LOCUS       sequence-272785-        6309 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 6309)
  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 6309)
  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..6309
                     /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    complement(697..730)
                     /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(781..814)
                     /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     930..951
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      993..1002
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1009..1042
                     /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    1093..1126
                     /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    1222..1255
                     /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(1529..1562)
                     /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(1839..2090)
                     /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(1881..1902)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2230..2249
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    2436..3024
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2489..2509)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2907..2918)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3056..3532
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3255..3274)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3572..3712
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3572..3701
                     /label=AAV2 ITR
     rep_origin      3787..4242
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3874..3893)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4084..4105
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4259..4278)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4378..4400
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4438..4456)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4524..4628
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4629..4697,4698..5489)
                     /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(4847..4866)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5660..6248
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6149..6168
                     /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 ggatccataa cttcgtatag gatactttat
      721 acgaagttat gtagctggag caatttgaaa agattgttgt attacctcta gctgcgctta
      781 ataacttcgt atagtatacc ttatacgaag ttatggcgcg ccaaccccga cacttacctt
      841 gccggtggtg cagatgaact tcagggtcag cttgccgtag gtggcatcgc cctcgccctc
      901 gccggacacg ctgaacttgt ggccgtttac gtcgccgtcc agctcgacca ggatgggcac
      961 caccccggtg aacagctcct cgcccttgct caccatggtg gcaccggtat aacttcgtat
     1021 aaagtatcct atacgaagtt atttgcctta gttattctta gatagaccca gaaattatca
     1081 cttgtgcaaa gaataacttc gtataaggta tactatacga agttatgagc tctgtgcccc
     1141 cccttttttt tataaaattg tattaatgtt atatacatat ctcctgtatg tgacccatgt
     1201 gcttatgact ctattaatta agaagttcct attcttcaaa tagtatagga acttcgcaga
     1261 atggtagctg gattgtagct gctattagca atatgaaacc tcttagaagt tcctattctt
     1321 caaaaggtat aggaacttca ctagtcctcc tgtactcacc ttgaagaaga tggtgcgctc
     1381 ctggacgtag ccttcgggca tggcggactt gaagaagtcg tgctgcttca tgtggtcggg
     1441 gtagcgggcg aagcactgca ggccgtagcc gaaggtggtc acgagggtgg gccagggcac
     1501 gggcagctaa acacatgaga aagtcgacga agttcctata ctatttgaag aataggaact
     1561 tcttgcctta acccagaaat tatcactgtt attctttaga atggtgcaaa gagaagttcc
     1621 tatacctttt gaagaatagg aacttcgcta gctggagagt ggccagccct tctcatgttc
     1681 agagaacatg gttaactggt taagtcatgt ctcaatttct gagaactgtc attctcggaa
     1741 attgagtagc tggttgaaga ttgctctagt attacagcaa tttgaactgc gcttatcaat
     1801 ttctgagaag tgtctttctc ggaaattgag gtaccttact tgtacagctc gtccatgccg
     1861 agagtgatcc cggcggcggt cacgaactcc agcaggacca tgtgatcgcg cttctcgttg
     1921 gggtctttgc tcagggcgga ctggtagctc aggtagtggt tgtcgggcag cagcacgggg
     1981 ccgtcgccga tgggggtgtt ctgctggtag tggtcggcga gctgcacgct gccgtcctcg
     2041 atgttgtggc ggatcttgaa gttcaccttg atgccgttct tctgcttgtc ggccatgata
     2101 tagacgttgt ggctgttgta gttgtactcc agcttgtgcc ccaggatgtt gccgtcctcc
     2161 ttgaagtcga tgcccttcag ctcgatgcgg ttcaccaggg tgtcgccctc gaacttcacc
     2221 tcggcgcggg tcttgtagtt gccgtcgtcc tgtgggatct agagtcaaga ttgctccagc
     2281 aatcttgtaa tactcaattt ccgagaatga cagttctcag aaattgataa gcagacaatc
     2341 ttgtaataca tgcagctagt caagattgct ccagctatca atttccgaga aagacacttc
     2401 tcagaaattg agaattcgat atcaagctta tcgataatca acctctggat tacaaaattt
     2461 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
     2521 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
     2581 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg
     2641 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc
     2701 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg
     2761 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt
     2821 tgtcggggaa atcatcgtcc tttccttggc tgctcgccta tgttgccacc tggattctgc
     2881 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg
     2941 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga
     3001 tctccctttg ggccgcctcc ccgcatcgat accgagcgct gctcgagaga tctacgggtg
     3061 gcatccctgt gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc
     3121 caccagcctt gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta
     3181 taatattatg gggtggaggg gggtggtatg gagcaagggg caagttggga agacaacctg
     3241 tagggcctgc ggggtctatt gggaaccaag ctggagtgca gtggcacaat cttggctcac
     3301 tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct cagcctcccg agttgttggg
     3361 attccaggca tgcatgacca ggctcagcta atttttgttt ttttggtaga gacggggttt
     3421 caccatattg gccaggctgg tctccaactc ctaatctcag gtgatctacc caccttggcc
     3481 tcccaaattg ctgggattac aggcgtgaac cactgctccc ttccctgtcc ttctgatttt
     3541 gtaggtaacc acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact
     3601 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg
     3661 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg
     3721 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac
     3781 catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     3841 tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc
     3901 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     3961 gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta
     4021 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     4081 atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg
     4141 atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa
     4201 aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta
     4261 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     4321 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     4381 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
     4441 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     4501 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     4561 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     4621 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     4681 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     4741 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     4801 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     4861 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     4921 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     4981 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     5041 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     5101 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     5161 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     5221 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     5281 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     5341 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     5401 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     5461 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     5521 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     5581 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     5641 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     5701 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     5761 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     5821 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     5881 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     5941 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     6001 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     6061 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     6121 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     6181 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     6241 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     6301 ctcacatgt
//
