LOCUS       Exported                6970 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Intersectional viral expression of ChR2-EYFP in cells expressing Cre
            AND NOT Flp.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-nEF-Con/Foff 2.0-ChR2-EYFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6970)
  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 6970)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6970
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    1279..1312
                     /label=loxN
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GTATACCT). loxN sites are compatible with each 
                     other, but incompatible with loxP or lox2272 sites."
     protein_bind    1363..1396
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     primer_bind     1509..1530
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    complement(2196..2229)
                     /label=loxN
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GTATACCT). loxN sites are compatible with each 
                     other, but incompatible with loxP or lox2272 sites."
     protein_bind    complement(2280..2313)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     primer_bind     complement(2535..2554)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2694..2945
                     /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     2882..2903
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2955..2988)
                     /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    3097..3685
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3150..3170)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3568..3579)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3717..4193
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3916..3935)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4233..4373
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4233..4362
                     /label=AAV2 ITR
     rep_origin      4448..4903
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4535..4554)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4745..4766
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4920..4939)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5039..5061
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5099..5117)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5185..5289
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5290..6150
                     /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(5508..5527)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6321..6909
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6810..6829
                     /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 atgcagagtg gctgctcact tgtcctgtca tccttatccg 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 tcgccgtcca gctcgaccag gatgggcacc accccggtga acagctcctc
     1561 gcccttgctc acggcggccg ctggcacggc tccggcctcg gcttcgtctt cgacgagagt
     1621 ctcgacctcg atctccgttc cgccgatgtt cagtttggtg gttttgcgga tatctccgtg
     1681 aatcaatatg tgctcgtgga tcaggacgcg caggtagtgt cccaacaacc cccaacaatt
     1741 tttactcatc agatcaataa tcgtgtgacc tacggtggag ccatagacgc tcaggacgcc
     1801 aaaaccttcg ggccccaaaa tgaagagaat tgggaacata ccccagctca cgaaaaacag
     1861 ccatgccatg ccggtcacga cctggcggca ccgacccttt ggcacagtat gataaccctc
     1921 gatatatgct ttggcggcgt gaaaaaatgt gttcgcgcca tagcacaatc caagacaaaa
     1981 gaagatgact ttaacatagc cggttgccat ggcgctggta gccccccaca cgatagtccc
     2041 gatgtctgag acaaggagtc ccatggttct cctgctgtag tcgttgctca ggcctaaaca
     2101 catgagaaat agagtcataa gcacatgggt cacatacagg agatatgtat ataacattaa
     2161 tacaatttat aaaaaaaagg gggggcacaa ctagtataac ttcgtataag gtatactata
     2221 cgaagttatt aagaggtttc atattgctaa tagcagctac aatccagcta ccattctgca
     2281 taacttcgta taaagtatcc tatacgaagt tatgtcgact ggagagtggc cagcccttct
     2341 catgttcaga gaacatggtt aactggttaa gtcatgtcgt cccacagctg cccgtgccct
     2401 ggcccaccct cgtgaccacc ttcggctacg gcctgcagtg cttcgcccgc taccccgacc
     2461 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     2521 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     2581 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     2641 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     2701 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     2761 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     2821 acaaccacta cctgagctac cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
     2881 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     2941 acaagtaagc tagcgaagtt cctatacttt ctagagaata ggaacttcta agcagacaat
     3001 cttgtaatac atcagcagct agtagattgc tccagctaga agttcctata ccttttgaag
     3061 aataggaact tcgaattcga tatcaagctt atcgataatc aacctctgga ttacaaaatt
     3121 tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct
     3181 gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg
     3241 tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc
     3301 gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt
     3361 cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc
     3421 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg
     3481 ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg
     3541 cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc
     3601 ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg
     3661 atctcccttt gggccgcctc cccgcatcga taccgagcgc tgctcgagag atctacgggt
     3721 ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc actccagtgc
     3781 ccaccagcct tgtcctaata aaattaagtt gcatcatttt gtctgactag gtgtccttct
     3841 ataatattat ggggtggagg ggggtggtat ggagcaaggg gcaagttggg aagacaacct
     3901 gtagggcctg cggggtctat tgggaaccaa gctggagtgc agtggcacaa tcttggctca
     3961 ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc tcagcctccc gagttgttgg
     4021 gattccaggc atgcatgacc aggctcagct aatttttgtt tttttggtag agacggggtt
     4081 tcaccatatt ggccaggctg gtctccaact cctaatctca ggtgatctac ccaccttggc
     4141 ctcccaaatt gctgggatta caggcgtgaa ccactgctcc cttccctgtc cttctgattt
     4201 tgtaggtaac cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg agttggccac
     4261 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     4321 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct
     4381 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa
     4441 ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     4501 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     4561 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     4621 cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt
     4681 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     4741 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggg ctattctttt
     4801 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     4861 aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt
     4921 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     4981 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     5041 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     5101 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     5161 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     5221 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     5281 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     5341 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     5401 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     5461 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     5521 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     5581 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     5641 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     5701 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     5761 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     5821 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     5881 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     5941 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     6001 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     6061 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     6121 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     6181 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     6241 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     6301 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     6361 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     6421 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     6481 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     6541 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     6601 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     6661 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     6721 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     6781 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     6841 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     6901 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     6961 gctcacatgt
//
