LOCUS       sequence-388595-        6494 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To tag endogenous mouse GFAP with mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6494)
  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 6494)
  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-mCherry
FEATURES             Location/Qualifiers
     source          1..6494
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(443..463)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(484..502)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(545..564)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          547..679
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     CDS             complement(1523..2227)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(1798..1817)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2067..2085
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     misc_RNA        complement(2265..2340)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(2362..2610)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2420..2439)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2590..2610)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   2625..2754
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(2816..3404)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2896..2915)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3575..4366,4367..4435))
                     /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(4436..4540)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4608..4626
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4664..4686)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4786..4805
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(4822..5277)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4959..4980)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5171..5190
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5404..5426
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5418..5435
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5419..5435
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   5454..5583
                     /label=AAV2 ITR
     polyA_signal    5745..5866
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5782..5801)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5836..5855
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tcagggatct gacctggtcg aaatcagtcc gctcaaaagc gagagcctgc ttagccctct
       61 cacccgcatc tacgttttcc tttctaattc tcctcataac aagggagact gcgtttgaat
      121 cgctaggccg ggggagcccg ctgcgtcgat gcagcatatt cagttggccc aagtgctgct
      181 ggatagtctt gacagccagc cctcttgcct ggagatacag caggtaatcg cgcacgtcct
      241 ccggctcggc ggggaaccat ttgcgattgt tcagcttaca ccaggctgcc caggatcggc
      301 acacgctaag cagcattttc caggtgtgtt cagaaaacgc ctgtctatcc ctaaacatat
      361 ccatgagatt tttcctgacc tcatcgctag tggcatctac tggcagcgca gggaggttct
      421 ggtgaacagt caggagattg gacaccttcc tcttcttctt gggcatggtg gcggtaccgc
      481 tagcctatag tgagtcgtat taagtactct agccttaaga gctgtaattg aactgggagt
      541 ggacacctgt ggagagaaag gcaaagtgga tgtcagtaag accaataggt gcctatcaga
      601 aacgcaagag tcttctctgt ctcgacaagc ccagtttcta ttggtctcct taaacctgtc
      661 ttgtaacctt gatacttacc tgcccagtgc ctcacgacca acttctgcag cttaagttcg
      721 agactgttgt gtcagaagca ctgactgcgt tagcaattta actgtgataa actaccgcaa
      781 taaagcttgc gagcagcgga ggtgatgcgt ctcctctcca acctgctctg gctctgctcg
      841 ctcctgggat gcgagggctt tatgaaggag tgggctagac tggcgatgcc cgggtgcccc
      901 tggcaacacc ccctggcata gcctgagggg gtggggttgg gccgcagccc agctatgggg
      961 agagctctcc cctcacccat ttgtgtcctt ggacttctgg gaagcagaca gccctcctct
     1021 ccccccctgc cacccactgt ccatgcctgg gcatcccctg atcccctttc ctgggcacag
     1081 gctgaataga gccttgttct ccaccgccgg tggcgccaca cagcaagggc agcccctagg
     1141 gggctgtgct gcttttatcc caagatgcca ggctgtcagg ctggggtgca atgcaagccc
     1201 cctgctcaat gggcttctcg gaaggcattt gaggggggct ggggtgcggc gggaagcagg
     1261 cactgtgccc accccgaaca ttgtgtctgt gccaaggtga gtcattcact gggcatgaag
     1321 aggaggccct ggggggcggc cagcccaatg ctgccaggtc tgcttgcaga caaggcctgg
     1381 ctgtctgcct cctccccagc tcacagagcc agctcaggcc cccgagcctc tgtcagagca
     1441 gcgtccccta ctccacacca ggatatgtta gatctccctc tgcagtctag agtcgacccg
     1501 agacggtggt caggggcctc atcttgtaca gctcgtccat gccgccggtg gagtggcggc
     1561 cctcggcgcg ttcgtactgt tccacgatgg tgtagtcctc gttgtgggag gtgatgtcca
     1621 acttgatgtt gacgttgtag gcgccgggca gctgcacggg cttcttggcc ttgtaggtgg
     1681 tcttgacctc agcgtcgtag tggccgccgt ccttcagctt cagcctctgc ttgatctcgc
     1741 ccttcagggc gccgtcctcg gggtacatcc gctcggagga ggcctcccag cccatggtct
     1801 tcttctgcat tacggggccg tcggagggga agttggtgcc gcgcagcttc accttgtaga
     1861 tgaactcgcc gtcctgcagg gaggagtcct gggtcacggt caccacgccg ccgtcctcga
     1921 agttcatcac gcgctcccac ttgaagccct cggggaagga cagcttcaag tagtcgggga
     1981 tgtcggcggg gtgcttcacg taggccttgg agccgtacat gaactgaggg gacaggatgt
     2041 cccaggcgaa gggcaggggg ccacccttgg tcaccttcag cttggcggtc tgggtgccct
     2101 cgtaggggcg gccctcgccc tcgccctcga tctcgaactc gtggccgttc acggagccct
     2161 ccatgtgcac cttgaagcgc atgaactcct tgatgatggc catgttatcc tcctcgccct
     2221 tgctcacatc cgagacggtg gtcaggggcc tcggatccaa aaaagcaccg actcggtgcc
     2281 actttttcaa gttgataacg gactagcctt attttaactt gctatttcta gctctaaaac
     2341 agacggtggt caggggcctc cggtgtttcg tcctttccac aagatatata aagccaagaa
     2401 atcgaaatac tttcaagtta cggtaagcat atgatagtcc attttaaaac ataattttaa
     2461 aactgcaaac tacccaagaa attattactt tctacgtcac gtattttgta ctaatatctt
     2521 tgtgtttaca gtcaaattaa ttccaattat ctctctaaca gccttgtatc gtatatgcaa
     2581 atatgaagga atcatgggaa ataggccctc acgcgtgcgg ccgcaggaac ccctagtgat
     2641 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
     2701 cgcccgacgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggacatgt
     2761 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     2821 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     2881 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     2941 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3001 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3061 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3121 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3181 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3241 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3301 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3361 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     3421 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     3481 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     3541 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     3601 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     3661 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     3721 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     3781 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     3841 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     3901 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     3961 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4021 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4081 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4141 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     4201 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4261 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4321 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4381 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     4441 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     4501 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     4561 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     4621 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     4681 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     4741 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga
     4801 ttgtactgag agtgcaccat aaaattgtaa acgttaatat tttgttaaaa ttcgcgttaa
     4861 atttttgtta aatcagctca ttttttaacc aatagaccga aatcggcaaa atcccttata
     4921 aatcaaaaga atagcccgag atagagttga gtgttgttcc agtttggaac aagagtccac
     4981 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     5041 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     5101 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     5161 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     5221 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc
     5281 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     5341 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
     5401 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt aggttaatta aggctgcgcg
     5461 ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc
     5521 cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt
     5581 ccttgtagtt aatgattaac ccgccatgct acttatctac gtagccatgc tctaggaaga
     5641 tccttatcga ttttaccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacatc
     5701 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
     5761 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     5821 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgctcga
     5881 agcggccgca agcttgatat cgaattccta gtcaccatct tcgagcagtc tcaccatggc
     5941 cccggtttca gagtcaagat tgcggatata gttcataacg atgttcacat ttgtccaacc
     6001 tccagcttgc ataatctctg gtatggagac tcctgcccgc gccatgtctc gtgcggcccc
     6061 caccctagca gaatgacctg accaggcgag atacctctgg ccactgtcat ccttggctcc
     6121 gtatatgagt cggtgggttg cctcaaagat cccttcaagg gcccgtgtac tcaattggct
     6181 ggtggcagag ggcgcggcga ctccgttttt tctcactcga cagaacaagt aattattagg
     6241 gtcgtcagcc acgccggaca ctgagatcca tctttccacc agcttcgtta cgcccaggga
     6301 caaggccttt tcaacgccag cggtgctgac caatgtcttt gtgcgtccaa tatgaataag
     6361 catccggccg ccgtccgttc gactgatgtc tttcactctg atccgggcga tctcagctat
     6421 gcgcagaagt gtgttatatg caattcccag aaatgcgagg ttcctgatgt cctggcaccg
     6481 atcgctgttc tcca
//
