LOCUS       sequence-455020-        8030 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of CasRx-NES-HA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8030)
  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 8030)
  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-NES-HA (pKW387)
FEATURES             Location/Qualifiers
     source          1..8030
                     /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             4544..4570
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(4639..4659)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5057..5068)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    5144..5265
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5155..5174)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5209..5228
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      5446..5901
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5533..5552)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5743..5764
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5968..5987)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6087..6109
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6147..6165)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6233..6337
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6338..6406,6407..7198)
                     /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(6556..6575)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7369..7957
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7858..7877
                     /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 actgtaccct gagcggctgc ggcggatcct gaccgcggcc gcttacccat acgatgttcc
     4561 agattacgct taatctagag ggcccaatca acctctggat tacaaaattt gtgaaagatt
     4621 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     4681 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     4741 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
     4801 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
     4861 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
     4921 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
     4981 gctgacgtcc tttccatggc tgctcgcctg tgttgctacc tggattctgc gcgggacgtc
     5041 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
     5101 ggctctgcgg cctcttccgc gtcttcgctt cgagcagaca tgataagata cattgatgag
     5161 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     5221 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     5281 attcatttta tgtttcaggt tcagggggag atgtgggagg ttttttaaag caagtaaaac
     5341 ctctacaaat gtggtaaaat cgataaggat cagggctggc gtaatagcga agaggcccgc
     5401 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggacgcg ccctgtagcg
     5461 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     5521 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     5581 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     5641 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     5701 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     5761 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     5821 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     5881 aaatattaac gcttacaatt tcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     5941 ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     6001 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     6061 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     6121 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     6181 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     6241 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     6301 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     6361 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     6421 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     6481 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     6541 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     6601 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     6661 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     6721 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     6781 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     6841 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     6901 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     6961 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     7021 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     7081 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     7141 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     7201 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     7261 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     7321 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     7381 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     7441 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     7501 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     7561 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     7621 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     7681 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     7741 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     7801 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     7861 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     7921 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     7981 acggttcctg gccttttgct ggccttttgc tcacatggct cgacagatct
//
