LOCUS       sequence-355055-        7527 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector for Cre- and Flp-dependent transgene expression (CRE-ON /
            FLP-ON) 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 7527)
  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 7527)
  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-VTKD4-ChR2-YFP
FEATURES             Location/Qualifiers
     source          1..7527
                     /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)"
     polyA_signal    1127..1175
                     /note="synthetic polyadenylation signal"
     polyA_signal    1184..1660
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(1383..1402)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(1695..1714)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1739..1787
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    1947..1980
                     /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(1987..2020)
                     /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(2071..2104)
                     /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(2125..2838,2839..2841,2842..2844))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2170..2191)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2519..2538
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2778..2799
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2841..2850
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2860..3786)
                     /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      3783..3792
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3842..3875
                     /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    3926..3959
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3968..4556
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4021..4041)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4439..4450)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4564..4788
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4790..4930
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4790..4919
                     /label=AAV2 ITR
     rep_origin      5005..5460
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5092..5111)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5302..5323
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5477..5496)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5596..5618
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5656..5674)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5742..5846
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5847..5915,5916..6707)
                     /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(6065..6084)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6878..7466
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7367..7386
                     /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 ccctcagcac catggctagc ggcagcctcg gagtttgaat agatagaata aaatatcttt
     1141 attttcattc catctgtgtg ttggtttttt gtgtgagatc tacgggtggc atccctgtga
     1201 cccctcccca gtgcctctcc tggccctgga agttgccact ccagtgccca ccagccttgt
     1261 cctaataaaa ttaagttgca tcattttgtc tgactaggtg tccttctata atattatggg
     1321 gtggaggggg gtggtatgga gcaaggggca agttgggaag acaacctgta gggcctgcgg
     1381 ggtctattgg gaaccaagct ggagtgcagt ggcacaatct tggctcactg caatctccgc
     1441 ctcctgggtt caagcgattc tcctgcctca ggctcccgag ttgttgggat tccaggcatg
     1501 catgaccagg ctcagctaat ttttgttttt ttggtagaga cggggtttca ccatattggc
     1561 caggctggtc tccaactcct aatctcaggt gatctaccca ccttggcctc ccaaattgct
     1621 gggattacag gcgtgaacca ctgctccctt ccctgtcctt ctgcctcagc taattgagta
     1681 ggggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg
     1741 gcaataaaaa gacagaataa aacgcacggg tgttgggtcg tttgttcata aacgcggggt
     1801 tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc
     1861 ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc
     1921 agccaacgtc ggggcggcag gccgtggaag ttcctattct ctagaaagta taggaacttc
     1981 tccggaataa cttcgtatag gatactttat acgaagttat gcagaatggt agctggattg
     2041 tagctgctat tagcaatatg aaacctctta ataacttcgt atagcataca ttatacgaag
     2101 ttataacgcg ccactagtga attcttactt gtacagctcg tccatgccga gagtgatccc
     2161 ggcggcggtc acgaactcca gcaggaccat gtgatcgcgc ttctcgttgg ggtctttgct
     2221 cagggcggac tggtagctca ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat
     2281 gggggtgttc tgctggtagt ggtcggcgag ctgcacgctg ccgtcctcga tgttgtggcg
     2341 gatcttgaag ttcaccttga tgccgttctt ctgcttgtcg gccatgatat agacgttgtg
     2401 gctgttgtag ttgtactcca gcttgtgccc caggatgttg ccgtcctcct tgaagtcgat
     2461 gcccttcagc tcgatgcggt tcaccagggt gtcgccctcg aacttcacct cggcgcgggt
     2521 cttgtagttg ccgtcgtcct tgaagaagat ggtgcgctcc tggacgtagc cttcgggcat
     2581 ggcggacttg aagaagtcgt gctgcttcat gtggtcgggg tagcgggcga agcactgcag
     2641 gccgtagccg aaggtggtca cgagggtggg ccagggcacg ggcagcttgc cggtggtgca
     2701 gatgaacttc agggtcagct tgccgtaggt ggcatcgccc tcgccctcgc cggacacgct
     2761 gaacttgtgg ccgtttacgt cgccgtccag ctcgaccagg atgggcacca ccccggtgaa
     2821 cagctcctcg cccttgctca ccatggtggc ggccgctggc acggctccgg cctcggcttc
     2881 gtcttcgacg agagtctcga cctcgatctc cgttccgccg atgttcagtt tggtggtttt
     2941 gcggatatct ccgtgaatca atatgtgctc gtggatcagg acgcgcaggt agtgtcccaa
     3001 caacccccaa caatttttac tcatcagatc aataatcgtg tgacctacgg tggagccata
     3061 gacgctcagg acgccaaaac cttcgggccc caaaatgaag agaattggga acatacccca
     3121 gctcacgaaa aacagccatg ccatgccggt cacgacctgg cggcaccgac cctttggcac
     3181 agtatgataa ccctcgatat atgctttggc ggcgtgaaaa aatgtgttcg cgccatagca
     3241 caatccaaga caaaagaaga tgactttaac atagccggtt gccatggcgc tggtagcccc
     3301 ccacacgata gtcccgatgt ctgagacaag gagtcccatg gttctcctgc tgtagtcgtt
     3361 gctcaggccg gtgaggttgc tcaggcggat aaggatgaca ggacaagtga gcagccactc
     3421 tgcatagcgc agccactgca cccggtgtcc tgtggcaagg tagagcatag agggattctt
     3481 aaactcaaaa aagaactcga gaatcacctt aaccatttca atggcgcaca catagatctc
     3541 ctcccagccg catgtagatt tccaggtttg gtaggcatag aacatcagca gcaaaatgct
     3601 gaatcctgct gcaagccact gcaggacatt tgacgcggtc tgagcgccgt tcgtgccgcg
     3661 agattcaatc catccggcac agtaacattg atcctcaggg accaggacgg acccgttcac
     3721 caccacagga ttagtaacga acaaaagttc gcgtccgacg gcagacaaag cgccgccata
     3781 gtccatggtg gctccggagt cgacctgcag gcatgcaagc tttactagtg gtggcgctag
     3841 cataacttcg tataaagtat cctatacgaa gttatttgcc ttaacccaga aattatcact
     3901 gttattcttt agaatggtgc aaagaataac ttcgtataat gtatgctata cgaagttatg
     3961 gcgcgccaat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     4021 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     4081 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     4141 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     4201 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     4261 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     4321 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aagctgacgt cctttccatg
     4381 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     4441 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     4501 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcctgg
     4561 atcctgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     4621 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     4681 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     4741 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggca ggaaccccta
     4801 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
     4861 aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc
     4921 tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     4981 cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     5041 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg
     5101 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     5161 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     5221 tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     5281 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta
     5341 tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa
     5401 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     5461 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     5521 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     5581 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     5641 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     5701 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     5761 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     5821 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     5881 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     5941 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     6001 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     6061 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     6121 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     6181 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     6241 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     6301 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     6361 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     6421 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     6481 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     6541 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     6601 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     6661 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     6721 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     6781 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     6841 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     6901 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     6961 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     7021 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     7081 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     7141 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     7201 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     7261 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     7321 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     7381 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     7441 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     7501 ccttttgctg gccttttgct cacatgt
//
