LOCUS       Exported                7623 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Can be used to generate AAV virus that will express cytoplasmic 
            tdTomato and presynaptic (synaptophysin-fused) EGFP in the presence 
            of Cre in neurons from the synapsin promoter.
ACCESSION   .
VERSION     .
KEYWORDS    AAV phSyn1(S)-FLEX-tdTomato-T2A-SypEGFP-WPRE
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7623)
  AUTHORS   Oh SW, Harris JA, Ng L, Winslow B, Cain N, Mihalas S, Wang Q, Lau C,
            Kuan L, Henry AM, Mortrud MT, Ouellette B, Nguyen TN, Sorensen SA, 
            Slaughterbeck CR, Wakeman W, Li Y, Feng D, Ho A, Nicholas E, 
            Hirokawa KE, Bohn P, Joines KM, Peng H, Hawrylycz MJ, Phillips JW, 
            Hohmann JG, Wohnoutka P, Gerfen CR, Koch C, Bernard A, Dang C, Jones
            AR, Zeng H
  TITLE     A mesoscale connectome of the mouse brain.
  JOURNAL   Nature. 2014 Apr 10;508(7495):207-14. doi: 10.1038/nature13186. Epub
            2014 Apr 2.
  PUBMED    24695228
REFERENCE   2  (bases 1 to 7623)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7623
                     /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        166..613
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    646..679
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     protein_bind    690..723
                     /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)."
     CDS             complement(740..1459)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(743..1459)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(785..806)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1134..1153
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1393..1414
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(2408..2461)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     primer_bind     complement(3277..3300)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(3475..3494)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3744..3762
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     protein_bind    complement(3926..3959)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     protein_bind    complement(3970..4003)
                     /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)."
     misc_feature    4027..4615
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4080..4100)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4498..4509)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4650..4667)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4656..4863
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4886..5026
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4886..5015
                     /label=AAV2 ITR
     rep_origin      5101..5556
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5188..5207)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5398..5419
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5573..5592)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5692..5714
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5752..5770)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5838..5942
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5943..6803
                     /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(6161..6180)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6974..7562
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7463..7482
                     /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 gcgtctgcag agggccctgc gtatgagtgc aagtgggttt
      181 taggaccagg atgaggcggg gtgggggtgc ctacctgacg accgaccccg acccactgga
      241 caagcaccca acccccattc cccaaattgc gcatccccta tcagagaggg ggaggggaaa
      301 caggatgcgg cgaggcgcgt gcgcactgcc agcttcagca ccgcggacag tgccttcgcc
      361 cccgcctggc ggcgcgcgcc accgccgcct cagcactgaa ggcgcgctga cgtcactcgc
      421 cggtcccccg caaactcccc ttcccggcca ccttggtcgc gtccgcgccg ccgccggccc
      481 agccggaccg caccacgcga ggcgcgagat aggggggcac gggcgcgacc atctgcgctg
      541 cggcgccggc gactcagcgc tgcctcagtc tgcggtgggc agcggaggag tcgtgtcgtg
      601 cctgagagcg cagacgcgtg gacggtaccc ttcaggtcga cgtcgataac ttcgtatagc
      661 atacattata cgaagttatc cgaatcgcaa taacttcgta taggatactt tatacgaagt
      721 tatttggcgc gcctctagat tacttgtaca gctcgtccat gccgagagtg atcccggcgg
      781 cggtcacgaa ctccagcagg accatgtgat cgcgcttctc gttggggtct ttgctcaggg
      841 cggactgggt gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg ccgatggggg
      901 tgttctgctg gtagtggtcg gcgagctgca cgctgccgtc ctcgatgttg tggcggatct
      961 tgaagttcac cttgatgccg ttcttctgct tgtcggccat gatatagacg ttgtggctgt
     1021 tgtagttgta ctccagcttg tgccccagga tgttgccgtc ctccttgaag tcgatgccct
     1081 tcagctcgat gcggttcacc agggtgtcgc cctcgaactt cacctcggcg cgggtcttgt
     1141 agttgccgtc gtccttgaag aagatggtgc gctcctggac gtagccttcg ggcatggcgg
     1201 acttgaagaa gtcgtgctgc ttcatgtggt cggggtagcg gctgaagcac tgcacgccgt
     1261 aggtcagggt ggtcacgagg gtgggccagg gcacgggcag cttgccggtg gtgcagatga
     1321 acttcagggt cagcttgccg taggtggcat cgccctcgcc ctcgccggac acgctgaact
     1381 tgtggccgtt tacgtcgccg tccagctcga ccaggatggg caccaccccg gtgaacagct
     1441 cctcgccctt gctcaccatt cgcgacatct gattggagaa ggaggtgggc gcaccctgtt
     1501 ggccgtagcc ttgctgccca tagtcgccct gaggcccgta gccaccgcca ccgccgctgg
     1561 ctggctgccc gtaatcgggt tgataaccac cctgaggccc gtaggagtcc tggggcccat
     1621 agcctccggg gccctgccca tagcccgcat cgccgtaggc atcgccagga gctggttgct
     1681 tttctggggc gcctggaggt gcgcgcatga atggggcggc ccagcctgtc tccttgaaca
     1741 cgaaccatag gttgccaacc cagagcacca ggttcaggaa gccaaacacc accgaggtgt
     1801 tgagtcctga agtcacaggg tccctcagtt ccttgcatgt gtttcctgtc tggcggcaca
     1861 taggcatctc cttgataatg ttctctgggt cagtggccat cttcacatcg gacaggcctt
     1921 tggcccaggc ggatgagcta actagccaca tgaaagcgaa cactgctgtg gccaggaagt
     1981 ccatcattgg ccctttgttg ttctctcggt acttgttctg caggaagatg taggtggcca
     2041 gggcccccat ggagtagagg aaggcaaaca cagccacggt gacaaagaat tcagccgagg
     2101 aggagtagtc accaactagg aagatcttgg tagtgccccc tttaacgcag gagggtgcat
     2161 caaagtacac ttggtgcagc ctgaatgggt actcaaattc gacttcgatg ttgagggcac
     2221 tctccgtctt gttggcacac tccacgctca gccgaagctc tccggtgtag ctgccgcacg
     2281 tagcaaaggc gaagatggca aagacccact gcagcacctt cacgaagcca aggggctcct
     2341 tgaccacccg gaactgaccc ccagccacca gctgattcac cacgtccatt ctagatgagc
     2401 ctggggcagg gccgggattc tcttccacgt ctccgcatgt cagaagactt cctctgccct
     2461 cactagtctt gtacagctcg tccatgccgt acagaaacag atggtgcctc ccctcgctcc
     2521 gctcatactg ctccacaatg gtgtagtctt cgttatggct agtaatatcc agctttgtgt
     2581 cgacgtaata gtagccaggc agctgcacgg gtttcttggc catgtagatg gttttgaatt
     2641 cgaccagata atgccctcca tctttcagct tcagtgcctg gtggatctct cccttcagca
     2701 cgccgtctct ggggtacagc ctttctgttg aagcctccca gcccattgtc ttcttctgca
     2761 tgactgggcc atcaggggga aaattggtgc cgcgcatttt caccttatag atcagagtgc
     2821 cgtcctgcag agagctgtcc tgggtgacag tcaccaggcc cccgtcttcg aagttcatca
     2881 ctctttccca cttaaacccc tctgggaagc tcagtttctt gtaatctgga atgtcagcag
     2941 ggtgtttcac gtaggcttta cttccataca taaactgagg agacaggatg tcccatgcga
     3001 atggcagagg gccgcccttt gtcactttca gctttgctgt ctgggtgccc tcatagggcc
     3061 tcccttcccc ctctccttca atctcaaact cgtgcccgtt catgctgccc tccatcctga
     3121 ctttaaacct cataaactcc ttaatcacgg ccatgttgtt gtcctcggag gaggcggtgc
     3181 cggagctgcc gctgccggtg ctgccggtac catgccccag gaacaggtgg tggcggccct
     3241 cggagcgctc gtactgttcc acgatggtgt agtcctcgtt gtgggaggtg atgtccagct
     3301 tggtgtccac gtagtagtag ccgggcagtt gcacgggctt cttggccatg tagatggtct
     3361 tgaactccac caggtagtgg ccgccgtcct tcagcttcag ggcctggtgg atctcgccct
     3421 tcagcacgcc gtcgcggggg tacaggcgct cggtggaggc ctcccagccc atggtcttct
     3481 tctgcattac ggggccgtcg ggggggaagt tggtgccgcg catcttcacc ttgtagatca
     3541 gcgtgccgtc ctgcagggag gagtcctggg tcacggtcac cagaccgccg tcctcgaagt
     3601 tcatcacgcg ctcccacttg aagccctcgg ggaaggacag cttcttgtaa tcggggatgt
     3661 cggcggggtg cttcacgtac gccttggagc cgtacatgaa ctggggggac aggatgtccc
     3721 aggcgaaggg cagggggccg cccttggtca ccttcagctt ggcggtctgg gtgccctcgt
     3781 aggggcggcc ctcgccctcg ccctcgatct cgaactcgtg gccgttcatg gagccctcca
     3841 tgcgcacctt gaagcgcatg aactctttga tgacctcctc gcccttgctc accatactag
     3901 tggcgcgcca acctggccgg ccttgataac ttcgtataat gtatgctata cgaagttatc
     3961 cgaatcgcaa taacttcgta taaagtatcc tatacgaagt tatcgaagat catcaagctt
     4021 atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     4081 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     4141 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     4201 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     4261 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     4321 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     4381 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     4441 ctgctcgcct atgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     4501 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     4561 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga
     4621 taccgagcgc tgctcgagag atcgatctgc ctcgactgtg ccttctagtt gccagccatc
     4681 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     4741 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     4801 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     4861 ggacacgtgc ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct
     4921 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt
     4981 gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg
     5041 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt
     5101 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     5161 ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     5221 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     5281 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
     5341 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     5401 gactcttgtt ccaaactgga acaacactca actctatctc gggctattct tttgatttat
     5461 aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta
     5521 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
     5581 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     5641 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     5701 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     5761 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     5821 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     5881 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     5941 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     6001 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     6061 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     6121 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     6181 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     6241 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     6301 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     6361 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     6421 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     6481 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     6541 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     6601 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     6661 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     6721 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     6781 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     6841 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     6901 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     6961 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     7021 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     7081 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     7141 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     7201 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     7261 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     7321 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     7381 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     7441 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     7501 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     7561 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     7621 tgt
//
