LOCUS       sequence-265097-        6617 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intersectional viral expression of BFP in cells expressing Flp AND 
            NOT Cre.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6617)
  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 6617)
  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-BFP
FEATURES             Location/Qualifiers
     source          1..6617
                     /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."
     protein_bind    1796..1829
                     /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    1880..1913
                     /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    1920..1953
                     /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)"
     protein_bind    complement(2602..2635)
                     /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    2744..3332
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2797..2817)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3215..3226)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3364..3840
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3563..3582)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3880..4020
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3880..4009
                     /label=AAV2 ITR
     rep_origin      4095..4550
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4182..4201)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4392..4413
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4567..4586)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4686..4708
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4746..4764)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4832..4936
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4937..5005,5006..5797)
                     /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(5155..5174)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5968..6556
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6457..6476
                     /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 tatactagtg ccaccatgag
     1561 cgagctgatt aaggagaaca tgcacatgaa gctgtacatg gagggcaccg tggacaacca
     1621 tcacttcaag tgcacatccg agggcgaagg caagccctac gagggcaccc agaccatgag
     1681 aatcaaggtg gtcgagggcg gccctctccc cttcgccttc gacatcctgg ctactagctt
     1741 cctctacggc agcaagacct tcatcaacca cacccgtgag tacaggaggg ctagcataac
     1801 ttcgtataaa gtatcctata cgaagttatt tgccttaacc cagaaattat cactgttatt
     1861 ctttagatag gtgcaaagaa taacttcgta taaggtatac tatacgaagt tatggtaccg
     1921 aagttcctat tcttcaaata gtataggaac ttcaattgtt attggtgcaa agaatcactt
     1981 ccttaagtct ttatactgcc caggaagttc ctattcttca aaaggtatag gaacttcggc
     2041 gcgccttaat taagcttgtg ccccagtttg ctagggaggt cgcagtatct ggccactgcc
     2101 acctcgtgct gctcgacgta ggtctcgttg ttggcctcct tgattctttc cagtctgtag
     2161 tccacatagt agacgccagg catcttgagg ttcttagcgg gtttcttgga tctatatgtg
     2221 gtcttgatgt ttgcgatcag atggctcccg cccacgagct tcagggccat gtcgtttctg
     2281 ccttccaggc cgccgtcagc ggggtacagc gtctcggtga aggcctccca gccgagtgtt
     2341 ttcttctgca tcacagggcc gttggatgtg aagttcaccc ctctgatctt gacgttgtag
     2401 atgaggcagc cgtcctggag gctggtgtcc tgggtagcgg tcagcacgcc cccgtcttcg
     2461 tatgtggtga ctctctccca tgtgaagccc tcagggaagg actgcttgaa gaagtcgggg
     2521 atgccctctg tgggacgaca tgacttaacc agttaaccat gttctctgaa catgagaagg
     2581 gctggccact ctccagagct cgaagttcct atactatttg aagaatagga acttcttgcc
     2641 ttaacccaga aattatcact gttattcttt agaatggtgc aaagagaagt tcctatacct
     2701 tttgaagaat aggaacttcg aattcgatat caagcttatc gataatcaac ctctggatta
     2761 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     2821 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     2881 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     2941 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     3001 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     3061 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     3121 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctatg ttgccacctg
     3181 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     3241 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     3301 gagtcggatc tccctttggg ccgcctcccc gcatcgatac cgagcgctgc tcgagagatc
     3361 tacgggtggc atccctgtga cccctcccca gtgcctctcc tggccctgga agttgccact
     3421 ccagtgccca ccagccttgt cctaataaaa ttaagttgca tcattttgtc tgactaggtg
     3481 tccttctata atattatggg gtggaggggg gtggtatgga gcaaggggca agttgggaag
     3541 acaacctgta gggcctgcgg ggtctattgg gaaccaagct ggagtgcagt ggcacaatct
     3601 tggctcactg caatctccgc ctcctgggtt caagcgattc tcctgcctca gcctcccgag
     3661 ttgttgggat tccaggcatg catgaccagg ctcagctaat ttttgttttt ttggtagaga
     3721 cggggtttca ccatattggc caggctggtc tccaactcct aatctcaggt gatctaccca
     3781 ccttggcctc ccaaattgct gggattacag gcgtgaacca ctgctccctt ccctgtcctt
     3841 ctgattttgt aggtaaccac gtgcggaccg agcggccgca ggaaccccta gtgatggagt
     3901 tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc
     3961 gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg
     4021 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc
     4081 aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     4141 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     4201 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     4261 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg
     4321 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     4381 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta
     4441 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     4501 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac
     4561 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     4621 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     4681 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     4741 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     4801 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     4861 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     4921 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     4981 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     5041 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     5101 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     5161 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     5221 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     5281 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     5341 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     5401 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     5461 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     5521 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     5581 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     5641 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     5701 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     5761 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     5821 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     5881 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     5941 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     6001 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     6061 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     6121 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     6181 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     6241 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     6301 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     6361 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     6421 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     6481 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     6541 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     6601 gccttttgct cacatgt
//
