LOCUS       sequence-272761-        7017 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 7017)
  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 7017)
  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..7017
                     /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(826..1077)
                     /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(868..889)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1217..1236
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    complement(1458..1491)
                     /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(1542..1575)
                     /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     1688..1709
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    2341..2374
                     /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    2425..2458
                     /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      2986..2995
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3002..3035)
                     /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    3144..3732
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3197..3217)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3615..3626)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3764..4240
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3963..3982)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4280..4420
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4280..4409
                     /label=AAV2 ITR
     rep_origin      4495..4950
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4582..4601)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4792..4813
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4967..4986)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5086..5108
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5146..5164)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5232..5336
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5337..5405,5406..6197)
                     /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(5555..5574)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6368..6956
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6857..6876
                     /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 ccttacttgt acagctcgtc
      841 catgccgaga gtgatcccgg cggcggtcac gaactccagc aggaccatgt gatcgcgctt
      901 ctcgttgggg tctttgctca gggcggactg gtagctcagg tagtggttgt cgggcagcag
      961 cacggggccg tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct gcacgctgcc
     1021 gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg ccgttcttct gcttgtcggc
     1081 catgatatag acgttgtggc tgttgtagtt gtactccagc ttgtgcccca ggatgttgcc
     1141 gtcctccttg aagtcgatgc ccttcagctc gatgcggttc accagggtgt cgccctcgaa
     1201 cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg tgcgctcctg
     1261 gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt ggtcggggta
     1321 gcgggcgaag cactgcaggc cgtagccgaa ggtggtcacg agggtgggcc agggcacggg
     1381 cagctgtggg acgacatgac ttaaccagtt aaccatgttc tctgaacatg agaagggctg
     1441 gccactctcc agtcgacata acttcgtata ggatacttta tacgaagtta tgcagaatgg
     1501 tagctggatt gtagctgcta ttagcaatat gaaacctctt aataacttcg tatagtatac
     1561 cttatacgaa gttatactag tcctcctgta ctcaccttgc cggtggtgca gatgaacttc
     1621 agggtcagct tgccgtaggt ggcatcgccc tcgccctcgc cggacacgct gaacttgtgg
     1681 ccgtttacgt cgccgtccag ctcgaccagg atgggcacca ccccggtgaa cagctcctcg
     1741 cccttgctca ccacgttgat gtcgatctgg tccaggggga tgtactcgcc ctcgctggtg
     1801 atcctgctct tggcggccgc gaccgcgcca gcctcggcct cgtcctccac cagcgtctcg
     1861 acctcaatct cagtgccacc aatgttcaat ttggtggtct tgcgaatgtc gccgtggatg
     1921 aggatatggc tgtggatcag cacgcgcagg tagtggccga gcagacccca gcactgcttc
     1981 gacatcaggt caatgatggt gtggccgacg ttggagccgt acctgctcag gacgccgaag
     2041 ccctcggggc cgaggatgaa caggatgggg aacatacccc atgatacgaa gaagagccaa
     2101 gccatgccag tcaccacctg gcgacaccgg cccttcggca cggtgtggta cgcctcaatg
     2161 tagaccttgg ctgcgttgaa gaatgtgtag atgccgtagc acaggcccat caggaagaaa
     2221 atgacacgga cgtatcccta aacacatgag aaatagagtc ataagcacat gggtcacata
     2281 caggagatat gtatataaca ttaatacaat tttataaaaa aaaggggggg cacaggtacc
     2341 ataacttcgt ataaagtatc ctatacgaag ttatttgcct taacccagaa attatcactg
     2401 ttattcttta gaatggtgca aagaataact tcgtataagg tatactatac gaagttatgc
     2461 tagcaacccc gacacttacc ttggacagcg cggccgtggt gccccacacg atcgtgccga
     2521 tatctgacac cagcagaccc atggtacgct tgttatagtc gttcgccaga cccgtaaggt
     2581 tgctcaggtg gataagaatg acagggcagg tcagcagcca gctcgcgtaa cgaagccacc
     2641 tggtcttgtt gccgttggat gagtagatca ccgcaggttc gtcaaagcta tggaaatact
     2701 cgatgatgaa cttgatcatc tggatcgtgg ccacgtaaat attctcccag ccgcaagtag
     2761 acttccaggt ctggtagccg tagaacatca ggcagagcgc tgaaagcgca aaagaaatcc
     2821 actgcagaat gttggcagcc aacttctcgg catttgttcc gttggattta agccaagcca
     2881 agcagaagca ctggccgttg ttcgggatgc aaataacaga accattgttc tcaagagtgt
     2941 atgaggtttg gaagagtccg acggcagaca aagcgccgcc atagtccatg gtggcgagct
     3001 cgaagttcct atactatttg aagaatagga acttcttgcc ttaacccaga aattatcact
     3061 gttattcttt agaatggtgc aaagagaagt tcctatacct tttgaagaat aggaacttcg
     3121 aattcgatat caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga
     3181 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     3241 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     3301 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     3361 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     3421 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     3481 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
     3541 catcgtcctt tccttggctg ctcgcctatg ttgccacctg gattctgcgc gggacgtcct
     3601 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     3661 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
     3721 ccgcctcccc gcatcgatac cgagcgctgc tcgagagatc tacgggtggc atccctgtga
     3781 cccctcccca gtgcctctcc tggccctgga agttgccact ccagtgccca ccagccttgt
     3841 cctaataaaa ttaagttgca tcattttgtc tgactaggtg tccttctata atattatggg
     3901 gtggaggggg gtggtatgga gcaaggggca agttgggaag acaacctgta gggcctgcgg
     3961 ggtctattgg gaaccaagct ggagtgcagt ggcacaatct tggctcactg caatctccgc
     4021 ctcctgggtt caagcgattc tcctgcctca gcctcccgag ttgttgggat tccaggcatg
     4081 catgaccagg ctcagctaat ttttgttttt ttggtagaga cggggtttca ccatattggc
     4141 caggctggtc tccaactcct aatctcaggt gatctaccca ccttggcctc ccaaattgct
     4201 gggattacag gcgtgaacca ctgctccctt ccctgtcctt ctgattttgt aggtaaccac
     4261 gtgcggaccg agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc
     4321 gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg
     4381 gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt
     4441 ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc
     4501 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     4561 ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     4621 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     4681 tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc
     4741 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     4801 tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga
     4861 ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     4921 attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg
     4981 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     5041 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     5101 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     5161 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     5221 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     5281 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     5341 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     5401 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     5461 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     5521 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     5581 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     5641 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     5701 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     5761 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     5821 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     5881 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     5941 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     6001 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     6061 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     6121 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     6181 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     6241 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     6301 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     6361 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     6421 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     6481 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     6541 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     6601 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     6661 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     6721 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     6781 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     6841 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     6901 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     6961 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt
//
