LOCUS       sequence-239070-        7252 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Flp-Dependent ChR2-EYFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7252)
  AUTHORS   Fenno LE, Mattis J, Ramakrishnan C, Hyun M, Lee SY, He M, Tucciarone
            J, Selimbeyoglu A, Berndt A, Grosenick L, Zalocusky KA, Bernstein H,
            Swanson H, Perry C, Diester I, Boyce FM, Bass CE, Neve R, Huang ZJ, 
            Deisseroth K
  TITLE     Targeting cells with single vectors using multiple-feature Boolean 
            logic.
  JOURNAL   Nat Methods. 2014 Jul;11(7):763-72. doi: 10.1038/nmeth.2996. Epub 
            2014 Jun 8.
  PUBMED    24908100
REFERENCE   2  (bases 1 to 7252)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/nmeth.2996"; journalName: "Nat Methods"; date: "2014-07"; 
            volume: "11"; issue: "7"; pages: "763-72"
COMMENT     Sequence Label: pAAV-Ef1a-fDIO hChR2(H134R)-EYFP
FEATURES             Location/Qualifiers
     source          1..7252
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..126
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        227..1405
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          458..1396
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1353..1373
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1436..1469
                     /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)"
     CDS             complement(join(1565..2278,2279..2281,2282..2284))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1610..1631)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1959..1978
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2218..2239
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2281..2290
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2300..3226)
                     /codon_start=1
                     /product="H134R mutant of an N-terminal fragment of 
                     channelrhodopsin-2 from the alga Chlamydomonas reinhardtii 
                     (Nagel et al., 2005)"
                     /label=ChR2(H134R)
                     /note="blue light-activated cation channel"
                     /translation="MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGT
                     NGAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKN
                     PSMLYLATGHRVQWLRYAEWLLTCPVILIRLSNLTGLSNDYSRRTMGLLVSDIGTIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     regulatory      3223..3232
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3237..3270)
                     /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    3379..3967
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3432..3452)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3850..3861)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3999..4475
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4198..4217)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4515..4655
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4515..4644
                     /label=AAV2 ITR
     rep_origin      4730..5185
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4817..4836)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5027..5048
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5202..5221)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5321..5343
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5381..5399)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5467..5571
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5572..5640,5641..6432)
                     /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(5790..5809)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6603..7191
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7092..7111
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgcgca gagagggagt ggccaactcc atcactaggg
      121 gttcctgcgg ccgcacgcgt aagctttgca aagatggata aagttttaaa cagagaggaa
      181 tctttgcagc taatggacct tctaggtctt gaaaggagtg ggaattggct ccggtgcccg
      241 tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa
      301 ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg
      361 gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa
      421 cgttcttttt cgcaacgggt ttgccgccag aacacaggta agtgccgtgt gtggttcccg
      481 cgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactt ccacctggct
      541 gcagtacgtg attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc
      601 ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg
      661 ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct
      721 agccatttaa aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt
      781 aaatgcgggc caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg
      841 ggcccgtgcg tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag
      901 aatcggacgg gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc
      961 gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga
     1021 aagatggccg cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg
     1081 agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc
     1141 ttcatgtgac tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt
     1201 ttggagtacg tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac
     1261 tgagtgggtg gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt
     1321 tgcccttttt gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt
     1381 ttttcttcca tttcaggtgt cgtgaggtac cggatcctct agagtcgact ccggagaagt
     1441 tcctattctc tagaaagtat aggaacttcg cagaatggta gctggattgt agctgctatt
     1501 agcaatatga aacctcttag aagttcctat tcttcaaaag gtataggaac ttcggcgcgc
     1561 cctattactt gtacagctcg tccatgccga gagtgatccc ggcggcggtc acgaactcca
     1621 gcaggaccat gtgatcgcgc ttctcgttgg ggtctttgct cagggcggac tggtagctca
     1681 ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat gggggtgttc tgctggtagt
     1741 ggtcggcgag ctgcacgctg ccgtcctcga tgttgtggcg gatcttgaag ttcaccttga
     1801 tgccgttctt ctgcttgtcg gccatgatat agacgttgtg gctgttgtag ttgtactcca
     1861 gcttgtgccc caggatgttg ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt
     1921 tcaccagggt gtcgccctcg aacttcacct cggcgcgggt cttgtagttg ccgtcgtcct
     1981 tgaagaagat ggtgcgctcc tggacgtagc cttcgggcat ggcggacttg aagaagtcgt
     2041 gctgcttcat gtggtcgggg tagcgggcga agcactgcag gccgtagccg aaggtggtca
     2101 cgagggtggg ccagggcacg ggcagcttgc cggtggtgca gatgaacttc agggtcagct
     2161 tgccgtaggt ggcatcgccc tcgccctcgc cggacacgct gaacttgtgg ccgtttacgt
     2221 cgccgtccag ctcgaccagg atgggcacca ccccggtgaa cagctcctcg cccttgctca
     2281 ccatggtggc ggccgctggc acggctccgg cctcggcttc gtcttcgacg agagtctcga
     2341 cctcgatctc cgttccgccg atgttcagtt tggtggtttt gcggatatct ccgtgaatca
     2401 atatgtgctc gtggatcagg acgcgcaggt agtgtcccaa caacccccaa caatttttac
     2461 tcatcagatc aataatcgtg tgacctacgg tggagccata gacgctcagg acgccaaaac
     2521 cttcgggccc caaaatgaag agaattggga acatacccca gctcacgaaa aacagccatg
     2581 ccatgccggt cacgacctgg cggcaccgac cctttggcac agtatgataa ccctcgatat
     2641 atgctttggc ggcgtgaaaa aatgtgttcg cgccatagca caatccaaga caaaagaaga
     2701 tgactttaac atagccggtt gccatggcgc tggtagcccc ccacacgata gtcccgatgt
     2761 ctgagacaag gagtcccatg gttctcctgc tgtagtcgtt gctcaggccg gtgaggttgc
     2821 tcaggcggat aaggatgaca ggacaagtga gcagccactc tgcatagcgc agccactgca
     2881 cccggtgtcc tgtggcaagg tagagcatag agggattctt aaactcaaaa aagaactcga
     2941 gaatcacctt aaccatttca atggcgcaca catagatctc ctcccagccg catgtagatt
     3001 tccaggtttg gtaggcatag aacatcagca gcaaaatgct gaatcctgct gcaagccact
     3061 gcaggacatt tgacgcggtc tgagcgccgt tcgtgccgcg agattcaatc catccggcac
     3121 agtaacattg atcctcaggg accaggacgg acccgttcac caccacagga ttagtaacga
     3181 acaaaagttc gcgtccgacg gcagacaaag cgccgccata gtccatggtg gctagcgaag
     3241 ttcctatact ttctagagaa taggaacttc ttgccttaac ccagaaatta tcactgttat
     3301 tctttagaat ggtgcaaaga gaagttccta taccttttga agaataggaa cttcgaattc
     3361 gatatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     3421 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     3481 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
     3541 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
     3601 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
     3661 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     3721 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg
     3781 tcctttcctt ggctgctcgc ctatgttgcc acctggattc tgcgcgggac gtccttctgc
     3841 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
     3901 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
     3961 tccccgcatc gataccgagc gctgctcgag agatctacgg gtggcatccc tgtgacccct
     4021 ccccagtgcc tctcctggcc ctggaagttg ccactccagt gcccaccagc cttgtcctaa
     4081 taaaattaag ttgcatcatt ttgtctgact aggtgtcctt ctataatatt atggggtgga
     4141 ggggggtggt atggagcaag gggcaagttg ggaagacaac ctgtagggcc tgcggggtct
     4201 attgggaacc aagctggagt gcagtggcac aatcttggct cactgcaatc tccgcctcct
     4261 gggttcaagc gattctcctg cctcagcctc ccgagttgtt gggattccag gcatgcatga
     4321 ccaggctcag ctaatttttg tttttttggt agagacgggg tttcaccata ttggccaggc
     4381 tggtctccaa ctcctaatct caggtgatct acccaccttg gcctcccaaa ttgctgggat
     4441 tacaggcgtg aaccactgct cccttccctg tccttctgat tttgtaggta accacgtgcg
     4501 gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
     4561 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc
     4621 ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct
     4681 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt
     4741 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     4801 agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     4861 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     4921 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga
     4981 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     5041 caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg
     5101 ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     5161 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc
     5221 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     5281 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     5341 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     5401 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     5461 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     5521 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     5581 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     5641 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     5701 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     5761 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     5821 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     5881 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     5941 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     6001 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     6061 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     6121 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     6181 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     6241 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     6301 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     6361 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     6421 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     6481 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     6541 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6601 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6661 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6721 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     6781 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6841 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6901 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6961 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     7021 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     7081 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     7141 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     7201 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gt
//
