LOCUS       sequence-265129-        6320 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intersectional viral expression of EYFP in cells expressing Cre AND 
            Flp AND Vcre.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6320)
  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 6320)
  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/Von eYFP
FEATURES             Location/Qualifiers
     source          1..6320
                     /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    complement(708..741)
                     /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(792..825)
                     /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     941..962
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1004..1013
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1020..1053
                     /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    1104..1137
                     /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    1233..1266
                     /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)"
     protein_bind    complement(1540..1573)
                     /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(1850..2101)
                     /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(1892..1913)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2241..2260
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    2447..3035
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2500..2520)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2918..2929)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3067..3543
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3266..3285)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3583..3723
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3583..3712
                     /label=AAV2 ITR
     rep_origin      3798..4253
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3885..3904)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4095..4116
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4270..4289)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4389..4411
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4449..4467)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4535..4639
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4640..4708,4709..5500)
                     /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(4858..4877)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5671..6259
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6160..6179
                     /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 cggatccata acttcgtata
      721 ggatacttta tacgaagtta tgtagctgga gcaatttgaa aagattgttg tattacctct
      781 agctgcgctt aataacttcg tatagtatac cttatacgaa gttatggcgc gccaaccccg
      841 acacttacct tgccggtggt gcagatgaac ttcagggtca gcttgccgta ggtggcatcg
      901 ccctcgccct cgccggacac gctgaacttg tggccgttta cgtcgccgtc cagctcgacc
      961 aggatgggca ccaccccggt gaacagctcc tcgcccttgc tcaccatggt ggcaccggta
     1021 taacttcgta taaagtatcc tatacgaagt tatttgcctt agttattctt agatagaccc
     1081 agaaattatc acttgtgcaa agaataactt cgtataaggt atactatacg aagttatgag
     1141 ctctgtgccc cccctttttt ttataaaatt gtattaatgt tatatacata tctcctgtat
     1201 gtgacccatg tgcttatgac tctattaatt aagaagttcc tattcttcaa atagtatagg
     1261 aacttcgcag aatggtagct ggattgtagc tgctattagc aatatgaaac ctcttagaag
     1321 ttcctattct tcaaaaggta taggaacttc actagtcctc ctgtactcac cttgaagaag
     1381 atggtgcgct cctggacgta gccttcgggc atggcggact tgaagaagtc gtgctgcttc
     1441 atgtggtcgg ggtagcgggc gaagcactgc aggccgtagc cgaaggtggt cacgagggtg
     1501 ggccagggca cgggcagcta aacacatgag aaagtcgacg aagttcctat actatttgaa
     1561 gaataggaac ttcttgcctt aacccagaaa ttatcactgt tattctttag aatggtgcaa
     1621 agagaagttc ctataccttt tgaagaatag gaacttcgct agctggagag tggccagccc
     1681 ttctcatgtt cagagaacat ggttaactgg ttaagtcatg tctcaatttc tgagaactgt
     1741 cattctcgga aattgagtag ctggttgaag attgctctag tattacagca atttgaactg
     1801 cgcttatcaa tttctgagaa gtgtctttct cggaaattga ggtaccttac ttgtacagct
     1861 cgtccatgcc gagagtgatc ccggcggcgg tcacgaactc cagcaggacc atgtgatcgc
     1921 gcttctcgtt ggggtctttg ctcagggcgg actggtagct caggtagtgg ttgtcgggca
     1981 gcagcacggg gccgtcgccg atgggggtgt tctgctggta gtggtcggcg agctgcacgc
     2041 tgccgtcctc gatgttgtgg cggatcttga agttcacctt gatgccgttc ttctgcttgt
     2101 cggccatgat atagacgttg tggctgttgt agttgtactc cagcttgtgc cccaggatgt
     2161 tgccgtcctc cttgaagtcg atgcccttca gctcgatgcg gttcaccagg gtgtcgccct
     2221 cgaacttcac ctcggcgcgg gtcttgtagt tgccgtcgtc ctgtgggatc tagagtcaag
     2281 attgctccag caatcttgta atactcaatt tccgagaatg acagttctca gaaattgata
     2341 agcagacaat cttgtaatac atgcagctag tcaagattgc tccagctatc aatttccgag
     2401 aaagacactt ctcagaaatt gagaattcga tatcaagctt atcgataatc aacctctgga
     2461 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     2521 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     2581 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     2641 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     2701 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     2761 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     2821 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct atgttgccac
     2881 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     2941 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
     3001 gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgagcgc tgctcgagag
     3061 atctacgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc
     3121 actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt gtctgactag
     3181 gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg gcaagttggg
     3241 aagacaacct gtagggcctg cggggtctat tgggaaccaa gctggagtgc agtggcacaa
     3301 tcttggctca ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc tcagcctccc
     3361 gagttgttgg gattccaggc atgcatgacc aggctcagct aatttttgtt tttttggtag
     3421 agacggggtt tcaccatatt ggccaggctg gtctccaact cctaatctca ggtgatctac
     3481 ccaccttggc ctcccaaatt gctgggatta caggcgtgaa ccactgctcc cttccctgtc
     3541 cttctgattt tgtaggtaac cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg
     3601 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg
     3661 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc
     3721 aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     3781 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     3841 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     3901 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3961 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     4021 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     4081 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggg
     4141 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     4201 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
     4261 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     4321 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     4381 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     4441 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     4501 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     4561 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     4621 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     4681 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     4741 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     4801 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     4861 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     4921 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     4981 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     5041 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     5101 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     5161 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     5221 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     5281 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     5341 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     5401 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     5461 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     5521 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     5581 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     5641 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     5701 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     5761 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     5821 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     5881 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     5941 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     6001 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     6061 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     6121 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     6181 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     6241 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     6301 ctggcctttt gctcacatgt
//
