LOCUS       sequence-265130-        7569 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intersectional viral expression of GCaMP6M in cells expressing Cre 
            AND Flp AND Vcre.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7569)
  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 7569)
  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-Ef1a-Con/Fon/Von GCaMP 6m
FEATURES             Location/Qualifiers
     source          1..7569
                     /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        242..1419
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          473..1410
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1367..1387
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1426..1459)
                     /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(1510..1543)
                     /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     complement(1639..1660)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             complement(1873..1929)
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     regulatory      1941..1950
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1957..1990
                     /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    2041..2074
                     /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    2170..2203
                     /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)"
     primer_bind     2547..2568
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    complement(2624..2657)
                     /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)"
     primer_bind     3490..3509
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    3696..4284
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3749..3769)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4167..4178)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4316..4792
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4515..4534)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4832..4972
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4832..4961
                     /label=AAV2 ITR
     rep_origin      5047..5502
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5134..5153)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5344..5365
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5519..5538)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5638..5660
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5698..5716)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5784..5888
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5889..5957,5958..6749)
                     /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(6107..6126)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6920..7508
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7409..7428
                     /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 cgcgtaagct ttgcaaagat ggataaagtt
      181 ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat
      241 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
      301 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
      361 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
      421 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc
      481 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat
      541 tacttccact ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      601 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      661 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      721 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      781 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      841 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      901 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      961 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
     1021 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
     1081 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
     1141 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
     1201 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1261 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1321 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1381 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gatccataac ttcgtatagg
     1441 atactttata cgaagttatg tagctggagc aatttgaaaa gattgttgta ttacctctag
     1501 ctgcgcttaa taacttcgta tagtatacct tatacgaagt tatggcgcgc caaccccgac
     1561 acttaccttg ctcaccatgc tccctccggt accgcccttg tacagctcgt ccatgccgag
     1621 agtgatcccg gcggcggtca cgaactccag caggaccatg tgatcgcgct tctcgttggg
     1681 gtctttcgaa agtttggact gcacgctcag gtagtggttg tcgggcagca gcacggggcc
     1741 gtcgccgatg ggggtgttct gctggtagtg gtaggcgagc tgcacgccgc cgtcctcgat
     1801 gttgtggcgg atcttgaagt tcgccttgat gccgttcttc tgcttgtcgg ccttgatata
     1861 gacgttctcg agtgagctca gccgacctat agctctgact gcgtgacctg tcttattcca
     1921 cttacgacgt gatgagtcca ccatggtggc accggtataa cttcgtataa agtatcctat
     1981 acgaagttat ttgccttagt tattcttaga tagacccaga aattatcact tgtgcaaaga
     2041 ataacttcgt ataaggtata ctatacgaag ttatgagctc tgtgcccccc ctttttttta
     2101 taaaattgta ttaatgttat atacatatct cctgtatgtg acccatgtgc ttatgactct
     2161 attaattaag aagttcctat tcttcaaata gtataggaac ttcgcagaat ggtagctgga
     2221 ttgtagctgc tattagcaat atgaaacctc ttagaagttc ctattcttca aaaggtatag
     2281 gaacttcact agtcctcctg tactcacctt gaagaagatg gtgcgctctt ggatgtagcc
     2341 ctcgggcatg gcggacttga agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa
     2401 gcactgcacg ccgtaggtca gggtggtcac gagggtgggc cagggcacgg gcagcttgcc
     2461 ggtggtgcag atgaacttca gggtcagctt gccgtaggtg gcatcgccct caccctcgcc
     2521 ggacacgctg aacttgtggc cgtttacgtc gccgtccagc tcgaccagga tgggcaccac
     2581 cccggtgaac agctcctcgc cctaaacaca tgagaaagtc gacgaagttc ctatactatt
     2641 tgaagaatag gaacttcttg ccttaaccca gaaattatca ctgttattct ttagaatggt
     2701 gcaaagagaa gttcctatac cttttgaaga ataggaactt cgctagctgg agagtggcca
     2761 gcccttctca tgttcagaga acatggttaa ctggttaagt catgtctcaa tttctgagaa
     2821 ctgtcattct cggaaattga gtagctggtt gaagattgct ctagtattac agcaatttga
     2881 actgcgctta tcaatttctg agaagtgtct ttctcggaaa ttgaggtacc tcacttcgct
     2941 gtcatcattt gtacaaactc ttcgtagttt acctgaccat ccccatcgat gtctgcttcc
     3001 ctgatcattt catcaacctc ttcatctgtt aacttctctc caaggtttgt catcacgtgg
     3061 cgaagctctg ctgcactgat gtagccattg ccatccttat caaacacacc gaacgcttct
     3121 ctaatttctt cttccgtgtc cctgtagctc ccttttcttg ccatcattgt caggaactca
     3181 gggaagtcga ttgtgccgtc accgtcggca tctacttcat tgatcatgtc ctgcagctct
     3241 gcttctgtgg ggttctgccc cagagaccgc atcaccgtcc ccagctcctt ggttgttatt
     3301 gtcccatccc cgtccttgtc aaatagggag aaagcctctt taaattctgc gatctgctct
     3361 tcagtcagtt ggtccggcag gttgtactcc agcttgtgcc ccaggatgtt gccgtcctcc
     3421 ttgaagtcga tgcccttcag ctcgatgcgg ttcaccaggg tgtcgccctc gaacttcacc
     3481 tcggcgcggg tcttgtagtt gccgtcgtcc tgtgggatct agagtcaaga ttgctccagc
     3541 aatcttgtaa tactcaattt ccgagaatga cagttctcag aaattgataa gcagacaatc
     3601 ttgtaataca tgcagctagt caagattgct ccagctatca atttccgaga aagacacttc
     3661 tcagaaattg agaattcgat atcaagctta tcgataatca acctctggat tacaaaattt
     3721 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
     3781 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
     3841 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg
     3901 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc
     3961 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg
     4021 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt
     4081 tgtcggggaa atcatcgtcc tttccttggc tgctcgccta tgttgccacc tggattctgc
     4141 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg
     4201 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga
     4261 tctccctttg ggccgcctcc ccgcatcgat accgagcgct gctcgagaga tctacgggtg
     4321 gcatccctgt gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc
     4381 caccagcctt gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta
     4441 taatattatg gggtggaggg gggtggtatg gagcaagggg caagttggga agacaacctg
     4501 tagggcctgc ggggtctatt gggaaccaag ctggagtgca gtggcacaat cttggctcac
     4561 tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct cagcctcccg agttgttggg
     4621 attccaggca tgcatgacca ggctcagcta atttttgttt ttttggtaga gacggggttt
     4681 caccatattg gccaggctgg tctccaactc ctaatctcag gtgatctacc caccttggcc
     4741 tcccaaattg ctgggattac aggcgtgaac cactgctccc ttccctgtcc ttctgatttt
     4801 gtaggtaacc acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact
     4861 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg
     4921 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg
     4981 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac
     5041 catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     5101 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     5161 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     5221 gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta
     5281 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     5341 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcgggc tattcttttg
     5401 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     5461 aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta
     5521 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     5581 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     5641 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
     5701 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     5761 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     5821 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     5881 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     5941 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     6001 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     6061 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     6121 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     6181 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     6241 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     6301 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     6361 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     6421 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     6481 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     6541 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     6601 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     6661 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     6721 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     6781 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     6841 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     6901 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     6961 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     7021 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     7081 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     7141 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     7201 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     7261 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     7321 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     7381 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     7441 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     7501 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     7561 ctcacatgt
//
