LOCUS       sequence-355104-        6662 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector for Cre- and Flp-dependent transgene expression (CRE-ON /
            FLP-OFF) of ChR2-YFP in cortical interneurons under the control of 
            the Dlx enhancer (Dimidschstein et al 2016).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6662)
  AUTHORS   Pouchelon G, Vergara J, McMahon J, Gorissen BL, Lin JD, 
            Vormstein-Schneider D, Niehaus JL, Burbridge TJ, Wester JC, Sherer 
            M, Fernandez-Otero M, Allaway KC, Pelkey K, Chittajallu R, McBain 
            CJ, Fan M, Nasse JS, Wildenberg GA, Fishell G, Dimidschstein J
  TITLE     A versatile viral toolkit for functional discovery in the nervous 
            system.
  JOURNAL   Cell Rep Methods. 2022 May 26;2(6):100225. doi: 
            10.1016/j.crmeth.2022.100225. eCollection 2022 Jun 20.
  PUBMED    35784651
REFERENCE   2  (bases 1 to 6662)
  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: "Cell Rep 
            Methods."; date: "2022-05-26"; volume: "2(6):100225. doi"; pages: " 
            10.1016/j.crmeth.2022.100225. eCollection 2022 Jun 20"
COMMENT     Sequence Label: pAAV-VTKD5-ChR2-YFP
FEATURES             Location/Qualifiers
     source          1..6662
                     /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          873..1005
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     988..1007
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     protein_bind    1048..1081
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    complement(1088..1121)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(1172..1205)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(1226..1939,1940..1942,1943..1945))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1271..1292)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1620..1639
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1879..1900
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1942..1951
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(1961..2887)
                     /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"
     regulatory      2884..2893
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2943..2976
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    3027..3060
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3061..3094
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     misc_feature    3103..3691
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3156..3176)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3574..3585)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3699..3923
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3925..4065
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3925..4054
                     /label=AAV2 ITR
     rep_origin      4140..4595
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4227..4246)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4437..4458
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4612..4631)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4731..4753
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4791..4809)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4877..4981
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4982..5050,5051..5842)
                     /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(5200..5219)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6013..6601
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6502..6521
                     /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 cgtttattca gaatgttatg
      181 cactcacagt ggtttggcat gcatctggtg aatttttttt aacgaaaaat tagtgttggt
      241 ttcgatgtat ggtagcattc tccctaacgt aatttgaata attcagcaaa gccccactac
      301 cagctgtact tctgcagcct cttccattct tttcagcatt ataattttgg ttaattttca
      361 attttaggtc ctacgtctct gcaatttgtg tatgaataac agaataattt ccctcttttg
      421 tttcgccttt cctgttcctg aatctaaata aagatggctt tttagtatta aaagtggaag
      481 aaaattacag gtaattatct ttgacggtaa aaacgctgta atcagcgggc tacatgaaaa
      541 attactctaa ttatggctgc atttaagaga atggaaaaaa accttcttgt ggataaaaac
      601 cttaaattgt ccccaatgtc tgcttcaaat tggatggcac tgcagctgga ggctttgttc
      661 agaattgatc ctggggagct acgaacccaa agtttcacag taggaagcta gatatcgttt
      721 aaacttcctg cagcccgggc tgggcataaa agtcagggca gagccatcta ttgcttacat
      781 ttgcttctag cctgcaagtc gaggagcgca gccttccaga agcagagcgc ggcgccttaa
      841 gctgcagaag ttggtcgtga ggcactgggc acgtaagtat caaggttaca agacaggttt
      901 aaggagacca atagaaactg ggcttgtcga gacagagaag actcttgcgt ttctgatagg
      961 cacctattgg tcttactgac atccactttg cctttctctc cacaggtgtc cactcccagt
     1021 tcaattacag ctcttaagaa agctagcgaa gttcctattc tctagaaagt ataggaactt
     1081 ctccggaata acttcgtata ggatacttta tacgaagtta tgcagaatgg tagctggatt
     1141 gtagctgcta ttagcaatat gaaacctctt aataacttcg tatagcatac attatacgaa
     1201 gttataacgc gccactagtg aattcttact tgtacagctc gtccatgccg agagtgatcc
     1261 cggcggcggt cacgaactcc agcaggacca tgtgatcgcg cttctcgttg gggtctttgc
     1321 tcagggcgga ctggtagctc aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga
     1381 tgggggtgtt ctgctggtag tggtcggcga gctgcacgct gccgtcctcg atgttgtggc
     1441 ggatcttgaa gttcaccttg atgccgttct tctgcttgtc ggccatgata tagacgttgt
     1501 ggctgttgta gttgtactcc agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga
     1561 tgcccttcag ctcgatgcgg ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg
     1621 tcttgtagtt gccgtcgtcc ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca
     1681 tggcggactt gaagaagtcg tgctgcttca tgtggtcggg gtagcgggcg aagcactgca
     1741 ggccgtagcc gaaggtggtc acgagggtgg gccagggcac gggcagcttg ccggtggtgc
     1801 agatgaactt cagggtcagc ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc
     1861 tgaacttgtg gccgtttacg tcgccgtcca gctcgaccag gatgggcacc accccggtga
     1921 acagctcctc gcccttgctc accatggtgg cggccgctgg cacggctccg gcctcggctt
     1981 cgtcttcgac gagagtctcg acctcgatct ccgttccgcc gatgttcagt ttggtggttt
     2041 tgcggatatc tccgtgaatc aatatgtgct cgtggatcag gacgcgcagg tagtgtccca
     2101 acaaccccca acaattttta ctcatcagat caataatcgt gtgacctacg gtggagccat
     2161 agacgctcag gacgccaaaa ccttcgggcc ccaaaatgaa gagaattggg aacatacccc
     2221 agctcacgaa aaacagccat gccatgccgg tcacgacctg gcggcaccga ccctttggca
     2281 cagtatgata accctcgata tatgctttgg cggcgtgaaa aaatgtgttc gcgccatagc
     2341 acaatccaag acaaaagaag atgactttaa catagccggt tgccatggcg ctggtagccc
     2401 cccacacgat agtcccgatg tctgagacaa ggagtcccat ggttctcctg ctgtagtcgt
     2461 tgctcaggcc ggtgaggttg ctcaggcgga taaggatgac aggacaagtg agcagccact
     2521 ctgcatagcg cagccactgc acccggtgtc ctgtggcaag gtagagcata gagggattct
     2581 taaactcaaa aaagaactcg agaatcacct taaccatttc aatggcgcac acatagatct
     2641 cctcccagcc gcatgtagat ttccaggttt ggtaggcata gaacatcagc agcaaaatgc
     2701 tgaatcctgc tgcaagccac tgcaggacat ttgacgcggt ctgagcgccg ttcgtgccgc
     2761 gagattcaat ccatccggca cagtaacatt gatcctcagg gaccaggacg gacccgttca
     2821 ccaccacagg attagtaacg aacaaaagtt cgcgtccgac ggcagacaaa gcgccgccat
     2881 agtccatggt ggctccggag tcgacctgca ggcatgcaag ctttactagt ggtggcgcta
     2941 gcataacttc gtataaagta tcctatacga agttatttgc cttaacccag aaattatcac
     3001 tgttattctt tagaatggtg caaagaataa cttcgtataa tgtatgctat acgaagttat
     3061 gaagttccta ttctctagaa agtataggaa cttcggcgcg ccaatcaacc tctggattac
     3121 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     3181 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     3241 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     3301 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     3361 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
     3421 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     3481 gtggtgttgt cggggaagct gacgtccttt ccatggctgc tcgcctgtgt tgccacctgg
     3541 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     3601 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     3661 agtcggatct ccctttgggc cgcctccccg cctggatcct gtgccttcta gttgccagcc
     3721 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     3781 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     3841 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     3901 tggggatgcg gtgggctcta tggcaggaac ccctagtgat ggagttggcc actccctctc
     3961 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
     4021 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt
     4081 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta
     4141 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     4201 tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     4261 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     4321 tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc
     4381 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     4441 actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata
     4501 agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     4561 cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg
     4621 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     4681 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     4741 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     4801 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     4861 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     4921 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     4981 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     5041 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     5101 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     5161 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     5221 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     5281 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     5341 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     5401 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     5461 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     5521 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     5581 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     5641 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     5701 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     5761 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     5821 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     5881 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     5941 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6001 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6061 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6121 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     6181 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6241 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     6301 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     6361 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     6421 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     6481 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     6541 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     6601 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     6661 gt
//
