LOCUS       sequence-217431-        5295 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gsk3b targeting gRNA cloned into px552 (SpGuide) plasmid..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5295)
  AUTHORS   Khlghatyan J, Evstratova A, Chamberland S, Marakhovskaia A, 
            Bahremand A, Toth K, Beaulieu JM
  TITLE     Mental Illnesses-Associated Fxr1 and Its Negative Regulator Gsk3beta
            Are Modulators of Anxiety and Glutamatergic Neurotransmission.
  JOURNAL   Front Mol Neurosci. 2018 Apr 12;11:119. doi: 
            10.3389/fnmol.2018.00119. eCollection 2018.
  PUBMED    29706865
REFERENCE   2  (bases 1 to 5295)
  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: "Front Mol 
            Neurosci."; date: "2018-04-12"; volume: "11:119. doi"; pages: " 
            10.3389/fnmol.2018.00119. eCollection 2018"
COMMENT     Sequence Label: pAAV Gsk3 sgRNA/GFP
FEATURES             Location/Qualifiers
     source          1..5295
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   23..152
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        167..415
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     167..187
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     338..357
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        437..512
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        546..993
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      1025..1034
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1031..1033,1034..1036,1037..1747)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1031..1033,1034..1036,1037..1747)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1076..1097)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1337..1356)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1684..1705
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2064..2540
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2263..2282)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2580..2720
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2580..2709
                     /label=AAV2 ITR
     rep_origin      2795..3250
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2882..2901)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3092..3113
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3267..3286)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3386..3408
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3446..3464)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3532..3636
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3637..3705,3706..4497)
                     /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"
     primer_bind     complement(3855..3874)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4668..5256
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5157..5176
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgctggcctt ttgctcacat gtcctgcagg cagctgcgcg ctcgctcgct cactgaggcc
       61 gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga
      121 gggagtggcc aactccatca ctaggggttc ctgcggccgc acgcgtgagg gcctatttcc
      181 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa
      241 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct
      301 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac
      361 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgactg
      421 taacatagtc cgactggttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
      481 atcaacttga aaaagtggca ccgagtcggt gctttttttc tagactgcag agggccctgc
      541 gtatgagtgc aagtgggttt taggaccagg atgaggcggg gtgggggtgc ctacctgacg
      601 accgaccccg acccactgga caagcaccca acccccattc cccaaattgc gcatccccta
      661 tcagagaggg ggaggggaaa caggatgcgg cgaggcgcgt gcgcactgcc agcttcagca
      721 ccgcggacag tgccttcgcc cccgcctggc ggcgcgcgcc accgccgcct cagcactgaa
      781 ggcgcgctga cgtcactcgc cggtcccccg caaactcccc ttcccggcca ccttggtcgc
      841 gtccgcgccg ccgccggccc agccggaccg caccacgcga ggcgcgagat aggggggcac
      901 gggcgcgacc atctgcgctg cggcgccggc gactcagcgc tgcctcagtc tgcggtgggc
      961 agcggaggag tcgtgtcgtg cctgagagcg cagtcgagaa ggtaccggat cctctagagt
     1021 cgacgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     1081 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     1141 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     1201 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     1261 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     1321 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     1381 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     1441 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     1501 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     1561 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     1621 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     1681 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     1741 gtacaagtcc ggactcagat ctcgagagga ggaggaggag acagacagca ggatgcccca
     1801 cctcgacagc cccggcagct cccagccgag acgctccttc ctctcaaggg tgatcagggc
     1861 agcgctaccg ttgcagctgc ttctgctgct gctgctgctc ctggcctgcc tgctacctgc
     1921 ctctgaagat gactacagct gcacccaggc caacaacttt gcccgatcct tctaccccat
     1981 gctgcggtac accaacgggc cacctcccac ctaggaattc gatatcaagc ttatcgatac
     2041 cgagcgctgc tcgagagatc tacgggtggc atccctgtga cccctcccca gtgcctctcc
     2101 tggccctgga agttgccact ccagtgccca ccagccttgt cctaataaaa ttaagttgca
     2161 tcattttgtc tgactaggtg tccttctata atattatggg gtggaggggg gtggtatgga
     2221 gcaaggggca agttgggaag acaacctgta gggcctgcgg ggtctattgg gaaccaagct
     2281 ggagtgcagt ggcacaatct tggctcactg caatctccgc ctcctgggtt caagcgattc
     2341 tcctgcctca gcctcccgag ttgttgggat tccaggcatg catgaccagg ctcagctaat
     2401 ttttgttttt ttggtagaga cggggtttca ccatattggc caggctggtc tccaactcct
     2461 aatctcaggt gatctaccca ccttggcctc ccaaattgct gggattacag gcgtgaacca
     2521 ctgctccctt ccctgtcctt ctgattttgt aggtaaccac gtgcggaccg agcggccgca
     2581 ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc
     2641 cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg
     2701 agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     2761 tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc
     2821 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc
     2881 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     2941 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     3001 aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     3061 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     3121 ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat
     3181 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     3241 tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     3301 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     3361 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     3421 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     3481 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     3541 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     3601 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     3661 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     3721 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     3781 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     3841 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     3901 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     3961 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     4021 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     4081 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     4141 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     4201 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     4261 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     4321 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     4381 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     4441 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     4501 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     4561 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     4621 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     4681 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4741 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4801 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4861 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4921 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4981 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     5041 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     5101 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     5161 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     5221 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     5281 cggttcctgg ccttt
//
