LOCUS       sequence-271969-        7559 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 7559)
  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 7559)
  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..7559
                     /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        231..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          462..1400
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1357..1377
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1416..1449)
                     /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(1500..1533)
                     /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(1629..1650)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             complement(1863..1919)
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     regulatory      1931..1940
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1947..1980
                     /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    2031..2064
                     /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    2160..2193
                     /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     2537..2558
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    complement(2614..2647)
                     /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     3480..3499
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    3686..4274
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3739..3759)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4157..4168)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4306..4782
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4505..4524)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4822..4962
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4822..4951
                     /label=AAV2 ITR
     rep_origin      5037..5492
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5124..5143)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5334..5355
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5509..5528)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5628..5650
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5688..5706)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5774..5878
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5879..5947,5948..6739)
                     /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(6097..6116)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6910..7498
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7399..7418
                     /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 gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgag gatccataac ttcgtatagg atactttata
     1441 cgaagttatg tagctggagc aatttgaaaa gattgttgta ttacctctag ctgcgcttaa
     1501 taacttcgta tagtatacct tatacgaagt tatggcgcgc caaccccgac acttaccttg
     1561 ctcaccatgc tccctccggt accgcccttg tacagctcgt ccatgccgag agtgatcccg
     1621 gcggcggtca cgaactccag caggaccatg tgatcgcgct tctcgttggg gtctttcgaa
     1681 agtttggact gcacgctcag gtagtggttg tcgggcagca gcacggggcc gtcgccgatg
     1741 ggggtgttct gctggtagtg gtaggcgagc tgcacgccgc cgtcctcgat gttgtggcgg
     1801 atcttgaagt tcgccttgat gccgttcttc tgcttgtcgg ccttgatata gacgttctcg
     1861 agtgagctca gccgacctat agctctgact gcgtgacctg tcttattcca cttacgacgt
     1921 gatgagtcca ccatggtggc accggtataa cttcgtataa agtatcctat acgaagttat
     1981 ttgccttagt tattcttaga tagacccaga aattatcact tgtgcaaaga ataacttcgt
     2041 ataaggtata ctatacgaag ttatgagctc tgtgcccccc ctttttttta taaaattgta
     2101 ttaatgttat atacatatct cctgtatgtg acccatgtgc ttatgactct attaattaag
     2161 aagttcctat tcttcaaata gtataggaac ttcgcagaat ggtagctgga ttgtagctgc
     2221 tattagcaat atgaaacctc ttagaagttc ctattcttca aaaggtatag gaacttcact
     2281 agtcctcctg tactcacctt gaagaagatg gtgcgctctt ggatgtagcc ctcgggcatg
     2341 gcggacttga agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa gcactgcacg
     2401 ccgtaggtca gggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag
     2461 atgaacttca gggtcagctt gccgtaggtg gcatcgccct caccctcgcc ggacacgctg
     2521 aacttgtggc cgtttacgtc gccgtccagc tcgaccagga tgggcaccac cccggtgaac
     2581 agctcctcgc cctaaacaca tgagaaagtc gacgaagttc ctatactatt tgaagaatag
     2641 gaacttcttg ccttaaccca gaaattatca ctgttattct ttagaatggt gcaaagagaa
     2701 gttcctatac cttttgaaga ataggaactt cgctagctgg agagtggcca gcccttctca
     2761 tgttcagaga acatggttaa ctggttaagt catgtctcaa tttctgagaa ctgtcattct
     2821 cggaaattga gtagctggtt gaagattgct ctagtattac agcaatttga actgcgctta
     2881 tcaatttctg agaagtgtct ttctcggaaa ttgaggtacc tcacttcgct gtcatcattt
     2941 gtacaaactc ttcgtagttt acctgaccat ccccatcgat gtctgcttcc ctgatcattt
     3001 catcaacctc ttcatctgtt aacttctctc caaggtttgt catcacgtgg cgaagctctg
     3061 ctgcactgat gtagccattg ccatccttat caaacacacc gaacgcttct ctaatttctt
     3121 cttccgtgtc cctgtagctc ccttttcttg ccatcattgt caggaactca gggaagtcga
     3181 ttgtgccgtc accgtcggca tctacttcat tgatcatgtc ctgcagctct gcttctgtgg
     3241 ggttctgccc cagagaccgc atcaccgtcc ccagctcctt ggttgttatt gtcccatccc
     3301 cgtccttgtc aaatagggag aaagcctctt taaattctgc gatctgctct tcagtcagtt
     3361 ggtccggcag gttgtactcc agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga
     3421 tgcccttcag ctcgatgcgg ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg
     3481 tcttgtagtt gccgtcgtcc tgtgggatct agagtcaaga ttgctccagc aatcttgtaa
     3541 tactcaattt ccgagaatga cagttctcag aaattgataa gcagacaatc ttgtaataca
     3601 tgcagctagt caagattgct ccagctatca atttccgaga aagacacttc tcagaaattg
     3661 agaattcgat atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt
     3721 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     3781 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     3841 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
     3901 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
     3961 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
     4021 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
     4081 atcatcgtcc tttccttggc tgctcgccta tgttgccacc tggattctgc gcgggacgtc
     4141 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
     4201 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg
     4261 ggccgcctcc ccgcatcgat accgagcgct gctcgagaga tctacgggtg gcatccctgt
     4321 gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc caccagcctt
     4381 gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta taatattatg
     4441 gggtggaggg gggtggtatg gagcaagggg caagttggga agacaacctg tagggcctgc
     4501 ggggtctatt gggaaccaag ctggagtgca gtggcacaat cttggctcac tgcaatctcc
     4561 gcctcctggg ttcaagcgat tctcctgcct cagcctcccg agttgttggg attccaggca
     4621 tgcatgacca ggctcagcta atttttgttt ttttggtaga gacggggttt caccatattg
     4681 gccaggctgg tctccaactc ctaatctcag gtgatctacc caccttggcc tcccaaattg
     4741 ctgggattac aggcgtgaac cactgctccc ttccctgtcc ttctgatttt gtaggtaacc
     4801 acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc
     4861 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     4921 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt
     4981 ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc
     5041 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     5101 acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     5161 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     5221 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc
     5281 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     5341 cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg
     5401 gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     5461 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc
     5521 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     5581 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     5641 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     5701 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     5761 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     5821 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     5881 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     5941 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     6001 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     6061 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     6121 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     6181 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     6241 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     6301 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     6361 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     6421 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     6481 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     6541 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     6601 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     6661 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     6721 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     6781 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     6841 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     6901 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     6961 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     7021 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     7081 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     7141 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     7201 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     7261 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     7321 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     7381 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     7441 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     7501 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt
//
