LOCUS       sequence-265117-        7061 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intersectional viral expression of NpHR3.3-EYFP in cells expressing 
            Flp AND NOT Cre.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7061)
  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 7061)
  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-nEF-Coff/Fon-NpHR3.3-EYFP
FEATURES             Location/Qualifiers
     source          1..7061
                     /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        188..399
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             412..680
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     protein_bind    708..741
                     /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)"
     CDS             complement(858..1109)
                     /codon_start=1
                     /product="C-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VC155
                     /translation="DKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
                     YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK"
     primer_bind     complement(900..921)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1249..1268
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    complement(1490..1523)
                     /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(1574..1607)
                     /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     1720..1741
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(1784..1840)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     protein_bind    2645..2678
                     /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    2729..2762
                     /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    complement(3046..3079)
                     /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    3188..3776
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3241..3261)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3659..3670)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3808..4284
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4007..4026)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4324..4464
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4324..4453
                     /label=AAV2 ITR
     rep_origin      4539..4994
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4626..4645)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4836..4857
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5011..5030)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5130..5152
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5190..5208)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5276..5380
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5381..5449,5450..6241)
                     /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(5599..5618)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6412..7000
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6901..6920
                     /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 cgcgtaagga tctgcgatcg ctccggtgcc
      181 cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc
      241 aattgaacgg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac
      301 tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg
      361 aacgttcttt ttcgcaacgg gtttgccgcc agaacacagc tgaagcttcg aggggctcgc
      421 atctctcctt cacgcgcccg ccgccctacc tgaggccgcc atccacgccg gttgagtcgc
      481 gttctgccgc ctcccgcctg tggtgcctcc tgaactgcgt ccgccgtcta ggtaagttta
      541 aagctcaggt cgagaccggg cctttgtccg gcgctccctt ggagcctacc tagactcagc
      601 cggctctcca cgctttgcct gaccctgctt gctcaactct acgtctttgt ttcgttttct
      661 gttctgcgcc gttacagatc caagctgtga ccggcgccta cggatccgaa gttcctattc
      721 ttcaaatagt ataggaactt cgcagagtag tagctggatt gtagctgcta ttagcaatct
      781 aaaacctctt agaagttcct attcttcaaa aggtatagga acttcggcgc gccttacacc
      841 tcgttctcgt agcagaactt gtacagctcg tccatgccga gagtgatccc ggcggcggtc
      901 acgaactcca gcaggaccat gtgatcgcgc ttctcgttgg ggtctttgct cagggcggac
      961 tggtagctca ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat gggggtgttc
     1021 tgctggtagt ggtcggcgag ctgcacgctg ccgtcctcga tgttgtggcg gatcttgaag
     1081 ttcaccttga tgccgttctt ctgcttgtcg gccatgatat agacgttgtg gctgttgtag
     1141 ttgtactcca gcttgtgccc caggatgttg ccgtcctcct tgaagtcgat gcccttcagc
     1201 tcgatgcggt tcaccagggt gtcgccctcg aacttcacct cggcgcgggt cttgtagttg
     1261 ccgtcgtcct tgaagaagat ggtgcgctcc tggacgtagc cttcgggcat ggcggacttg
     1321 aagaagtcgt gctgcttcat gtggtcgggg tagcgggcga agcactgcag gccgtagccg
     1381 aaggtggtca cgagggtggg ccagggcacg ggcagctgtg ggacgacatg acttaaccag
     1441 ttaaccatgt tctctgaaca tgagaagggc tggccactct ccagtcgaca taacttcgta
     1501 taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc tattagcaat
     1561 atgaaacctc ttaataactt cgtatagtat accttatacg aagttatact agtcctcctg
     1621 tactcacctt gccggtggtg cagatgaact tcagggtcag cttgccgtag gtggcatcgc
     1681 cctcgccctc gccggacacg ctgaacttgt ggccgtttac gtcgccgtcc agctcgacca
     1741 ggatgggcac caccccggtg aacagctcct cgcccttgct cacgggaccg gggttttctt
     1801 ccacgtctcc tgcttgcttt aacagagaga agttcgtggc tccggagccg ggcgcgccca
     1861 cgttgatgtc gatctggtcc agggggatgt actcgccctc gctggtgatc ctgctcttat
     1921 catcagccgg ggtcccagaa gcagatggaa catccaaaat gctgccggac acgacagatt
     1981 cgtttgacgt cagatagttc aggagcaaga atgcgaaaat gtactttgcc acgatatcca
     2041 ggaaagaata tccccagctc gtcaccccaa cgggcaacac cgcgataccc tcgactccaa
     2101 gagcccacac aattggatac cccagccaca tcactactgt caacagcttc agggtattga
     2161 acatgtcagc ggttcccgcg gctttggcgt cctgtgccca ctccaccagc aggatataca
     2221 agaccaccag aaagcatgca caactgatag cgtaaaagaa ccatctcatg aggtgggagg
     2281 aggtagtcag ggccgcggca aggccagtca cgcacatagc gatatcgaaa gtgatagctg
     2341 tgaagagctt tgtagcattg cttccggcca ggagacccag agcgaggaga atcatgggcg
     2401 tggaaagtgc ccaggtgaga taccgtcccc acatggtgac tacaccatct acctcttctc
     2461 ctcccagcat cactgagctg ccttctgcaa aatggcccgc tggcatttca agaacggaaa
     2521 ttgtcaggcc gctcgccaat cctaaacaca tgagaaatag agtcataagc acatgggtca
     2581 catacaggag atatgtatat aacattaata caattttata aaaaaaaggg ggggcacagg
     2641 taccataact tcgtataaag tatcctatac gaagttattt gccttaaccc agaaattatc
     2701 actgttattc tttagaatgg tgcaaagaat aacttcgtat aaggtatact atacgaagtt
     2761 atgctagcaa ccccgacact tacctgtgta ggaggcaatg ctgacgacag gcacaaggat
     2821 ggttgacaca gcaataagtt ttgcccgtgg atcatcgagt cctcgggtca taaaaacgaa
     2881 cagcagtata ctcagtcctg caagtgcgat gttgatatag agactgcttg caagcaaagg
     2941 gtcgttcagc acgaactcga acaactccct ttgggtaacc tcggcttgaa gggccacggc
     3001 actctcggtc acgggaggca gggtctctgt catggtggcg agctcgaagt tcctatacta
     3061 tttgaagaat aggaacttct tgccttaacc cagaaattat cactgttatt ctttagaatg
     3121 gtgcaaagag aagttcctat accttttgaa gaataggaac ttcgaattcg atatcaagct
     3181 tatcgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     3241 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     3301 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     3361 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     3421 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     3481 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     3541 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
     3601 gctgctcgcc tatgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     3661 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     3721 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg
     3781 ataccgagcg ctgctcgaga gatctacggg tggcatccct gtgacccctc cccagtgcct
     3841 ctcctggccc tggaagttgc cactccagtg cccaccagcc ttgtcctaat aaaattaagt
     3901 tgcatcattt tgtctgacta ggtgtccttc tataatatta tggggtggag gggggtggta
     3961 tggagcaagg ggcaagttgg gaagacaacc tgtagggcct gcggggtcta ttgggaacca
     4021 agctggagtg cagtggcaca atcttggctc actgcaatct ccgcctcctg ggttcaagcg
     4081 attctcctgc ctcagcctcc cgagttgttg ggattccagg catgcatgac caggctcagc
     4141 taatttttgt ttttttggta gagacggggt ttcaccatat tggccaggct ggtctccaac
     4201 tcctaatctc aggtgatcta cccaccttgg cctcccaaat tgctgggatt acaggcgtga
     4261 accactgctc ccttccctgt ccttctgatt ttgtaggtaa ccacgtgcgg accgagcggc
     4321 cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg
     4381 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg
     4441 agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt
     4501 gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt
     4561 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     4621 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     4681 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     4741 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     4801 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     4861 aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggc
     4921 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     4981 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     5041 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     5101 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     5161 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     5221 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     5281 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     5341 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     5401 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     5461 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     5521 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     5581 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     5641 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     5701 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     5761 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     5821 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     5881 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     5941 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     6001 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     6061 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     6121 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     6181 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     6241 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     6301 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     6361 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     6421 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     6481 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     6541 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     6601 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     6661 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     6721 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     6781 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     6841 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     6901 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     6961 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     7021 tttacggttc ctggcctttt gctggccttt tgctcacatg t
//
