LOCUS       Exported                7634 bp ds-DNA     circular SYN 15-MAR-2024
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 7634)
  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 7634)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Mar 15, 2024 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7634
                     /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        177..624
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    657..690
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     protein_bind    701..734
                     /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(751..1470)
                     /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(754..1470)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(796..817)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1145..1164
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1404..1425
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(2419..2472)
                     /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(3288..3311)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(3486..3505)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3755..3773
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     protein_bind    complement(3937..3970)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     protein_bind    complement(3981..4014)
                     /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    4038..4626
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4091..4111)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4509..4520)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4661..4678)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4667..4874
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4897..5037
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4897..5026
                     /label=AAV2 ITR
     rep_origin      5112..5567
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5199..5218)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5409..5430
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5584..5603)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5703..5725
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5763..5781)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5849..5953
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5954..6814
                     /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(6172..6191)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6985..7573
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7474..7493
                     /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 cgcgtctgca gagggccctg cgtatgagtg
      181 caagtgggtt ttaggaccag gatgaggcgg ggtgggggtg cctacctgac gaccgacccc
      241 gacccactgg acaagcaccc aacccccatt ccccaaattg cgcatcccct atcagagagg
      301 gggaggggaa acaggatgcg gcgaggcgcg tgcgcactgc cagcttcagc accgcggaca
      361 gtgccttcgc ccccgcctgg cggcgcgcgc caccgccgcc tcagcactga aggcgcgctg
      421 acgtcactcg ccggtccccc gcaaactccc cttcccggcc accttggtcg cgtccgcgcc
      481 gccgccggcc cagccggacc gcaccacgcg aggcgcgaga taggggggca cgggcgcgac
      541 catctgcgct gcggcgccgg cgactcagcg ctgcctcagt ctgcggtggg cagcggagga
      601 gtcgtgtcgt gcctgagagc gcagacgcgt ggacggtacc cttcaggtcg acgtcgataa
      661 cttcgtatag catacattat acgaagttat ccgaatcgca ataacttcgt ataggatact
      721 ttatacgaag ttatttggcg cgcctctaga ttacttgtac agctcgtcca tgccgagagt
      781 gatcccggcg gcggtcacga actccagcag gaccatgtga tcgcgcttct cgttggggtc
      841 tttgctcagg gcggactggg tgctcaggta gtggttgtcg ggcagcagca cggggccgtc
      901 gccgatgggg gtgttctgct ggtagtggtc ggcgagctgc acgctgccgt cctcgatgtt
      961 gtggcggatc ttgaagttca ccttgatgcc gttcttctgc ttgtcggcca tgatatagac
     1021 gttgtggctg ttgtagttgt actccagctt gtgccccagg atgttgccgt cctccttgaa
     1081 gtcgatgccc ttcagctcga tgcggttcac cagggtgtcg ccctcgaact tcacctcggc
     1141 gcgggtcttg tagttgccgt cgtccttgaa gaagatggtg cgctcctgga cgtagccttc
     1201 gggcatggcg gacttgaaga agtcgtgctg cttcatgtgg tcggggtagc ggctgaagca
     1261 ctgcacgccg taggtcaggg tggtcacgag ggtgggccag ggcacgggca gcttgccggt
     1321 ggtgcagatg aacttcaggg tcagcttgcc gtaggtggca tcgccctcgc cctcgccgga
     1381 cacgctgaac ttgtggccgt ttacgtcgcc gtccagctcg accaggatgg gcaccacccc
     1441 ggtgaacagc tcctcgccct tgctcaccat tcgcgacatc tgattggaga aggaggtggg
     1501 cgcaccctgt tggccgtagc cttgctgccc atagtcgccc tgaggcccgt agccaccgcc
     1561 accgccgctg gctggctgcc cgtaatcggg ttgataacca ccctgaggcc cgtaggagtc
     1621 ctggggccca tagcctccgg ggccctgccc atagcccgca tcgccgtagg catcgccagg
     1681 agctggttgc ttttctgggg cgcctggagg tgcgcgcatg aatggggcgg cccagcctgt
     1741 ctccttgaac acgaaccata ggttgccaac ccagagcacc aggttcagga agccaaacac
     1801 caccgaggtg ttgagtcctg aagtcacagg gtccctcagt tccttgcatg tgtttcctgt
     1861 ctggcggcac ataggcatct ccttgataat gttctctggg tcagtggcca tcttcacatc
     1921 ggacaggcct ttggcccagg cggatgagct aactagccac atgaaagcga acactgctgt
     1981 ggccaggaag tccatcattg gccctttgtt gttctctcgg tacttgttct gcaggaagat
     2041 gtaggtggcc agggccccca tggagtagag gaaggcaaac acagccacgg tgacaaagaa
     2101 ttcagccgag gaggagtagt caccaactag gaagatcttg gtagtgcccc ctttaacgca
     2161 ggagggtgca tcaaagtaca cttggtgcag cctgaatggg tactcaaatt cgacttcgat
     2221 gttgagggca ctctccgtct tgttggcaca ctccacgctc agccgaagct ctccggtgta
     2281 gctgccgcac gtagcaaagg cgaagatggc aaagacccac tgcagcacct tcacgaagcc
     2341 aaggggctcc ttgaccaccc ggaactgacc cccagccacc agctgattca ccacgtccat
     2401 tctagatgag cctggggcag ggccgggatt ctcttccacg tctccgcatg tcagaagact
     2461 tcctctgccc tcactagtct tgtacagctc gtccatgccg tacagaaaca gatggtgcct
     2521 cccctcgctc cgctcatact gctccacaat ggtgtagtct tcgttatggc tagtaatatc
     2581 cagctttgtg tcgacgtaat agtagccagg cagctgcacg ggtttcttgg ccatgtagat
     2641 ggttttgaat tcgaccagat aatgccctcc atctttcagc ttcagtgcct ggtggatctc
     2701 tcccttcagc acgccgtctc tggggtacag cctttctgtt gaagcctccc agcccattgt
     2761 cttcttctgc atgactgggc catcaggggg aaaattggtg ccgcgcattt tcaccttata
     2821 gatcagagtg ccgtcctgca gagagctgtc ctgggtgaca gtcaccaggc ccccgtcttc
     2881 gaagttcatc actctttccc acttaaaccc ctctgggaag ctcagtttct tgtaatctgg
     2941 aatgtcagca gggtgtttca cgtaggcttt acttccatac ataaactgag gagacaggat
     3001 gtcccatgcg aatggcagag ggccgccctt tgtcactttc agctttgctg tctgggtgcc
     3061 ctcatagggc ctcccttccc cctctccttc aatctcaaac tcgtgcccgt tcatgctgcc
     3121 ctccatcctg actttaaacc tcataaactc cttaatcacg gccatgttgt tgtcctcgga
     3181 ggaggcggtg ccggagctgc cgctgccggt gctgccggta ccatgcccca ggaacaggtg
     3241 gtggcggccc tcggagcgct cgtactgttc cacgatggtg tagtcctcgt tgtgggaggt
     3301 gatgtccagc ttggtgtcca cgtagtagta gccgggcagt tgcacgggct tcttggccat
     3361 gtagatggtc ttgaactcca ccaggtagtg gccgccgtcc ttcagcttca gggcctggtg
     3421 gatctcgccc ttcagcacgc cgtcgcgggg gtacaggcgc tcggtggagg cctcccagcc
     3481 catggtcttc ttctgcatta cggggccgtc gggggggaag ttggtgccgc gcatcttcac
     3541 cttgtagatc agcgtgccgt cctgcaggga ggagtcctgg gtcacggtca ccagaccgcc
     3601 gtcctcgaag ttcatcacgc gctcccactt gaagccctcg gggaaggaca gcttcttgta
     3661 atcggggatg tcggcggggt gcttcacgta cgccttggag ccgtacatga actgggggga
     3721 caggatgtcc caggcgaagg gcagggggcc gcccttggtc accttcagct tggcggtctg
     3781 ggtgccctcg taggggcggc cctcgccctc gccctcgatc tcgaactcgt ggccgttcat
     3841 ggagccctcc atgcgcacct tgaagcgcat gaactctttg atgacctcct cgcccttgct
     3901 caccatacta gtggcgcgcc aacctggccg gccttgataa cttcgtataa tgtatgctat
     3961 acgaagttat ccgaatcgca ataacttcgt ataaagtatc ctatacgaag ttatcgaaga
     4021 tcatcaagct tatcgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta
     4081 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
     4141 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
     4201 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
     4261 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
     4321 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
     4381 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt
     4441 cctttccttg gctgctcgcc tatgttgcca cctggattct gcgcgggacg tccttctgct
     4501 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc
     4561 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct
     4621 ccccgcatcg ataccgagcg ctgctcgaga gatcgatctg cctcgactgt gccttctagt
     4681 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     4741 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     4801 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     4861 aggcatgctg gggacacgtg cggaccgagc ggccgcagga acccctagtg atggagttgg
     4921 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
     4981 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc
     5041 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa
     5101 gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     5161 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     5221 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     5281 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc
     5341 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     5401 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cgggctattc
     5461 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     5521 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct
     5581 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     5641 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     5701 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa
     5761 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     5821 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     5881 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     5941 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     6001 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     6061 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     6121 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     6181 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     6241 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     6301 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     6361 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     6421 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     6481 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     6541 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     6601 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     6661 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     6721 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     6781 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     6841 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     6901 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     6961 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     7021 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     7081 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     7141 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     7201 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     7261 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     7321 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     7381 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     7441 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     7501 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     7561 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     7621 ttttgctcac atgt
//
