LOCUS       sequence-272725-        6965 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intersectional viral expression of ChR2(ET/TC)-EYFP in cells 
            expressing Cre AND NOT 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/Foff 2.0-ChR2(ET/TC)-EYFP
FEATURES             Location/Qualifiers
     source          1..6965
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        177..388
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             401..669
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     protein_bind    697..730
                     /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)"
     regulatory      823..832
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(1268..1301)
                     /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."
     protein_bind    complement(1352..1385)
                     /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)."
     regulatory      1561..1570
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2191..2224
                     /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."
     protein_bind    2275..2308
                     /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)."
     primer_bind     complement(2530..2549)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2689..2940
                     /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     2877..2898
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2950..2983)
                     /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    3092..3680
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3145..3165)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3563..3574)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3712..4188
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3911..3930)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4228..4368
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4228..4357
                     /label=AAV2 ITR
     rep_origin      4443..4898
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4530..4549)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4740..4761
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4915..4934)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5034..5056
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5094..5112)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5180..5284
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5285..5353,5354..6145)
                     /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(5503..5522)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6316..6904
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6805..6824
                     /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 gcggccgcac gcgtaaggat ctgcgatcgc tccggtgccc gtcagtgggc
      181 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaacggg
      241 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
      301 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
      361 tcgcaacggg tttgccgcca gaacacagct gaagcttcga ggggctcgca tctctccttc
      421 acgcgcccgc cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc
      481 tcccgcctgt ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc
      541 gagaccgggc ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac
      601 gctttgcctg accctgcttg ctcaactcta cgtctttgtt tcgttttctg ttctgcgccg
      661 ttacagatcc aagctgtgac cggcgcctac ggatccgaag ttcctattct ctagaaagta
      721 taggaacttc gcagagtagt agctggattg tagctgctat tagcaatcta aaacctctta
      781 gaagttccta ttcttcaaaa ggtataggaa cttcggcgcg ccgccaccat ggactatggc
      841 ggcgctttgt ctgccgtcgg acgcgaactt ttgttcgtta ctaatcctgt ggtggtgaac
      901 gggtccgtcc tggtccctga ggatcaatgt tactgtgccg gatggattga atctcgcggc
      961 acgaacggcg ctcagaccgc gtcaaatgtc ctgcagtggc ttgcagcagg attcagcatt
     1021 ttgctgctga tgttctatgc ctaccaaacc tggaaatcta catgcggctg ggaggagatc
     1081 tatgtgtgcg ccattgaaat ggttaaggtg attctcgagt tcttttttga gtttaagaat
     1141 ccctctatgc tctaccttgc cacaggacac cgggtgcagt ggctgcgcta tgcaacctgg
     1201 ctgctcactt gtcctgtcat ccttatccac ctgagcaacc tcaccggtaa gtgtcggggt
     1261 tgagctcata acttcgtata gtatacctta tacgaagtta ttctttgcac cattctaaag
     1321 aataacagtg ataatttctg ggttaaggca aataacttcg tataggatac tttatacgaa
     1381 gttatggtac ccctcctgta ctcaccttgc cggtggtgca gatgaacttc agggtcagct
     1441 tgccgtaggt ggcatcgccc tcgccctcgc cggacacgct gaacttgtgg ccgtttacgt
     1501 caccgtccag ctcgaccagg atgggcacca ccccggtgaa cagctcctcg cccttgctca
     1561 ccatggtggc ggccgctggc acggctccgg cctcggcttc gtcttcgacg agagtctcga
     1621 cctcgatctc cgttccgccg atgttcagtt tggtggtttt gcggatatct ccgtgaatca
     1681 atatgtgctc gtggatcagg acgcgcaggt agtgtcccaa caacccccaa caatttttac
     1741 tcatcagatc aataatcgtg tgacctacgg tggagccata gacgctcagg acgccaaaac
     1801 cttcgggccc caaaatgaag agaattggga acatacccca gctcacgaaa aacagccatg
     1861 ccatgccggt cacgacctgg cggcaccgac cctttggcac agtatgataa ccctcgatat
     1921 atgctttggc ggcgtgaaaa aatgtgttcg cgccatagca caatccaaga caaaagaaga
     1981 tgactttaac atagccggtt gccatggcgc tggtagcccc ccacacgata cacccgatgt
     2041 ctgagacaag gagtcccatg gttctcctgc tgtagtcgtt gctcaggcct aaacacatga
     2101 gaaatagagt cataagcaca tgggtcacat acaggagata tgtatataac attaatacaa
     2161 tttataaaaa aaaggggggg cacaactagt ataacttcgt ataaggtata ctatacgaag
     2221 ttattaagag gtttcatatt gctaatagca gctacaatcc agctaccatt ctgcataact
     2281 tcgtataaag tatcctatac gaagttatgt cgactggaga gtggccagcc cttctcatgt
     2341 tcagagaaca tggttaactg gttaagtcat gtcgtcccac agctgcccgt gccctggccc
     2401 accctcgtga ccaccttcgg ctacggcctg cagtgcttcg cccgctaccc cgaccacatg
     2461 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     2521 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     2581 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     2641 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     2701 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     2761 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     2821 cactacctga gctaccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     2881 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     2941 taagctagcg aagttcctat actttctaga gaataggaac ttctaagcag acaatcttgt
     3001 aatacatcag cagctagtag attgctccag ctagaagttc ctataccttt tgaagaatag
     3061 gaacttcgaa ttcgatatca agcttatcga taatcaacct ctggattaca aaatttgtga
     3121 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     3181 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     3241 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     3301 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     3361 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     3421 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     3481 ggggaaatca tcgtcctttc cttggctgct cgcctatgtt gccacctgga ttctgcgcgg
     3541 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     3601 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     3661 cctttgggcc gcctccccgc atcgataccg agcgctgctc gagagatcta cgggtggcat
     3721 ccctgtgacc cctccccagt gcctctcctg gccctggaag ttgccactcc agtgcccacc
     3781 agccttgtcc taataaaatt aagttgcatc attttgtctg actaggtgtc cttctataat
     3841 attatggggt ggaggggggt ggtatggagc aaggggcaag ttgggaagac aacctgtagg
     3901 gcctgcgggg tctattggga accaagctgg agtgcagtgg cacaatcttg gctcactgca
     3961 atctccgcct cctgggttca agcgattctc ctgcctcagc ctcccgagtt gttgggattc
     4021 caggcatgca tgaccaggct cagctaattt ttgttttttt ggtagagacg gggtttcacc
     4081 atattggcca ggctggtctc caactcctaa tctcaggtga tctacccacc ttggcctccc
     4141 aaattgctgg gattacaggc gtgaaccact gctcccttcc ctgtccttct gattttgtag
     4201 gtaaccacgt gcggaccgag cggccgcagg aacccctagt gatggagttg gccactccct
     4261 ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct
     4321 ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc
     4381 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata
     4441 gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     4501 cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     4561 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     4621 tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg
     4681 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     4741 tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt
     4801 ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt
     4861 taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat
     4921 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     4981 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     5041 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
     5101 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     5161 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     5221 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     5281 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     5341 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     5401 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     5461 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     5521 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     5581 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     5641 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     5701 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     5761 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     5821 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     5881 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     5941 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     6001 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     6061 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     6121 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     6181 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     6241 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     6301 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     6361 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     6421 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     6481 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     6541 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     6601 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     6661 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     6721 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     6781 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     6841 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     6901 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     6961 catgt
//
