LOCUS       sequence-272742-        6950 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intersectional viral expression of Arch3.3-EYFP in cells expressing 
            Flp AND NOT Cre.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6950)
  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 6950)
  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-Arch3.3-p2a-EYFP
FEATURES             Location/Qualifiers
     source          1..6950
                     /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    2509..2542
                     /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    2593..2626
                     /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."
     regulatory      2919..2928
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2935..2968)
                     /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    3077..3665
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3130..3150)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3548..3559)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3697..4173
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3896..3915)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4213..4353
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4213..4342
                     /label=AAV2 ITR
     rep_origin      4428..4883
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4515..4534)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4725..4746
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4900..4919)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5019..5041
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5079..5097)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5165..5269
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5270..5338,5339..6130)
                     /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(5488..5507)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6301..6889
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6790..6809
                     /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 tgctcttgtc ggcggcactg
     1921 acatcggcac cggcactggg ttctggtgcc tcggtgtcgc ccagaatagc ccgggatctc
     1981 aacaggataa agccaaagcc gaccttggca gtcacgtcca acaccataaa cagcagagtt
     2041 tcgatgccca ggcccaccac gccagcgccc tcagtgccta tgatccacag gatagggtaa
     2101 gcggtccaca gcaccaagac cagagctgtc agggtgttaa aggtagatgc cacctcgggg
     2161 ccccgctcct ttgcagcaga tcgcagggat gtagccagaa aatagagcac cactatcatg
     2221 caaattgtag agaacaacca ccaactgtat ctggctatgg ccgtgtggct caaggctccg
     2281 atgaggccag tgacgatcat cagggcgtcc acacccacca gggtgccgat ggtcacccga
     2341 tccaccttag cgagaagggc cagatccagc agcagaagtg gggtggtaaa cagccagtcg
     2401 gcgtacctaa acacatgaga aatagagtca taagcacatg ggtcacatac aggagattgt
     2461 atataacatt aatacaattt tataaaaaaa agggggggca caggtaccat aacttcgtat
     2521 aaagtatcct atacgaagtt atttgcctta acccagaaat tatcactgtt attctttaga
     2581 atggtgcaaa gaataacttc gtataaggta tactatacga agttatgcta gcaaccccga
     2641 cacttacctg gcataataga tatccaacat ttcgcccccg acggtcacct cagtaagccc
     2701 gataccaaag aacatagaca gatatgcggc ggatgcgatt ccgggcacca ggatagtcac
     2761 agcgtaatat tcccgggcat ccttatcggt gactccccat ccgcggacca gaaagtagaa
     2821 ggttccaatc agcatcagca gagtgccgat gcccagccac agagtttcag gtctgccgtc
     2881 acccagcagg tcgtaaccag cctgcagagc gatggggtcc atggtggcga gctcgaagtt
     2941 cctatactat ttgaagaata ggaacttctt gccttaaccc agaaattatc actgttattc
     3001 tttagaatgg tgcaaagaga agttcctata ccttttgaag aataggaact tcgaattcga
     3061 tatcaagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat
     3121 tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca
     3181 tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc
     3241 tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc
     3301 tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt
     3361 cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg
     3421 gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc
     3481 ctttccttgg ctgctcgcct atgttgccac ctggattctg cgcgggacgt ccttctgcta
     3541 cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg
     3601 gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc
     3661 cccgcatcga taccgagcgc tgctcgagag atctacgggt ggcatccctg tgacccctcc
     3721 ccagtgcctc tcctggccct ggaagttgcc actccagtgc ccaccagcct tgtcctaata
     3781 aaattaagtt gcatcatttt gtctgactag gtgtccttct ataatattat ggggtggagg
     3841 ggggtggtat ggagcaaggg gcaagttggg aagacaacct gtagggcctg cggggtctat
     3901 tgggaaccaa gctggagtgc agtggcacaa tcttggctca ctgcaatctc cgcctcctgg
     3961 gttcaagcga ttctcctgcc tcagcctccc gagttgttgg gattccaggc atgcatgacc
     4021 aggctcagct aatttttgtt tttttggtag agacggggtt tcaccatatt ggccaggctg
     4081 gtctccaact cctaatctca ggtgatctac ccaccttggc ctcccaaatt gctgggatta
     4141 caggcgtgaa ccactgctcc cttccctgtc cttctgattt tgtaggtaac cacgtgcgga
     4201 ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct
     4261 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct
     4321 cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta
     4381 cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag
     4441 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     4501 cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     4561 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     4621 cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata
     4681 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     4741 aactggaaca acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc
     4801 gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     4861 caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc
     4921 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     4981 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     5041 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
     5101 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     5161 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     5221 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     5281 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     5341 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     5401 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     5461 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     5521 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     5581 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     5641 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     5701 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     5761 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     5821 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     5881 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     5941 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     6001 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     6061 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     6121 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     6181 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     6241 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     6301 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     6361 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     6421 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     6481 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     6541 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     6601 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     6661 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     6721 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     6781 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     6841 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     6901 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
