LOCUS       sequence-297794-        6729 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express CasRx for AAV package.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6729)
  AUTHORS   Zhou H, Su J, Hu X, Zhou C, Li H, Chen Z, Xiao Q, Wang B, Wu W, Sun 
            Y, Zhou Y, Tang C, Liu F, Wang L, Feng C, Liu M, Li S, Zhang Y, Xu 
            H, Yao H, Shi L, Yang H
  TITLE     Glia-to-Neuron Conversion by CRISPR-CasRx Alleviates Symptoms of 
            Neurological Disease in Mice.
  JOURNAL   Cell. 2020 Apr 30;181(3):590-603.e16. doi: 
            10.1016/j.cell.2020.03.024. Epub 2020 Apr 8.
  PUBMED    32272060
REFERENCE   2  (bases 1 to 6729)
  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.cell.2020.03.024"; journalName: "Cell"; date: 
            "2020-04-30- 30"; volume: "181"; issue: "3"; pages: "590-603.e16"
COMMENT     Sequence Label: AAV-GFAP-NLS-CasRx-NLS-FLAG
FEATURES             Location/Qualifiers
     source          1..6729
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     CDS             839..859
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             869..3766
                     /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             3776..3796
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3806..3829
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    3842..3976
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3879..3898)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3933..3952
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   3992..4132
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3992..4121
                     /label=AAV2 ITR
     rep_origin      4207..4662
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4294..4313)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4504..4525
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4679..4698)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4798..4820
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4858..4876)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4944..5048
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5049..5117,5118..5909)
                     /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"
     rep_origin      6080..6668
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6569..6588
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gaaacatatc ctggtgtgga gtaggggacg ctgctctgac
      181 agaggctcgg gggcctgagc tggctctgtg agctggggag gaggcagaca gccaggcctt
      241 gtctgcaagc agacctggca gcattgggct ggccgccccc cagggcctcc tcttcatgcc
      301 cagtgaatga ctcaccttgg cacagacaca atgttcgggg tgggcacagt gcctgcttcc
      361 cgccgcaccc cagcccccct caaatgcctt ccgagaagcc cattgagcag ggggcttgca
      421 ttgcacccca gcctgacagc ctggcatctt gggataaaag cagcacagcc ccctaggggc
      481 tgcccttgct gtgtggcgcc accggcggtg gagaacaagg ctctattcag cctgtgccca
      541 ggaaagggga tcaggggatg cccaggcatg gacagtgggt ggcagggggg gagaggaggg
      601 ctgtctgctt cccagaagtc caaggacaca aatgggtgag gggagagctc tccccatagc
      661 tgggctgcgg cccaacccca ccccctcagg ctatgccagg gggtgttgcc aggggcaccc
      721 gggcatcgcc agtctagccc actccttcat aaagccctcg catcccagga gcgagcagag
      781 ccagagcagg ttggagagga gacgcatcac ctccgctgct cgcaccggtg ccaccatgcc
      841 taaaaagaaa agaaaggtgg gttctggtat cgagaagaag aagagcttcg ccaagggcat
      901 gggagtgaag agcaccctgg tgtccggctc taaggtgtac atgaccacat ttgctgaggg
      961 aagcgacgcc aggctggaga agatcgtgga gggcgatagc atcagatccg tgaacgaggg
     1021 agaggctttc agcgccgaga tggctgacaa gaacgctggc tacaagatcg gaaacgccaa
     1081 gttttcccac ccaaagggct acgccgtggt ggctaacaac ccactgtaca ccggaccagt
     1141 gcagcaggac atgctgggac tgaaggagac actggagaag aggtacttcg gcgagtccgc
     1201 cgacggaaac gataacatct gcatccaggt catccacaac atcctggata tcgagaagat
     1261 cctggctgag tacatcacaa acgccgctta cgccgtgaac aacatctccg gcctggacaa
     1321 ggatatcatc ggcttcggaa agttttctac cgtgtacaca tacgacgagt tcaaggatcc
     1381 agagcaccac cgggccgctt ttaacaacaa cgacaagctg atcaacgcca tcaaggctca
     1441 gtacgacgag ttcgataact ttctggataa ccccaggctg ggctacttcg gacaggcttt
     1501 cttttctaag gagggcagaa actacatcat caactacgga aacgagtgtt acgacatcct
     1561 ggccctgctg agcggactga ggcactgggt ggtgcacaac aacgaggagg agtctcggat
     1621 cagccgcacc tggctgtaca acctggacaa gaacctggat aacgagtaca tctccacact
     1681 gaactacctg tacgacagga tcaccaacga gctgacaaac agcttctcca agaactctgc
     1741 cgctaacgtg aactacatcg ctgagaccct gggcatcaac ccagctgagt tcgctgagca
     1801 gtacttcaga ttttccatca tgaaggagca gaagaacctg ggcttcaaca tcacaaagct
     1861 gagagaagtg atgctggaca gaaaggatat gtccgagatc aggaagaacc acaaggtgtt
     1921 cgattctatc agaaccaagg tgtacacaat gatggacttt gtgatctaca ggtactacat
     1981 cgaggaggat gccaaggtgg ccgctgccaa caagagcctg cccgacaacg agaagtctct
     2041 gagcgagaag gatatcttcg tgatcaacct gagaggctcc tttaacgacg atcagaagga
     2101 cgctctgtac tacgatgagg ccaacaggat ctggagaaag ctggagaaca tcatgcacaa
     2161 catcaaggag ttccggggaa acaagacccg cgagtacaag aagaaggacg ctccaaggct
     2221 gcctaggatc ctgcctgctg gaagggacgt gagcgccttc agcaagctga tgtacgccct
     2281 gacaatgttt ctggacggaa aggagatcaa cgatctgctg accacactga tcaacaagtt
     2341 cgacaacatc cagtcttttc tgaaagtgat gcctctgatc ggcgtgaacg ctaagttcgt
     2401 ggaggagtac gccttcttta aggacagcgc caagatcgct gatgagctgc ggctgatcaa
     2461 gtcctttgcc aggatgggag agccaatcgc tgacgctagg agagctatgt acatcgatgc
     2521 catccggatc ctgggaacca acctgtctta cgacgagctg aaggctctgg ccgacacctt
     2581 cagcctggat gagaacggca acaagctgaa gaagggcaag cacggaatgc gcaacttcat
     2641 catcaacaac gtgatcagca acaagcggtt tcactacctg atcagatacg gcgacccagc
     2701 tcacctgcac gagatcgcta agaacgaggc cgtggtgaag ttcgtgctgg gacggatcgc
     2761 cgatatccag aagaagcagg gccagaacgg aaagaaccag atcgaccgct actacgagac
     2821 ctgcatcggc aaggataagg gaaagtccgt gtctgagaag gtggacgctc tgaccaagat
     2881 catcacaggc atgaactacg accagttcga taagaagaga tctgtgatcg aggacaccgg
     2941 aagggagaac gccgagagag agaagtttaa gaagatcatc agcctgtacc tgacagtgat
     3001 ctaccacatc ctgaagaaca tcgtgaacat caacgctaga tacgtgatcg gcttccactg
     3061 cgtggagcgc gatgcccagc tgtacaagga gaagggatac gacatcaacc tgaagaagct
     3121 ggaggagaag ggctttagct ccgtgaccaa gctgtgcgct ggaatcgacg agacagcccc
     3181 cgacaagagg aaggatgtgg agaaggagat ggccgagaga gctaaggaga gcatcgactc
     3241 cctggagtct gctaacccta agctgtacgc caactacatc aagtactccg atgagaagaa
     3301 ggccgaggag ttcaccaggc agatcaacag agagaaggcc aagaccgctc tgaacgccta
     3361 cctgaggaac acaaagtgga acgtgatcat ccgggaggac ctgctgcgca tcgataacaa
     3421 gacctgtaca ctgttccgga acaaggctgt gcacctggag gtggctcgct acgtgcacgc
     3481 ctacatcaac gacatcgccg aggtgaactc ctactttcag ctgtaccact acatcatgca
     3541 gaggatcatc atgaacgaga gatacgagaa gtctagcggc aaggtgtctg agtacttcga
     3601 cgccgtgaac gatgagaaga agtacaacga tagactgctg aagctgctgt gcgtgccttt
     3661 cggatactgt atcccacggt ttaagaacct gagcatcgag gccctgttcg accgcaacga
     3721 ggctgccaag tttgataagg agaagaagaa ggtgagcggc aactccggtt ctggtcccaa
     3781 gaagaagcgc aaggtggcgg ccgctgatta caaagacgat gacgataagt aaagatccta
     3841 aaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     3901 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     3961 ttatcatgtc tggatcgcgg ccgagggccg caggaacccc tagtgatgga gttggccact
     4021 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg
     4081 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg
     4141 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac
     4201 catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     4261 tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc
     4321 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     4381 gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta
     4441 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     4501 atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg
     4561 atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa
     4621 aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta
     4681 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     4741 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     4801 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
     4861 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     4921 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     4981 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     5041 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     5101 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     5161 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     5221 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     5281 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     5341 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     5401 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     5461 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     5521 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     5581 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     5641 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     5701 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     5761 gtggaagccg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     5821 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     5881 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     5941 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     6001 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     6061 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     6121 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     6181 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     6241 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     6301 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     6361 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     6421 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     6481 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     6541 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     6601 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     6661 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     6721 ctcacatgt
//
