LOCUS       sequence-455014-        7997 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of CasRx-HA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7997)
  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 7997)
  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-HA (pKW88)
FEATURES             Location/Qualifiers
     source          1..7997
                     /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             1595..4492
                     /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             4511..4537
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(4606..4626)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5024..5035)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    5111..5232
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5122..5141)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5176..5195
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      5413..5868
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5500..5519)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5710..5731
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5935..5954)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6054..6076
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6114..6132)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6200..6304
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6305..6373,6374..7165)
                     /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(6523..6542)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7336..7924
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7825..7844
                     /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 tgagcgaggc cagcatcgaa aaaaaaaagt ccttcgccaa
     1621 gggcatgggc gtgaagtcca cactcgtgtc cggctccaaa gtgtacatga caaccttcgc
     1681 cgaaggcagc gacgccaggc tggaaaagat cgtggagggc gacagcatca ggagcgtgaa
     1741 tgagggcgag gccttcagcg ctgaaatggc cgataaaaac gccggctata agatcggcaa
     1801 cgccaaattc agccatccta agggctacgc cgtggtggct aacaaccctc tgtatacagg
     1861 acccgtccag caggatatgc tcggcctgaa ggaaactctg gaaaagaggt acttcggcga
     1921 gagcgctgat ggcaatgaca atatttgtat ccaggtgatc cataacatcc tggacattga
     1981 aaaaatcctc gccgaataca ttaccaacgc cgcctacgcc gtcaacaata tctccggcct
     2041 ggataaggac attattggat tcggcaagtt ctccacagtg tatacctacg acgaattcaa
     2101 agaccccgag caccataggg ccgctttcaa caataacgat aagctcatca acgccatcaa
     2161 ggcccagtat gacgagttcg acaacttcct cgataacccc agactcggct atttcggcca
     2221 ggcctttttc agcaaggagg gcagaaatta catcatcaat tacggcaacg aatgctatga
     2281 cattctggcc ctcctgagcg gactgaggca ctgggtggtc cataacaacg aagaagagtc
     2341 caggatctcc aggacctggc tctacaacct cgataagaac ctcgacaacg aatacatctc
     2401 caccctcaac tacctctacg acaggatcac caatgagctg accaactcct tctccaagaa
     2461 ctccgccgcc aacgtgaact atattgccga aactctggga atcaaccctg ccgaattcgc
     2521 cgaacaatat ttcagattca gcattatgaa agagcagaaa aacctcggat tcaatatcac
     2581 caagctcagg gaagtgatgc tggacaggaa ggatatgtcc gagatcagga aaaatcataa
     2641 ggtgttcgac tccatcagga ccaaggtcta caccatgatg gactttgtga tttataggta
     2701 ttacatcgaa gaggatgcca aggtggctgc cgccaataag tccctccccg ataatgagaa
     2761 gtccctgagc gagaaggata tctttgtgat taacctgagg ggctccttca acgacgacca
     2821 gaaggatgcc ctctactacg atgaagctaa tagaatttgg agaaagctcg aaaatatcat
     2881 gcacaacatc aaggaattta ggggaaacaa gacaagagag tataagaaga aggacgcccc
     2941 tagactgccc agaatcctgc ccgctggccg tgatgtttcc gccttcagca aactcatgta
     3001 tgccctgacc atgttcctgg atggcaagga gatcaacgac ctcctgacca ccctgattaa
     3061 taaattcgat aacatccaga gcttcctgaa ggtgatgcct ctcatcggag tcaacgctaa
     3121 gttcgtggag gaatacgcct ttttcaaaga ctccgccaag atcgccgatg agctgaggct
     3181 gatcaagtcc ttcgctagaa tgggagaacc tattgccgat gccaggaggg ccatgtatat
     3241 cgacgccatc cgtattttag gaaccaacct gtcctatgat gagctcaagg ccctcgccga
     3301 caccttttcc ctggacgaga acggaaacaa gctcaagaaa ggcaagcacg gcatgagaaa
     3361 tttcattatt aataacgtga tcagcaataa aaggttccac tacctgatca gatacggtga
     3421 tcctgcccac ctccatgaga tcgccaaaaa cgaggccgtg gtgaagttcg tgctcggcag
     3481 gatcgctgac atccagaaaa aacagggcca gaacggcaag aaccagatcg acaggtacta
     3541 cgaaacttgt atcggaaagg ataagggcaa gagcgtgagc gaaaaggtgg acgctctcac
     3601 aaagatcatc accggaatga actacgacca attcgacaag aaaaggagcg tcattgagga
     3661 caccggcagg gaaaacgccg agagggagaa gtttaaaaag atcatcagcc tgtacctcac
     3721 cgtgatctac cacatcctca agaatattgt caatatcaac gccaggtacg tcatcggatt
     3781 ccattgcgtc gagcgtgatg ctcaactgta caaggagaaa ggctacgaca tcaatctcaa
     3841 gaaactggaa gagaagggat tcagctccgt caccaagctc tgcgctggca ttgatgaaac
     3901 tgcccccgat aagagaaagg acgtggaaaa ggagatggct gaaagagcca aggagagcat
     3961 tgacagcctc gagagcgcca accccaagct gtatgccaat tacatcaaat acagcgacga
     4021 gaagaaagcc gaggagttca ccaggcagat taacagggag aaggccaaaa ccgccctgaa
     4081 cgcctacctg aggaacacca agtggaatgt gatcatcagg gaggacctcc tgagaattga
     4141 caacaagaca tgtaccctgt tcagaaacaa ggccgtccac ctggaagtgg ccaggtatgt
     4201 ccacgcctat atcaacgaca ttgccgaggt caattcctac ttccaactgt accattacat
     4261 catgcagaga attatcatga atgagaggta cgagaaaagc agcggaaagg tgtccgagta
     4321 cttcgacgct gtgaatgacg agaagaagta caacgatagg ctcctgaaac tgctgtgtgt
     4381 gcctttcggc tactgtatcc ccaggtttaa gaacctgagc atcgaggccc tgttcgatag
     4441 gaacgaggcc gccaagttcg acaaggagaa aaagaaggtg tccggcaatt ccggatccgg
     4501 agcggccgct tacccatacg atgttccaga ttacgcttaa tctagagggc ccaatcaacc
     4561 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     4621 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     4681 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     4741 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     4801 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     4861 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     4921 tgacaattcc gtggtgttgt cggggaagct gacgtccttt ccatggctgc tcgcctgtgt
     4981 tgctacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     5041 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgcttcga
     5101 gcagacatga taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa
     5161 aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc
     5221 aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggagatg
     5281 tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtaaaatcga taaggatcag
     5341 ggctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     5401 aatggcgaat ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     5461 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     5521 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     5581 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     5641 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     5701 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     5761 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     5821 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttcc tgatgcggta
     5881 ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat
     5941 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     6001 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     6061 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
     6121 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     6181 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     6241 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     6301 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     6361 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     6421 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     6481 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     6541 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     6601 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     6661 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     6721 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     6781 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     6841 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     6901 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     6961 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     7021 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     7081 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     7141 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     7201 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     7261 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     7321 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     7381 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     7441 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     7501 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     7561 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     7621 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     7681 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     7741 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     7801 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     7861 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     7921 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     7981 catggctcga cagatct
//
