LOCUS       Exported                6959 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 6959)
  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 6959)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6959
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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=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      823..832
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    1268..1301
                     /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    1352..1385
                     /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     1498..1519
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    complement(2185..2218)
                     /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(2269..2302)
                     /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(2524..2543)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2683..2934
                     /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     2871..2892
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2944..2977)
                     /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    3086..3674
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3139..3159)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3557..3568)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3706..4182
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3905..3924)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4222..4362
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4222..4351
                     /label=AAV2 ITR
     rep_origin      4437..4892
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4524..4543)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4734..4755
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4909..4928)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5028..5050
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5088..5106)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5174..5278
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5279..6139
                     /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(5497..5516)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6310..6898
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6799..6818
                     /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 ctagaaagta
      721 taggaacttc gcagagtagt agctggattg tagctgctat tagcaatcta aaacctctta
      781 gaagttccta ttcttcaaaa ggtataggaa cttcggcgcg ccgccaccat ggactatggc
      841 ggcgctttgt ctgccgtcgg acgcgaactt ttgttcgtta ctaatcctgt ggtggtgaac
      901 gggtccgtcc tggtccctga ggatcaatgt tactgtgccg gatggattga atctcgcggc
      961 acgaacggcg ctcagaccgc gtcaaatgtc ctgcagtggc ttgcagcagg attcagcatt
     1021 ttgctgctga tgttctatgc ctaccaaacc tggaaatcta catgcggctg ggaggagatc
     1081 tatgtgtgcg ccattgaaat ggttaaggtg attctcgagt tcttttttga gtttaagaat
     1141 ccctctatgc tctaccttgc cacaggacac cgggtgcagt ggctgcgcta tgcagagtgg
     1201 ctgctcactt gtcctgtcat ccttatccgc ctgagcaacc tcaccggtaa gtgtcggggt
     1261 tgagctcata acttcgtata gtatacctta tacgaagtta ttctttgcac cattctaaag
     1321 aataacagtg ataatttctg ggttaaggca aataacttcg tataggatac tttatacgaa
     1381 gttatggtac ccctcctgta ctcaccttgc cggtggtgca gatgaacttc agggtcagct
     1441 tgccgtaggt ggcatcgccc tcgccctcgc cggacacgct gaacttgtgg ccgtttacgt
     1501 cgccgtccag ctcgaccagg atgggcacca ccccggtgaa cagctcctcg cccttgctca
     1561 cggcggccgc tggcacggct ccggcctcgg cttcgtcttc gacgagagtc tcgacctcga
     1621 tctccgttcc gccgatgttc agtttggtgg ttttgcggat atctccgtga atcaatatgt
     1681 gctcgtggat caggacgcgc aggtagtgtc ccaacaaccc ccaacaattt ttactcatca
     1741 gatcaataat cgtgtgacct acggtggagc catagacgct caggacgcca aaaccttcgg
     1801 gccccaaaat gaagagaatt gggaacatac cccagctcac gaaaaacagc catgccatgc
     1861 cggtcacgac ctggcggcac cgaccctttg gcacagtatg ataaccctcg atatatgctt
     1921 tggcggcgtg aaaaaatgtg ttcgcgccat agcacaatcc aagacaaaag aagatgactt
     1981 taacatagcc ggttgccatg gcgctggtag ccccccacac gatagtcccg atgtctgaga
     2041 caaggagtcc catggttctc ctgctgtagt cgttgctcag gcctaaacac atgagaaata
     2101 gagtcataag cacatgggtc acatacagga gatatgtata taacattaat acaatttata
     2161 aaaaaaaggg ggggcacaac tagtataact tcgtataagg tatactatac gaagttatta
     2221 agaggtttca tattgctaat agcagctaca atccagctac cattctgcat aacttcgtat
     2281 aaagtatcct atacgaagtt atgtcgactg gagagtggcc agcccttctc atgttcagag
     2341 aacatggtta actggttaag tcatgtcgtc ccacagctgc ccgtgccctg gcccaccctc
     2401 gtgaccacct tcggctacgg cctgcagtgc ttcgcccgct accccgacca catgaagcag
     2461 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     2521 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     2581 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     2641 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     2701 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     2761 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     2821 ctgagctacc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     2881 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaagct
     2941 agcgaagttc ctatactttc tagagaatag gaacttctaa gcagacaatc ttgtaataca
     3001 tcagcagcta gtagattgct ccagctagaa gttcctatac cttttgaaga ataggaactt
     3061 cgaattcgat atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt
     3121 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     3181 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     3241 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
     3301 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
     3361 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
     3421 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
     3481 atcatcgtcc tttccttggc tgctcgccta tgttgccacc tggattctgc gcgggacgtc
     3541 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
     3601 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg
     3661 ggccgcctcc ccgcatcgat accgagcgct gctcgagaga tctacgggtg gcatccctgt
     3721 gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc caccagcctt
     3781 gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta taatattatg
     3841 gggtggaggg gggtggtatg gagcaagggg caagttggga agacaacctg tagggcctgc
     3901 ggggtctatt gggaaccaag ctggagtgca gtggcacaat cttggctcac tgcaatctcc
     3961 gcctcctggg ttcaagcgat tctcctgcct cagcctcccg agttgttggg attccaggca
     4021 tgcatgacca ggctcagcta atttttgttt ttttggtaga gacggggttt caccatattg
     4081 gccaggctgg tctccaactc ctaatctcag gtgatctacc caccttggcc tcccaaattg
     4141 ctgggattac aggcgtgaac cactgctccc ttccctgtcc ttctgatttt gtaggtaacc
     4201 acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc
     4261 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     4321 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt
     4381 ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc
     4441 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     4501 acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     4561 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     4621 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc
     4681 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     4741 cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg
     4801 gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     4861 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc
     4921 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     4981 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     5041 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     5101 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     5161 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     5221 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     5281 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     5341 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     5401 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     5461 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     5521 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     5581 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     5641 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     5701 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     5761 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     5821 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     5881 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     5941 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     6001 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     6061 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     6121 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     6181 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     6241 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     6301 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     6361 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     6421 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     6481 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6541 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6601 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6661 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     6721 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     6781 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     6841 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     6901 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt
//
