LOCUS       sequence-265110-        7016 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intersectional viral expression of bREACHes-EYFP in cells expressing
            Flp AND NOT Cre.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7016)
  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 7016)
  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-bREACHes-EYFP
FEATURES             Location/Qualifiers
     source          1..7016
                     /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     1699..1720
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    2391..2424
                     /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    2475..2508
                     /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      2985..2994
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3001..3034)
                     /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    3143..3731
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3196..3216)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3614..3625)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3763..4239
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3962..3981)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4279..4419
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4279..4408
                     /label=AAV2 ITR
     rep_origin      4494..4949
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4581..4600)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4791..4812
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4966..4985)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5085..5107
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5145..5163)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5231..5335
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5336..5404,5405..6196)
                     /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(5554..5573)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6367..6955
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6856..6875
                     /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 gtcctcctgt actcaccttg ccggtggtgc
     1621 agatgaactt cagggtcagc ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc
     1681 tgaacttgtg gccgtttacg tcgccgtcca gctcgaccag gatgggcacc accccggtga
     1741 acagctcctc gcccttgctc accacgttga tgtcgatctg gtccaggggg atgtactcgc
     1801 cctcgctggt gatcctgctc ttggcggccg catcttcttc ttccgccacc agggtttcca
     1861 cttccatttc ctggcccgca atggtaattt tctgtttttt gcgaatatcg ccatacagca
     1921 gaatatgttc atgaattttc acgcgcagat agttgcccag cacgccccac atgttcttcg
     1981 caatcagatc cagaatgcta tggccaatcg cgctgccata cgggctaata tggccgaagc
     2041 cctcggggcc cagcaggaac agcacgggga acatgcccca gctcacgaag aacagccagg
     2101 ccatggctct caccagctgt ctgcacaggc ctttcggcac ggtatgaaac gcttcaatat
     2161 acactttggc cgcatgaaaa taggtataca tgccatagct caggctaatc agaaaaaaca
     2221 gaattttggt ccagccggtg cacatcgcgc tggtcgcgcc ccacacaatg cagcccacgt
     2281 cgctcaccta aacacatgag aaatagagtc ataagcacat gggtcacata caggagatat
     2341 gtatataaca ttaatacaat tttataaaaa aaaggggggg cacaggtacc ataacttcgt
     2401 ataaagtatc ctatacgaag ttatttgcct taacccagaa attatcactg ttattcttta
     2461 gaatggtgca aagaataact tcgtataagg tatactatac gaagttatgc tagcaacccc
     2521 gacacttacc agcaggccca tggtgcgttt gctataatca tctttcaggc cggtcaggtt
     2581 gctcagatga atcagaatca cggggcaggt cagcagccat gagccatagc gcatccacac
     2641 cacgccgttg ccgctgctca gccacagggt cgccgggcta tcaaattcat gaaacgcttc
     2701 aataatgctt ttcatcattt caatcagcgc cacatacact tcttcccagc cgcaggtcgc
     2761 gcgccacgcc tgatacgcat accagcccag gcacgccacg ctcagcgcaa acaccaccca
     2821 ctgcaggatg ttggcggcca gcttctcggc gttggtgccg ttactcttca gccaggccag
     2881 gcagaagcac tggccgttgt tggggatgca gatcacgctg ccgttgttct ccagggtgta
     2941 gctggtctgg aacagtccga cggcagacaa agcgccgcca tagtccatgg tggcgagctc
     3001 gaagttccta tactatttga agaataggaa cttcttgcct taacccagaa attatcactg
     3061 ttattcttta gaatggtgca aagagaagtt cctatacctt ttgaagaata ggaacttcga
     3121 attcgatatc aagcttatcg ataatcaacc tctggattac aaaatttgtg aaagattgac
     3181 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
     3241 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
     3301 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
     3361 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
     3421 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
     3481 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc
     3541 atcgtccttt ccttggctgc tcgcctatgt tgccacctgg attctgcgcg ggacgtcctt
     3601 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
     3661 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
     3721 cgcctccccg catcgatacc gagcgctgct cgagagatct acgggtggca tccctgtgac
     3781 ccctccccag tgcctctcct ggccctggaa gttgccactc cagtgcccac cagccttgtc
     3841 ctaataaaat taagttgcat cattttgtct gactaggtgt ccttctataa tattatgggg
     3901 tggagggggg tggtatggag caaggggcaa gttgggaaga caacctgtag ggcctgcggg
     3961 gtctattggg aaccaagctg gagtgcagtg gcacaatctt ggctcactgc aatctccgcc
     4021 tcctgggttc aagcgattct cctgcctcag cctcccgagt tgttgggatt ccaggcatgc
     4081 atgaccaggc tcagctaatt tttgtttttt tggtagagac ggggtttcac catattggcc
     4141 aggctggtct ccaactccta atctcaggtg atctacccac cttggcctcc caaattgctg
     4201 ggattacagg cgtgaaccac tgctcccttc cctgtccttc tgattttgta ggtaaccacg
     4261 tgcggaccga gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg
     4321 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg
     4381 cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc
     4441 tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc
     4501 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     4561 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     4621 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     4681 acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc
     4741 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     4801 gttccaaact ggaacaacac tcaaccctat ctcgggctat tcttttgatt tataagggat
     4861 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     4921 ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga
     4981 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     5041 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     5101 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     5161 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     5221 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     5281 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     5341 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     5401 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     5461 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     5521 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     5581 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     5641 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     5701 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     5761 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     5821 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     5881 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     5941 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     6001 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     6061 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     6121 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     6181 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     6241 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     6301 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     6361 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     6421 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     6481 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     6541 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     6601 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     6661 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     6721 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     6781 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     6841 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     6901 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     6961 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt
//
