LOCUS       sequence-437147-        6769 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An adeno-associated viral vector expressing nuclear 
            envelope-embedded eGFP and a guide RNA for mGrid1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6769)
  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 6769)
  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: pOTTC1706 - pAAV mGrid1 390F gRNA EF1a EGFP-KASH
FEATURES             Location/Qualifiers
     source          1..6769
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        156..469
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     388..408
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     misc_RNA        492..567
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        684..1862
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          915..1853
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1810..1830
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1882..1891
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1888..1890,1891..1893,1894..2604)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1888..1890,1891..1893,1894..2604)
                     /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(1933..1954)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2194..2213)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2541..2562
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2896..3484
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2949..2969)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3367..3378)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3516..3992
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3715..3734)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4032..4172
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4032..4161
                     /label=AAV2 ITR
     rep_origin      4247..4702
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4334..4353)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4544..4565
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4719..4738)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4838..4860
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4898..4916)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4984..5088
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5089..5157,5158..5949)
                     /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(5307..5326)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6120..6708
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6609..6628
                     /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 gcgtcgtctc agatagatcc gacgccgcca tctctaggcc
      181 cgcgccggcc ccctcgcaca gacttgtggg agaagctcgg ctactcccct gccccggtta
      241 atttgcatat aatatttcct agtaactata gaggcttaat gtgcgataaa agacagataa
      301 tctgttcttt ttaatactag ctacatttta catgataggc ttggatttct ataagagata
      361 caaatactaa attattattt taaaaaacag cacaaaagga aactcaccct aactgtaaag
      421 taattgtgtg ttttgagact ataaatatcc cttggagaaa agccttgttt ggaaccctag
      481 ccctgacggc ggttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
      541 cttgaaaaag tggcaccgag tcggtgcttt ttttgtactg agacgccgcg gtcgcgtaag
      601 ctttgcaaag atggataaag ttttaaacag agaggaatct ttgcagctaa tggaccttct
      661 aggtcttgaa aggagtggga attggctccg gtgcccgtca gtgggcagag cgcacatcgc
      721 ccacagtccc cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt
      781 ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg
      841 ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg
      901 ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt
      961 atggcccttg cgtgccttga attacttcca cctggctgca gtacgtgatt cttgatcccg
     1021 agcttcgggt tggaagtggg tgggagagtt cgaggccttg cgcttaagga gccccttcgc
     1081 ctcgtgcttg agttgaggcc tggcctgggc gctggggccg ccgcgtgcga atctggtggc
     1141 accttcgcgc ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgac
     1201 ctgctgcgac gctttttttc tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca
     1261 ctggtatttc ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat
     1321 gttcggcgag gcggggcctg cgagcgcggc caccgagaat cggacggggg tagtctcaag
     1381 ctggccggcc tgctctggtg cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg
     1441 caaggctggc ccggtcggca ccagttgcgt gagcggaaag atggccgctt cccggccctg
     1501 ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt gagtcaccca
     1561 cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc
     1621 gggcgccgtc caggcacctc gattagttct cgagcttttg gagtacgtcg tctttaggtt
     1681 ggggggaggg gttttatgcg atggagtttc cccacactga gtgggtggag actgaagtta
     1741 ggccagcttg gcacttgatg taattctcct tggaatttgc cctttttgag tttggatctt
     1801 ggttcattct caagcctcag acagtggttc aaagtttttt tcttccattt caggtgtcgt
     1861 gaggtaccgg atccaccggt cgccaccatg gtgagcaagg gcgaggagct gttcaccggg
     1921 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     1981 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
     2041 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
     2101 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     2161 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     2221 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     2281 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc
     2341 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
     2401 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
     2461 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac
     2521 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
     2581 ctcggcatgg acgagctgta caagtccgga ctcagatctc gagaggagga ggaggagaca
     2641 gacagcagga tgccccacct cgacagcccc ggcagctccc agccgagacg ctccttcctc
     2701 tcaagggtga tcagggcagc gctaccgttg cagctgcttc tgctgctgct gctgctcctg
     2761 gcctgcctgc tacctgcctc tgaagatgac tacagctgca cccaggccaa caactttgcc
     2821 cgatccttct accccatgct gcggtacacc aacgggccac ctcccaccta ggaattcgat
     2881 atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     2941 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     3001 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct
     3061 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct
     3121 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc
     3181 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg
     3241 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc
     3301 tttccttggc tgctcgccta tgttgccacc tggattctgc gcgggacgtc cttctgctac
     3361 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg
     3421 cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc
     3481 ccgcatcgat accgagcgct gctcgagaga tctacgggtg gcatccctgt gacccctccc
     3541 cagtgcctct cctggccctg gaagttgcca ctccagtgcc caccagcctt gtcctaataa
     3601 aattaagttg catcattttg tctgactagg tgtccttcta taatattatg gggtggaggg
     3661 gggtggtatg gagcaagggg caagttggga agacaacctg tagggcctgc ggggtctatt
     3721 gggaaccaag ctggagtgca gtggcacaat cttggctcac tgcaatctcc gcctcctggg
     3781 ttcaagcgat tctcctgcct cagcctcccg agttgttggg attccaggca tgcatgacca
     3841 ggctcagcta atttttgttt ttttggtaga gacggggttt caccatattg gccaggctgg
     3901 tctccaactc ctaatctcag gtgatctacc caccttggcc tcccaaattg ctgggattac
     3961 aggcgtgaac cactgctccc ttccctgtcc ttctgatttt gtaggtaacc acgtgcggac
     4021 cgagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     4081 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc
     4141 agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac
     4201 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc
     4261 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     4321 gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     4381 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     4441 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag
     4501 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     4561 actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg
     4621 atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     4681 aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca
     4741 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     4801 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     4861 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
     4921 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     4981 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     5041 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     5101 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     5161 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     5221 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     5281 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     5341 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     5401 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     5461 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     5521 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     5581 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     5641 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     5701 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     5761 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     5821 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     5881 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     5941 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     6001 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     6061 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     6121 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     6181 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     6241 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     6301 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     6361 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     6421 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     6481 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     6541 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     6601 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     6661 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     6721 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt
//
