LOCUS       sequence-305844-        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   102..231
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     CDS             940..960
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             970..3867
                     /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             3877..3897
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3907..3930
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    3943..4077
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3980..3999)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4034..4053
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        4084..4332
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4084..4104
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4255..4274
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4334..4369
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        4400..4435
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        4466..4501
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     repeat_region   4533..4673
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4533..4662
                     /label=AAV2 ITR
     rep_origin      4748..5203
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4835..4854)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5045..5066
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5220..5239)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5339..5361
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5399..5417)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5485..5589
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5590..5658,5659..6450)
                     /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      join(6621..7169,1..40)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7110..7129
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
       61 tttacggttc ctggcctttt gctggccttt tgctcacatg tcctgcaggc agctgcgcgc
      121 tcgctcgctc actgaggccg cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga
      181 gcgagcgagc gcgcagagag ggagtggcca actccatcac taggggttcc tgcggcctct
      241 agaaacatat cctggtgtgg agtaggggac gctgctctga cagaggctcg ggggcctgag
      301 ctggctctgt gagctgggga ggaggcagac agccaggcct tgtctgcaag cagacctggc
      361 agcattgggc tggccgcccc ccagggcctc ctcttcatgc ccagtgaatg actcaccttg
      421 gcacagacac aatgttcggg gtgggcacag tgcctgcttc ccgccgcacc ccagcccccc
      481 tcaaatgcct tccgagaagc ccattgagca gggggcttgc attgcacccc agcctgacag
      541 cctggcatct tgggataaaa gcagcacagc cccctagggg ctgcccttgc tgtgtggcgc
      601 caccggcggt ggagaacaag gctctattca gcctgtgccc aggaaagggg atcaggggat
      661 gcccaggcat ggacagtggg tggcaggggg ggagaggagg gctgtctgct tcccagaagt
      721 ccaaggacac aaatgggtga ggggagagct ctccccatag ctgggctgcg gcccaacccc
      781 accccctcag gctatgccag ggggtgttgc caggggcacc cgggcatcgc cagtctagcc
      841 cactccttca taaagccctc gcatcccagg agcgagcaga gccagagcag gttggagagg
      901 agacgcatca cctccgctgc tcgcaccggt gccaccatgc ctaaaaagaa aagaaaggtg
      961 ggttctggta tcgagaagaa gaagagcttc gccaagggca tgggagtgaa gagcaccctg
     1021 gtgtccggct ctaaggtgta catgaccaca tttgctgagg gaagcgacgc caggctggag
     1081 aagatcgtgg agggcgatag catcagatcc gtgaacgagg gagaggcttt cagcgccgag
     1141 atggctgaca agaacgctgg ctacaagatc ggaaacgcca agttttccca cccaaagggc
     1201 tacgccgtgg tggctaacaa cccactgtac accggaccag tgcagcagga catgctggga
     1261 ctgaaggaga cactggagaa gaggtacttc ggcgagtccg ccgacggaaa cgataacatc
     1321 tgcatccagg tcatccacaa catcctggat atcgagaaga tcctggctga gtacatcaca
     1381 aacgccgctt acgccgtgaa caacatctcc ggcctggaca aggatatcat cggcttcgga
     1441 aagttttcta ccgtgtacac atacgacgag ttcaaggatc cagagcacca ccgggccgct
     1501 tttaacaaca acgacaagct gatcaacgcc atcaaggctc agtacgacga gttcgataac
     1561 tttctggata accccaggct gggctacttc ggacaggctt tcttttctaa ggagggcaga
     1621 aactacatca tcaactacgg aaacgagtgt tacgacatcc tggccctgct gagcggactg
     1681 aggcactggg tggtgcacaa caacgaggag gagtctcgga tcagccgcac ctggctgtac
     1741 aacctggaca agaacctgga taacgagtac atctccacac tgaactacct gtacgacagg
     1801 atcaccaacg agctgacaaa cagcttctcc aagaactctg ccgctaacgt gaactacatc
     1861 gctgagaccc tgggcatcaa cccagctgag ttcgctgagc agtacttcag attttccatc
     1921 atgaaggagc agaagaacct gggcttcaac atcacaaagc tgagagaagt gatgctggac
     1981 agaaaggata tgtccgagat caggaagaac cacaaggtgt tcgattctat cagaaccaag
     2041 gtgtacacaa tgatggactt tgtgatctac aggtactaca tcgaggagga tgccaaggtg
     2101 gccgctgcca acaagagcct gcccgacaac gagaagtctc tgagcgagaa ggatatcttc
     2161 gtgatcaacc tgagaggctc ctttaacgac gatcagaagg acgctctgta ctacgatgag
     2221 gccaacagga tctggagaaa gctggagaac atcatgcaca acatcaagga gttccgggga
     2281 aacaagaccc gcgagtacaa gaagaaggac gctccaaggc tgcctaggat cctgcctgct
     2341 ggaagggacg tgagcgcctt cagcaagctg atgtacgccc tgacaatgtt tctggacgga
     2401 aaggagatca acgatctgct gaccacactg atcaacaagt tcgacaacat ccagtctttt
     2461 ctgaaagtga tgcctctgat cggcgtgaac gctaagttcg tggaggagta cgccttcttt
     2521 aaggacagcg ccaagatcgc tgatgagctg cggctgatca agtcctttgc caggatggga
     2581 gagccaatcg ctgacgctag gagagctatg tacatcgatg ccatccggat cctgggaacc
     2641 aacctgtctt acgacgagct gaaggctctg gccgacacct tcagcctgga tgagaacggc
     2701 aacaagctga agaagggcaa gcacggaatg cgcaacttca tcatcaacaa cgtgatcagc
     2761 aacaagcggt ttcactacct gatcagatac ggcgacccag ctcacctgca cgagatcgct
     2821 aagaacgagg ccgtggtgaa gttcgtgctg ggacggatcg ccgatatcca gaagaagcag
     2881 ggccagaacg gaaagaacca gatcgaccgc tactacgaga cctgcatcgg caaggataag
     2941 ggaaagtccg tgtctgagaa ggtggacgct ctgaccaaga tcatcacagg catgaactac
     3001 gaccagttcg ataagaagag atctgtgatc gaggacaccg gaagggagaa cgccgagaga
     3061 gagaagttta agaagatcat cagcctgtac ctgacagtga tctaccacat cctgaagaac
     3121 atcgtgaaca tcaacgctag atacgtgatc ggcttccact gcgtggagcg cgatgcccag
     3181 ctgtacaagg agaagggata cgacatcaac ctgaagaagc tggaggagaa gggctttagc
     3241 tccgtgacca agctgtgcgc tggaatcgac gagacagccc ccgacaagag gaaggatgtg
     3301 gagaaggaga tggccgagag agctaaggag agcatcgact ccctggagtc tgctaaccct
     3361 aagctgtacg ccaactacat caagtactcc gatgagaaga aggccgagga gttcaccagg
     3421 cagatcaaca gagagaaggc caagaccgct ctgaacgcct acctgaggaa cacaaagtgg
     3481 aacgtgatca tccgggagga cctgctgcgc atcgataaca agacctgtac actgttccgg
     3541 aacaaggctg tgcacctgga ggtggctcgc tacgtgcacg cctacatcaa cgacatcgcc
     3601 gaggtgaact cctactttca gctgtaccac tacatcatgc agaggatcat catgaacgag
     3661 agatacgaga agtctagcgg caaggtgtct gagtacttcg acgccgtgaa cgatgagaag
     3721 aagtacaacg atagactgct gaagctgctg tgcgtgcctt tcggatactg tatcccacgg
     3781 tttaagaacc tgagcatcga ggccctgttc gaccgcaacg aggctgccaa gtttgataag
     3841 gagaagaaga aggtgagcgg caactccggt tctggtccca agaagaagcg caaggtggcg
     3901 gccgctgatt acaaagacga tgacgataag taaagatcct aaaacttgtt tattgcagct
     3961 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     4021 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatcgcg
     4081 gccgagggcc tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga
     4141 gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt
     4201 agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat
     4261 catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa
     4321 ggacgaaaca ccgcaagtaa acccctacca actggtcggg gtttgaaact gtagatgggc
     4381 tgtccacgaa gcactggcgc aagtaaaccc ctaccaactg gtcggggttt gaaacgcttg
     4441 gagaagtcga tgcgcagcgt gcagccaagt aaacccctac caactggtcg gggtttgaaa
     4501 ctttttttga attctgatgc ggtggcggcc gcaggaaccc ctagtgatgg agttggccac
     4561 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     4621 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct
     4681 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa
     4741 ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     4801 gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt
     4861 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     4921 cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt
     4981 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     5041 aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt
     5101 gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa
     5161 aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt
     5221 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     5281 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     5341 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     5401 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     5461 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     5521 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     5581 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     5641 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     5701 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     5761 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     5821 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     5881 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     5941 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     6001 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     6061 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     6121 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     6181 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     6241 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     6301 cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     6361 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     6421 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     6481 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     6541 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     6601 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     6661 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     6721 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     6781 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     6841 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     6901 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     6961 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     7021 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     7081 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     7141 gggtttcgcc acctctgact tgagcgtcg
//
