LOCUS       sequence-271941-        7154 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intersectional viral expression of GCaMP6F in cells expressing both 
            Cre AND Flp.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7154)
  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 7154)
  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-GCaMP6F
FEATURES             Location/Qualifiers
     source          1..7154
                     /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(1626..1647)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             complement(1860..1916)
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     regulatory      1928..1937
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1942..1975
                     /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    2026..2059
                     /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    2066..2099
                     /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     2751..2770
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3010..3031
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    complement(3139..3172)
                     /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)"
     misc_feature    3281..3869
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3334..3354)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3752..3763)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3901..4377
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4100..4119)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4417..4557
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4417..4546
                     /label=AAV2 ITR
     rep_origin      4632..5087
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4719..4738)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4929..4950
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5104..5123)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5223..5245
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5283..5301)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5369..5473
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5474..5542,5543..6334)
                     /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(5692..5711)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6505..7093
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6994..7013
                     /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 tatgtcgacc ctcctgtact caccttgctc
     1561 accatgctcc ctccggtacc gcccttgtac agctcgtcca tgccgagagt gatcccggcg
     1621 gcggtcacga actccagcag gaccatgtga tcgcgcttct cgttggggtc tttcgaaagt
     1681 ttggactgca cgctcaggta gtggttgtcg ggcagcagca cggggccgtc gccgatgggg
     1741 gtgttctgct ggtagtggta ggcgagctgc acgccgccgt cctcgatgtt gtggcggatc
     1801 ttgaagttcg ccttgatgcc gttcttctgc ttgtcggcct tgatatagac gttctcgagt
     1861 gagctcagcc gacctatagc tctgactgcg tgacctgtct tattccactt acgacgtgat
     1921 gagtccacca tggtggctag cataacttcg tataaagtat cctatacgaa gttatttgcc
     1981 ttaacccaga aattatcact gttattcttt agataggtgc aaagaataac ttcgtataag
     2041 gtatactata cgaagttatg gtaccgaagt tcctattctt caaatagtat aggaacttca
     2101 attgttattg gtgcaaagaa tcacttcctt aagtctttat actgcccagg aagttcctat
     2161 tcttcaaaag gtataggaac ttcggcgcgc ctcacttcgc tgtcatcatt tgtacaaact
     2221 cttcgtagtt tacctgacca tccccatcga tgtctgcttc cctgatcatt tcatcaacct
     2281 cttcatctgt taacttctct ccaaggtttg tcatcacgtg gcgaagctct gctgcactga
     2341 tgtagccatt gccatcctta tcaaacacac cgaacgcttc tctaatttct tcttccgtgt
     2401 ccctgtattt catttttctt gccatcattg tcaggaactc agggaagtcg attgtgccgt
     2461 caccgtcggc atctacttca ttgatcatgt cctgcagctc tgcttctgtg gggttctgcc
     2521 ccagagaccg catcaccgtc cccagctcct tggttgttat tgtcccatcc ccgtccttgt
     2581 caaataggga gaattcctct ttaaattctg cgatctgctc ttcagtcagt tggtccggca
     2641 ggttgtactc cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg atgcccttca
     2701 gctcgatgcg gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg gtcttgtagt
     2761 tgccgtcgtc cttgaagaag atggtgcgct cctggatgta gccttcgggc atggcggact
     2821 tgaagaagtc gtgctgcttc atgtggtcgg ggtagcggct gaagcactgc acgccgtagg
     2881 tcagggtggt cacgagggtg ggccagggca cgggcagctt gccggtggtg cagatgaact
     2941 tcagggtcag cttgccgtag gtggcatcgc cctcaccctc gccggacacg ctgaacttgt
     3001 ggccgtttac gtcgccgtcc agctcgacca ggatgggcac caccccggtg aacagctcct
     3061 cgccctgtgg gacgacatga cttaaccagt taaccatgtt ctctgaacat gagaagggct
     3121 ggccactctc catctagaga agttcctata ctatttgaag aataggaact tcttgcctta
     3181 acccagaaat tatcactgtt attctttaga atagtgcaaa gagaagttcc tatacctttt
     3241 gaagaatagg aacttcgaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa
     3301 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     3361 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     3421 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
     3481 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
     3541 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
     3601 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     3661 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctatgttg ccacctggat
     3721 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
     3781 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag
     3841 tcggatctcc ctttgggccg cctccccgca tcgataccga gcgctgctcg agagatctac
     3901 gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca
     3961 gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc
     4021 ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca
     4081 acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg
     4141 ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg
     4201 ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg
     4261 ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct
     4321 tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc tgtccttctg
     4381 attttgtagg taaccacgtg cggaccgagc ggccgcagga acccctagtg atggagttgg
     4441 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
     4501 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc
     4561 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa
     4621 gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     4681 cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc
     4741 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     4801 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc
     4861 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     4921 ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc
     4981 ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta
     5041 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct
     5101 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     5161 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     5221 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa
     5281 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     5341 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     5401 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     5461 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     5521 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     5581 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     5641 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     5701 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     5761 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     5821 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     5881 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     5941 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     6001 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     6061 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     6121 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     6181 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     6241 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     6301 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     6361 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     6421 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     6481 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     6541 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     6601 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     6661 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     6721 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     6781 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     6841 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     6901 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     6961 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     7021 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     7081 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     7141 ttttgctcac atgt
//
