LOCUS       sequence-271943-        7004 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 7004)
  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 7004)
  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..7004
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        172..383
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             396..664
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     protein_bind    692..725
                     /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(821..1072)
                     /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(863..884)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1212..1231
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    complement(1453..1486)
                     /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(1537..1570)
                     /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     1683..1704
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    2374..2407
                     /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    2458..2491
                     /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      2968..2977
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2984..3017)
                     /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    3126..3714
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3179..3199)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3597..3608)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3746..4222
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3945..3964)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4262..4402
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4262..4391
                     /label=AAV2 ITR
     rep_origin      4477..4932
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4564..4583)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4774..4795
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4949..4968)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5068..5090
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5128..5146)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5214..5318
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5319..5387,5388..6179)
                     /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(5537..5556)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6350..6938
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6839..6858
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(7000..7004,1..125)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 aggcagctgc gcgctcgctc gctcactgag gccgcccggg cgtcgggcga cctttggtcg
       61 cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg
      121 ttcctgcggc cgcacgcgta aggatctgcg atcgctccgg tgcccgtcag tgggcagagc
      181 gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga acgggtgcct
      241 agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc
      301 ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca
      361 acgggtttgc cgccagaaca cagctgaagc ttcgaggggc tcgcatctct ccttcacgcg
      421 cccgccgccc tacctgaggc cgccatccac gccggttgag tcgcgttctg ccgcctcccg
      481 cctgtggtgc ctcctgaact gcgtccgccg tctaggtaag tttaaagctc aggtcgagac
      541 cgggcctttg tccggcgctc ccttggagcc tacctagact cagccggctc tccacgcttt
      601 gcctgaccct gcttgctcaa ctctacgtct ttgtttcgtt ttctgttctg cgccgttaca
      661 gatccaagct gtgaccggcg cctacggatc cgaagttcct attcttcaaa tagtatagga
      721 acttcgcaga gtagtagctg gattgtagct gctattagca atctaaaacc tcttagaagt
      781 tcctattctt caaaaggtat aggaacttcg gcgcgcctta cttgtacagc tcgtccatgc
      841 cgagagtgat cccggcggcg gtcacgaact ccagcaggac catgtgatcg cgcttctcgt
      901 tggggtcttt gctcagggcg gactggtagc tcaggtagtg gttgtcgggc agcagcacgg
      961 ggccgtcgcc gatgggggtg ttctgctggt agtggtcggc gagctgcacg ctgccgtcct
     1021 cgatgttgtg gcggatcttg aagttcacct tgatgccgtt cttctgcttg tcggccatga
     1081 tatagacgtt gtggctgttg tagttgtact ccagcttgtg ccccaggatg ttgccgtcct
     1141 ccttgaagtc gatgcccttc agctcgatgc ggttcaccag ggtgtcgccc tcgaacttca
     1201 cctcggcgcg ggtcttgtag ttgccgtcgt ccttgaagaa gatggtgcgc tcctggacgt
     1261 agccttcggg catggcggac ttgaagaagt cgtgctgctt catgtggtcg gggtagcggg
     1321 cgaagcactg caggccgtag ccgaaggtgg tcacgagggt gggccagggc acgggcagct
     1381 gtgggacgac atgacttaac cagttaacca tgttctctga acatgagaag ggctggccac
     1441 tctccagtcg acataacttc gtataggata ctttatacga agttatgcag aatggtagct
     1501 ggattgtagc tgctattagc aatatgaaac ctcttaataa cttcgtatag tataccttat
     1561 acgaagttat actagtcctc ctgtactcac cttgccggtg gtgcagatga acttcagggt
     1621 cagcttgccg taggtggcat cgccctcgcc ctcgccggac acgctgaact tgtggccgtt
     1681 tacgtcgccg tccagctcga ccaggatggg caccaccccg gtgaacagct cctcgccctt
     1741 gctcaccacg ttgatgtcga tctggtccag ggggatgtac tcgccctcgc tggtgatcct
     1801 gctcttggcg gccgcatctt cttcttccgc caccagggtt tccacttcca tttcctggcc
     1861 cgcaatggta attttctgtt ttttgcgaat atcgccatac agcagaatat gttcatgaat
     1921 tttcacgcgc agatagttgc ccagcacgcc ccacatgttc ttcgcaatca gatccagaat
     1981 gctatggcca atcgcgctgc catacgggct aatatggccg aagccctcgg ggcccagcag
     2041 gaacagcacg gggaacatgc cccagctcac gaagaacagc caggccatgg ctctcaccag
     2101 ctgtctgcac aggcctttcg gcacggtatg aaacgcttca atatacactt tggccgcatg
     2161 aaaataggta tacatgccat agctcaggct aatcagaaaa aacagaattt tggtccagcc
     2221 ggtgcacatc gcgctggtcg cgccccacac aatgcagccc acgtcgctca cctaaacaca
     2281 tgagaaatag agtcataagc acatgggtca catacaggag atatgtatat aacattaata
     2341 caatttataa aaaaaagggg gggcacaggt accataactt cgtataaagt atcctatacg
     2401 aagttatttg ccttaaccca gaaattatca ctgttattct ttagaatggt gcaaagaata
     2461 acttcgtata aggtatacta tacgaagtta tgctagcaac cccgacactt accagcaggc
     2521 ccatggtgcg tttgctataa tcatctttca ggccggtcag gttgctcaga tgaatcagaa
     2581 tcacggggca ggtcagcagc catgagccat agcgcatcca caccacgccg ttgccgctgc
     2641 tcagccacag ggtcgccggg ctatcaaatt catgaaacgc ttcaataatg cttttcatca
     2701 tttcaatcag cgccacatac acttcttccc agccgcaggt cgcgcgccac gcctgatacg
     2761 cataccagcc caggcacgcc acgctcagcg caaacaccac ccactgcagg atgttggcgg
     2821 ccagcttctc ggcgttggtg ccgttactct tcagccaggc caggcagaag cactggccgt
     2881 tgttggggat gcagatcacg ctgccgttgt tctccagggt gtagctggtc tggaacagtc
     2941 cgacggcaga caaagcgccg ccatagtcca tggtggcgag ctcgaagttc ctatactatt
     3001 tgaagaatag gaacttcttg ccttaaccca gaaattatca ctgttattct ttagaatggt
     3061 gcaaagagaa gttcctatac cttttgaaga ataggaactt cgaattcgat atcaagctta
     3121 tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     3181 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     3241 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
     3301 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
     3361 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
     3421 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     3481 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc
     3541 tgctcgccta tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
     3601 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
     3661 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat
     3721 accgagcgct gctcgagaga tctacgggtg gcatccctgt gacccctccc cagtgcctct
     3781 cctggccctg gaagttgcca ctccagtgcc caccagcctt gtcctaataa aattaagttg
     3841 catcattttg tctgactagg tgtccttcta taatattatg gggtggaggg gggtggtatg
     3901 gagcaagggg caagttggga agacaacctg tagggcctgc ggggtctatt gggaaccaag
     3961 ctggagtgca gtggcacaat cttggctcac tgcaatctcc gcctcctggg ttcaagcgat
     4021 tctcctgcct cagcctcccg agttgttggg attccaggca tgcatgacca ggctcagcta
     4081 atttttgttt ttttggtaga gacggggttt caccatattg gccaggctgg tctccaactc
     4141 ctaatctcag gtgatctacc caccttggcc tcccaaattg ctgggattac aggcgtgaac
     4201 cactgctccc ttccctgtcc ttctgatttt gtaggtaacc acgtgcggac cgagcggccg
     4261 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     4321 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     4381 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     4441 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     4501 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc
     4561 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     4621 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     4681 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     4741 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     4801 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
     4861 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     4921 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     4981 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     5041 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     5101 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     5161 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     5221 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     5281 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     5341 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     5401 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     5461 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     5521 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     5581 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     5641 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     5701 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     5761 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     5821 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     5881 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     5941 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     6001 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     6061 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     6121 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     6181 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     6241 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     6301 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     6361 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     6421 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     6481 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     6541 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     6601 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     6661 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     6721 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     6781 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     6841 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     6901 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     6961 tacggttcct ggccttttgc tggccttttg ctcacatgtc ctgc
//
