LOCUS       sequence-381233-        5747 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To KO Gfap in mouse astrocytes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5747)
  AUTHORS   Bindu DS, Tan CX, Savage JT, Eroglu C
  TITLE     GEARBOCS: An Adeno Associated Virus Tool for In Vivo Gene Editing in
            Astrocytes.
  JOURNAL   bioRxiv. 2023 Jan 19:2023.01.17.524433. doi: 
            10.1101/2023.01.17.524433. Preprint.
  PUBMED    36711516
REFERENCE   2  (bases 1 to 5747)
  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: "bioRxiv. 
            2023 Jan 19:2023.01.17.524433. doi: 10.1101/2023.01.17.524433. 
            Preprint."
COMMENT     Sequence Label: pGEARBOCS-v0-Gfap-KO
FEATURES             Location/Qualifiers
     source          1..5747
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     7..26
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(1108..1128)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(1149..1167)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1210..1229)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          1212..1344
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     misc_RNA        complement(2183..2258)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(2280..2528)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2338..2357)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2508..2528)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   2543..2672
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(2734..3322)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2814..2833)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3493..4284,4285..4353))
                     /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"
     promoter        complement(4354..4458)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4526..4544
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4582..4604)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4704..4723
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(4740..5195)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4877..4898)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5089..5108
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5322..5344
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5336..5353
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5337..5353
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   5372..5501
                     /label=AAV2 ITR
     polyA_signal    join(5663..5747,1..37)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5700..5719)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
ORIGIN
        1 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgctcg aagcggccgc
       61 aagcttgata tcgaattcct agtcaccatc ttcgagcagt ctcaccatgg ccccggtttc
      121 agagtcaaga ttgcggatat agttcataac gatgttcaca tttgtccaac ctccagcttg
      181 cataatctct ggtatggaga ctcctgcccg cgccatgtct cgtgcggccc ccaccctagc
      241 agaatgacct gaccaggcga gatacctctg gccactgtca tccttggctc cgtatatgag
      301 tcggtgggtt gcctcaaaga tcccttcaag ggcccgtgta ctcaattggc tggtggcaga
      361 gggcgcggcg actccgtttt ttctcactcg acagaacaag taattattag ggtcgtcagc
      421 cacgccggac actgagatcc atctttccac cagcttcgtt acgcccaggg acaaggcctt
      481 ttcaacgcca gcggtgctga ccaatgtctt tgtgcgtcca atatgaataa gcatccggcc
      541 gccgtccgtt cgactgatgt ctttcactct gatccgggcg atctcagcta tgcgcagaag
      601 tgtgttatat gcaattccca gaaatgcgag gttcctgatg tcctggcacc gatcgctgtt
      661 ctccatcagg gatctgacct ggtcgaaatc agtccgctca aaagcgagag cctgcttagc
      721 cctctcaccc gcatctacgt tttcctttct aattctcctc ataacaaggg agactgcgtt
      781 tgaatcgcta ggccggggga gcccgctgcg tcgatgcagc atattcagtt ggcccaagtg
      841 ctgctggata gtcttgacag ccagccctct tgcctggaga tacagcaggt aatcgcgcac
      901 gtcctccggc tcggcgggga accatttgcg attgttcagc ttacaccagg ctgcccagga
      961 tcggcacacg ctaagcagca ttttccaggt gtgttcagaa aacgcctgtc tatccctaaa
     1021 catatccatg agatttttcc tgacctcatc gctagtggca tctactggca gcgcagggag
     1081 gttctggtga acagtcagga gattggacac cttcctcttc ttcttgggca tggtggcggt
     1141 accgctagcc tatagtgagt cgtattaagt actctagcct taagagctgt aattgaactg
     1201 ggagtggaca cctgtggaga gaaaggcaaa gtggatgtca gtaagaccaa taggtgccta
     1261 tcagaaacgc aagagtcttc tctgtctcga caagcccagt ttctattggt ctccttaaac
     1321 ctgtcttgta accttgatac ttacctgccc agtgcctcac gaccaacttc tgcagcttaa
     1381 gttcgagact gttgtgtcag aagcactgac tgcgttagca atttaactgt gataaactac
     1441 cgcaataaag cttgcgagca gcggaggtga tgcgtctcct ctccaacctg ctctggctct
     1501 gctcgctcct gggatgcgag ggctttatga aggagtgggc tagactggcg atgcccgggt
     1561 gccctcggca acaccccccg gcatagcctg agggggtggg gttgggccgc agcccagcta
     1621 tggggagagc tctcccctca cccatttgtg tccttggact tctgggaagc agacagccct
     1681 cctctccccc cctgccaccc actgtccatg cctgggcatc ccctgatccc ctttcctggg
     1741 cacaggctga atagagcctt gttctccacc gccggtggcg ccacacagca agggcagccc
     1801 ctagggggct gtgctgcttt tatcccaaga tgccaggctg tcaggctggg gtgcaatgca
     1861 agccccctgc tcaatgggct tctcggaagg catttgaggg gggctggggt gcggcgggaa
     1921 gcaggcactg tgcccacccc gaacattgtg tctgtgccaa ggtgagtcat tcactgggca
     1981 tgaagaggag gccctggggg gcggccagcc caatgctgcc aggtctgctt gcagacaagg
     2041 cctggctgtc tgcctcctcc ccagctcaca gagccagctc aggcccccga gcctctgtca
     2101 gagcagcgtc ccctactcca caccaggata tgttagatct ccctctgcag tctagagtcg
     2161 acttgtagat ggatccaaaa aagcaccgac tcggtgccac tttttcaagt tgataacgga
     2221 ctagccttat tttaacttgc tatttctagc tctaaaacag acggtggtca ggggcctccg
     2281 gtgtttcgtc ctttccacaa gatatataaa gccaagaaat cgaaatactt tcaagttacg
     2341 gtaagcatat gatagtccat tttaaaacat aattttaaaa ctgcaaacta cccaagaaat
     2401 tattactttc tacgtcacgt attttgtact aatatctttg tgtttacagt caaattaatt
     2461 ccaattatct ctctaacagc cttgtatcgt atatgcaaat atgaaggaat catgggaaat
     2521 aggccctcac gcgtgcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg
     2581 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggcggcctc
     2641 agtgagcgag cgagcgcgca gctgcctgca ggacatgtga gcaaaaggcc agcaaaaggc
     2701 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     2761 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     2821 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     2881 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     2941 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     3001 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     3061 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     3121 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     3181 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     3241 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     3301 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     3361 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     3421 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     3481 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     3541 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     3601 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     3661 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     3721 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     3781 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     3841 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     3901 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     3961 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     4021 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     4081 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     4141 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     4201 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     4261 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     4321 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     4381 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     4441 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     4501 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg
     4561 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     4621 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     4681 gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccataa
     4741 aattgtaaac gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     4801 ttttaaccaa tagaccgaaa tcggcaaaat cccttataaa tcaaaagaat agcccgagat
     4861 agagttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     4921 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     4981 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     5041 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     5101 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     5161 acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa
     5221 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
     5281 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
     5341 aacgacggcc agtgaattag gttaattaag gctgcgcgct cgctcgctca ctgaggccgc
     5401 ccgggcaaag cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc
     5461 gcgcagagag ggagtggcca actccatcac taggggttcc ttgtagttaa tgattaaccc
     5521 gccatgctac ttatctacgt agccatgctc taggaagatc cttatcgatt ttaccacatt
     5581 tgtagaggtt ttacttgctt taaaaaacct cccacatctc cccctgaacc tgaaacataa
     5641 aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag
     5701 caatagcatc acaaatttca caaataaagc atttttttca ctgcatt
//
