LOCUS       sequence-271955-        7053 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 7053)
  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 7053)
  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..7053
                     /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=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(1100..1133)
                     /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(1184..1217)
                     /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     1330..1351
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(1394..1450)
                     /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    2255..2288
                     /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    2339..2372
                     /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(2594..2613)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2753..3004
                     /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     2941..2962
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(3038..3071)
                     /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    3180..3768
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3233..3253)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3651..3662)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3800..4276
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3999..4018)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4316..4456
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4316..4445
                     /label=AAV2 ITR
     rep_origin      4531..4986
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4618..4637)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4828..4849
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5003..5022)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5122..5144
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5182..5200)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5268..5372
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5373..5441,5442..6233)
                     /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(5591..5610)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6404..6992
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6893..6912
                     /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 ctagaaagta
      721 taggaacttc gcagagtagt agctggattg tagctgctat tagcaatcta aaacctctta
      781 gaagttccta ttcttcaaaa ggtataggaa cttcggcgcg ccgccaccat gacagagacc
      841 ctgcctcccg tgaccgagag tgccgtggcc cttcaagccg aggttaccca aagggagttg
      901 ttcgagttcg tgctgaacga ccctttgctt gcaagcagtc tctatatcaa catcgcactt
      961 gcaggactga gtatactgct gttcgttttt atgacccgag gactcgatga tccacgggca
     1021 aaacttattg ctgtgtcaac catccttgtg cctgtcgtca gcattgcctc ctacacaggt
     1081 aagtgtcggg gttgagctca taacttcgta tagtatacct tatacgaagt tattctttgc
     1141 accattctaa agaataacag tgataatttc tgggttaagg caaataactt cgtataggat
     1201 actttatacg aagttatggt acccctcctg tactcacctt gccggtggtg cagatgaact
     1261 tcagggtcag cttgccgtag gtggcatcgc cctcgccctc gccggacacg ctgaacttgt
     1321 ggccgtttac gtcgccgtcc agctcgacca ggatgggcac caccccggtg aacagctcct
     1381 cgcccttgct cacgggaccg gggttttctt ccacgtctcc tgcttgcttt aacagagaga
     1441 agttcgtggc tccggagccg ggcgcgccca cgttgatgtc gatctggtcc agggggatgt
     1501 actcgccctc gctggtgatc ctgctcttat catcagccgg ggtcccagaa gcagatggaa
     1561 catccaaaat gctgccggac acgacagatt cgtttgacgt cagatagttc aggagcaaga
     1621 atgcgaaaat gtactttgcc acgatatcca ggaaagaata tccccagctc gtcaccccaa
     1681 cgggcaacac cgcgataccc tcgactccaa gagcccacac aattggatac cccagccaca
     1741 tcactactgt caacagcttc agggtattga acatgtcagc ggttcccgcg gctttggcgt
     1801 cctgtgccca ctccaccagc aggatataca agaccaccag aaagcatgca caactgatag
     1861 cgtaaaagaa ccatctcatg aggtgggagg aggtagtcag ggccgcggca aggccagtca
     1921 cgcacatagc gatatcgaaa gtgatagctg tgaagagctt tgtagcattg cttccggcca
     1981 ggagacccag agcgaggaga atcatgggcg tggaaagtgc ccaggtgaga taccgtcccc
     2041 acatggtgac tacaccatct acctcttctc ctcccagcat cactgagctg ccttctgcaa
     2101 aatggcccgc tggcatttca agaacggaaa ttgtcaggcc gctcgccaat cctaaacaca
     2161 tgagaaatag agtcataagc acatgggtca catacaggag atatgtatat aacattaata
     2221 caattttata aaaaaaaggg ggggcacaac tagtataact tcgtataagg tatactatac
     2281 gaagttatta agaggtttca tattgctaat agcagctaca atccagctac cattctgcat
     2341 aacttcgtat aaagtatcct atacgaagtt atgtcgactg gagagtggcc agcccttctc
     2401 atgttcagag aacatggtta actggttaag tcatgtcgtc ccacagctgc ccgtgccctg
     2461 gcccaccctc gtgaccacct tcggctacgg cctgcagtgc ttcgcccgct accccgacca
     2521 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     2581 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     2641 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     2701 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     2761 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     2821 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     2881 caaccactac ctgagctacc agtccgccct gagcaaagac cccaacgaga agcgcgatca
     2941 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     3001 caagttctgc tacgagaacg aggtgtaatg agctagcgaa gttcctatac tttctagaga
     3061 ataggaactt ctaagcagac aatcttgtaa tacatcagca gctagtagat tgctccagct
     3121 agaagttcct ataccttttg aagaatagga acttcgaatt cgatatcaag cttatcgata
     3181 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc
     3241 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta
     3301 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt
     3361 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg
     3421 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta
     3481 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt
     3541 tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg
     3601 cctatgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca
     3661 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc
     3721 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgag
     3781 cgctgctcga gagatctacg ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc
     3841 cctggaagtt gccactccag tgcccaccag ccttgtccta ataaaattaa gttgcatcat
     3901 tttgtctgac taggtgtcct tctataatat tatggggtgg aggggggtgg tatggagcaa
     3961 ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc tattgggaac caagctggag
     4021 tgcagtggca caatcttggc tcactgcaat ctccgcctcc tgggttcaag cgattctcct
     4081 gcctcagcct cccgagttgt tgggattcca ggcatgcatg accaggctca gctaattttt
     4141 gtttttttgg tagagacggg gtttcaccat attggccagg ctggtctcca actcctaatc
     4201 tcaggtgatc tacccacctt ggcctcccaa attgctggga ttacaggcgt gaaccactgc
     4261 tcccttccct gtccttctga ttttgtaggt aaccacgtgc ggaccgagcg gccgcaggaa
     4321 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
     4381 cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg
     4441 cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     4501 tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg
     4561 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc
     4621 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     4681 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     4741 ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     4801 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     4861 actctatctc gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt
     4921 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     4981 caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     5041 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     5101 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     5161 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     5221 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     5281 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     5341 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     5401 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     5461 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     5521 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     5581 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     5641 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     5701 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     5761 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     5821 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     5881 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     5941 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     6001 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     6061 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     6121 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     6181 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     6241 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     6301 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     6361 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     6421 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     6481 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     6541 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     6601 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     6661 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     6721 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     6781 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     6841 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     6901 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     6961 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     7021 tcctggcctt ttgctggcct tttgctcaca tgt
//
