LOCUS       sequence-271940-        7156 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intersectional viral expression of GCaMP6F in cells expressing Flp 
            AND NOT Cre.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7156)
  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 7156)
  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-Coff/Fon-GCaMP6F
FEATURES             Location/Qualifiers
     source          1..7156
                     /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."
     regulatory      1540..1549
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1561..1617
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     primer_bind     1830..1851
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    1944..1977
                     /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    2028..2061
                     /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    2068..2101
                     /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     2753..2772
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3012..3033
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    complement(3141..3174)
                     /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    3283..3871
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3336..3356)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3754..3765)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3903..4379
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4102..4121)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4419..4559
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4419..4548
                     /label=AAV2 ITR
     rep_origin      4634..5089
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4721..4740)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4931..4952
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5106..5125)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5225..5247
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5285..5303)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5371..5475
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5476..5544,5545..6336)
                     /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(5694..5713)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6507..7095
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6996..7015
                     /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 tatgtcgacg ccaccatggt ggactcatca
     1561 cgtcgtaagt ggaataagac aggtcacgca gtcagagcta taggtcggct gagctcactc
     1621 gagaacgtct atatcaaggc cgacaagcag aagaacggca tcaaggcgaa cttcaagatc
     1681 cgccacaaca tcgaggacgg cggcgtgcag ctcgcctacc actaccagca gaacaccccc
     1741 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcgtgca gtccaaactt
     1801 tcgaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     1861 gggatcactc tcggcatgga cgagctgtac aagggcggta ccggagggag catggtgagc
     1921 aaggtgagta caggagggct agcataactt cgtataaagt atcctatacg aagttatttg
     1981 ccttaaccca gaaattatca ctgttattct ttagataggt gcaaagaata acttcgtata
     2041 aggtatacta tacgaagtta tggtaccgaa gttcctattc ttcaaatagt ataggaactt
     2101 caattgttat tggtgcaaag aatcacttcc ttaagtcttt atactgccca ggaagttcct
     2161 attcttcaaa aggtatagga acttcggcgc gcctcacttc gctgtcatca tttgtacaaa
     2221 ctcttcgtag tttacctgac catccccatc gatgtctgct tccctgatca tttcatcaac
     2281 ctcttcatct gttaacttct ctccaaggtt tgtcatcacg tggcgaagct ctgctgcact
     2341 gatgtagcca ttgccatcct tatcaaacac accgaacgct tctctaattt cttcttccgt
     2401 gtccctgtat ttcatttttc ttgccatcat tgtcaggaac tcagggaagt cgattgtgcc
     2461 gtcaccgtcg gcatctactt cattgatcat gtcctgcagc tctgcttctg tggggttctg
     2521 ccccagagac cgcatcaccg tccccagctc cttggttgtt attgtcccat ccccgtcctt
     2581 gtcaaatagg gagaattcct ctttaaattc tgcgatctgc tcttcagtca gttggtccgg
     2641 caggttgtac tccagcttgt gccccaggat gttgccgtcc tccttgaagt cgatgccctt
     2701 cagctcgatg cggttcacca gggtgtcgcc ctcgaacttc acctcggcgc gggtcttgta
     2761 gttgccgtcg tccttgaaga agatggtgcg ctcctggatg tagccttcgg gcatggcgga
     2821 cttgaagaag tcgtgctgct tcatgtggtc ggggtagcgg ctgaagcact gcacgccgta
     2881 ggtcagggtg gtcacgaggg tgggccaggg cacgggcagc ttgccggtgg tgcagatgaa
     2941 cttcagggtc agcttgccgt aggtggcatc gccctcaccc tcgccggaca cgctgaactt
     3001 gtggccgttt acgtcgccgt ccagctcgac caggatgggc accaccccgg tgaacagctc
     3061 ctcgccctgt gggacgacat gacttaacca gttaaccatg ttctctgaac atgagaaggg
     3121 ctggccactc tccatctaga gaagttccta tactatttga agaataggaa cttcttgcct
     3181 taacccagaa attatcactg ttattcttta gaatagtgca aagagaagtt cctatacctt
     3241 ttgaagaata ggaacttcga attcgatatc aagcttatcg ataatcaacc tctggattac
     3301 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     3361 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     3421 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     3481 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     3541 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
     3601 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     3661 gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctatgt tgccacctgg
     3721 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     3781 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     3841 agtcggatct ccctttgggc cgcctccccg catcgatacc gagcgctgct cgagagatct
     3901 acgggtggca tccctgtgac ccctccccag tgcctctcct ggccctggaa gttgccactc
     3961 cagtgcccac cagccttgtc ctaataaaat taagttgcat cattttgtct gactaggtgt
     4021 ccttctataa tattatgggg tggagggggg tggtatggag caaggggcaa gttgggaaga
     4081 caacctgtag ggcctgcggg gtctattggg aaccaagctg gagtgcagtg gcacaatctt
     4141 ggctcactgc aatctccgcc tcctgggttc aagcgattct cctgcctcag cctcccgagt
     4201 tgttgggatt ccaggcatgc atgaccaggc tcagctaatt tttgtttttt tggtagagac
     4261 ggggtttcac catattggcc aggctggtct ccaactccta atctcaggtg atctacccac
     4321 cttggcctcc caaattgctg ggattacagg cgtgaaccac tgctcccttc cctgtccttc
     4381 tgattttgta ggtaaccacg tgcggaccga gcggccgcag gaacccctag tgatggagtt
     4441 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
     4501 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg
     4561 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca
     4621 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     4681 cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct
     4741 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     4801 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt
     4861 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     4921 ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat
     4981 tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt
     5041 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact
     5101 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     5161 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     5221 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     5281 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     5341 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     5401 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     5461 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     5521 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     5581 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     5641 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     5701 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     5761 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     5821 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     5881 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     5941 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     6001 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     6061 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     6121 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     6181 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     6241 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     6301 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     6361 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     6421 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     6481 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     6541 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     6601 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     6661 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6721 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     6781 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     6841 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     6901 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     6961 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     7021 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     7081 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     7141 ccttttgctc acatgt
//
