LOCUS       sequence-271952-        7017 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intersectional viral expression of ic++-EYFP in cells expressing Cre
            AND NOT Flp.
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-Con/Foff 2.0-iC++-EYFP
FEATURES             Location/Qualifiers
     source          1..7017
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        47..258
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             271..539
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     protein_bind    567..600
                     /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)"
     regulatory      693..702
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(1230..1263)
                     /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    complement(1314..1347)
                     /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)."
     primer_bind     1460..1481
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    2113..2146
                     /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    2197..2230
                     /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)."
     primer_bind     complement(2452..2471)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2611..2862
                     /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     2799..2820
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2872..2905)
                     /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    3014..3602
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3067..3087)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3485..3496)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3634..4110
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3833..3852)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4150..4290
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4150..4279
                     /label=AAV2 ITR
     rep_origin      4365..4820
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4452..4471)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4662..4683
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4837..4856)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4956..4978
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5016..5034)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5102..5206
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5207..5275,5276..6067)
                     /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(5425..5444)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6238..6826
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6727..6746
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   6888..7017
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 gcggccgcac gcgtaaggat ctgcgatcgc tccggtgccc gtcagtgggc agagcgcaca
       61 tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaacggg tgcctagaga
      121 aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag
      181 ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg
      241 tttgccgcca gaacacagct gaagcttcga ggggctcgca tctctccttc acgcgcccgc
      301 cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt
      361 ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc gagaccgggc
      421 ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg
      481 accctgcttg ctcaactcta cgtctttgtt tcgttttctg ttctgcgccg ttacagatcc
      541 aagctgtgac cggcgcctac ggatccgaag ttcctattct ctagaaagta taggaacttc
      601 gcagagtagt agctggattg tagctgctat tagcaatcta aaacctctta gaagttccta
      661 ttcttcaaaa ggtataggaa cttcggcgcg ccgccaccat ggactatggc ggcgctttgt
      721 ctgccgtcgg actcttccaa acctcataca ctcttgagaa caatggttct gttatttgca
      781 tcccgaacaa cggccagtgc ttctgcttgg cttggcttaa atccaacgga acaaatgccg
      841 agaagttggc tgccaacatt ctgcagtgga tttcttttgc gctttcagcg ctctgcctga
      901 tgttctacgg ctaccagacc tggaagtcta cttgcggctg ggagaatatt tacgtggcca
      961 cgatccagat gatcaagttc atcatcgagt atttccatag ctttgacgaa cctgcggtga
     1021 tctactcatc caacggcaac aagaccaggt ggcttcgtta cgcgagctgg ctgctgacct
     1081 gccctgtcat tcttatccac ctgagcaacc ttacgggtct ggcgaacgac tataacaagc
     1141 gtaccatggg tctgctggtg tcagatatcg gcacgatcgt gtggggcacc acggccgcgc
     1201 tgtccaaggt aagtgtcggg gttgagctca taacttcgta tagtatacct tatacgaagt
     1261 tattctttgc accattctaa agaataacag tgataatttc tgggttaagg caaataactt
     1321 cgtataggat actttatacg aagttatggt acccctcctg tactcacctt gccggtggtg
     1381 cagatgaact tcagggtcag cttgccgtag gtggcatcgc cctcgccctc gccggacacg
     1441 ctgaacttgt ggccgtttac gtcgccgtcc agctcgacca ggatgggcac caccccggtg
     1501 aacagctcct cgcccttgct caccacgttg atgtcgatct ggtccagggg gatgtactcg
     1561 ccctcgctgg tgatcctgct cttggcggcc gcgaccgcgc cagcctcggc ctcgtcctcc
     1621 accagcgtct cgacctcaat ctcagtgcca ccaatgttca atttggtggt cttgcgaatg
     1681 tcgccgtgga tgaggatatg gctgtggatc agcacgcgca ggtagtggcc gagcagaccc
     1741 cagcactgct tcgacatcag gtcaatgatg gtgtggccga cgttggagcc gtacctgctc
     1801 aggacgccga agccctcggg gccgaggatg aacaggatgg ggaacatacc ccatgatacg
     1861 aagaagagcc aagccatgcc agtcaccacc tggcgacacc ggcccttcgg cacggtgtgg
     1921 tacgcctcaa tgtagacctt ggctgcgttg aagaatgtgt agatgccgta gcacaggccc
     1981 atcaggaaga aaatgacacg gacgtatccc taaacacatg agaaatagag tcataagcac
     2041 atgggtcaca tacaggagat atgtatataa cattaataca attttataaa aaaaaggggg
     2101 ggcacaacta gtataacttc gtataaggta tactatacga agttattaag aggtttcata
     2161 ttgctaatag cagctacaat ccagctacca ttctgcataa cttcgtataa agtatcctat
     2221 acgaagttat gtcgactgga gagtggccag cccttctcat gttcagagaa catggttaac
     2281 tggttaagtc atgtcgtccc acagctgccc gtgccctggc ccaccctcgt gaccaccttc
     2341 ggctacggcc tgcagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc
     2401 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     2461 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     2521 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     2581 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     2641 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     2701 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagctaccag
     2761 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     2821 accgccgccg ggatcactct cggcatggac gagctgtaca agtaagctag cgaagttcct
     2881 atactttcta gagaatagga acttctaagc agacaatctt gtaatacatc agcagctagt
     2941 agattgctcc agctagaagt tcctatacct tttgaagaat aggaacttcg aattcgatat
     3001 caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct
     3061 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
     3121 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
     3181 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga
     3241 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
     3301 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
     3361 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt
     3421 tccttggctg ctcgcctatg ttgccacctg gattctgcgc gggacgtcct tctgctacgt
     3481 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
     3541 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc
     3601 gcatcgatac cgagcgctgc tcgagagatc tacgggtggc atccctgtga cccctcccca
     3661 gtgcctctcc tggccctgga agttgccact ccagtgccca ccagccttgt cctaataaaa
     3721 ttaagttgca tcattttgtc tgactaggtg tccttctata atattatggg gtggaggggg
     3781 gtggtatgga gcaaggggca agttgggaag acaacctgta gggcctgcgg ggtctattgg
     3841 gaaccaagct ggagtgcagt ggcacaatct tggctcactg caatctccgc ctcctgggtt
     3901 caagcgattc tcctgcctca gcctcccgag ttgttgggat tccaggcatg catgaccagg
     3961 ctcagctaat ttttgttttt ttggtagaga cggggtttca ccatattggc caggctggtc
     4021 tccaactcct aatctcaggt gatctaccca ccttggcctc ccaaattgct gggattacag
     4081 gcgtgaacca ctgctccctt ccctgtcctt ctgattttgt aggtaaccac gtgcggaccg
     4141 agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc
     4201 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
     4261 tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc
     4321 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg
     4381 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     4441 cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     4501 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     4561 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
     4621 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     4681 tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat
     4741 ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     4801 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata
     4861 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     4921 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     4981 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     5041 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     5101 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     5161 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     5221 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     5281 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     5341 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     5401 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     5461 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     5521 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     5581 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     5641 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     5701 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     5761 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     5821 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     5881 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     5941 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     6001 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     6061 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     6121 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     6181 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     6241 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     6301 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     6361 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     6421 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     6481 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     6541 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     6601 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     6661 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     6721 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     6781 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     6841 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgtcct gcaggcagct
     6901 gcgcgctcgc tcgctcactg aggccgcccg ggcgtcgggc gacctttggt cgcccggcct
     6961 cagtgagcga gcgagcgcgc agagagggag tggccaactc catcactagg ggttcct
//
