LOCUS       sequence-265120-        7028 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intersectional viral expression of iC++-EYFP in cells expressing Flp
            AND NOT Cre.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7028)
  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 7028)
  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-iC++-EYFP
FEATURES             Location/Qualifiers
     source          1..7028
                     /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(837..1088)
                     /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(879..900)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1228..1247
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    complement(1469..1502)
                     /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(1553..1586)
                     /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     1699..1720
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    2352..2385
                     /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    2436..2469
                     /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      2997..3006
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3013..3046)
                     /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    3155..3743
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3208..3228)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3626..3637)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3775..4251
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3974..3993)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4291..4431
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4291..4420
                     /label=AAV2 ITR
     rep_origin      4506..4961
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4593..4612)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4803..4824
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4978..4997)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5097..5119
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5157..5175)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5243..5347
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5348..5416,5417..6208)
                     /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(5566..5585)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6379..6967
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6868..6887
                     /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 gccttacttg
      841 tacagctcgt ccatgccgag agtgatcccg gcggcggtca cgaactccag caggaccatg
      901 tgatcgcgct tctcgttggg gtctttgctc agggcggact ggtagctcag gtagtggttg
      961 tcgggcagca gcacggggcc gtcgccgatg ggggtgttct gctggtagtg gtcggcgagc
     1021 tgcacgctgc cgtcctcgat gttgtggcgg atcttgaagt tcaccttgat gccgttcttc
     1081 tgcttgtcgg ccatgatata gacgttgtgg ctgttgtagt tgtactccag cttgtgcccc
     1141 aggatgttgc cgtcctcctt gaagtcgatg cccttcagct cgatgcggtt caccagggtg
     1201 tcgccctcga acttcacctc ggcgcgggtc ttgtagttgc cgtcgtcctt gaagaagatg
     1261 gtgcgctcct ggacgtagcc ttcgggcatg gcggacttga agaagtcgtg ctgcttcatg
     1321 tggtcggggt agcgggcgaa gcactgcagg ccgtagccga aggtggtcac gagggtgggc
     1381 cagggcacgg gcagctgtgg gacgacatga cttaaccagt taaccatgtt ctctgaacat
     1441 gagaagggct ggccactctc cagtcgacat aacttcgtat aggatacttt atacgaagtt
     1501 atgcagaatg gtagctggat tgtagctgct attagcaata tgaaacctct taataacttc
     1561 gtatagtata ccttatacga agttatacta gtcctcctgt actcaccttg ccggtggtgc
     1621 agatgaactt cagggtcagc ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc
     1681 tgaacttgtg gccgtttacg tcgccgtcca gctcgaccag gatgggcacc accccggtga
     1741 acagctcctc gcccttgctc accacgttga tgtcgatctg gtccaggggg atgtactcgc
     1801 cctcgctggt gatcctgctc ttggcggccg cgaccgcgcc agcctcggcc tcgtcctcca
     1861 ccagcgtctc gacctcaatc tcagtgccac caatgttcaa tttggtggtc ttgcgaatgt
     1921 cgccgtggat gaggatatgg ctgtggatca gcacgcgcag gtagtggccg agcagacccc
     1981 agcactgctt cgacatcagg tcaatgatgg tgtggccgac gttggagccg tacctgctca
     2041 ggacgccgaa gccctcgggg ccgaggatga acaggatggg gaacataccc catgatacga
     2101 agaagagcca agccatgcca gtcaccacct ggcgacaccg gcccttcggc acggtgtggt
     2161 acgcctcaat gtagaccttg gctgcgttga agaatgtgta gatgccgtag cacaggccca
     2221 tcaggaagaa aatgacacgg acgtatccct aaacacatga gaaatagagt cataagcaca
     2281 tgggtcacat acaggagata tgtatataac attaatacaa ttttataaaa aaaagggggg
     2341 gcacaggtac cataacttcg tataaagtat cctatacgaa gttatttgcc ttaacccaga
     2401 aattatcact gttattcttt agaatggtgc aaagaataac ttcgtataag gtatactata
     2461 cgaagttatg ctagcaaccc cgacacttac cttggacagc gcggccgtgg tgccccacac
     2521 gatcgtgccg atatctgaca ccagcagacc catggtacgc ttgttatagt cgttcgccag
     2581 acccgtaagg ttgctcaggt ggataagaat gacagggcag gtcagcagcc agctcgcgta
     2641 acgaagccac ctggtcttgt tgccgttgga tgagtagatc accgcaggtt cgtcaaagct
     2701 atggaaatac tcgatgatga acttgatcat ctggatcgtg gccacgtaaa tattctccca
     2761 gccgcaagta gacttccagg tctggtagcc gtagaacatc aggcagagcg ctgaaagcgc
     2821 aaaagaaatc cactgcagaa tgttggcagc caacttctcg gcatttgttc cgttggattt
     2881 aagccaagcc aagcagaagc actggccgtt gttcgggatg caaataacag aaccattgtt
     2941 ctcaagagtg tatgaggttt ggaagagtcc gacggcagac aaagcgccgc catagtccat
     3001 ggtggcgagc tcgaagttcc tatactattt gaagaatagg aacttcttgc cttaacccag
     3061 aaattatcac tgttattctt tagaatggtg caaagagaag ttcctatacc ttttgaagaa
     3121 taggaacttc gaattcgata tcaagcttat cgataatcaa cctctggatt acaaaatttg
     3181 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     3241 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta
     3301 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt
     3361 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca
     3421 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc
     3481 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt
     3541 gtcggggaaa tcatcgtcct ttccttggct gctcgcctat gttgccacct ggattctgcg
     3601 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg
     3661 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat
     3721 ctccctttgg gccgcctccc cgcatcgata ccgagcgctg ctcgagagat ctacgggtgg
     3781 catccctgtg acccctcccc agtgcctctc ctggccctgg aagttgccac tccagtgccc
     3841 accagccttg tcctaataaa attaagttgc atcattttgt ctgactaggt gtccttctat
     3901 aatattatgg ggtggagggg ggtggtatgg agcaaggggc aagttgggaa gacaacctgt
     3961 agggcctgcg gggtctattg ggaaccaagc tggagtgcag tggcacaatc ttggctcact
     4021 gcaatctccg cctcctgggt tcaagcgatt ctcctgcctc agcctcccga gttgttggga
     4081 ttccaggcat gcatgaccag gctcagctaa tttttgtttt tttggtagag acggggtttc
     4141 accatattgg ccaggctggt ctccaactcc taatctcagg tgatctaccc accttggcct
     4201 cccaaattgc tgggattaca ggcgtgaacc actgctccct tccctgtcct tctgattttg
     4261 taggtaacca cgtgcggacc gagcggccgc aggaacccct agtgatggag ttggccactc
     4321 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg
     4381 gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga
     4441 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc
     4501 atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     4561 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     4621 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     4681 atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag
     4741 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     4801 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcgggct attcttttga
     4861 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     4921 atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac
     4981 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc
     5041 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     5101 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct
     5161 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     5221 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     5281 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     5341 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     5401 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     5461 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     5521 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     5581 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     5641 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     5701 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     5761 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     5821 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     5881 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     5941 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     6001 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     6061 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     6121 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     6181 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     6241 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     6301 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     6361 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     6421 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     6481 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     6541 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     6601 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     6661 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     6721 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     6781 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     6841 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     6901 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     6961 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     7021 tcacatgt
//
