LOCUS       sequence-272740-        7004 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intersectional viral expression of bREACHes-EYFP in cells expressing
            Cre AND NOT Flp.
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-Con/Foff 2.0-bREACHes-EYFP
FEATURES             Location/Qualifiers
     source          1..7004
                     /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(1309..1342)
                     /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(1393..1426)
                     /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     1539..1560
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    2230..2263
                     /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    2314..2347
                     /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(2569..2588)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2728..2979
                     /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     2916..2937
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2989..3022)
                     /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    3131..3719
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3184..3204)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3602..3613)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3751..4227
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3950..3969)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4267..4407
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4267..4396
                     /label=AAV2 ITR
     rep_origin      4482..4937
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4569..4588)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4779..4800
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4954..4973)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5073..5095
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5133..5151)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5219..5323
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5324..5392,5393..6184)
                     /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(5542..5561)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6355..6943
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6844..6863
                     /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 actgttccag accagctaca ccctggagaa caacggcagc
      901 gtgatctgca tccccaacaa cggccagtgc ttctgcctgg cctggctgaa gagtaacggc
      961 accaacgccg agaagctggc cgccaacatc ctgcagtggg tggtgtttgc gctgagcgtg
     1021 gcgtgcctgg gctggtatgc gtatcaggcg tggcgcgcga cctgcggctg ggaagaagtg
     1081 tatgtggcgc tgattgaaat gatgaaaagc attattgaag cgtttcatga atttgatagc
     1141 ccggcgaccc tgtggctgag cagcggcaac ggcgtggtgt ggatgcgcta tggctcatgg
     1201 ctgctgacct gccccgtgat tctgattcat ctgagcaacc tgaccggcct gaaagatgat
     1261 tatagcaaac gcaccatggg cctgctggta agtgtcgggg ttgagctcat aacttcgtat
     1321 agtatacctt atacgaagtt attctttgca ccattctaaa gaataacagt gataatttct
     1381 gggttaaggc aaataacttc gtataggata ctttatacga agttatggta cccctcctgt
     1441 actcaccttg ccggtggtgc agatgaactt cagggtcagc ttgccgtagg tggcatcgcc
     1501 ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg tcgccgtcca gctcgaccag
     1561 gatgggcacc accccggtga acagctcctc gcccttgctc accacgttga tgtcgatctg
     1621 gtccaggggg atgtactcgc cctcgctggt gatcctgctc ttggcggccg catcttcttc
     1681 ttccgccacc agggtttcca cttccatttc ctggcccgca atggtaattt tctgtttttt
     1741 gcgaatatcg ccatacagca gaatatgttc atgaattttc acgcgcagat agttgcccag
     1801 cacgccccac atgttcttcg caatcagatc cagaatgcta tggccaatcg cgctgccata
     1861 cgggctaata tggccgaagc cctcggggcc cagcaggaac agcacgggga acatgcccca
     1921 gctcacgaag aacagccagg ccatggctct caccagctgt ctgcacaggc ctttcggcac
     1981 ggtatgaaac gcttcaatat acactttggc cgcatgaaaa taggtataca tgccatagct
     2041 caggctaatc agaaaaaaca gaattttggt ccagccggtg cacatcgcgc tggtcgcgcc
     2101 ccacacaatg cagcccacgt cgctcaccta aacacatgag aaatagagtc ataagcacat
     2161 gggtcacata caggagatat gtatataaca ttaatacaat ttataaaaaa aagggggggc
     2221 acaactagta taacttcgta taaggtatac tatacgaagt tattaagagg tttcatattg
     2281 ctaatagcag ctacaatcca gctaccattc tgcataactt cgtataaagt atcctatacg
     2341 aagttatgtc gactggagag tggccagccc ttctcatgtt cagagaacat ggttaactgg
     2401 ttaagtcatg tcgtcccaca gctgcccgtg ccctggccca ccctcgtgac caccttcggc
     2461 tacggcctgc agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag
     2521 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
     2581 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     2641 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
     2701 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc
     2761 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     2821 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag ctaccagtcc
     2881 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     2941 gccgccggga tcactctcgg catggacgag ctgtacaagt aagctagcga agttcctata
     3001 ctttctagag aataggaact tctaagcaga caatcttgta atacatcagc agctagtaga
     3061 ttgctccagc tagaagttcc tatacctttt gaagaatagg aacttcgaat tcgatatcaa
     3121 gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     3181 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     3241 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
     3301 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
     3361 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
     3421 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     3481 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
     3541 ttggctgctc gcctatgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
     3601 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
     3661 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca
     3721 tcgataccga gcgctgctcg agagatctac gggtggcatc cctgtgaccc ctccccagtg
     3781 cctctcctgg ccctggaagt tgccactcca gtgcccacca gccttgtcct aataaaatta
     3841 agttgcatca ttttgtctga ctaggtgtcc ttctataata ttatggggtg gaggggggtg
     3901 gtatggagca aggggcaagt tgggaagaca acctgtaggg cctgcggggt ctattgggaa
     3961 ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa
     4021 gcgattctcc tgcctcagcc tcccgagttg ttgggattcc aggcatgcat gaccaggctc
     4081 agctaatttt tgtttttttg gtagagacgg ggtttcacca tattggccag gctggtctcc
     4141 aactcctaat ctcaggtgat ctacccacct tggcctccca aattgctggg attacaggcg
     4201 tgaaccactg ctcccttccc tgtccttctg attttgtagg taaccacgtg cggaccgagc
     4261 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
     4321 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
     4381 gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc
     4441 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc
     4501 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt
     4561 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     4621 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     4681 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     4741 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     4801 aacaacactc aactctatct cgggctattc ttttgattta taagggattt tgccgatttc
     4861 ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     4921 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     4981 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     5041 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     5101 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     5161 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     5221 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     5281 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     5341 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     5401 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     5461 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     5521 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     5581 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     5641 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     5701 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     5761 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     5821 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     5881 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     5941 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     6001 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     6061 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6121 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6181 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6241 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     6301 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     6361 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     6421 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     6481 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     6541 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     6601 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     6661 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     6721 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     6781 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     6841 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     6901 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     6961 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgt
//
