LOCUS       sequence-437146-        7071 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An adeno-associated viral vector expressing nuclear 
            envelope-embedded eGFP and a Cre-dependent guide RNA for mGrid1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7071)
  AUTHORS   Gantz SC, Moussawi K, Hake HS
  TITLE     Delta glutamate receptor conductance drives excitation of mouse 
            dorsal raphe neurons.
  JOURNAL   Elife. 2020 Apr 1;9. pii: 56054. doi: 10.7554/eLife.56054.
  PUBMED    32234214
REFERENCE   2  (bases 1 to 7071)
  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: "Elife."; 
            date: "2020-04-1"; volume: "9. pii: 56054. doi"; pages: " 
            10.7554/eLife.56054"
COMMENT     Sequence Label: pOTTC1789 - pAAV (flox-stop) mGrid1 390F gRNA ...
FEATURES             Location/Qualifiers
     source          1..7071
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     misc_RNA        complement(183..258)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    291..324
                     /label=loxP511
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATAC) (Shaw et al., 2021). loxP511 sites are 
                     compatible with each other, but incompatible with wild-type
                     loxP sites."
     protein_bind    555..588
                     /label=loxP511
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATAC) (Shaw et al., 2021). loxP511 sites are 
                     compatible with each other, but incompatible with wild-type
                     loxP sites."
     promoter        986..2164
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1217..2155
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2112..2132
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      2184..2193
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2190..2192,2193..2195,2196..2906)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2190..2192,2193..2195,2196..2906)
                     /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(2235..2256)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2496..2515)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2843..2864
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3198..3786
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3251..3271)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3669..3680)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3818..4294
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4017..4036)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4334..4474
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4334..4463
                     /label=AAV2 ITR
     rep_origin      4549..5004
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4636..4655)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4846..4867
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5021..5040)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5140..5162
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5200..5218)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5286..5390
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5391..5459,5460..6251)
                     /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(5609..5628)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6422..7010
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6911..6930
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtaagctg gagctccacc gcggcgtctc agtacaaaaa
      181 aagcaccgac tcggtgccac tttttcaagt tgataacgga ctagccttat tttaacttgc
      241 tatttctagc tctaaaaccg ccgtcagggc tagggttcca aacaaggctt ataacttcgt
      301 ataatttata ctatacgaag ttatggatcc cccgggcgtg gcttggtggt acacctctgt
      361 catcccagta ggtaggtaga ggcagtcact ggagaacctc atcagtgtca ggccaacctg
      421 agctatacag tgagcaacga gctctgctgt caagaaacaa aatagaaaaa acgtgagtga
      481 gcctccaata ggatgttagg gtgcttgtca gttcctctga ggaacgaatt caaaaatcgc
      541 aaaaaacaag gcttataact tcgtataatt tatactatac gaagttatac taactttaca
      601 gttagggtga gttctcgagt gtgctgtttt ttaaaataat aatttagtat ttgtatctct
      661 tatagaaatc caagcctatc atgtaaaatg tagctagtat taaaaagaac agattatctg
      721 tcttttatcg cacattaagc ctctatagtt actaggaaat attatatgca aattaaccgg
      781 ggcaggggag tagccgagct tctcccacaa gtctgtgcga gggggccggc gcgggcctag
      841 agatggcggc gtcggatcta tctgagacgg gtacccaatt cgccctatag tgagcgcgta
      901 agctttgcaa agatggataa agttttaaac agagaggaat ctttgcagct aatggacctt
      961 ctaggtcttg aaaggagtgg gaattggctc cggtgcccgt cagtgggcag agcgcacatc
     1021 gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag
     1081 gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg
     1141 tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt
     1201 tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc tctttacggg
     1261 ttatggccct tgcgtgcctt gaattacttc cacctggctg cagtacgtga ttcttgatcc
     1321 cgagcttcgg gttggaagtg ggtgggagag ttcgaggcct tgcgcttaag gagccccttc
     1381 gcctcgtgct tgagttgagg cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg
     1441 gcaccttcgc gcctgtctcg ctgctttcga taagtctcta gccatttaaa atttttgatg
     1501 acctgctgcg acgctttttt tctggcaaga tagtcttgta aatgcgggcc aagatctgca
     1561 cactggtatt tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac
     1621 atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga atcggacggg ggtagtctca
     1681 agctggccgg cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc
     1741 ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa agatggccgc ttcccggccc
     1801 tgctgcaggg agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc
     1861 cacacaaagg aaaagggcct ttccgtcctc agccgtcgct tcatgtgact ccacggagta
     1921 ccgggcgccg tccaggcacc tcgattagtt ctcgagcttt tggagtacgt cgtctttagg
     1981 ttggggggag gggttttatg cgatggagtt tccccacact gagtgggtgg agactgaagt
     2041 taggccagct tggcacttga tgtaattctc cttggaattt gccctttttg agtttggatc
     2101 ttggttcatt ctcaagcctc agacagtggt tcaaagtttt tttcttccat ttcaggtgtc
     2161 gtgaggtacc ggatccaccg gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg
     2221 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     2281 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     2341 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     2401 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     2461 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     2521 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     2581 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     2641 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     2701 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     2761 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     2821 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     2881 ctctcggcat ggacgagctg tacaagtccg gactcagatc tcgagaggag gaggaggaga
     2941 cagacagcag gatgccccac ctcgacagcc ccggcagctc ccagccgaga cgctccttcc
     3001 tctcaagggt gatcagggca gcgctaccgt tgcagctgct tctgctgctg ctgctgctcc
     3061 tggcctgcct gctacctgcc tctgaagatg actacagctg cacccaggcc aacaactttg
     3121 cccgatcctt ctaccccatg ctgcggtaca ccaacgggcc acctcccacc taggaattcg
     3181 atatcaagct tatcgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta
     3241 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
     3301 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
     3361 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
     3421 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
     3481 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
     3541 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt
     3601 cctttccttg gctgctcgcc tatgttgcca cctggattct gcgcgggacg tccttctgct
     3661 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc
     3721 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct
     3781 ccccgcatcg ataccgagcg ctgctcgaga gatctacggg tggcatccct gtgacccctc
     3841 cccagtgcct ctcctggccc tggaagttgc cactccagtg cccaccagcc ttgtcctaat
     3901 aaaattaagt tgcatcattt tgtctgacta ggtgtccttc tataatatta tggggtggag
     3961 gggggtggta tggagcaagg ggcaagttgg gaagacaacc tgtagggcct gcggggtcta
     4021 ttgggaacca agctggagtg cagtggcaca atcttggctc actgcaatct ccgcctcctg
     4081 ggttcaagcg attctcctgc ctcagcctcc cgagttgttg ggattccagg catgcatgac
     4141 caggctcagc taatttttgt ttttttggta gagacggggt ttcaccatat tggccaggct
     4201 ggtctccaac tcctaatctc aggtgatcta cccaccttgg cctcccaaat tgctgggatt
     4261 acaggcgtga accactgctc ccttccctgt ccttctgatt ttgtaggtaa ccacgtgcgg
     4321 accgagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc
     4381 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc
     4441 tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt
     4501 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     4561 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     4621 gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     4681 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     4741 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     4801 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     4861 aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc
     4921 cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     4981 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     5041 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     5101 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     5161 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     5221 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     5281 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     5341 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     5401 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     5461 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     5521 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     5581 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     5641 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     5701 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     5761 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     5821 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     5881 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     5941 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     6001 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     6061 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     6121 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     6181 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     6241 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     6301 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     6361 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     6421 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     6481 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     6541 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     6601 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     6661 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     6721 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     6781 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     6841 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     6901 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     6961 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     7021 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg t
//
