LOCUS       sequence-388531-        5744 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR tool to target astrocytes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5744)
  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 5744)
  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
FEATURES             Location/Qualifiers
     source          1..5744
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(441..461)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(482..500)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(543..562)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          545..677
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     misc_RNA        complement(1516..1591)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1610..1858)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1668..1687)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1838..1858)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   1873..2002
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(2064..2652)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2144..2163)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2823..3614,3615..3683))
                     /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(3684..3788)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3856..3874
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3912..3934)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4034..4053
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(4070..4525)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4207..4228)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4419..4438
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4652..4674
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4666..4683
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4667..4683
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   4702..4831
                     /label=AAV2 ITR
     polyA_signal    4993..5114
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5030..5049)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5084..5103
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 agggatctga cctggtcgaa atcagtccgc tcaaaagcga gagcctgctt agccctctca
       61 cccgcatcta cgttttcctt tctaattctc ctcataacaa gggagactgc gtttgaatcg
      121 ctaggccggg ggagcccgct gcgtcgatgc agcatattca gttggcccaa gtgctgctgg
      181 atagtcttga cagccagccc tcttgcctgg agatacagca ggtaatcgcg cacgtcctcc
      241 ggctcggcgg ggaaccattt gcgattgttc agcttacacc aggctgccca ggatcggcac
      301 acgctaagca gcattttcca ggtgtgttca gaaaacgcct gtctatccct aaacatatcc
      361 atgagatttt tcctgacctc atcgctagtg gcatctactg gcagcgcagg gaggttctgg
      421 tgaacagtca ggagattgga caccttcctc ttcttcttgg gcatggtggc ggtaccgcta
      481 gcctatagtg agtcgtatta agtactctag ccttaagagc tgtaattgaa ctgggagtgg
      541 acacctgtgg agagaaaggc aaagtggatg tcagtaagac caataggtgc ctatcagaaa
      601 cgcaagagtc ttctctgtct cgacaagccc agtttctatt ggtctcctta aacctgtctt
      661 gtaaccttga tacttacctg cccagtgcct cacgaccaac ttctgcagct taagttcgag
      721 actgttgtgt cagaagcact gactgcgtta gcaatttaac tgtgataaac taccgcaata
      781 aagcttgcga gcagcggagg tgatgcgtct cctctccaac ctgctctggc tctgctcgct
      841 cctgggatgc gagggcttta tgaaggagtg ggctagactg gcgatgcccg ggtgcccctg
      901 gcaacacccc ctggcatagc ctgagggggt ggggttgggc cgcagcccag ctatggggag
      961 agctctcccc tcacccattt gtgtccttgg acttctggga agcagacagc cctcctctcc
     1021 ccccctgcca cccactgtcc atgcctgggc atcccctgat cccctttcct gggcacaggc
     1081 tgaatagagc cttgttctcc accgccggtg gcgccacaca gcaagggcag cccctagggg
     1141 gctgtgctgc ttttatccca agatgccagg ctgtcaggct ggggtgcaat gcaagccccc
     1201 tgctcaatgg gcttctcgga aggcatttga ggggggctgg ggtgcggcgg gaagcaggca
     1261 ctgtgcccac cccgaacatt gtgtctgtgc caaggtgagt cattcactgg gcatgaagag
     1321 gaggccctgg ggggcggcca gcccaatgct gccaggtctg cttgcagaca aggcctggct
     1381 gtctgcctcc tccccagctc acagagccag ctcaggcccc cgagcctctg tcagagcagc
     1441 gtcccctact ccacaccagg atatgttaga tctccctctg cagtctagag tcgacttgta
     1501 gatggatcca aaaaagcacc gactcggtgc cactttttca agttgataac ggactagcct
     1561 tattttaact tgctatttct agctctaaaa cagaagagct cgctcttccg gtgtttcgtc
     1621 ctttccacaa gatatataaa gccaagaaat cgaaatactt tcaagttacg gtaagcatat
     1681 gatagtccat tttaaaacat aattttaaaa ctgcaaacta cccaagaaat tattactttc
     1741 tacgtcacgt attttgtact aatatctttg tgtttacagt caaattaatt ccaattatct
     1801 ctctaacagc cttgtatcgt atatgcaaat atgaaggaat catgggaaat aggccctcac
     1861 gcgtgcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct
     1921 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggcggcctc agtgagcgag
     1981 cgagcgcgca gctgcctgca ggacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     2041 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     2101 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     2161 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     2221 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     2281 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     2341 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     2401 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     2461 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     2521 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     2581 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     2641 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     2701 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     2761 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     2821 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     2881 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     2941 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     3001 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     3061 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     3121 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     3181 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     3241 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     3301 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     3361 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     3421 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     3481 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     3541 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     3601 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     3661 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     3721 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     3781 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg
     3841 acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat
     3901 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
     3961 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
     4021 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccataa aattgtaaac
     4081 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     4141 tagaccgaaa tcggcaaaat cccttataaa tcaaaagaat agcccgagat agagttgagt
     4201 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     4261 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     4321 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     4381 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     4441 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     4501 cttaatgcgc cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa
     4561 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca
     4621 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc
     4681 agtgaattag gttaattaag gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag
     4741 cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag
     4801 ggagtggcca actccatcac taggggttcc ttgtagttaa tgattaaccc gccatgctac
     4861 ttatctacgt agccatgctc taggaagatc cttatcgatt ttaccacatt tgtagaggtt
     4921 ttacttgctt taaaaaacct cccacatctc cccctgaacc tgaaacataa aatgaatgca
     4981 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     5041 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     5101 atcaatgtat cttatcatgt ctgctcgaag cggccgcaag cttgatatcg aattcctagt
     5161 caccatcttc gagcagtctc accatggccc cggtttcaga gtcaagattg cggatatagt
     5221 tcataacgat gttcacattt gtccaacctc cagcttgcat aatctctggt atggagactc
     5281 ctgcccgcgc catgtctcgt gcggccccca ccctagcaga atgacctgac caggcgagat
     5341 acctctggcc actgtcatcc ttggctccgt atatgagtcg gtgggttgcc tcaaagatcc
     5401 cttcaagggc ccgtgtactc aattggctgg tggcagaggg cgcggcgact ccgttttttc
     5461 tcactcgaca gaacaagtaa ttattagggt cgtcagccac gccggacact gagatccatc
     5521 tttccaccag cttcgttacg cccagggaca aggccttttc aacgccagcg gtgctgacca
     5581 atgtctttgt gcgtccaata tgaataagca tccggccgcc gtccgttcga ctgatgtctt
     5641 tcactctgat ccgggcgatc tcagctatgc gcagaagtgt gttatatgca attcccagaa
     5701 atgcgaggtt cctgatgtcc tggcaccgat cgctgttctc catc
//
