LOCUS       sequence-265089-        7164 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 7164)
  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 7164)
  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..7164
                     /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(1636..1657)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             complement(1870..1926)
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     regulatory      1938..1947
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1952..1985
                     /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    2036..2069
                     /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    2076..2109
                     /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     2761..2780
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3020..3041
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    complement(3149..3182)
                     /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    3291..3879
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3344..3364)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3762..3773)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3911..4387
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4110..4129)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4427..4567
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4427..4556
                     /label=AAV2 ITR
     rep_origin      4642..5097
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4729..4748)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4939..4960
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5114..5133)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5233..5255
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5293..5311)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5379..5483
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5484..5552,5553..6344)
                     /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(5702..5721)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6515..7103
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7004..7023
                     /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 tatgtcgacc ctcctgtact
     1561 caccttgctc accatgctcc ctccggtacc gcccttgtac agctcgtcca tgccgagagt
     1621 gatcccggcg gcggtcacga actccagcag gaccatgtga tcgcgcttct cgttggggtc
     1681 tttcgaaagt ttggactgca cgctcaggta gtggttgtcg ggcagcagca cggggccgtc
     1741 gccgatgggg gtgttctgct ggtagtggta ggcgagctgc acgccgccgt cctcgatgtt
     1801 gtggcggatc ttgaagttcg ccttgatgcc gttcttctgc ttgtcggcct tgatatagac
     1861 gttctcgagt gagctcagcc gacctatagc tctgactgcg tgacctgtct tattccactt
     1921 acgacgtgat gagtccacca tggtggctag cataacttcg tataaagtat cctatacgaa
     1981 gttatttgcc ttaacccaga aattatcact gttattcttt agataggtgc aaagaataac
     2041 ttcgtataag gtatactata cgaagttatg gtaccgaagt tcctattctt caaatagtat
     2101 aggaacttca attgttattg gtgcaaagaa tcacttcctt aagtctttat actgcccagg
     2161 aagttcctat tcttcaaaag gtataggaac ttcggcgcgc ctcacttcgc tgtcatcatt
     2221 tgtacaaact cttcgtagtt tacctgacca tccccatcga tgtctgcttc cctgatcatt
     2281 tcatcaacct cttcatctgt taacttctct ccaaggtttg tcatcacgtg gcgaagctct
     2341 gctgcactga tgtagccatt gccatcctta tcaaacacac cgaacgcttc tctaatttct
     2401 tcttccgtgt ccctgtattt catttttctt gccatcattg tcaggaactc agggaagtcg
     2461 attgtgccgt caccgtcggc atctacttca ttgatcatgt cctgcagctc tgcttctgtg
     2521 gggttctgcc ccagagaccg catcaccgtc cccagctcct tggttgttat tgtcccatcc
     2581 ccgtccttgt caaataggga gaattcctct ttaaattctg cgatctgctc ttcagtcagt
     2641 tggtccggca ggttgtactc cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg
     2701 atgcccttca gctcgatgcg gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg
     2761 gtcttgtagt tgccgtcgtc cttgaagaag atggtgcgct cctggatgta gccttcgggc
     2821 atggcggact tgaagaagtc gtgctgcttc atgtggtcgg ggtagcggct gaagcactgc
     2881 acgccgtagg tcagggtggt cacgagggtg ggccagggca cgggcagctt gccggtggtg
     2941 cagatgaact tcagggtcag cttgccgtag gtggcatcgc cctcaccctc gccggacacg
     3001 ctgaacttgt ggccgtttac gtcgccgtcc agctcgacca ggatgggcac caccccggtg
     3061 aacagctcct cgccctgtgg gacgacatga cttaaccagt taaccatgtt ctctgaacat
     3121 gagaagggct ggccactctc catctagaga agttcctata ctatttgaag aataggaact
     3181 tcttgcctta acccagaaat tatcactgtt attctttaga atagtgcaaa gagaagttcc
     3241 tatacctttt gaagaatagg aacttcgaat tcgatatcaa gcttatcgat aatcaacctc
     3301 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     3361 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     3421 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
     3481 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
     3541 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
     3601 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
     3661 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctatgttg
     3721 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
     3781 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
     3841 ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccga gcgctgctcg
     3901 agagatctac gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt
     3961 tgccactcca gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga
     4021 ctaggtgtcc ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt
     4081 tgggaagaca acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc
     4141 acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc
     4201 tcccgagttg ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg
     4261 gtagagacgg ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat
     4321 ctacccacct tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc
     4381 tgtccttctg attttgtagg taaccacgtg cggaccgagc ggccgcagga acccctagtg
     4441 atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag
     4501 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc
     4561 ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     4621 atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     4681 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     4741 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     4801 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg
     4861 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     4921 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     4981 cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     5041 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat
     5101 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     5161 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     5221 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     5281 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     5341 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     5401 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     5461 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     5521 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     5581 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     5641 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     5701 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     5761 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     5821 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     5881 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     5941 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     6001 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     6061 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     6121 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     6181 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     6241 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     6301 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     6361 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     6421 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     6481 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     6541 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     6601 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     6661 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     6721 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     6781 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     6841 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     6901 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     6961 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     7021 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     7081 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     7141 tttgctggcc ttttgctcac atgt
//
