LOCUS       sequence-430686-        6332 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 6332)
  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 6332)
  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: GEARBOCS
FEATURES             Location/Qualifiers
     source          1..6332
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        145..393
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     145..165
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     316..335
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        412..487
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     intron          1326..1458
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1441..1460
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1503..1521
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1542..1562
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3221..3342
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3232..3251)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3286..3305
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3504..3633
                     /label=AAV2 ITR
     primer_bind     complement(3652..3669)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3652..3668)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3661..3683)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3810..4265
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3897..3916)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4107..4128
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4282..4301)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4401..4423
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4461..4479)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4547..4651
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4652..4720,4721..5512)
                     /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      5683..6271
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6172..6191
                     /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 gcggccgcac gcgtgagggc ctatttccca tgattccttc atatttgcat
      181 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
      241 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      301 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      361 gctttatata tcttgtggaa aggacgaaac accggaagag cgagctcttc tgttttagag
      421 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
      481 tcggtgcttt tttggatcca tctacaagtc gactctagac tgcagaggga gatctaacat
      541 atcctggtgt ggagtagggg acgctgctct gacagaggct cgggggcctg agctggctct
      601 gtgagctggg gaggaggcag acagccaggc cttgtctgca agcagacctg gcagcattgg
      661 gctggccgcc ccccagggcc tcctcttcat gcccagtgaa tgactcacct tggcacagac
      721 acaatgttcg gggtgggcac agtgcctgct tcccgccgca ccccagcccc cctcaaatgc
      781 cttccgagaa gcccattgag cagggggctt gcattgcacc ccagcctgac agcctggcat
      841 cttgggataa aagcagcaca gccccctagg ggctgccctt gctgtgtggc gccaccggcg
      901 gtggagaaca aggctctatt cagcctgtgc ccaggaaagg ggatcagggg atgcccaggc
      961 atggacagtg ggtggcaggg ggggagagga gggctgtctg cttcccagaa gtccaaggac
     1021 acaaatgggt gaggggagag ctctccccat agctgggctg cggcccaacc ccaccccctc
     1081 aggctatgcc agggggtgtt gccaggggca cccgggcatc gccagtctag cccactcctt
     1141 cataaagccc tcgcatccca ggagcgagca gagccagagc aggttggaga ggagacgcat
     1201 cacctccgct gctcgcaagc tttattgcgg tagtttatca cagttaaatt gctaacgcag
     1261 tcagtgcttc tgacacaaca gtctcgaact taagctgcag aagttggtcg tgaggcactg
     1321 ggcaggtaag tatcaaggtt acaagacagg tttaaggaga ccaatagaaa ctgggcttgt
     1381 cgagacagag aagactcttg cgtttctgat aggcacctat tggtcttact gacatccact
     1441 ttgcctttct ctccacaggt gtccactccc agttcaatta cagctcttaa ggctagagta
     1501 cttaatacga ctcactatag gctagcggta ccgccaccat gcccaagaag aagaggaagg
     1561 tgtccaatct cctgactgtt caccagaacc tccctgcgct gccagtagat gccactagcg
     1621 atgaggtcag gaaaaatctc atggatatgt ttagggatag acaggcgttt tctgaacaca
     1681 cctggaaaat gctgcttagc gtgtgccgat cctgggcagc ctggtgtaag ctgaacaatc
     1741 gcaaatggtt ccccgccgag ccggaggacg tgcgcgatta cctgctgtat ctccaggcaa
     1801 gagggctggc tgtcaagact atccagcagc acttgggcca actgaatatg ctgcatcgac
     1861 gcagcgggct cccccggcct agcgattcaa acgcagtctc ccttgttatg aggagaatta
     1921 gaaaggaaaa cgtagatgcg ggtgagaggg ctaagcaggc tctcgctttt gagcggactg
     1981 atttcgacca ggtcagatcc ctgatggaga acagcgatcg gtgccaggac atcaggaacc
     2041 tcgcatttct gggaattgca tataacacac ttctgcgcat agctgagatc gcccggatca
     2101 gagtgaaaga catcagtcga acggacggcg gccggatgct tattcatatt ggacgcacaa
     2161 agacattggt cagcaccgct ggcgttgaaa aggccttgtc cctgggcgta acgaagctgg
     2221 tggaaagatg gatctcagtg tccggcgtgg ctgacgaccc taataattac ttgttctgtc
     2281 gagtgagaaa aaacggagtc gccgcgccct ctgccaccag ccaattgagt acacgggccc
     2341 ttgaagggat ctttgaggca acccaccgac tcatatacgg agccaaggat gacagtggcc
     2401 agaggtatct cgcctggtca ggtcattctg ctagggtggg ggccgcacga gacatggcgc
     2461 gggcaggagt ctccatacca gagattatgc aagctggagg ttggacaaat gtgaacatcg
     2521 ttatgaacta tatccgcaat cttgactctg aaaccggggc catggtgaga ctgctcgaag
     2581 atggtgacta ggaattcgat atctgctgga agatggcgat tagcatatgg gcattcaccg
     2641 cgtgccttac tacgcgtatt cttaagcaat aagcgaatat aacacggtcg atctaaaaag
     2701 gttagctggg tgtgttagaa gccctgcaag acgacctgcg cattattact cacggtacga
     2761 cgcattatta ctcacggtac gaggcattca ccgcgtgcct tactacgcgt attcttaagc
     2821 aataagcgaa tataacacgg tcgatctaaa aaggttagct gggtgtgtta gaagccctgc
     2881 aagacgacct gggcattcac cgcgtgcctt actacgcgta ttcttaagca ataagcgaat
     2941 ataacacggt cgatctaaaa aggttagctg ggtgtgttag aagccctgca agacgacctg
     3001 ggtaccgcat tattactcac ggtacgacgc attattactc acggtacgag gcattcaccg
     3061 cgtgccttac tacgcgtatt cttaagcaat aagcgaatat aacacggtcg atctaaaaag
     3121 gttagctggg tgtgttagaa gccctgcaag acgacctggg taccaggtaa agcggccgcg
     3181 actctgatat caagcttgcg gccgcttcga gcagacatga taagatacat tgatgagttt
     3241 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     3301 attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt
     3361 cattttatgt ttcaggttca gggggagatg tgggaggttt tttaaagcaa gtaaaacctc
     3421 tacaaatgtg gtaaaatcga taaggatctt cctagagcat ggctacgtag ataagtagca
     3481 tggcgggtta atcattaact acaaggaacc cctagtgatg gagttggcca ctccctctct
     3541 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc
     3601 ccgggcggcc tcagtgagcg agcgagcgcg cagccttaat taacctaatt cactggccgt
     3661 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
     3721 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     3781 acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     3841 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     3901 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3961 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     4021 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     4081 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     4141 ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt
     4201 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
     4261 aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     4321 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     4381 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     4441 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     4501 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     4561 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     4621 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     4681 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     4741 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     4801 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     4861 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     4921 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     4981 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     5041 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     5101 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     5161 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     5221 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     5281 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     5341 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     5401 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     5461 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     5521 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     5581 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     5641 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     5701 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     5761 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     5821 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     5881 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     5941 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     6001 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     6061 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     6121 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     6181 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     6241 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     6301 cctggccttt tgctggcctt ttgctcacat gt
//
