LOCUS       sequence-388350-        6657 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To KO and genetrap mouse GFAP simultaneously.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6657)
  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 6657)
  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-GeneTRAP
FEATURES             Location/Qualifiers
     source          1..6657
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(427..447)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(468..486)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(529..548)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          531..663
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     polyA_signal    1507..1628
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(1670..2374)
                     /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(1945..1964)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2214..2232
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     misc_RNA        complement(2412..2487)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(2509..2757)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2567..2586)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2737..2757)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   2772..2901
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(2963..3551)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3043..3062)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3722..4513,4514..4582))
                     /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(4583..4687)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4755..4773
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4811..4833)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4933..4952
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(4969..5424)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5106..5127)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5318..5337
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5551..5573
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5565..5582
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5566..5582
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   5601..5730
                     /label=AAV2 ITR
     polyA_signal    5892..6013
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
ORIGIN
        1 gtcgaaatca gtccgctcaa aagcgagagc ctgcttagcc ctctcacccg catctacgtt
       61 ttcctttcta attctcctca taacaaggga gactgcgttt gaatcgctag gccgggggag
      121 cccgctgcgt cgatgcagca tattcagttg gcccaagtgc tgctggatag tcttgacagc
      181 cagccctctt gcctggagat acagcaggta atcgcgcacg tcctccggct cggcggggaa
      241 ccatttgcga ttgttcagct tacaccaggc tgcccaggat cggcacacgc taagcagcat
      301 tttccaggtg tgttcagaaa acgcctgtct atccctaaac atatccatga gatttttcct
      361 gacctcatcg ctagtggcat ctactggcag cgcagggagg ttctggtgaa cagtcaggag
      421 attggacacc ttcctcttct tcttgggcat ggtggcggta ccgctagcct atagtgagtc
      481 gtattaagta ctctagcctt aagagctgta attgaactgg gagtggacac ctgtggagag
      541 aaaggcaaag tggatgtcag taagaccaat aggtgcctat cagaaacgca agagtcttct
      601 ctgtctcgac aagcccagtt tctattggtc tccttaaacc tgtcttgtaa ccttgatact
      661 tacctgccca gtgcctcacg accaacttct gcagcttaag ttcgagactg ttgtgtcaga
      721 agcactgact gcgttagcaa tttaactgtg ataaactacc gcaataaagc ttgcgagcag
      781 cggaggtgat gcgtctcctc tccaacctgc tctggctctg ctcgctcctg ggatgcgagg
      841 gctttatgaa ggagtgggct agactggcga tgcccgggtg cccctggcaa caccccctgg
      901 catagcctga gggggtgggg ttgggccgca gcccagctat ggggagagct ctcccctcac
      961 ccatttgtgt ccttggactt ctgggaagca gacagccctc ctctcccccc ctgccaccca
     1021 ctgtccatgc ctgggcatcc cctgatcccc tttcctgggc acaggctgaa tagagccttg
     1081 ttctccaccg ccggtggcgc cacacagcaa gggcagcccc tagggggctg tgctgctttt
     1141 atcccaagat gccaggctgt caggctgggg tgcaatgcaa gccccctgct caatgggctt
     1201 ctcggaaggc atttgagggg ggctggggtg cggcgggaag caggcactgt gcccaccccg
     1261 aacattgtgt ctgtgccaag gtgagtcatt cactgggcat gaagaggagg ccctgggggg
     1321 cggccagccc aatgctgcca ggtctgcttg cagacaaggc ctggctgtct gcctcctccc
     1381 cagctcacag agccagctca ggcccccgag cctctgtcag agcagcgtcc cctactccac
     1441 accaggatat gttagatctc cctctgcagt ctagagtcga cccgagacgg tggtcagggg
     1501 cctcataact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     1561 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     1621 gtatcttatc atgtctgctc gaagcggccg cttacaggat gatgcagcac ttgtacagct
     1681 cgtccatgcc gccggtggag tggcggccct cggcgcgttc gtactgttcc acgatggtgt
     1741 agtcctcgtt gtgggaggtg atgtccaact tgatgttgac gttgtaggcg ccgggcagct
     1801 gcacgggctt cttggccttg taggtggtct tgacctcagc gtcgtagtgg ccgccgtcct
     1861 tcagcttcag cctctgcttg atctcgccct tcagggcgcc gtcctcgggg tacatccgct
     1921 cggaggaggc ctcccagccc atggtcttct tctgcattac ggggccgtcg gaggggaagt
     1981 tggtgccgcg cagcttcacc ttgtagatga actcgccgtc ctgcagggag gagtcctggg
     2041 tcacggtcac cacgccgccg tcctcgaagt tcatcacgcg ctcccacttg aagccctcgg
     2101 ggaaggacag cttcaagtag tcggggatgt cggcggggtg cttcacgtag gccttggagc
     2161 cgtacatgaa ctgaggggac aggatgtccc aggcgaaggg cagggggcca cccttggtca
     2221 ccttcagctt ggcggtctgg gtgccctcgt aggggcggcc ctcgccctcg ccctcgatct
     2281 cgaactcgtg gccgttcacg gagccctcca tgtgcacctt gaagcgcatg aactccttga
     2341 tgatggccat gttatcctcc tcgcccttgc tcacatccga gacggtggtc aggggcctcg
     2401 gatccaaaaa agcaccgact cggtgccact ttttcaagtt gataacggac tagccttatt
     2461 ttaacttgct atttctagct ctaaaacaga cggtggtcag gggcctccgg tgtttcgtcc
     2521 tttccacaag atatataaag ccaagaaatc gaaatacttt caagttacgg taagcatatg
     2581 atagtccatt ttaaaacata attttaaaac tgcaaactac ccaagaaatt attactttct
     2641 acgtcacgta ttttgtacta atatctttgt gtttacagtc aaattaattc caattatctc
     2701 tctaacagcc ttgtatcgta tatgcaaata tgaaggaatc atgggaaata ggccctcacg
     2761 cgtgcggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     2821 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggcggcctca gtgagcgagc
     2881 gagcgcgcag ctgcctgcag gacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     2941 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     3001 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     3061 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     3121 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     3181 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     3241 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     3301 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     3361 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     3421 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     3481 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     3541 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     3601 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     3661 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     3721 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     3781 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     3841 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     3901 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     3961 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     4021 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     4081 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     4141 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     4201 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     4261 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     4321 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     4381 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     4441 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     4501 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     4561 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     4621 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     4681 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     4741 cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg
     4801 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     4861 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct
     4921 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccataaa attgtaaacg
     4981 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     5041 agaccgaaat cggcaaaatc ccttataaat caaaagaata gcccgagata gagttgagtg
     5101 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     5161 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     5221 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     5281 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     5341 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     5401 ttaatgcgcc gctacagggc gcgtcccatt cgccattcag gctgcgcaac tgttgggaag
     5461 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
     5521 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
     5581 gtgaattagg ttaattaagg ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc
     5641 ccgggcgtcg ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg
     5701 gagtggccaa ctccatcact aggggttcct tgtagttaat gattaacccg ccatgctact
     5761 tatctacgta gccatgctct aggaagatcc ttatcgattt taccacattt gtagaggttt
     5821 tacttgcttt aaaaaacctc ccacatctcc ccctgaacct gaaacataaa atgaatgcaa
     5881 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     5941 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     6001 tcaatgtatc ttatcatgtc tgctcgaagc ggccgcaagc ttgatatcga attcctagtc
     6061 accatcttcg agcagtctca ccatggcccc ggtttcagag tcaagattgc ggatatagtt
     6121 cataacgatg ttcacatttg tccaacctcc agcttgcata atctctggta tggagactcc
     6181 tgcccgcgcc atgtctcgtg cggcccccac cctagcagaa tgacctgacc aggcgagata
     6241 cctctggcca ctgtcatcct tggctccgta tatgagtcgg tgggttgcct caaagatccc
     6301 ttcaagggcc cgtgtactca attggctggt ggcagagggc gcggcgactc cgttttttct
     6361 cactcgacag aacaagtaat tattagggtc gtcagccacg ccggacactg agatccatct
     6421 ttccaccagc ttcgttacgc ccagggacaa ggccttttca acgccagcgg tgctgaccaa
     6481 tgtctttgtg cgtccaatat gaataagcat ccggccgccg tccgttcgac tgatgtcttt
     6541 cactctgatc cgggcgatct cagctatgcg cagaagtgtg ttatatgcaa ttcccagaaa
     6601 tgcgaggttc ctgatgtcct ggcaccgatc gctgttctcc atcagggatc tgacctg
//
