LOCUS       sequence-272675-        6616 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intersectional viral expression of mCherry in cells expressing Flp 
            AND NOT Cre.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6616)
  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 6616)
  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-mCherry
FEATURES             Location/Qualifiers
     source          1..6616
                     /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"
     primer_bind     complement(1691..1709)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     protein_bind    1908..1941
                     /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    1992..2025
                     /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    2032..2065
                     /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     complement(2436..2455)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(2601..2634)
                     /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    2743..3331
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2796..2816)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3214..3225)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3363..3839
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3562..3581)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3879..4019
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3879..4008
                     /label=AAV2 ITR
     rep_origin      4094..4549
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4181..4200)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4391..4412
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4566..4585)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4685..4707
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4745..4763)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4831..4935
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4936..5004,5005..5796)
                     /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(5154..5173)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5967..6555
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6456..6475
                     /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 tatactagtg ccaccatggt gagcaagggc
     1561 gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc
     1621 tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
     1681 acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc
     1741 ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc
     1801 gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag
     1861 gacggcggcg tggtgaccgt gacccaggtg agtacaggag ggctagcata acttcgtata
     1921 aagtatccta tacgaagtta tttgccttaa cccagaaatt atcactgtta ttctttagat
     1981 aggtgcaaag aataacttcg tataaggtat actatacgaa gttatggtac cgaagttcct
     2041 attcttcaaa tagtatagga acttcaattg ttattggtgc aaagaatcac ttccttaagt
     2101 ctttatactg cccaggaagt tcctattctt caaaaggtat aggaacttcg gcgcgcctta
     2161 cttgtacagc tcgtccatgc cgccggtgga gtggcggccc tcggcgcgtt cgtactgttc
     2221 cacgatggtg tagtcctcgt tgtgggaggt gatgtccaac ttgatgttga cgttgtaggc
     2281 gccgggcagc tgcacgggct tcttggcctt gtaggtggtc ttgacctcag cgtcgtagtg
     2341 gccgccgtcc ttcagcttca gcctctgctt gatctcgccc ttcagggcgc cgtcctcggg
     2401 gtacatccgc tcggaggagg cctcccagcc catggtcttc ttctgcatta cggggccgtc
     2461 ggaggggaag ttggtgccgc gcagcttcac cttgtagatg aactcgccgt cctgcaggga
     2521 ggagtcctgt gggacgacat gacttaacca gttaaccatg ttctctgaac atgagaaggg
     2581 ctggccactc tccagagctc gaagttccta tactatttga agaataggaa cttcttgcct
     2641 taacccagaa attatcactg ttattcttta gaatggtgca aagagaagtt cctatacctt
     2701 ttgaagaata ggaacttcga attcgatatc aagcttatcg ataatcaacc tctggattac
     2761 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     2821 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     2881 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     2941 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     3001 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
     3061 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     3121 gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctatgt tgccacctgg
     3181 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     3241 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     3301 agtcggatct ccctttgggc cgcctccccg catcgatacc gagcgctgct cgagagatct
     3361 acgggtggca tccctgtgac ccctccccag tgcctctcct ggccctggaa gttgccactc
     3421 cagtgcccac cagccttgtc ctaataaaat taagttgcat cattttgtct gactaggtgt
     3481 ccttctataa tattatgggg tggagggggg tggtatggag caaggggcaa gttgggaaga
     3541 caacctgtag ggcctgcggg gtctattggg aaccaagctg gagtgcagtg gcacaatctt
     3601 ggctcactgc aatctccgcc tcctgggttc aagcgattct cctgcctcag cctcccgagt
     3661 tgttgggatt ccaggcatgc atgaccaggc tcagctaatt tttgtttttt tggtagagac
     3721 ggggtttcac catattggcc aggctggtct ccaactccta atctcaggtg atctacccac
     3781 cttggcctcc caaattgctg ggattacagg cgtgaaccac tgctcccttc cctgtccttc
     3841 tgattttgta ggtaaccacg tgcggaccga gcggccgcag gaacccctag tgatggagtt
     3901 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
     3961 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg
     4021 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca
     4081 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     4141 cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct
     4201 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     4261 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt
     4321 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     4381 ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat
     4441 tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt
     4501 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact
     4561 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     4621 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     4681 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     4741 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     4801 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     4861 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     4921 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     4981 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     5041 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     5101 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     5161 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     5221 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     5281 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     5341 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     5401 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     5461 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     5521 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     5581 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     5641 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     5701 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     5761 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     5821 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     5881 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     5941 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     6001 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     6061 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     6121 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6181 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     6241 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     6301 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     6361 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     6421 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     6481 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     6541 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     6601 ccttttgctc acatgt
//
