LOCUS       sequence-265116-        7064 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intersectional viral expression of NpHR3.3-EYFP in cells expressing 
            Cre AND NOT Flp.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7064)
  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 7064)
  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-Con/Foff 2.0-NpHR3.3-EYFP
FEATURES             Location/Qualifiers
     source          1..7064
                     /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=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    complement(1111..1144)
                     /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(1195..1228)
                     /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)."
     primer_bind     1341..1362
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(1405..1461)
                     /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    2266..2299
                     /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    2350..2383
                     /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)."
     primer_bind     complement(2605..2624)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2764..3015
                     /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     2952..2973
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(3049..3082)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     misc_feature    3191..3779
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3244..3264)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3662..3673)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3811..4287
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4010..4029)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4327..4467
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4327..4456
                     /label=AAV2 ITR
     rep_origin      4542..4997
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4629..4648)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4839..4860
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5014..5033)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5133..5155
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5193..5211)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5279..5383
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5384..5452,5453..6244)
                     /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(5602..5621)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6415..7003
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6904..6923
                     /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 tctagaaagt ataggaactt cgcagagtag tagctggatt gtagctgcta ttagcaatct
      781 aaaacctctt agaagttcct attcttcaaa aggtatagga acttcggcgc gccgccacca
      841 tgacagagac cctgcctccc gtgaccgaga gtgccgtggc ccttcaagcc gaggttaccc
      901 aaagggagtt gttcgagttc gtgctgaacg accctttgct tgcaagcagt ctctatatca
      961 acatcgcact tgcaggactg agtatactgc tgttcgtttt tatgacccga ggactcgatg
     1021 atccacgggc aaaacttatt gctgtgtcaa ccatccttgt gcctgtcgtc agcattgcct
     1081 cctacacagg taagtgtcgg ggttgagctc ataacttcgt atagtatacc ttatacgaag
     1141 ttattctttg caccattcta aagaataaca gtgataattt ctgggttaag gcaaataact
     1201 tcgtatagga tactttatac gaagttatgg tacccctcct gtactcacct tgccggtggt
     1261 gcagatgaac ttcagggtca gcttgccgta ggtggcatcg ccctcgccct cgccggacac
     1321 gctgaacttg tggccgttta cgtcgccgtc cagctcgacc aggatgggca ccaccccggt
     1381 gaacagctcc tcgcccttgc tcacgggacc ggggttttct tccacgtctc ctgcttgctt
     1441 taacagagag aagttcgtgg ctccggagcc gggcgcgccc acgttgatgt cgatctggtc
     1501 cagggggatg tactcgccct cgctggtgat cctgctctta tcatcagccg gggtcccaga
     1561 agcagatgga acatccaaaa tgctgccgga cacgacagat tcgtttgacg tcagatagtt
     1621 caggagcaag aatgcgaaaa tgtactttgc cacgatatcc aggaaagaat atccccagct
     1681 cgtcacccca acgggcaaca ccgcgatacc ctcgactcca agagcccaca caattggata
     1741 ccccagccac atcactactg tcaacagctt cagggtattg aacatgtcag cggttcccgc
     1801 ggctttggcg tcctgtgccc actccaccag caggatatac aagaccacca gaaagcatgc
     1861 acaactgata gcgtaaaaga accatctcat gaggtgggag gaggtagtca gggccgcggc
     1921 aaggccagtc acgcacatag cgatatcgaa agtgatagct gtgaagagct ttgtagcatt
     1981 gcttccggcc aggagaccca gagcgaggag aatcatgggc gtggaaagtg cccaggtgag
     2041 ataccgtccc cacatggtga ctacaccatc tacctcttct cctcccagca tcactgagct
     2101 gccttctgca aaatggcccg ctggcatttc aagaacggaa attgtcaggc cgctcgccaa
     2161 tcctaaacac atgagaaata gagtcataag cacatgggtc acatacagga gatatgtata
     2221 taacattaat acaattttat aaaaaaaagg gggggcacaa ctagtataac ttcgtataag
     2281 gtatactata cgaagttatt aagaggtttc atattggtaa tagcagctac aatccagcta
     2341 ccattctgca taacttcgta taaagtatcc tatacgaagt tatgtcgact ggagagtggc
     2401 cagcccttct catgttcaga gaacatggtt aactggttaa gtcatgtcgt cccacagctg
     2461 cccgtgccct ggcccaccct cgtgaccacc ttcggctacg gcctgcagtg cttcgcccgc
     2521 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     2581 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     2641 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     2701 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     2761 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     2821 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     2881 ctgctgcccg acaaccacta cctgagctac cagtccgccc tgagcaaaga ccccaacgag
     2941 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     3001 gacgagctgt acaagttctg ctacgagaac gaggtgtaat gagctagcga agttcctata
     3061 ctttctagag aataggaact tctaagcaga caatcttgta atacatcagc agctagtaga
     3121 ttgctccagc tagaagttcc tatacctttt gaagaatagg aacttcgaat tcgatatcaa
     3181 gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     3241 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     3301 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
     3361 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
     3421 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
     3481 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     3541 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
     3601 ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
     3661 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
     3721 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca
     3781 tcgataccga gcgctgctcg agagatctac gggtggcatc cctgtgaccc ctccccagtg
     3841 cctctcctgg ccctggaagt tgccactcca gtgcccacca gccttgtcct aataaaatta
     3901 agttgcatca ttttgtctga ctaggtgtcc ttctataata ttatggggtg gaggggggtg
     3961 gtatggagca aggggcaagt tgggaagaca acctgtaggg cctgcggggt ctattgggaa
     4021 ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa
     4081 gcgattctcc tgcctcagcc tcccgagttg ttgggattcc aggcatgcat gaccaggctc
     4141 agctaatttt tgtttttttg gtagagacgg ggtttcacca tattggccag gctggtctcc
     4201 aactcctaat ctcaggtgat ctacccacct tggcctccca aattgctggg attacaggcg
     4261 tgaaccactg ctcccttccc tgtccttctg attttgtagg taaccacgtg cggaccgagc
     4321 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
     4381 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
     4441 gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc
     4501 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc
     4561 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     4621 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     4681 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     4741 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     4801 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     4861 aacaacactc aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc
     4921 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     4981 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     5041 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     5101 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     5161 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     5221 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     5281 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     5341 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     5401 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     5461 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     5521 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     5581 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     5641 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     5701 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     5761 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     5821 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     5881 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     5941 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     6001 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     6061 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     6121 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6181 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6241 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6301 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     6361 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     6421 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     6481 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     6541 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     6601 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     6661 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     6721 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     6781 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     6841 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     6901 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     6961 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     7021 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgt
//
