LOCUS       sequence-272783-        6625 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intersectional viral expression of EYFP in cells expressing Cre AND 
            NOT Flp.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6625)
  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 6625)
  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-Ef1a-Con/Foff 2.0-EYFP
FEATURES             Location/Qualifiers
     source          1..6625
                     /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        231..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          462..1400
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1357..1377
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1416..1449)
                     /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(1500..1533)
                     /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     1646..1667
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1709..1718
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1725..1758
                     /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    1809..1842
                     /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    1849..1882
                     /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)"
     primer_bind     complement(2190..2209)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2349..2600
                     /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     2537..2558
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2610..2643)
                     /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    2752..3340
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2805..2825)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3223..3234)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3372..3848
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3571..3590)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3888..4028
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3888..4017
                     /label=AAV2 ITR
     rep_origin      4103..4558
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4190..4209)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4400..4421
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4575..4594)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4694..4716
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4754..4772)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4840..4944
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4945..5013,5014..5805)
                     /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(5163..5182)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5976..6564
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6465..6484
                     /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 gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgag gatccataac ttcgtatagg atactttata
     1441 cgaagttatg tagctggagc aatttgaaaa gattgttgta ttacctctag ctgcgcttaa
     1501 taacttcgta tagtatacct tatacgaagt tatgtcgacc ctcctgtact caccttgccg
     1561 gtggtgcaga tgaacttcag ggtcagcttg ccgtaggtgg catcgccctc gccctcgccg
     1621 gacacgctga acttgtggcc gtttacgtcg ccgtccagct cgaccaggat gggcaccacc
     1681 ccggtgaaca gctcctcgcc cttgctcacc atggtggcac tagtataact tcgtataaag
     1741 tatcctatac gaagttattt gccttaaccc agaaattatc actgttattc tttagatagg
     1801 tgcaaagaat aacttcgtat aaggtatact atacgaagtt atggtaccga agttcctatt
     1861 ctctagaaag tataggaact tcgcagaatg gtagctggat tgtagctgct attagcaata
     1921 tgaaacctct tagaagttcc tattcttcaa aaggtatagg aacttcggcg cgcctggaga
     1981 gtggccagcc cttctcatgt tcagagaaca tggttaactg gttaagtcat gtcgtcccac
     2041 agctgcccgt gccctggccc accctcgtga ccaccttcgg ctacggcctg cagtgcttcg
     2101 cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     2161 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     2221 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     2281 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     2341 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     2401 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     2461 ccgtgctgct gcccgacaac cactacctga gctaccagtc cgccctgagc aaagacccca
     2521 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     2581 gcatggacga gctgtacaag taagctagcg aagttcctat actttctaga gaataggaac
     2641 ttctaagcag acaatcttgt aatacatcag cagctagtag attgctccag ctagaagttc
     2701 ctataccttt tgaagaatag gaacttcgaa ttcgatatca agcttatcga taatcaacct
     2761 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     2821 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
     2881 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
     2941 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
     3001 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
     3061 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
     3121 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctatgtt
     3181 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
     3241 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
     3301 cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg agcgctgctc
     3361 gagagatcta cgggtggcat ccctgtgacc cctccccagt gcctctcctg gccctggaag
     3421 ttgccactcc agtgcccacc agccttgtcc taataaaatt aagttgcatc attttgtctg
     3481 actaggtgtc cttctataat attatggggt ggaggggggt ggtatggagc aaggggcaag
     3541 ttgggaagac aacctgtagg gcctgcgggg tctattggga accaagctgg agtgcagtgg
     3601 cacaatcttg gctcactgca atctccgcct cctgggttca agcgattctc ctgcctcagc
     3661 ctcccgagtt gttgggattc caggcatgca tgaccaggct cagctaattt ttgttttttt
     3721 ggtagagacg gggtttcacc atattggcca ggctggtctc caactcctaa tctcaggtga
     3781 tctacccacc ttggcctccc aaattgctgg gattacaggc gtgaaccact gctcccttcc
     3841 ctgtccttct gattttgtag gtaaccacgt gcggaccgag cggccgcagg aacccctagt
     3901 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     3961 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     4021 cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     4081 catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     4141 gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct
     4201 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     4261 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg
     4321 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     4381 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc
     4441 tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat
     4501 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta
     4561 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     4621 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     4681 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     4741 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     4801 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     4861 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     4921 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     4981 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     5041 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     5101 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     5161 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     5221 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     5281 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     5341 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     5401 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     5461 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     5521 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     5581 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     5641 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     5701 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     5761 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     5821 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     5881 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     5941 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     6001 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     6061 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     6121 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     6181 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     6241 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     6301 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     6361 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     6421 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     6481 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     6541 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     6601 ttttgctggc cttttgctca catgt
//
