LOCUS       sequence-271939-        7156 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intersectional viral expression of GCaMP6M 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-GCaMP6M
FEATURES             Location/Qualifiers
     source          1..7156
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        213..1391
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          444..1382
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1339..1359
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1398..1431)
                     /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(1482..1515)
                     /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      1522..1531
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1543..1599
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     primer_bind     1812..1833
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    1926..1959
                     /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    2010..2043
                     /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    2050..2083
                     /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     2735..2754
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2994..3015
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    complement(3123..3156)
                     /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    3265..3853
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3318..3338)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3736..3747)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3885..4361
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4084..4103)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4401..4541
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4401..4530
                     /label=AAV2 ITR
     rep_origin      4616..5071
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4703..4722)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4913..4934
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5088..5107)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5207..5229
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5267..5285)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5353..5457
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5458..5526,5527..6318)
                     /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(5676..5695)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6489..7077
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6978..6997
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(7139..7156,1..112)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 ctcgctcgct cactgaggcc gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg
       61 agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc ctgcggccgc
      121 acgcgtaagc tttgcaaaga tggataaagt tttaaacaga gaggaatctt tgcagctaat
      181 ggaccttcta ggtcttgaaa ggagtgggaa ttggctccgg tgcccgtcag tgggcagagc
      241 gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct
      301 agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc
      361 ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca
      421 acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct
      481 ttacgggtta tggcccttgc gtgccttgaa ttacttccac ctggctgcag tacgtgattc
      541 ttgatcccga gcttcgggtt ggaagtgggt gggagagttc gaggccttgc gcttaaggag
      601 ccccttcgcc tcgtgcttga gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa
      661 tctggtggca ccttcgcgcc tgtctcgctg ctttcgataa gtctctagcc atttaaaatt
      721 tttgatgacc tgctgcgacg ctttttttct ggcaagatag tcttgtaaat gcgggccaag
      781 atctgcacac tggtatttcg gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc
      841 agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc accgagaatc ggacgggggt
      901 agtctcaagc tggccggcct gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc
      961 cctgggcggc aaggctggcc cggtcggcac cagttgcgtg agcggaaaga tggccgcttc
     1021 ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg
     1081 agtcacccac acaaaggaaa agggcctttc cgtcctcagc cgtcgcttca tgtgactcca
     1141 cggagtaccg ggcgccgtcc aggcacctcg attagttctc gagcttttgg agtacgtcgt
     1201 ctttaggttg gggggagggg ttttatgcga tggagtttcc ccacactgag tgggtggaga
     1261 ctgaagttag gccagcttgg cacttgatgt aattctcctt ggaatttgcc ctttttgagt
     1321 ttggatcttg gttcattctc aagcctcaga cagtggttca aagttttttt cttccatttc
     1381 aggtgtcgtg aggatccata acttcgtata ggatacttta tacgaagtta tgtagctgga
     1441 gcaatttgaa aagattgttg tattacctct agctgcgctt aataacttcg tatagtatac
     1501 cttatacgaa gttatgtcga cgccaccatg gtggactcat cacgtcgtaa gtggaataag
     1561 acaggtcacg cagtcagagc tataggtcgg ctgagctcac tcgagaacgt ctatatcaag
     1621 gccgacaagc agaagaacgg catcaaggcg aacttcaaga tccgccacaa catcgaggac
     1681 ggcggcgtgc agctcgccta ccactaccag cagaacaccc ccatcggcga cggccccgtg
     1741 ctgctgcccg acaaccacta cctgagcgtg cagtccaaac tttcgaaaga ccccaacgag
     1801 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     1861 gacgagctgt acaagggcgg taccggaggg agcatggtga gcaaggtgag tacaggaggg
     1921 ctagcataac ttcgtataaa gtatcctata cgaagttatt tgccttaacc cagaaattat
     1981 cactgttatt ctttagatag gtgcaaagaa taacttcgta taaggtatac tatacgaagt
     2041 tatggtaccg aagttcctat tcttcaaata gtataggaac ttcaattgtt attggtgcaa
     2101 agaatcactt ccttaagtct ttatactgcc caggaagttc ctattcttca aaaggtatag
     2161 gaacttcggc gcgcctcact tcgctgtcat catttgtaca aactcttcgt agtttacctg
     2221 accatcccca tcgatgtctg cttccctgat catttcatca acctcttcat ctgttaactt
     2281 ctctccaagg tttgtcatca cgtggcgaag ctctgctgca ctgatgtagc cattgccatc
     2341 cttatcaaac acaccgaacg cttctctaat ttcttcttcc gtgtccctgt agctcccttt
     2401 tcttgccatc attgtcagga actcagggaa gtcgattgtg ccgtcaccgt cggcatctac
     2461 ttcattgatc atgtcctgca gctctgcttc tgtggggttc tgccccagag accgcatcac
     2521 cgtccccagc tccttggttg ttattgtccc atccccgtcc ttgtcaaata gggagaaagc
     2581 ctctttaaat tctgcgatct gctcttcagt cagttggtcc ggcaggttgt actccagctt
     2641 gtgccccagg atgttgccgt cctccttgaa gtcgatgccc ttcagctcga tgcggttcac
     2701 cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg tagttgccgt cgtccttgaa
     2761 gaagatggtg cgctcctgga tgtagccttc gggcatggcg gacttgaaga agtcgtgctg
     2821 cttcatgtgg tcggggtagc ggctgaagca ctgcacgccg taggtcaggg tggtcacgag
     2881 ggtgggccag ggcacgggca gcttgccggt ggtgcagatg aacttcaggg tcagcttgcc
     2941 gtaggtggca tcgccctcac cctcgccgga cacgctgaac ttgtggccgt ttacgtcgcc
     3001 gtccagctcg accaggatgg gcaccacccc ggtgaacagc tcctcgccct gtgggacgac
     3061 atgacttaac cagttaacca tgttctctga acatgagaag ggctggccac tctccatcta
     3121 gagaagttcc tatactattt gaagaatagg aacttcttgc cttaacccag aaattatcac
     3181 tgttattctt tagaatagtg caaagagaag ttcctatacc ttttgaagaa taggaacttc
     3241 gaattcgata tcaagcttat cgataatcaa cctctggatt acaaaatttg tgaaagattg
     3301 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     3361 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
     3421 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact
     3481 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc
     3541 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc
     3601 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa
     3661 tcatcgtcct ttccttggct gctcgcctat gttgccacct ggattctgcg cgggacgtcc
     3721 ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg
     3781 gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg
     3841 gccgcctccc cgcatcgata ccgagcgctg ctcgagagat ctacgggtgg catccctgtg
     3901 acccctcccc agtgcctctc ctggccctgg aagttgccac tccagtgccc accagccttg
     3961 tcctaataaa attaagttgc atcattttgt ctgactaggt gtccttctat aatattatgg
     4021 ggtggagggg ggtggtatgg agcaaggggc aagttgggaa gacaacctgt agggcctgcg
     4081 gggtctattg ggaaccaagc tggagtgcag tggcacaatc ttggctcact gcaatctccg
     4141 cctcctgggt tcaagcgatt ctcctgcctc agcctcccga gttgttggga ttccaggcat
     4201 gcatgaccag gctcagctaa tttttgtttt tttggtagag acggggtttc accatattgg
     4261 ccaggctggt ctccaactcc taatctcagg tgatctaccc accttggcct cccaaattgc
     4321 tgggattaca ggcgtgaacc actgctccct tccctgtcct tctgattttg taggtaacca
     4381 cgtgcggacc gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg
     4441 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
     4501 ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt
     4561 tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg
     4621 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     4681 cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     4741 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     4801 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg
     4861 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     4921 ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga tttataaggg
     4981 attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     5041 aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct
     5101 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     5161 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     5221 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     5281 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     5341 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5401 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     5461 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     5521 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     5581 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     5641 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     5701 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     5761 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     5821 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     5881 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     5941 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     6001 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     6061 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     6121 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     6181 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     6241 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     6301 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     6361 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     6421 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6481 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     6541 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     6601 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     6661 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     6721 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     6781 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     6841 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     6901 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     6961 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     7021 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     7081 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgtcc
     7141 tgcaggcagc tgcgcg
//
