LOCUS       sequence-388600-        5816 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To tag endogenous mouse GFAP with HA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5816)
  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 5816)
  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- TagIN-HA
FEATURES             Location/Qualifiers
     source          1..5816
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(512..532)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(553..571)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(614..633)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          616..748
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     CDS             complement(1592..1618)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_RNA        complement(1656..1731)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1753..2001)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1811..1830)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1981..2001)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   2016..2145
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(2207..2795)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2287..2306)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2966..3757,3758..3826))
                     /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(3827..3931)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3999..4017
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4055..4077)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4177..4196
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(4213..4668)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4350..4371)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4562..4581
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4795..4817
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4809..4826
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4810..4826
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   4845..4974
                     /label=AAV2 ITR
     polyA_signal    5136..5257
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5173..5192)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5227..5246
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 gaagtgtgtt atatgcaatt cccagaaatg cgaggttcct gatgtcctgg caccgatcgc
       61 tgttctccat cagggatctg acctggtcga aatcagtccg ctcaaaagcg agagcctgct
      121 tagccctctc acccgcatct acgttttcct ttctaattct cctcataaca agggagactg
      181 cgtttgaatc gctaggccgg gggagcccgc tgcgtcgatg cagcatattc agttggccca
      241 agtgctgctg gatagtcttg acagccagcc ctcttgcctg gagatacagc aggtaatcgc
      301 gcacgtcctc cggctcggcg gggaaccatt tgcgattgtt cagcttacac caggctgccc
      361 aggatcggca cacgctaagc agcattttcc aggtgtgttc agaaaacgcc tgtctatccc
      421 taaacatatc catgagattt ttcctgacct catcgctagt ggcatctact ggcagcgcag
      481 ggaggttctg gtgaacagtc aggagattgg acaccttcct cttcttcttg ggcatggtgg
      541 cggtaccgct agcctatagt gagtcgtatt aagtactcta gccttaagag ctgtaattga
      601 actgggagtg gacacctgtg gagagaaagg caaagtggat gtcagtaaga ccaataggtg
      661 cctatcagaa acgcaagagt cttctctgtc tcgacaagcc cagtttctat tggtctcctt
      721 aaacctgtct tgtaaccttg atacttacct gcccagtgcc tcacgaccaa cttctgcagc
      781 ttaagttcga gactgttgtg tcagaagcac tgactgcgtt agcaatttaa ctgtgataaa
      841 ctaccgcaat aaagcttgcg agcagcggag gtgatgcgtc tcctctccaa cctgctctgg
      901 ctctgctcgc tcctgggatg cgagggcttt atgaaggagt gggctagact ggcgatgccc
      961 gggtgcccct ggcaacaccc cctggcatag cctgaggggg tggggttggg ccgcagccca
     1021 gctatgggga gagctctccc ctcacccatt tgtgtccttg gacttctggg aagcagacag
     1081 ccctcctctc cccccctgcc acccactgtc catgcctggg catcccctga tcccctttcc
     1141 tgggcacagg ctgaatagag ccttgttctc caccgccggt ggcgccacac agcaagggca
     1201 gcccctaggg ggctgtgctg cttttatccc aagatgccag gctgtcaggc tggggtgcaa
     1261 tgcaagcccc ctgctcaatg ggcttctcgg aaggcatttg aggggggctg gggtgcggcg
     1321 ggaagcaggc actgtgccca ccccgaacat tgtgtctgtg ccaaggtgag tcattcactg
     1381 ggcatgaaga ggaggccctg gggggcggcc agcccaatgc tgccaggtct gcttgcagac
     1441 aaggcctggc tgtctgcctc ctccccagct cacagagcca gctcaggccc ccgagcctct
     1501 gtcagagcag cgtcccctac tccacaccag gatatgttag atctccctct gcagtctaga
     1561 gtcgacccct gccagctctc ccttagatat gagcgtagtc tggaacgtcg taagggtaat
     1621 ccctgccagc tctcccttag ataggatcca aaaaagcacc gactcggtgc cactttttca
     1681 agttgataac ggactagcct tattttaact tgctatttct agctctaaaa ctgccagctc
     1741 tcccttagat acggtgtttc gtcctttcca caagatatat aaagccaaga aatcgaaata
     1801 ctttcaagtt acggtaagca tatgatagtc cattttaaaa cataatttta aaactgcaaa
     1861 ctacccaaga aattattact ttctacgtca cgtattttgt actaatatct ttgtgtttac
     1921 agtcaaatta attccaatta tctctctaac agccttgtat cgtatatgca aatatgaagg
     1981 aatcatggga aataggccct cacgcgtgcg gccgcaggaa cccctagtga tggagttggc
     2041 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     2101 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggacatg tgagcaaaag
     2161 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     2221 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     2281 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     2341 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     2401 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     2461 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     2521 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     2581 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     2641 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     2701 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     2761 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     2821 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     2881 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     2941 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     3001 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     3061 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     3121 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     3181 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     3241 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     3301 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     3361 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     3421 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     3481 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     3541 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     3601 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     3661 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     3721 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     3781 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     3841 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     3901 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     3961 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
     4021 ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
     4081 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
     4141 cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga
     4201 gagtgcacca taaaattgta aacgttaata ttttgttaaa attcgcgtta aatttttgtt
     4261 aaatcagctc attttttaac caatagaccg aaatcggcaa aatcccttat aaatcaaaag
     4321 aatagcccga gatagagttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     4381 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     4441 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     4501 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     4561 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     4621 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat
     4681 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
     4741 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt
     4801 cacgacgttg taaaacgacg gccagtgaat taggttaatt aaggctgcgc gctcgctcgc
     4861 tcactgaggc cgcccgggca aagcccgggc gtcgggcgac ctttggtcgc ccggcctcag
     4921 tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt tccttgtagt
     4981 taatgattaa cccgccatgc tacttatcta cgtagccatg ctctaggaag atccttatcg
     5041 attttaccac atttgtagag gttttacttg ctttaaaaaa cctcccacat ctccccctga
     5101 acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg
     5161 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     5221 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgctcg aagcggccgc
     5281 aagcttgata tcgaattcct agtcaccatc ttcgagcagt ctcaccatgg ccccggtttc
     5341 agagtcaaga ttgcggatat agttcataac gatgttcaca tttgtccaac ctccagcttg
     5401 cataatctct ggtatggaga ctcctgcccg cgccatgtct cgtgcggccc ccaccctagc
     5461 agaatgacct gaccaggcga gatacctctg gccactgtca tccttggctc cgtatatgag
     5521 tcggtgggtt gcctcaaaga tcccttcaag ggcccgtgta ctcaattggc tggtggcaga
     5581 gggcgcggcg actccgtttt ttctcactcg acagaacaag taattattag ggtcgtcagc
     5641 cacgccggac actgagatcc atctttccac cagcttcgtt acgcccaggg acaaggcctt
     5701 ttcaacgcca gcggtgctga ccaatgtctt tgtgcgtcca atatgaataa gcatccggcc
     5761 gccgtccgtt cgactgatgt ctttcactct gatccgggcg atctcagcta tgcgca
//
