LOCUS Exported 5295 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Gsk3b targeting gRNA cloned into px552 (SpGuide) plasmid.. ACCESSION . VERSION . KEYWORDS pAAV Gsk3 sgRNA/GFP 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 May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5295 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 23..152 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" promoter 167..407 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" 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 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1031..1747 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 1031..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 3637..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 //