LOCUS       sequence-272757-        7050 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 7050)
  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 7050)
  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..7050
                     /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        177..388
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             401..669
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     protein_bind    697..730
                     /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(847..1098)
                     /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(889..910)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1238..1257
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    complement(1479..1512)
                     /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(1563..1596)
                     /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     1709..1730
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(1773..1829)
                     /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    2634..2667
                     /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    2718..2751
                     /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(3035..3068)
                     /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    3177..3765
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3230..3250)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3648..3659)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3797..4273
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3996..4015)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4313..4453
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4313..4442
                     /label=AAV2 ITR
     rep_origin      4528..4983
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4615..4634)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4825..4846
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5000..5019)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5119..5141
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5179..5197)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5265..5369
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5370..5438,5439..6230)
                     /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(5588..5607)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6401..6989
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6890..6909
                     /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 gcgtaaggat ctgcgatcgc tccggtgccc gtcagtgggc
      181 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaacggg
      241 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
      301 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
      361 tcgcaacggg tttgccgcca gaacacagct gaagcttcga ggggctcgca tctctccttc
      421 acgcgcccgc cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc
      481 tcccgcctgt ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc
      541 gagaccgggc ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac
      601 gctttgcctg accctgcttg ctcaactcta cgtctttgtt tcgttttctg ttctgcgccg
      661 ttacagatcc aagctgtgac cggcgcctac ggatccgaag ttcctattct tcaaatagta
      721 taggaacttc gcagagtagt agctggattg tagctgctat tagcaatcta aaacctctta
      781 gaagttccta ttcttcaaaa ggtataggaa cttcggcgcg ccttacacct cgttctcgta
      841 gcagaacttg tacagctcgt ccatgccgag agtgatcccg gcggcggtca cgaactccag
      901 caggaccatg tgatcgcgct tctcgttggg gtctttgctc agggcggact ggtagctcag
      961 gtagtggttg tcgggcagca gcacggggcc gtcgccgatg ggggtgttct gctggtagtg
     1021 gtcggcgagc tgcacgctgc cgtcctcgat gttgtggcgg atcttgaagt tcaccttgat
     1081 gccgttcttc tgcttgtcgg ccatgatata gacgttgtgg ctgttgtagt tgtactccag
     1141 cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg cccttcagct cgatgcggtt
     1201 caccagggtg tcgccctcga acttcacctc ggcgcgggtc ttgtagttgc cgtcgtcctt
     1261 gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg gcggacttga agaagtcgtg
     1321 ctgcttcatg tggtcggggt agcgggcgaa gcactgcagg ccgtagccga aggtggtcac
     1381 gagggtgggc cagggcacgg gcagctgtgg gacgacatga cttaaccagt taaccatgtt
     1441 ctctgaacat gagaagggct ggccactctc cagtcgacat aacttcgtat aggatacttt
     1501 atacgaagtt atgcagaatg gtagctggat tgtagctgct attagcaata tgaaacctct
     1561 taataacttc gtatagtata ccttatacga agttatacta gtcctcctgt actcaccttg
     1621 ccggtggtgc agatgaactt cagggtcagc ttgccgtagg tggcatcgcc ctcgccctcg
     1681 ccggacacgc tgaacttgtg gccgtttacg tcgccgtcca gctcgaccag gatgggcacc
     1741 accccggtga acagctcctc gcccttgctc acgggaccgg ggttttcttc cacgtctcct
     1801 gcttgcttta acagagagaa gttcgtggct ccggagccgg gcgcgcccac gttgatgtcg
     1861 atctggtcca gggggatgta ctcgccctcg ctggtgatcc tgctcttatc atcagccggg
     1921 gtcccagaag cagatggaac atccaaaatg ctgccggaca cgacagattc gtttgacgtc
     1981 agatagttca ggagcaagaa tgcgaaaatg tactttgcca cgatatccag gaaagaatat
     2041 ccccagctcg tcaccccaac gggcaacacc gcgataccct cgactccaag agcccacaca
     2101 attggatacc ccagccacat cactactgtc aacagcttca gggtattgaa catgtcagcg
     2161 gttcccgcgg ctttggcgtc ctgtgcccac tccaccagca ggatatacaa gaccaccaga
     2221 aagcatgcac aactgatagc gtaaaagaac catctcatga ggtgggagga ggtagtcagg
     2281 gccgcggcaa ggccagtcac gcacatagcg atatcgaaag tgatagctgt gaagagcttt
     2341 gtagcattgc ttccggccag gagacccaga gcgaggagaa tcatgggcgt ggaaagtgcc
     2401 caggtgagat accgtcccca catggtgact acaccatcta cctcttctcc tcccagcatc
     2461 actgagctgc cttctgcaaa atggcccgct ggcatttcaa gaacggaaat tgtcaggccg
     2521 ctcgccaatc ctaaacacat gagaaataga gtcataagca catgggtcac atacaggaga
     2581 tatgtatata acattaatac aattttataa aaaaaagggg gggcacaggt accataactt
     2641 cgtataaagt atcctatacg aagttatttg ccttaaccca gaaattatca ctgttattct
     2701 ttagaatggt gcaaagaata acttcgtata aggtatacta tacgaagtta tgctagcaac
     2761 cccgacactt acctgtgtag gaggcaatgc tgacgacagg cacaaggatg gttgacacag
     2821 caataagttt tgcccgtgga tcatcgagtc ctcgggtcat aaaaacgaac agcagtatac
     2881 tcagtcctgc aagtgcgatg ttgatataga gactgcttgc aagcaaaggg tcgttcagca
     2941 cgaactcgaa caactccctt tgggtaacct cggcttgaag ggccacggca ctctcggtca
     3001 cgggaggcag ggtctctgtc atggtggcga gctcgaagtt cctatactat ttgaagaata
     3061 ggaacttctt gccttaaccc agaaattatc actgttattc tttagaatgg tgcaaagaga
     3121 agttcctata ccttttgaag aataggaact tcgaattcga tatcaagctt atcgataatc
     3181 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
     3241 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg
     3301 ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc
     3361 ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt
     3421 ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg
     3481 ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg
     3541 gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct
     3601 atgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc
     3661 cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc
     3721 ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgagcgc
     3781 tgctcgagag atctacgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct
     3841 ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt
     3901 gtctgactag gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg
     3961 gcaagttggg aagacaacct gtagggcctg cggggtctat tgggaaccaa gctggagtgc
     4021 agtggcacaa tcttggctca ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc
     4081 tcagcctccc gagttgttgg gattccaggc atgcatgacc aggctcagct aatttttgtt
     4141 tttttggtag agacggggtt tcaccatatt ggccaggctg gtctccaact cctaatctca
     4201 ggtgatctac ccaccttggc ctcccaaatt gctgggatta caggcgtgaa ccactgctcc
     4261 cttccctgtc cttctgattt tgtaggtaac cacgtgcgga ccgagcggcc gcaggaaccc
     4321 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     4381 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     4441 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     4501 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     4561 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt
     4621 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     4681 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     4741 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     4801 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact
     4861 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa
     4921 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     4981 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     5041 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     5101 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     5161 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     5221 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     5281 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     5341 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     5401 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     5461 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     5521 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     5581 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     5641 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     5701 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     5761 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     5821 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     5881 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     5941 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     6001 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     6061 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     6121 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     6181 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     6241 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     6301 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     6361 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     6421 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     6481 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     6541 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     6601 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     6661 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     6721 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     6781 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     6841 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     6901 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     6961 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     7021 tggccttttg ctggcctttt gctcacatgt
//
