LOCUS       Exported                6313 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Flp-Dependent EYFP.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-Ef1a-fDIO EYFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6313)
  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 6313)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6313
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..1179
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          232..1170
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1127..1147
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1210..1243
                     /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(1336..2055)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1381..1402)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1730..1749
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1989..2010
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2052..2061
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    complement(2068..2101)
                     /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    2210..2798
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2263..2283)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2681..2692)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2830..3306
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3029..3048)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3346..3486
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3346..3475
                     /label=AAV2 ITR
     rep_origin      3561..4016
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3648..3667)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3858..3879
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4033..4052)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4152..4174
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4212..4230)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4298..4402
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4403..5263
                     /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(4621..4640)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5434..6022
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5923..5942
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   6084..6213
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
       61 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
      121 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
      181 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc
      241 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt
      301 acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      361 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      421 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      481 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      541 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      601 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      661 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      721 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
      781 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
      841 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
      901 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
      961 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1021 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1081 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1141 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc
     1201 gactccggag aagttcctat tctctagaaa gtataggaac ttcgcagaat ggtagctgga
     1261 ttgtagctgc tattagcaat atgaaacctc ttagaagttc ctattcttca aaaggtatag
     1321 gaacttcggc gcgccttact tgtacagctc gtccatgccg agagtgatcc cggcggcggt
     1381 cacgaactcc agcaggacca tgtgatcgcg cttctcgttg gggtctttgc tcagggcgga
     1441 ctggtagctc aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt
     1501 ctgctggtag tggtcggcga gctgcacgct gccgtcctcg atgttgtggc ggatcttgaa
     1561 gttcaccttg atgccgttct tctgcttgtc ggccatgata tagacgttgt ggctgttgta
     1621 gttgtactcc agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag
     1681 ctcgatgcgg ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt
     1741 gccgtcgtcc ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca tggcggactt
     1801 gaagaagtcg tgctgcttca tgtggtcggg gtagcgggcg aagcactgca ggccgtagcc
     1861 gaaggtggtc acgagggtgg gccagggcac gggcagcttg ccggtggtgc agatgaactt
     1921 cagggtcagc ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg
     1981 gccgtttacg tcgccgtcca gctcgaccag gatgggcacc accccggtga acagctcctc
     2041 gcccttgctc accatggtgg cgctagcgaa gttcctatac tttctagaga ataggaactt
     2101 cttgccttaa cccagaaatt atcactgtta ttctttagaa tggtgcaaag agaagttcct
     2161 ataccttttg aagaatagga acttcgaatt cgatatcaag cttatcgata atcaacctct
     2221 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     2281 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     2341 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     2401 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     2461 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     2521 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     2581 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctatgttgc
     2641 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     2701 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     2761 tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgag cgctgctcga
     2821 gagatctacg ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc cctggaagtt
     2881 gccactccag tgcccaccag ccttgtccta ataaaattaa gttgcatcat tttgtctgac
     2941 taggtgtcct tctataatat tatggggtgg aggggggtgg tatggagcaa ggggcaagtt
     3001 gggaagacaa cctgtagggc ctgcggggtc tattgggaac caagctggag tgcagtggca
     3061 caatcttggc tcactgcaat ctccgcctcc tgggttcaag cgattctcct gcctcagcct
     3121 cccgagttgt tgggattcca ggcatgcatg accaggctca gctaattttt gtttttttgg
     3181 tagagacggg gtttcaccat attggccagg ctggtctcca actcctaatc tcaggtgatc
     3241 tacccacctt ggcctcccaa attgctggga ttacaggcgt gaaccactgc tcccttccct
     3301 gtccttctga ttttgtaggt aaccacgtgc ggaccgagcg gccgcaggaa cccctagtga
     3361 tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg
     3421 tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc
     3481 tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     3541 tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     3601 ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt
     3661 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     3721 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg
     3781 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     3841 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc
     3901 gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga
     3961 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg
     4021 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     4081 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     4141 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     4201 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     4261 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     4321 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     4381 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     4441 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     4501 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     4561 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     4621 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     4681 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     4741 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     4801 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     4861 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     4921 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     4981 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     5041 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     5101 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     5161 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     5221 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     5281 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     5341 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     5401 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     5461 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     5521 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     5581 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     5641 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     5701 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     5761 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     5821 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     5881 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     5941 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     6001 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     6061 ttgctggcct tttgctcaca tgtcctgcag gcagctgcgc gctcgctcgc tcactgaggc
     6121 cgcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag
     6181 agggagtggc caactccatc actaggggtt cctgcggccg cacgcgtaag ctttgcaaag
     6241 atggataaag ttttaaacag agaggaatct ttgcagctaa tggaccttct aggtcttgaa
     6301 aggagtggga att
//
