LOCUS       sequence-297792-        7169 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector expressed CasRx and sgRNA_Ptbp1 for AAV package.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7169)
  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 7169)
  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-U6-DR-SgRNA_Ptbp1-DR
FEATURES             Location/Qualifiers
     source          1..7169
                     /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"
     promoter        3983..4231
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3983..4003
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4154..4173
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4233..4268
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        4299..4334
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        4365..4400
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     repeat_region   4432..4572
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4432..4561
                     /label=AAV2 ITR
     rep_origin      4647..5102
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4734..4753)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4944..4965
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5119..5138)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5238..5260
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5298..5316)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5384..5488
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5489..5557,5558..6349)
                     /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      6520..7108
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7009..7028
                     /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 ccgagggcct atttcccatg attccttcat atttgcatat
     4021 acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt
     4081 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
     4141 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
     4201 tttatatatc ttgtggaaag gacgaaacac cgcaagtaaa cccctaccaa ctggtcgggg
     4261 tttgaaactg tagatgggct gtccacgaag cactggcgca agtaaacccc taccaactgg
     4321 tcggggtttg aaacgcttgg agaagtcgat gcgcagcgtg cagccaagta aacccctacc
     4381 aactggtcgg ggtttgaaac tttttttgaa ttctgatgcg gtggcggccg caggaacccc
     4441 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac
     4501 caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca
     4561 gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     4621 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg
     4681 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt
     4741 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     4801 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     4861 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     4921 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc
     4981 tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct attggttaaa
     5041 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     5101 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     5161 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     5221 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     5281 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     5341 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     5401 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     5461 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     5521 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     5581 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     5641 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     5701 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     5761 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     5821 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     5881 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     5941 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     6001 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     6061 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     6121 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     6181 taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg ggccagatgg
     6241 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     6301 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     6361 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     6421 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     6481 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     6541 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     6601 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     6661 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     6721 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     6781 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     6841 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     6901 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     6961 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     7021 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     7081 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     7141 ggccttttgc tggccttttg ctcacatgt
//
