LOCUS       sequence-301584-        6965 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intersectional viral expression of ChR2(ET/TC)-EYFP in cells 
            expressing Flp AND NOT Cre.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6965)
  AUTHORS   Fenno LE, Ramakrishnan C, Kim YS, Evans KE, Lo M, Vesuna S, Inoue M,
            Cheung KYM, Yuen E, Pichamoorthy N, Hong ASO, Deisseroth K
  TITLE     Comprehensive Dual- and Triple-Feature Intersectional Single-Vector 
            Delivery of Diverse Functional Payloads to Cells of Behaving 
            Mammals.
  JOURNAL   Neuron. 2020 Jun 20. pii: S0896-6273(20)30432-3. doi: 
            10.1016/j.neuron.2020.06.003.
  PUBMED    32574559
REFERENCE   2  (bases 1 to 6965)
  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: "Neuron. 
            2020 Jun 20. pii: S0896-6273(20)30432-3. doi: 
            10.1016/j.neuron.2020.06.003."
COMMENT     Sequence Label: pAAV-nEF-Coff/Fon-ChR2(ET/TC)-EYFP
FEATURES             Location/Qualifiers
     source          1..6965
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   120..249
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        296..507
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             520..788
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     protein_bind    816..849
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     CDS             complement(945..1196)
                     /codon_start=1
                     /product="C-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VC155
                     /translation="DKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
                     YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK"
     primer_bind     complement(987..1008)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1336..1355
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    complement(1577..1610)
                     /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(1661..1694)
                     /label=loxN
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AGGTATAC) (Shaw et al., 2021). loxN sites are 
                     compatible with each other, but incompatible with loxP or 
                     lox2272 sites."
     regulatory      1870..1879
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2500..2533
                     /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    2584..2617
                     /label=loxN
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AGGTATAC) (Shaw et al., 2021). loxN sites are 
                     compatible with each other, but incompatible with loxP or 
                     lox2272 sites."
     regulatory      3053..3062
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3069..3102)
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     misc_feature    3211..3799
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3264..3284)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3682..3693)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3831..4307
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4030..4049)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4347..4487
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4347..4476
                     /label=AAV2 ITR
     rep_origin      4562..5017
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4649..4668)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4859..4880
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5034..5053)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5153..5175
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5213..5231)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5299..5403
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5404..5472,5473..6264)
                     /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(5622..5641)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6435..6965,1..58)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6924..6943
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
       61 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtc
      121 ctgcaggcag ctgcgcgctc gctcgctcac tgaggccgcc cgggcgtcgg gcgacctttg
      181 gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta
      241 ggggttcctg cggccgcacg cgtaaggatc tgcgatcgct ccggtgcccg tcagtgggca
      301 gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaacgggt
      361 gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt
      421 tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt
      481 cgcaacgggt ttgccgccag aacacagctg aagcttcgag gggctcgcat ctctccttca
      541 cgcgcccgcc gccctacctg aggccgccat ccacgccggt tgagtcgcgt tctgccgcct
      601 cccgcctgtg gtgcctcctg aactgcgtcc gccgtctagg taagtttaaa gctcaggtcg
      661 agaccgggcc tttgtccggc gctcccttgg agcctaccta gactcagccg gctctccacg
      721 ctttgcctga ccctgcttgc tcaactctac gtctttgttt cgttttctgt tctgcgccgt
      781 tacagatcca agctgtgacc ggcgcctacg gatccgaagt tcctattctt caaatagtat
      841 aggaacttcg cagagtagta gctggattgt agctgctatt agcaatctaa aacctcttag
      901 aagttcctat tcttcaaaag gtataggaac ttcggcgcgc cttacttgta cagctcgtcc
      961 atgccgagag tgatcccggc ggcggtcacg aactccagca ggaccatgtg atcgcgcttc
     1021 tcgttggggt ctttgctcag ggcggactgg tagctcaggt agtggttgtc gggcagcagc
     1081 acggggccgt cgccgatggg ggtgttctgc tggtagtggt cggcgagctg cacgctgccg
     1141 tcctcgatgt tgtggcggat cttgaagttc accttgatgc cgttcttctg cttgtcggcc
     1201 atgatataga cgttgtggct gttgtagttg tactccagct tgtgccccag gatgttgccg
     1261 tcctccttga agtcgatgcc cttcagctcg atgcggttca ccagggtgtc gccctcgaac
     1321 ttcacctcgg cgcgggtctt gtagttgccg tcgtccttga agaagatggt gcgctcctgg
     1381 acgtagcctt cgggcatggc ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag
     1441 cgggcgaagc actgcaggcc gtagccgaag gtggtcacga gggtgggcca gggcacgggc
     1501 agctgtggga cgacatgact taaccagtta accatgttct ctgaacatga gaagggctgg
     1561 ccactctcca gtcgacataa cttcgtatag gatactttat acgaagttat gcagaatggt
     1621 agctggattg tagctgctat tagcaatatg aaacctctta ataacttcgt atagtatacc
     1681 ttatacgaag ttatactagt cctcctgtac tcaccttgcc ggtggtgcag atgaacttca
     1741 gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg aacttgtggc
     1801 cgtttacgtc accgtccagc tcgaccagga tgggcaccac cccggtgaac agctcctcgc
     1861 ccttgctcac catggtggcg gccgctggca cggctccggc ctcggcttcg tcttcgacga
     1921 gagtctcgac ctcgatctcc gttccgccga tgttcagttt ggtggttttg cggatatctc
     1981 cgtgaatcaa tatgtgctcg tggatcagga cgcgcaggta gtgtcccaac aacccccaac
     2041 aatttttact catcagatca ataatcgtgt gacctacggt ggagccatag acgctcagga
     2101 cgccaaaacc ttcgggcccc aaaatgaaga gaattgggaa cataccccag ctcacgaaaa
     2161 acagccatgc catgccggtc acgacctggc ggcaccgacc ctttggcaca gtatgataac
     2221 cctcgatata tgctttggcg gcgtgaaaaa atgtgttcgc gccatagcac aatccaagac
     2281 aaaagaagat gactttaaca tagccggttg ccatggcgct ggtagccccc cacacgatac
     2341 acccgatgtc tgagacaagg agtcccatgg ttctcctgct gtagtcgttg ctcaggccta
     2401 aacacatgag aaatagagtc ataagcacat gggtcacata caggagatat gtatataaca
     2461 ttaatacaat ttataaaaaa aagggggggc acaggtacca taacttcgta taaagtatcc
     2521 tatacgaagt tatttgcctt aacccagaaa ttatcactgt tattctttag aatggtgcaa
     2581 agaataactt cgtataaggt atactatacg aagttatgct agcaaccccg acacttaccg
     2641 gtgaggttgc tcaggtggat aaggatgaca ggacaagtga gcagccaggt tgcatagcgc
     2701 agccactgca cccggtgtcc tgtggcaagg tagagcatag agggattctt aaactcaaaa
     2761 aagaactcga gaatcacctt aaccatttca atggcgcaca catagatctc ctcccagccg
     2821 catgtagatt tccaggtttg gtaggcatag aacatcagca gcaaaatgct gaatcctgct
     2881 gcaagccact gcaggacatt tgacgcggtc tgagcgccgt tcgtgccgcg agattcaatc
     2941 catccggcac agtaacattg atcctcaggg accaggacgg acccgttcac caccacagga
     3001 ttagtaacga acaaaagttc gcgtccgacg gcagacaaag cgccgccata gtccatggtg
     3061 gcgagctcga agttcctata ctatttgaag aataggaact tcttgcctta acccagaaat
     3121 tatcactgtt attctttaga atggtgcaaa gagaagttcc tatacctttt gaagaatagg
     3181 aacttcgaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa
     3241 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     3301 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     3361 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     3421 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     3481 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     3541 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     3601 gggaaatcat cgtcctttcc ttggctgctc gcctatgttg ccacctggat tctgcgcggg
     3661 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     3721 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     3781 ctttgggccg cctccccgca tcgataccga gcgctgctcg agagatctac gggtggcatc
     3841 cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca gtgcccacca
     3901 gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc ttctataata
     3961 ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca acctgtaggg
     4021 cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa
     4081 tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg ttgggattcc
     4141 aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg ggtttcacca
     4201 tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct tggcctccca
     4261 aattgctggg attacaggcg tgaaccactg ctcccttccc tgtccttctg attttgtagg
     4321 taaccacgtg cggaccgagc ggccgcagga acccctagtg atggagttgg ccactccctc
     4381 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
     4441 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg
     4501 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag
     4561 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     4621 gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     4681 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     4741 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg
     4801 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     4861 ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta
     4921 taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt
     4981 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc
     5041 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     5101 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     5161 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     5221 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     5281 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     5341 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     5401 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     5461 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     5521 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     5581 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     5641 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     5701 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     5761 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     5821 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     5881 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     5941 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     6001 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     6061 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     6121 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     6181 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     6241 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     6301 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     6361 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     6421 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     6481 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     6541 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     6601 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     6661 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     6721 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     6781 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     6841 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     6901 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     6961 cgcca
//
