LOCUS       sequence-430690-        6335 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To KO Hevin in astrocytes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6335)
  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 6335)
  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-SPARCL1 KO
FEATURES             Location/Qualifiers
     source          1..6335
                     /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        415..490
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     intron          1329..1461
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1444..1463
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1506..1524
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1545..1565
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3224..3345
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3235..3254)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3289..3308
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3507..3636
                     /label=AAV2 ITR
     primer_bind     complement(3655..3672)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3655..3671)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3664..3686)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3813..4268
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3900..3919)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4110..4131
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4285..4304)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4404..4426
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4464..4482)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4550..4654
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4655..4723,4724..5515)
                     /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      5686..6274
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6175..6194
                     /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 accggtgcgc cttgggaacc gctggtttta
      421 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
      481 gagtcggtgc ttttttggat ccatctacaa gtcgactcta gactgcagag ggagatctaa
      541 catatcctgg tgtggagtag gggacgctgc tctgacagag gctcgggggc ctgagctggc
      601 tctgtgagct ggggaggagg cagacagcca ggccttgtct gcaagcagac ctggcagcat
      661 tgggctggcc gccccccagg gcctcctctt catgcccagt gaatgactca ccttggcaca
      721 gacacaatgt tcggggtggg cacagtgcct gcttcccgcc gcaccccagc ccccctcaaa
      781 tgccttccga gaagcccatt gagcaggggg cttgcattgc accccagcct gacagcctgg
      841 catcttggga taaaagcagc acagccccct aggggctgcc cttgctgtgt ggcgccaccg
      901 gcggtggaga acaaggctct attcagcctg tgcccaggaa aggggatcag gggatgccca
      961 ggcatggaca gtgggtggca gggggggaga ggagggctgt ctgcttccca gaagtccaag
     1021 gacacaaatg ggtgagggga gagctctccc catagctggg ctgcggccca accccacccc
     1081 ctcaggctat gccagggggt gttgccaggg gcacccgggc atcgccagtc tagcccactc
     1141 cttcataaag ccctcgcatc ccaggagcga gcagagccag agcaggttgg agaggagacg
     1201 catcacctcc gctgctcgca agctttattg cggtagttta tcacagttaa attgctaacg
     1261 cagtcagtgc ttctgacaca acagtctcga acttaagctg cagaagttgg tcgtgaggca
     1321 ctgggcaggt aagtatcaag gttacaagac aggtttaagg agaccaatag aaactgggct
     1381 tgtcgagaca gagaagactc ttgcgtttct gataggcacc tattggtctt actgacatcc
     1441 actttgcctt tctctccaca ggtgtccact cccagttcaa ttacagctct taaggctaga
     1501 gtacttaata cgactcacta taggctagcg gtaccgccac catgcccaag aagaagagga
     1561 aggtgtccaa tctcctgact gttcaccaga acctccctgc gctgccagta gatgccacta
     1621 gcgatgaggt caggaaaaat ctcatggata tgtttaggga tagacaggcg ttttctgaac
     1681 acacctggaa aatgctgctt agcgtgtgcc gatcctgggc agcctggtgt aagctgaaca
     1741 atcgcaaatg gttccccgcc gagccggagg acgtgcgcga ttacctgctg tatctccagg
     1801 caagagggct ggctgtcaag actatccagc agcacttggg ccaactgaat atgctgcatc
     1861 gacgcagcgg gctcccccgg cctagcgatt caaacgcagt ctcccttgtt atgaggagaa
     1921 ttagaaagga aaacgtagat gcgggtgaga gggctaagca ggctctcgct tttgagcgga
     1981 ctgatttcga ccaggtcaga tccctgatgg agaacagcga tcggtgccag gacatcagga
     2041 acctcgcatt tctgggaatt gcatataaca cacttctgcg catagctgag atcgcccgga
     2101 tcagagtgaa agacatcagt cgaacggacg gcggccggat gcttattcat attggacgca
     2161 caaagacatt ggtcagcacc gctggcgttg aaaaggcctt gtccctgggc gtaacgaagc
     2221 tggtggaaag atggatctca gtgtccggcg tggctgacga ccctaataat tacttgttct
     2281 gtcgagtgag aaaaaacgga gtcgccgcgc cctctgccac cagccaattg agtacacggg
     2341 cccttgaagg gatctttgag gcaacccacc gactcatata cggagccaag gatgacagtg
     2401 gccagaggta tctcgcctgg tcaggtcatt ctgctagggt gggggccgca cgagacatgg
     2461 cgcgggcagg agtctccata ccagagatta tgcaagctgg aggttggaca aatgtgaaca
     2521 tcgttatgaa ctatatccgc aatcttgact ctgaaaccgg ggccatggtg agactgctcg
     2581 aagatggtga ctaggaattc gatatctgct ggaagatggc gattagcata tgggcattca
     2641 ccgcgtgcct tactacgcgt attcttaagc aataagcgaa tataacacgg tcgatctaaa
     2701 aaggttagct gggtgtgtta gaagccctgc aagacgacct gcgcattatt actcacggta
     2761 cgacgcatta ttactcacgg tacgaggcat tcaccgcgtg ccttactacg cgtattctta
     2821 agcaataagc gaatataaca cggtcgatct aaaaaggtta gctgggtgtg ttagaagccc
     2881 tgcaagacga cctgggcatt caccgcgtgc cttactacgc gtattcttaa gcaataagcg
     2941 aatataacac ggtcgatcta aaaaggttag ctgggtgtgt tagaagccct gcaagacgac
     3001 ctgggtaccg cattattact cacggtacga cgcattatta ctcacggtac gaggcattca
     3061 ccgcgtgcct tactacgcgt attcttaagc aataagcgaa tataacacgg tcgatctaaa
     3121 aaggttagct gggtgtgtta gaagccctgc aagacgacct gggtaccagg taaagcggcc
     3181 gcgactctga tatcaagctt gcggccgctt cgagcagaca tgataagata cattgatgag
     3241 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     3301 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     3361 attcatttta tgtttcaggt tcagggggag atgtgggagg ttttttaaag caagtaaaac
     3421 ctctacaaat gtggtaaaat cgataaggat cttcctagag catggctacg tagataagta
     3481 gcatggcggg ttaatcatta actacaagga acccctagtg atggagttgg ccactccctc
     3541 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
     3601 tgcccgggcg gcctcagtga gcgagcgagc gcgcagcctt aattaaccta attcactggc
     3661 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     3721 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     3781 ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     3841 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     3901 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     3961 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     4021 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     4081 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     4141 caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg
     4201 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt
     4261 tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     4321 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     4381 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     4441 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     4501 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     4561 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     4621 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     4681 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     4741 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     4801 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     4861 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     4921 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     4981 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     5041 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     5101 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     5161 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     5221 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     5281 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     5341 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     5401 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     5461 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     5521 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     5581 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     5641 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     5701 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     5761 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     5821 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     5881 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     5941 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     6001 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     6061 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     6121 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     6181 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     6241 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     6301 gttcctggcc ttttgctggc cttttgctca catgt
//
