LOCUS       sequence-265108-        7015 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intersectional viral expression of bREACHes-EYFP in cells expressing
            both Cre AND Flp.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7015)
  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 7015)
  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-bREACHes-EYFP
FEATURES             Location/Qualifiers
     source          1..7015
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        188..399
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             412..680
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     protein_bind    708..741
                     /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(837..1088)
                     /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(879..900)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1228..1247
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    complement(1469..1502)
                     /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(1553..1586)
                     /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."
     primer_bind     complement(2256..2277)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    2390..2423
                     /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    2474..2507
                     /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      2984..2993
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3000..3033)
                     /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    3142..3730
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3195..3215)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3613..3624)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3762..4238
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3961..3980)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4278..4418
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4278..4407
                     /label=AAV2 ITR
     rep_origin      4493..4948
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4580..4599)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4790..4811
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4965..4984)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5084..5106
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5144..5162)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5230..5334
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5335..5403,5404..6195)
                     /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(5553..5572)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6366..6954
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6855..6874
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtaagga tctgcgatcg ctccggtgcc
      181 cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc
      241 aattgaacgg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac
      301 tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg
      361 aacgttcttt ttcgcaacgg gtttgccgcc agaacacagc tgaagcttcg aggggctcgc
      421 atctctcctt cacgcgcccg ccgccctacc tgaggccgcc atccacgccg gttgagtcgc
      481 gttctgccgc ctcccgcctg tggtgcctcc tgaactgcgt ccgccgtcta ggtaagttta
      541 aagctcaggt cgagaccggg cctttgtccg gcgctccctt ggagcctacc tagactcagc
      601 cggctctcca cgctttgcct gaccctgctt gctcaactct acgtctttgt ttcgttttct
      661 gttctgcgcc gttacagatc caagctgtga ccggcgccta cggatccgaa gttcctattc
      721 ttcaaatagt ataggaactt cgcagagtag tagctggatt gtagctgcta ttagcaatct
      781 aaaacctctt agaagttcct attcttcaaa aggtatagga acttcggcgc gccttacttg
      841 tacagctcgt ccatgccgag agtgatcccg gcggcggtca cgaactccag caggaccatg
      901 tgatcgcgct tctcgttggg gtctttgctc agggcggact ggtagctcag gtagtggttg
      961 tcgggcagca gcacggggcc gtcgccgatg ggggtgttct gctggtagtg gtcggcgagc
     1021 tgcacgctgc cgtcctcgat gttgtggcgg atcttgaagt tcaccttgat gccgttcttc
     1081 tgcttgtcgg ccatgatata gacgttgtgg ctgttgtagt tgtactccag cttgtgcccc
     1141 aggatgttgc cgtcctcctt gaagtcgatg cccttcagct cgatgcggtt caccagggtg
     1201 tcgccctcga acttcacctc ggcgcgggtc ttgtagttgc cgtcgtcctt gaagaagatg
     1261 gtgcgctcct ggacgtagcc ttcgggcatg gcggacttga agaagtcgtg ctgcttcatg
     1321 tggtcggggt agcgggcgaa gcactgcagg ccgtagccga aggtggtcac gagggtgggc
     1381 cagggcacgg gcagctgtgg gacgacatga cttaaccagt taaccatgtt ctctgaacat
     1441 gagaagggct ggccactctc cagtcgacat aacttcgtat aggatacttt atacgaagtt
     1501 atgcagaatg gtagctggat tgtagctgct attagcaata tgaaacctct taataacttc
     1561 gtatagtata ccttatacga agttatacta gttgtgcccc cccttttttt tataaattgt
     1621 attaatgtta tatacatatc tcctgtatgt gacccatgtg cttatgactc tatttctcat
     1681 gtgtttaggt gagcgacgtg ggctgcattg tgtggggcgc gaccagcgcg atgtgcaccg
     1741 gctggaccaa aattctgttt tttctgatta gcctgagcta tggcatgtat acctattttc
     1801 atgcggccaa agtgtatatt gaagcgtttc ataccgtgcc gaaaggcctg tgcagacagc
     1861 tggtgagagc catggcctgg ctgttcttcg tgagctgggg catgttcccc gtgctgttcc
     1921 tgctgggccc cgagggcttc ggccatatta gcccgtatgg cagcgcgatt ggccatagca
     1981 ttctggatct gattgcgaag aacatgtggg gcgtgctggg caactatctg cgcgtgaaaa
     2041 ttcatgaaca tattctgctg tatggcgata ttcgcaaaaa acagaaaatt accattgcgg
     2101 gccaggaaat ggaagtggaa accctggtgg cggaagaaga agatgcggcc gccaagagca
     2161 ggatcaccag cgagggcgag tacatccccc tggaccagat cgacatcaac gtggtgagca
     2221 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     2281 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     2341 ccctgaagtt catctgcacc accggcaagg tgagtacagg aggggtacca taacttcgta
     2401 taaagtatcc tatacgaagt tatttgcctt aacccagaaa ttatcactgt tattctttag
     2461 aatggtgcaa agaataactt cgtataaggt atactatacg aagttatgct agcaaccccg
     2521 acacttacca gcaggcccat ggtgcgtttg ctataatcat ctttcaggcc ggtcaggttg
     2581 ctcagatgaa tcagaatcac ggggcaggtc agcagccatg agccatagcg catccacacc
     2641 acgccgttgc cgctgctcag ccacagggtc gccgggctat caaattcatg aaacgcttca
     2701 ataatgcttt tcatcatttc aatcagcgcc acatacactt cttcccagcc gcaggtcgcg
     2761 cgccacgcct gatacgcata ccagcccagg cacgccacgc tcagcgcaaa caccacccac
     2821 tgcaggatgt tggcggccag cttctcggcg ttggtgccgt tactcttcag ccaggccagg
     2881 cagaagcact ggccgttgtt ggggatgcag atcacgctgc cgttgttctc cagggtgtag
     2941 ctggtctgga acagtccgac ggcagacaaa gcgccgccat agtccatggt ggcgagctcg
     3001 aagttcctat actatttgaa gaataggaac ttcttgcctt aacccagaaa ttatcactgt
     3061 tattctttag aatggtgcaa agagaagttc ctataccttt tgaagaatag gaacttcgaa
     3121 ttcgatatca agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact
     3181 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     3241 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     3301 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     3361 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     3421 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     3481 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca
     3541 tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     3601 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     3661 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     3721 gcctccccgc atcgataccg agcgctgctc gagagatcta cgggtggcat ccctgtgacc
     3781 cctccccagt gcctctcctg gccctggaag ttgccactcc agtgcccacc agccttgtcc
     3841 taataaaatt aagttgcatc attttgtctg actaggtgtc cttctataat attatggggt
     3901 ggaggggggt ggtatggagc aaggggcaag ttgggaagac aacctgtagg gcctgcgggg
     3961 tctattggga accaagctgg agtgcagtgg cacaatcttg gctcactgca atctccgcct
     4021 cctgggttca agcgattctc ctgcctcagc ctcccgagtt gttgggattc caggcatgca
     4081 tgaccaggct cagctaattt ttgttttttt ggtagagacg gggtttcacc atattggcca
     4141 ggctggtctc caactcctaa tctcaggtga tctacccacc ttggcctccc aaattgctgg
     4201 gattacaggc gtgaaccact gctcccttcc ctgtccttct gattttgtag gtaaccacgt
     4261 gcggaccgag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     4321 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     4381 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     4441 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     4501 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     4561 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     4621 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     4681 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     4741 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     4801 ttccaaactg gaacaacact caaccctatc tcgggctatt cttttgattt ataagggatt
     4861 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     4921 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     4981 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     5041 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     5101 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     5161 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     5221 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5281 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5341 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5401 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     5461 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     5521 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     5581 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     5641 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     5701 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     5761 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     5821 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     5881 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     5941 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     6001 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     6061 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6121 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6181 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6241 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6301 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6361 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6421 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6481 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     6541 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6601 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6661 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6721 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6781 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6841 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     6901 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     6961 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgt
//
