LOCUS       sequence-306040-        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"
     CDS             709..729
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             739..3636
                     /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             3646..3666
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3676..3699
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    3712..3846
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3749..3768)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3803..3822
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   3862..4002
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3862..3991
                     /label=AAV2 ITR
     rep_origin      4077..4532
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4164..4183)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4374..4395
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4549..4568)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4668..4690
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4728..4746)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4814..4918
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4919..4987,4988..5779)
                     /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      5950..6538
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6439..6458
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   6600..6729
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 gcggcctcta gaaacatatc ctggtgtgga gtaggggacg ctgctctgac agaggctcgg
       61 gggcctgagc tggctctgtg agctggggag gaggcagaca gccaggcctt gtctgcaagc
      121 agacctggca gcattgggct ggccgccccc cagggcctcc tcttcatgcc cagtgaatga
      181 ctcaccttgg cacagacaca atgttcgggg tgggcacagt gcctgcttcc cgccgcaccc
      241 cagcccccct caaatgcctt ccgagaagcc cattgagcag ggggcttgca ttgcacccca
      301 gcctgacagc ctggcatctt gggataaaag cagcacagcc ccctaggggc tgcccttgct
      361 gtgtggcgcc accggcggtg gagaacaagg ctctattcag cctgtgccca ggaaagggga
      421 tcaggggatg cccaggcatg gacagtgggt ggcagggggg gagaggaggg ctgtctgctt
      481 cccagaagtc caaggacaca aatgggtgag gggagagctc tccccatagc tgggctgcgg
      541 cccaacccca ccccctcagg ctatgccagg gggtgttgcc aggggcaccc gggcatcgcc
      601 agtctagccc actccttcat aaagccctcg catcccagga gcgagcagag ccagagcagg
      661 ttggagagga gacgcatcac ctccgctgct cgcaccggtg ccaccatgcc taaaaagaaa
      721 agaaaggtgg gttctggtat cgagaagaag aagagcttcg ccaagggcat gggagtgaag
      781 agcaccctgg tgtccggctc taaggtgtac atgaccacat ttgctgaggg aagcgacgcc
      841 aggctggaga agatcgtgga gggcgatagc atcagatccg tgaacgaggg agaggctttc
      901 agcgccgaga tggctgacaa gaacgctggc tacaagatcg gaaacgccaa gttttcccac
      961 ccaaagggct acgccgtggt ggctaacaac ccactgtaca ccggaccagt gcagcaggac
     1021 atgctgggac tgaaggagac actggagaag aggtacttcg gcgagtccgc cgacggaaac
     1081 gataacatct gcatccaggt catccacaac atcctggata tcgagaagat cctggctgag
     1141 tacatcacaa acgccgctta cgccgtgaac aacatctccg gcctggacaa ggatatcatc
     1201 ggcttcggaa agttttctac cgtgtacaca tacgacgagt tcaaggatcc agagcaccac
     1261 cgggccgctt ttaacaacaa cgacaagctg atcaacgcca tcaaggctca gtacgacgag
     1321 ttcgataact ttctggataa ccccaggctg ggctacttcg gacaggcttt cttttctaag
     1381 gagggcagaa actacatcat caactacgga aacgagtgtt acgacatcct ggccctgctg
     1441 agcggactga ggcactgggt ggtgcacaac aacgaggagg agtctcggat cagccgcacc
     1501 tggctgtaca acctggacaa gaacctggat aacgagtaca tctccacact gaactacctg
     1561 tacgacagga tcaccaacga gctgacaaac agcttctcca agaactctgc cgctaacgtg
     1621 aactacatcg ctgagaccct gggcatcaac ccagctgagt tcgctgagca gtacttcaga
     1681 ttttccatca tgaaggagca gaagaacctg ggcttcaaca tcacaaagct gagagaagtg
     1741 atgctggaca gaaaggatat gtccgagatc aggaagaacc acaaggtgtt cgattctatc
     1801 agaaccaagg tgtacacaat gatggacttt gtgatctaca ggtactacat cgaggaggat
     1861 gccaaggtgg ccgctgccaa caagagcctg cccgacaacg agaagtctct gagcgagaag
     1921 gatatcttcg tgatcaacct gagaggctcc tttaacgacg atcagaagga cgctctgtac
     1981 tacgatgagg ccaacaggat ctggagaaag ctggagaaca tcatgcacaa catcaaggag
     2041 ttccggggaa acaagacccg cgagtacaag aagaaggacg ctccaaggct gcctaggatc
     2101 ctgcctgctg gaagggacgt gagcgccttc agcaagctga tgtacgccct gacaatgttt
     2161 ctggacggaa aggagatcaa cgatctgctg accacactga tcaacaagtt cgacaacatc
     2221 cagtcttttc tgaaagtgat gcctctgatc ggcgtgaacg ctaagttcgt ggaggagtac
     2281 gccttcttta aggacagcgc caagatcgct gatgagctgc ggctgatcaa gtcctttgcc
     2341 aggatgggag agccaatcgc tgacgctagg agagctatgt acatcgatgc catccggatc
     2401 ctgggaacca acctgtctta cgacgagctg aaggctctgg ccgacacctt cagcctggat
     2461 gagaacggca acaagctgaa gaagggcaag cacggaatgc gcaacttcat catcaacaac
     2521 gtgatcagca acaagcggtt tcactacctg atcagatacg gcgacccagc tcacctgcac
     2581 gagatcgcta agaacgaggc cgtggtgaag ttcgtgctgg gacggatcgc cgatatccag
     2641 aagaagcagg gccagaacgg aaagaaccag atcgaccgct actacgagac ctgcatcggc
     2701 aaggataagg gaaagtccgt gtctgagaag gtggacgctc tgaccaagat catcacaggc
     2761 atgaactacg accagttcga taagaagaga tctgtgatcg aggacaccgg aagggagaac
     2821 gccgagagag agaagtttaa gaagatcatc agcctgtacc tgacagtgat ctaccacatc
     2881 ctgaagaaca tcgtgaacat caacgctaga tacgtgatcg gcttccactg cgtggagcgc
     2941 gatgcccagc tgtacaagga gaagggatac gacatcaacc tgaagaagct ggaggagaag
     3001 ggctttagct ccgtgaccaa gctgtgcgct ggaatcgacg agacagcccc cgacaagagg
     3061 aaggatgtgg agaaggagat ggccgagaga gctaaggaga gcatcgactc cctggagtct
     3121 gctaacccta agctgtacgc caactacatc aagtactccg atgagaagaa ggccgaggag
     3181 ttcaccaggc agatcaacag agagaaggcc aagaccgctc tgaacgccta cctgaggaac
     3241 acaaagtgga acgtgatcat ccgggaggac ctgctgcgca tcgataacaa gacctgtaca
     3301 ctgttccgga acaaggctgt gcacctggag gtggctcgct acgtgcacgc ctacatcaac
     3361 gacatcgccg aggtgaactc ctactttcag ctgtaccact acatcatgca gaggatcatc
     3421 atgaacgaga gatacgagaa gtctagcggc aaggtgtctg agtacttcga cgccgtgaac
     3481 gatgagaaga agtacaacga tagactgctg aagctgctgt gcgtgccttt cggatactgt
     3541 atcccacggt ttaagaacct gagcatcgag gccctgttcg accgcaacga ggctgccaag
     3601 tttgataagg agaagaagaa ggtgagcggc aactccggtt ctggtcccaa gaagaagcgc
     3661 aaggtggcgg ccgctgatta caaagacgat gacgataagt aaagatccta aaacttgttt
     3721 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     3781 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     3841 tggatcgcgg ccgagggccg caggaacccc tagtgatgga gttggccact ccctctctgc
     3901 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     3961 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt
     4021 ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc
     4081 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     4141 acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     4201 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     4261 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc
     4321 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     4381 cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg
     4441 gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     4501 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc
     4561 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     4621 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4681 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     4741 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     4801 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     4861 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     4921 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     4981 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     5041 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     5101 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     5161 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     5221 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     5281 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     5341 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     5401 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     5461 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     5521 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     5581 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtggaagccg
     5641 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     5701 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     5761 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     5821 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     5881 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     5941 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     6001 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     6061 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     6121 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6181 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6241 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6301 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     6361 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     6421 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     6481 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     6541 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtc
     6601 ctgcaggcag ctgcgcgctc gctcgctcac tgaggccgcc cgggcgtcgg gcgacctttg
     6661 gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta
     6721 ggggttcct
//
