LOCUS       sequence-265106-        6977 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intersectional viral expression of ChR2(ET/TC)-EYFP in cells 
            expressing Cre AND NOT Flp.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6977)
  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 6977)
  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-ChR2(ET/TC)-EYFP
FEATURES             Location/Qualifiers
     source          1..6977
                     /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=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      834..843
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(1279..1312)
                     /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(1363..1396)
                     /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)."
     regulatory      1572..1581
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2203..2236
                     /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    2287..2320
                     /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(2542..2561)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2701..2952
                     /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     2889..2910
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2962..2995)
                     /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    3104..3692
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3157..3177)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3575..3586)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3724..4200
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3923..3942)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4240..4380
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4240..4369
                     /label=AAV2 ITR
     rep_origin      4455..4910
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4542..4561)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4752..4773
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4927..4946)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5046..5068
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5106..5124)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5192..5296
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5297..5365,5366..6157)
                     /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(5515..5534)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6328..6916
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6817..6836
                     /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 tctagaaagt ataggaactt cgcagagtag tagctggatt gtagctgcta ttagcaatct
      781 aaaacctctt agaagttcct attcttcaaa aggtatagga acttcggcgc gccgccacca
      841 tggactatgg cggcgctttg tctgccgtcg gacgcgaact tttgttcgtt actaatcctg
      901 tggtggtgaa cgggtccgtc ctggtccctg aggatcaatg ttactgtgcc ggatggattg
      961 aatctcgcgg cacgaacggc gctcagaccg cgtcaaatgt cctgcagtgg cttgcagcag
     1021 gattcagcat tttgctgctg atgttctatg cctaccaaac ctggaaatct acatgcggct
     1081 gggaggagat ctatgtgtgc gccattgaaa tggttaaggt gattctcgag ttcttttttg
     1141 agtttaagaa tccctctatg ctctaccttg ccacaggaca ccgggtgcag tggctgcgct
     1201 atgcaacctg gctgctcact tgtcctgtca tccttatcca cctgagcaac ctcaccggta
     1261 agtgtcgggg ttgagctcat aacttcgtat agtatacctt atacgaagtt attctttgca
     1321 ccattctaaa gaataacagt gataatttct gggttaaggc aaataacttc gtataggata
     1381 ctttatacga agttatggta cccctcctgt actcaccttg ccggtggtgc agatgaactt
     1441 cagggtcagc ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg
     1501 gccgtttacg tcaccgtcca gctcgaccag gatgggcacc accccggtga acagctcctc
     1561 gcccttgctc accatggtgg cggccgctgg cacggctccg gcctcggctt cgtcttcgac
     1621 gagagtctcg acctcgatct ccgttccgcc gatgttcagt ttggtggttt tgcggatatc
     1681 tccgtgaatc aatatgtgct cgtggatcag gacgcgcagg tagtgtccca acaaccccca
     1741 acaattttta ctcatcagat caataatcgt gtgacctacg gtggagccat agacgctcag
     1801 gacgccaaaa ccttcgggcc ccaaaatgaa gagaattggg aacatacccc agctcacgaa
     1861 aaacagccat gccatgccgg tcacgacctg gcggcaccga ccctttggca cagtatgata
     1921 accctcgata tatgctttgg cggcgtgaaa aaatgtgttc gcgccatagc acaatccaag
     1981 acaaaagaag atgactttaa catagccggt tgccatggcg ctggtagccc cccacacgat
     2041 acacccgatg tctgagacaa ggagtcccat ggttctcctg ctgtagtcgt tgctcaggcc
     2101 taaacacatg agaaatagag tcataagcac atgggtcaca tacaggagat atgtatataa
     2161 cattaataca attttataaa aaaaaggggg ggcacaacta gtataacttc gtataaggta
     2221 tactatacga agttattaag aggtttcata ttgctaatag cagctacaat ccagctacca
     2281 ttctgcataa cttcgtataa agtatcctat acgaagttat gtcgactgga gagtggccag
     2341 cccttctcat gttcagagaa catggttaac tggttaagtc atgtcgtccc acagctgccc
     2401 gtgccctggc ccaccctcgt gaccaccttc ggctacggcc tgcagtgctt cgcccgctac
     2461 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     2521 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     2581 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     2641 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     2701 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     2761 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     2821 ctgcccgaca accactacct gagctaccag tccgccctga gcaaagaccc caacgagaag
     2881 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     2941 gagctgtaca agtaagctag cgaagttcct atactttcta gagaatagga acttctaagc
     3001 agacaatctt gtaatacatc agcagctagt agattgctcc agctagaagt tcctatacct
     3061 tttgaagaat aggaacttcg aattcgatat caagcttatc gataatcaac ctctggatta
     3121 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     3181 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     3241 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     3301 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     3361 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     3421 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     3481 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg
     3541 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     3601 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     3661 gagtcggatc tccctttggg ccgcctcccc gcatcgatac cgagcgctgc tcgagagatc
     3721 tacgggtggc atccctgtga cccctcccca gtgcctctcc tggccctgga agttgccact
     3781 ccagtgccca ccagccttgt cctaataaaa ttaagttgca tcattttgtc tgactaggtg
     3841 tccttctata atattatggg gtggaggggg gtggtatgga gcaaggggca agttgggaag
     3901 acaacctgta gggcctgcgg ggtctattgg gaaccaagct ggagtgcagt ggcacaatct
     3961 tggctcactg caatctccgc ctcctgggtt caagcgattc tcctgcctca gcctcccgag
     4021 ttgttgggat tccaggcatg catgaccagg ctcagctaat ttttgttttt ttggtagaga
     4081 cggggtttca ccatattggc caggctggtc tccaactcct aatctcaggt gatctaccca
     4141 ccttggcctc ccaaattgct gggattacag gcgtgaacca ctgctccctt ccctgtcctt
     4201 ctgattttgt aggtaaccac gtgcggaccg agcggccgca ggaaccccta gtgatggagt
     4261 tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc
     4321 gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg
     4381 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc
     4441 aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     4501 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     4561 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     4621 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg
     4681 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     4741 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta
     4801 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     4861 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac
     4921 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     4981 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     5041 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     5101 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     5161 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     5221 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     5281 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     5341 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     5401 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     5461 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     5521 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     5581 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     5641 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     5701 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     5761 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     5821 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     5881 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     5941 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     6001 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     6061 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     6121 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     6181 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     6241 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     6301 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     6361 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     6421 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     6481 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     6541 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     6601 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     6661 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     6721 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     6781 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     6841 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     6901 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     6961 gccttttgct cacatgt
//
