     5041 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg
     5101 cctgctgccg gctctgcggc ctcttccgcg tcttcgcttc gagcagacat gataagatac
     5161 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     5221 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     5281 aacaattgca ttcattttat gtttcaggtt cagggggaga tgtgggaggt tttttaaagc
     5341 aagtaaaacc tctacaaatg tggtaaaatc gataaggatc agggctggcg taatagcgaa
     5401 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggacgcgc
     5461 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     5521 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     5581 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     5641 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     5701 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     5761 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     5821 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     5881 attttaacaa aatattaacg cttacaattt cctgatgcgg tattttctcc ttacgcatct
     5941 gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata
     6001 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     6061 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     6121 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     6181 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     6241 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     6301 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     6361 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     6421 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     6481 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     6541 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     6601 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     6661 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     6721 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     6781 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     6841 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     6901 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     6961 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     7021 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     7081 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     7141 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     7201 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     7261 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     7321 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     7381 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     7441 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     7501 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     7561 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     7621 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     7681 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     7741 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     7801 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     7861 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     7921 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     7981 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatggctc gacagatct
//
