LOCUS       sequence-300335-        6994 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector to drive the expression of dChr2-GFP-P2A-GFP in PV 
            cortical interneuronsunder the control of the E22 regulatory 
            element.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6994)
  AUTHORS   Vormstein-Schneider D, Lin JD, Pelkey KA, Chittajallu R, Guo B, 
            Arias-Garcia MA, Allaway K, Sakopoulos S, Schneider G, Stevenson O, 
            Vergara J, Sharma J, Zhang Q, Franken TP, Smith J, Ibrahim LA, M 
            Astro KJ, Sabri E, Huang S, Favuzzi E, Burbridge T, Xu Q, Guo L, 
            Vogel I, Sanchez V, Saldi GA, Gorissen BL, Yuan X, Zaghloul KA, 
            Devinsky O, Sabatini BL, Batista-Brito R, Reynolds J, Feng G, Fu Z, 
            McBain CJ, Fishell G, Dimidschstein J
  TITLE     Viral manipulation of functionally distinct interneurons in mice, 
            non-human primates and humans.
  JOURNAL   Nat Neurosci. 2020 Dec;23(12):1629-1636. doi: 
            10.1038/s41593-020-0692-9. Epub 2020 Aug 17.
  PUBMED    32807948
REFERENCE   2  (bases 1 to 6994)
  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"; doi: 
            "10.1038/s41593-020-0692-9"; journalName: "Nat Neurosci"; date: 
            "2020-12"; volume: "23"; issue: "12"; pages: "1629-1636"
COMMENT     Sequence Label: pAAV-E22-ChR2GFP2x
FEATURES             Location/Qualifiers
     source          1..6994
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     intron          763..895
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     878..897
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      936..945
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             942..1868
                     /codon_start=1
                     /product="H134R mutant of an N-terminal fragment of 
                     channelrhodopsin-2 from the alga Chlamydomonas reinhardtii 
                     (Nagel et al., 2005)"
                     /label=ChR2(H134R)
                     /note="blue light-activated cation channel"
                     /translation="MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGT
                     NGAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKN
                     PSMLYLATGHRVQWLRYAEWLLTCPVILIRLSNLTGLSNDYSRRTMGLLVSDIGTIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     CDS             join(1884..1886,1887..2597)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1884..1886,1887..2597)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             2619..2675
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2688..2708
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2709..2711,2712..3425)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(2709..2711,2712..3422)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     misc_feature    3435..4023
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3488..3508)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3906..3917)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4031..4255
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4257..4397
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4257..4386
                     /label=AAV2 ITR
     rep_origin      4472..4927
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4559..4578)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4769..4790
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4944..4963)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5063..5085
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5123..5141)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5209..5313
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5314..5382,5383..6174)
                     /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(5532..5551)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6345..6933
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6834..6853
                     /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 gctagactgc agagggcccg cggccgcacg cgttcggggt ttaaccgttc
      181 agagtaattt tccattataa ttttgtgttc taaataaata gaaccagctt cactctagaa
      241 acgcttgtct gagtgctgta caatgcatgt ttaagtggct ctggtgaaat tactaatcat
      301 acccctgcag tggataaaat agaattgttt gtgagttttg ctcattcgat gggaggacgc
      361 taaatttata aaagtagact catgcccttg atagtgaatt taaataggtg catatgtaaa
      421 cagatactta ctcatttaga gacaccgtca tcagggagct ttgtgtgaga atgttttcta
      481 tgctccggct cctgctctgt ggtaggggaa ctaagcagac atcccaggtc catttcctaa
      541 cccagtgtct ctagttgagt aatcacacaa ggcagacatc ccagctggga ctctatattt
      601 aaacccgttt aaacttcctg cagcccgggc tgggcataaa agtcagggca gagccatcta
      661 ttgcttacat ttgcttctag cctgcaagtc gaggagcgca gccttccaga agcagagcgc
      721 ggcgccttaa gctgcagaag ttggtcgtga ggcactgggc acgtaagtat caaggttaca
      781 agacaggttt aaggagacca atagaaactg ggcttgtcga gacagagaag actcttgcgt
      841 ttctgatagg cacctattgg tcttactgac atccactttg cctttctctc cacaggtgtc
      901 cactcccagt tcaattacag ctcttaagaa agctagccac catggactat ggcggcgctt
      961 tgtctgccgt cggacgcgaa cttttgttcg ttactaatcc tgtggtggtg aacgggtccg
     1021 tcctggtccc tgaggatcaa tgttactgtg ccggatggat tgaatctcgc ggcacgaacg
     1081 gcgctcagac cgcgtcaaat gtcctgcagt ggcttgcagc aggattcagc attttgctgc
     1141 tgatgttcta tgcctaccaa acctggaaat ctacatgcgg ctgggaggag atctatgtgt
     1201 gcgccattga aatggttaag gtgattctcg agttcttttt tgagtttaag aatccctcta
     1261 tgctctacct tgccacagga caccgggtgc agtggctgcg ctatgcagag tggctgctca
     1321 cttgtcctgt catccttatc cgcctgagca acctcaccgg cctgagcaac gactacagca
     1381 ggagaaccat gggactcctt gtctcagaca tcgggactat cgtgtggggg gctaccagcg
     1441 ccatggcaac cggctatgtt aaagtcatct tcttttgtct tggattgtgc tatggcgcga
     1501 acacattttt tcacgccgcc aaagcatata tcgagggtta tcatactgtg ccaaagggtc
     1561 ggtgccgcca ggtcgtgacc ggcatggcat ggctgttttt cgtgagctgg ggtatgttcc
     1621 caattctctt cattttgggg cccgaaggtt ttggcgtcct gagcgtctat ggctccaccg
     1681 taggtcacac gattattgat ctgatgagta aaaattgttg ggggttgttg ggacactacc
     1741 tgcgcgtcct gatccacgag cacatattga ttcacggaga tatccgcaaa accaccaaac
     1801 tgaacatcgg cggaacggag atcgaggtcg agactctcgt cgaagacgaa gccgaggccg
     1861 gagccgtgcc agctgcagcc acagtgagca agggcgagga gctgttcacc ggggtggtgc
     1921 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
     1981 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     2041 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
     2101 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     2161 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     2221 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     2281 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     2341 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     2401 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     2461 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
     2521 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     2581 tggacgagct gtacaagtcc ggagataacg gaagcggagc cactaacttc tccctgttga
     2641 aacaagcagg ggatgtcgaa gagaatcccg ggccatctag aatggccccc aagaagaaga
     2701 ggaaggtggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     2761 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     2821 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     2881 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     2941 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     3001 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     3061 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     3121 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     3181 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     3241 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     3301 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     3361 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     3421 agtaatggcg cgccaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
     3481 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
     3541 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc
     3601 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg
     3661 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg
     3721 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga
     3781 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaag ctgacgtcct
     3841 ttccatggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg
     3901 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc
     3961 ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc
     4021 cgcctggatc ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     4081 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     4141 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     4201 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcagga
     4261 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     4321 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
     4381 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     4441 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     4501 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     4561 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     4621 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     4681 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     4741 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     4801 aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     4861 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     4921 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     4981 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     5041 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     5101 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     5161 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     5221 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     5281 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     5341 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     5401 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     5461 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     5521 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     5581 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     5641 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     5701 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     5761 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     5821 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     5881 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     5941 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     6001 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     6061 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     6121 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     6181 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     6241 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     6301 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     6361 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     6421 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6481 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6541 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     6601 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     6661 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     6721 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     6781 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     6841 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     6901 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     6961 ttcctggcct tttgctggcc ttttgctcac atgt
//
