LOCUS       sequence-301583-        6965 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intersectional viral expression of ChR2(ET/TC)-EYFP in cells 
            expressing both Cre AND Flp.
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-Con/Fon-ChR2(ET/TC)-EYFP
FEATURES             Location/Qualifiers
     source          1..6965
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   113..253
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   113..242
                     /label=AAV2 ITR
     rep_origin      328..783
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(415..434)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     625..646
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(800..819)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     919..941
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(979..997)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1065..1169
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1170..1238,1239..2030)
                     /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(1388..1407)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2201..2789
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2690..2709
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2851..2980
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        3027..3238
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             3251..3519
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     protein_bind    3547..3580
                     /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(3676..3927)
                     /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(3718..3739)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     4067..4086
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    complement(4308..4341)
                     /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(4392..4425)
                     /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      5046..5055
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    5231..5264
                     /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    5315..5348
                     /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      5784..5793
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(5800..5833)
                     /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    5942..6530
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5995..6015)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6413..6424)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    join(6562..6965,1..73)
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(6761..6780)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
ORIGIN
        1 ggtgatctac ccaccttggc ctcccaaatt gctgggatta caggcgtgaa ccactgctcc
       61 cttccctgtc cttctgattt tgtaggtaac cacgtgcgga ccgagcggcc gcaggaaccc
      121 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
      181 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
      241 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
      301 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
      361 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt
      421 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
      481 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
      541 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
      601 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact
      661 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa
      721 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
      781 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
      841 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
      901 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
      961 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     1021 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     1081 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     1141 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     1201 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     1261 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     1321 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     1381 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     1441 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     1501 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     1561 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     1621 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     1681 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     1741 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     1801 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     1861 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     1921 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     1981 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     2041 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     2101 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     2161 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     2221 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     2281 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     2341 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     2401 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     2461 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     2521 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     2581 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     2641 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     2701 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     2761 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     2821 tggccttttg ctggcctttt gctcacatgt cctgcaggca gctgcgcgct cgctcgctca
     2881 ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg
     2941 cgcagagagg gagtggccaa ctccatcact aggggttcct gcggccgcac gcgtaaggat
     3001 ctgcgatcgc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa
     3061 gttgggggga ggggtcggca attgaacggg tgcctagaga aggtggcgcg gggtaaactg
     3121 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat
     3181 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacagct
     3241 gaagcttcga ggggctcgca tctctccttc acgcgcccgc cgccctacct gaggccgcca
     3301 tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt ggtgcctcct gaactgcgtc
     3361 cgccgtctag gtaagtttaa agctcaggtc gagaccgggc ctttgtccgg cgctcccttg
     3421 gagcctacct agactcagcc ggctctccac gctttgcctg accctgcttg ctcaactcta
     3481 cgtctttgtt tcgttttctg ttctgcgccg ttacagatcc aagctgtgac cggcgcctac
     3541 ggatccgaag ttcctattct tcaaatagta taggaacttc gcagagtagt agctggattg
     3601 tagctgctat tagcaatcta aaacctctta gaagttccta ttcttcaaaa ggtataggaa
     3661 cttcggcgcg ccttacttgt acagctcgtc catgccgaga gtgatcccgg cggcggtcac
     3721 gaactccagc aggaccatgt gatcgcgctt ctcgttgggg tctttgctca gggcggactg
     3781 gtagctcagg tagtggttgt cgggcagcag cacggggccg tcgccgatgg gggtgttctg
     3841 ctggtagtgg tcggcgagct gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt
     3901 caccttgatg ccgttcttct gcttgtcggc catgatatag acgttgtggc tgttgtagtt
     3961 gtactccagc ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc
     4021 gatgcggttc accagggtgt cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc
     4081 gtcgtccttg aagaagatgg tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa
     4141 gaagtcgtgc tgcttcatgt ggtcggggta gcgggcgaag cactgcaggc cgtagccgaa
     4201 ggtggtcacg agggtgggcc agggcacggg cagctgtggg acgacatgac ttaaccagtt
     4261 aaccatgttc tctgaacatg agaagggctg gccactctcc agtcgacata acttcgtata
     4321 ggatacttta tacgaagtta tgcagaatgg tagctggatt gtagctgcta ttagcaatat
     4381 gaaacctctt aataacttcg tatagtatac cttatacgaa gttatactag ttgtgccccc
     4441 cctttttttt ataaattgta ttaatgttat atacatatct cctgtatgtg acccatgtgc
     4501 ttatgactct atttctcatg tgtttaggcc tgagcaacga ctacagcagg agaaccatgg
     4561 gactccttgt ctcagacatc gggtgtatcg tgtggggggc taccagcgcc atggcaaccg
     4621 gctatgttaa agtcatcttc ttttgtcttg gattgtgcta tggcgcgaac acattttttc
     4681 acgccgccaa agcatatatc gagggttatc atactgtgcc aaagggtcgg tgccgccagg
     4741 tcgtgaccgg catggcatgg ctgtttttcg tgagctgggg tatgttccca attctcttca
     4801 ttttggggcc cgaaggtttt ggcgtcctga gcgtctatgg ctccaccgta ggtcacacga
     4861 ttattgatct gatgagtaaa aattgttggg ggttgttggg acactacctg cgcgtcctga
     4921 tccacgagca catattgatt cacggagata tccgcaaaac caccaaactg aacatcggcg
     4981 gaacggagat cgaggtcgag actctcgtcg aagacgaagc cgaggccgga gccgtgccag
     5041 cggccgccac catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     5101 tcgagctgga cggtgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
     5161 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag gtgagtacag
     5221 gaggggtacc ataacttcgt ataaagtatc ctatacgaag ttatttgcct taacccagaa
     5281 attatcactg ttattcttta gaatggtgca aagaataact tcgtataagg tatactatac
     5341 gaagttatgc tagcaacccc gacacttacc ggtgaggttg ctcaggtgga taaggatgac
     5401 aggacaagtg agcagccagg ttgcatagcg cagccactgc acccggtgtc ctgtggcaag
     5461 gtagagcata gagggattct taaactcaaa aaagaactcg agaatcacct taaccatttc
     5521 aatggcgcac acatagatct cctcccagcc gcatgtagat ttccaggttt ggtaggcata
     5581 gaacatcagc agcaaaatgc tgaatcctgc tgcaagccac tgcaggacat ttgacgcggt
     5641 ctgagcgccg ttcgtgccgc gagattcaat ccatccggca cagtaacatt gatcctcagg
     5701 gaccaggacg gacccgttca ccaccacagg attagtaacg aacaaaagtt cgcgtccgac
     5761 ggcagacaaa gcgccgccat agtccatggt ggcgagctcg aagttcctat actatttgaa
     5821 gaataggaac ttcttgcctt aacccagaaa ttatcactgt tattctttag aatggtgcaa
     5881 agagaagttc ctataccttt tgaagaatag gaacttcgaa ttcgatatca agcttatcga
     5941 taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     6001 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     6061 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     6121 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     6181 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     6241 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     6301 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     6361 cgcctatgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     6421 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     6481 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg
     6541 agcgctgctc gagagatcta cgggtggcat ccctgtgacc cctccccagt gcctctcctg
     6601 gccctggaag ttgccactcc agtgcccacc agccttgtcc taataaaatt aagttgcatc
     6661 attttgtctg actaggtgtc cttctataat attatggggt ggaggggggt ggtatggagc
     6721 aaggggcaag ttgggaagac aacctgtagg gcctgcgggg tctattggga accaagctgg
     6781 agtgcagtgg cacaatcttg gctcactgca atctccgcct cctgggttca agcgattctc
     6841 ctgcctcagc ctcccgagtt gttgggattc caggcatgca tgaccaggct cagctaattt
     6901 ttgttttttt ggtagagacg gggtttcacc atattggcca ggctggtctc caactcctaa
     6961 tctca
//
