LOCUS       sequence-265113-        6961 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 6961)
  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 6961)
  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..6961
                     /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    2520..2553
                     /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    2604..2637
                     /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      2930..2939
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2946..2979)
                     /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    3088..3676
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3141..3161)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3559..3570)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3708..4184
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3907..3926)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4224..4364
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4224..4353
                     /label=AAV2 ITR
     rep_origin      4439..4894
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4526..4545)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4736..4757
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4911..4930)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5030..5052
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5090..5108)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5176..5280
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5281..5349,5350..6141)
                     /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(5499..5518)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6312..6900
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6801..6820
                     /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 ctgctcttgt
     1921 cggcggcact gacatcggca ccggcactgg gttctggtgc ctcggtgtcg cccagaatag
     1981 cccgggatct caacaggata aagccaaagc cgaccttggc agtcacgtcc aacaccataa
     2041 acagcagagt ttcgatgccc aggcccacca cgccagcgcc ctcagtgcct atgatccaca
     2101 ggatagggta agcggtccac agcaccaaga ccagagctgt cagggtgtta aaggtagatg
     2161 ccacctcggg gccccgctcc tttgcagcag atcgcaggga tgtagccaga aaatagagca
     2221 ccactatcat gcaaattgta gagaacaacc accaactgta tctggctatg gccgtgtggc
     2281 tcaaggctcc gatgaggcca gtgacgatca tcagggcgtc cacacccacc agggtgccga
     2341 tggtcacccg atccacctta gcgagaaggg ccagatccag cagcagaagt ggggtggtaa
     2401 acagccagtc ggcgtaccta aacacatgag aaatagagtc ataagcacat gggtcacata
     2461 caggagattg tatataacat taatacaatt ttataaaaaa aagggggggc acaggtacca
     2521 taacttcgta taaagtatcc tatacgaagt tatttgcctt aacccagaaa ttatcactgt
     2581 tattctttag aatggtgcaa agaataactt cgtataaggt atactatacg aagttatgct
     2641 agcaaccccg acacttacct ggcataatag atatccaaca tttcgccccc gacggtcacc
     2701 tcagtaagcc cgataccaaa gaacatagac agatatgcgg cggatgcgat tccgggcacc
     2761 aggatagtca cagcgtaata ttcccgggca tccttatcgg tgactcccca tccgcggacc
     2821 agaaagtaga aggttccaat cagcatcagc agagtgccga tgcccagcca cagagtttca
     2881 ggtctgccgt cacccagcag gtcgtaacca gcctgcagag cgatggggtc catggtggcg
     2941 agctcgaagt tcctatacta tttgaagaat aggaacttct tgccttaacc cagaaattat
     3001 cactgttatt ctttagaatg gtgcaaagag aagttcctat accttttgaa gaataggaac
     3061 ttcgaattcg atatcaagct tatcgataat caacctctgg attacaaaat ttgtgaaaga
     3121 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
     3181 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
     3241 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
     3301 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
     3361 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
     3421 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
     3481 aaatcatcgt cctttccttg gctgctcgcc tatgttgcca cctggattct gcgcgggacg
     3541 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
     3601 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
     3661 tgggccgcct ccccgcatcg ataccgagcg ctgctcgaga gatctacggg tggcatccct
     3721 gtgacccctc cccagtgcct ctcctggccc tggaagttgc cactccagtg cccaccagcc
     3781 ttgtcctaat aaaattaagt tgcatcattt tgtctgacta ggtgtccttc tataatatta
     3841 tggggtggag gggggtggta tggagcaagg ggcaagttgg gaagacaacc tgtagggcct
     3901 gcggggtcta ttgggaacca agctggagtg cagtggcaca atcttggctc actgcaatct
     3961 ccgcctcctg ggttcaagcg attctcctgc ctcagcctcc cgagttgttg ggattccagg
     4021 catgcatgac caggctcagc taatttttgt ttttttggta gagacggggt ttcaccatat
     4081 tggccaggct ggtctccaac tcctaatctc aggtgatcta cccaccttgg cctcccaaat
     4141 tgctgggatt acaggcgtga accactgctc ccttccctgt ccttctgatt ttgtaggtaa
     4201 ccacgtgcgg accgagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct
     4261 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc
     4321 ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta
     4381 ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac
     4441 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     4501 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     4561 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     4621 gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca
     4681 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     4741 ctcttgttcc aaactggaac aacactcaac cctatctcgg gctattcttt tgatttataa
     4801 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     4861 gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc
     4921 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     4981 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     5041 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata
     5101 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     5161 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     5221 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     5281 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     5341 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     5401 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     5461 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     5521 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5581 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5641 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     5701 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     5761 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     5821 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     5881 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     5941 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     6001 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     6061 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     6121 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     6181 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     6241 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     6301 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     6361 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     6421 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     6481 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     6541 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     6601 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6661 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6721 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     6781 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     6841 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     6901 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     6961 t
//
