LOCUS       sequence-425635-        7623 bp    DNA     circular SYN 15-DEC-2025
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    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/nature13186"; journalName: "Nature"; date: "2014-04-10- 
            10"; volume: "508"; issue: "7495"; pages: "207-14"
COMMENT     Sequence Label: AAV phSyn1(S)-FLEX-tdTomato-T2A-SypEGFP-WPRE
FEATURES             Location/Qualifiers
     source          1..7623
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(285..338)
                     /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(1154..1177)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(1352..1371)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1621..1639
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     protein_bind    1803..1836
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    1847..1880
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     misc_feature    1904..2492
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1957..1977)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2375..2386)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2527..2544)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2533..2740
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2763..2903
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2763..2892
                     /label=AAV2 ITR
     rep_origin      2978..3433
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3065..3084)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3275..3296
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3450..3469)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3569..3591
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3629..3647)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3715..3819
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3820..3888,3889..4680)
                     /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(4038..4057)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4851..5439
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5340..5359
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5501..5630
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        5666..6113
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(6146..6179)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(6190..6223)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     CDS             complement(join(6240..6953,6954..6956,6957..6959))
                     /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(join(6243..6953,6954..6956,6957..6959))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6285..6306)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     6634..6653
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6893..6914
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
ORIGIN
        1 atcttggtag tgcccccttt aacgcaggag ggtgcatcaa agtacacttg gtgcagcctg
       61 aatgggtact caaattcgac ttcgatgttg agggcactct ccgtcttgtt ggcacactcc
      121 acgctcagcc gaagctctcc ggtgtagctg ccgcacgtag caaaggcgaa gatggcaaag
      181 acccactgca gcaccttcac gaagccaagg ggctccttga ccacccggaa ctgaccccca
      241 gccaccagct gattcaccac gtccattcta gatgagcctg gggcagggcc gggattctct
      301 tccacgtctc cgcatgtcag aagacttcct ctgccctcac tagtcttgta cagctcgtcc
      361 atgccgtaca gaaacagatg gtgcctcccc tcgctccgct catactgctc cacaatggtg
      421 tagtcttcgt tatggctagt aatatccagc tttgtgtcga cgtaatagta gccaggcagc
      481 tgcacgggtt tcttggccat gtagatggtt ttgaattcga ccagataatg ccctccatct
      541 ttcagcttca gtgcctggtg gatctctccc ttcagcacgc cgtctctggg gtacagcctt
      601 tctgttgaag cctcccagcc cattgtcttc ttctgcatga ctgggccatc agggggaaaa
      661 ttggtgccgc gcattttcac cttatagatc agagtgccgt cctgcagaga gctgtcctgg
      721 gtgacagtca ccaggccccc gtcttcgaag ttcatcactc tttcccactt aaacccctct
      781 gggaagctca gtttcttgta atctggaatg tcagcagggt gtttcacgta ggctttactt
      841 ccatacataa actgaggaga caggatgtcc catgcgaatg gcagagggcc gccctttgtc
      901 actttcagct ttgctgtctg ggtgccctca tagggcctcc cttccccctc tccttcaatc
      961 tcaaactcgt gcccgttcat gctgccctcc atcctgactt taaacctcat aaactcctta
     1021 atcacggcca tgttgttgtc ctcggaggag gcggtgccgg agctgccgct gccggtgctg
     1081 ccggtaccat gccccaggaa caggtggtgg cggccctcgg agcgctcgta ctgttccacg
     1141 atggtgtagt cctcgttgtg ggaggtgatg tccagcttgg tgtccacgta gtagtagccg
     1201 ggcagttgca cgggcttctt ggccatgtag atggtcttga actccaccag gtagtggccg
     1261 ccgtccttca gcttcagggc ctggtggatc tcgcccttca gcacgccgtc gcgggggtac
     1321 aggcgctcgg tggaggcctc ccagcccatg gtcttcttct gcattacggg gccgtcgggg
     1381 gggaagttgg tgccgcgcat cttcaccttg tagatcagcg tgccgtcctg cagggaggag
     1441 tcctgggtca cggtcaccag accgccgtcc tcgaagttca tcacgcgctc ccacttgaag
     1501 ccctcgggga aggacagctt cttgtaatcg gggatgtcgg cggggtgctt cacgtacgcc
     1561 ttggagccgt acatgaactg gggggacagg atgtcccagg cgaagggcag ggggccgccc
     1621 ttggtcacct tcagcttggc ggtctgggtg ccctcgtagg ggcggccctc gccctcgccc
     1681 tcgatctcga actcgtggcc gttcatggag ccctccatgc gcaccttgaa gcgcatgaac
     1741 tctttgatga cctcctcgcc cttgctcacc atactagtgg cgcgccaacc tggccggcct
     1801 tgataacttc gtataatgta tgctatacga agttatccga atcgcaataa cttcgtataa
     1861 agtatcctat acgaagttat cgaagatcat caagcttatc gataatcaac ctctggatta
     1921 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     1981 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     2041 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     2101 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     2161 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     2221 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     2281 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctatg ttgccacctg
     2341 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     2401 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     2461 gagtcggatc tccctttggg ccgcctcccc gcatcgatac cgagcgctgc tcgagagatc
     2521 gatctgcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     2581 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     2641 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     2701 agggggagga ttgggaagac aatagcaggc atgctgggga cacgtgcgga ccgagcggcc
     2761 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     2821 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     2881 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     2941 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     3001 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg
     3061 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     3121 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     3181 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     3241 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     3301 acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc
     3361 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     3421 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     3481 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     3541 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     3601 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     3661 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     3721 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     3781 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     3841 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     3901 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     3961 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     4021 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     4081 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     4141 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     4201 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     4261 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     4321 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     4381 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     4441 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     4501 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     4561 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     4621 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     4681 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     4741 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     4801 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     4861 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     4921 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     4981 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     5041 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     5101 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     5161 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     5221 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     5281 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     5341 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     5401 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     5461 ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca gctgcgcgct
     5521 cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg cctcagtgag
     5581 cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct gcggccgcac
     5641 gcgtctgcag agggccctgc gtatgagtgc aagtgggttt taggaccagg atgaggcggg
     5701 gtgggggtgc ctacctgacg accgaccccg acccactgga caagcaccca acccccattc
     5761 cccaaattgc gcatccccta tcagagaggg ggaggggaaa caggatgcgg cgaggcgcgt
     5821 gcgcactgcc agcttcagca ccgcggacag tgccttcgcc cccgcctggc ggcgcgcgcc
     5881 accgccgcct cagcactgaa ggcgcgctga cgtcactcgc cggtcccccg caaactcccc
     5941 ttcccggcca ccttggtcgc gtccgcgccg ccgccggccc agccggaccg caccacgcga
     6001 ggcgcgagat aggggggcac gggcgcgacc atctgcgctg cggcgccggc gactcagcgc
     6061 tgcctcagtc tgcggtgggc agcggaggag tcgtgtcgtg cctgagagcg cagacgcgtg
     6121 gacggtaccc ttcaggtcga cgtcgataac ttcgtatagc atacattata cgaagttatc
     6181 cgaatcgcaa taacttcgta taggatactt tatacgaagt tatttggcgc gcctctagat
     6241 tacttgtaca gctcgtccat gccgagagtg atcccggcgg cggtcacgaa ctccagcagg
     6301 accatgtgat cgcgcttctc gttggggtct ttgctcaggg cggactgggt gctcaggtag
     6361 tggttgtcgg gcagcagcac ggggccgtcg ccgatggggg tgttctgctg gtagtggtcg
     6421 gcgagctgca cgctgccgtc ctcgatgttg tggcggatct tgaagttcac cttgatgccg
     6481 ttcttctgct tgtcggccat gatatagacg ttgtggctgt tgtagttgta ctccagcttg
     6541 tgccccagga tgttgccgtc ctccttgaag tcgatgccct tcagctcgat gcggttcacc
     6601 agggtgtcgc cctcgaactt cacctcggcg cgggtcttgt agttgccgtc gtccttgaag
     6661 aagatggtgc gctcctggac gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc
     6721 ttcatgtggt cggggtagcg gctgaagcac tgcacgccgt aggtcagggt ggtcacgagg
     6781 gtgggccagg gcacgggcag cttgccggtg gtgcagatga acttcagggt cagcttgccg
     6841 taggtggcat cgccctcgcc ctcgccggac acgctgaact tgtggccgtt tacgtcgccg
     6901 tccagctcga ccaggatggg caccaccccg gtgaacagct cctcgccctt gctcaccatt
     6961 cgcgacatct gattggagaa ggaggtgggc gcaccctgtt ggccgtagcc ttgctgccca
     7021 tagtcgccct gaggcccgta gccaccgcca ccgccgctgg ctggctgccc gtaatcgggt
     7081 tgataaccac cctgaggccc gtaggagtcc tggggcccat agcctccggg gccctgccca
     7141 tagcccgcat cgccgtaggc atcgccagga gctggttgct tttctggggc gcctggaggt
     7201 gcgcgcatga atggggcggc ccagcctgtc tccttgaaca cgaaccatag gttgccaacc
     7261 cagagcacca ggttcaggaa gccaaacacc accgaggtgt tgagtcctga agtcacaggg
     7321 tccctcagtt ccttgcatgt gtttcctgtc tggcggcaca taggcatctc cttgataatg
     7381 ttctctgggt cagtggccat cttcacatcg gacaggcctt tggcccaggc ggatgagcta
     7441 actagccaca tgaaagcgaa cactgctgtg gccaggaagt ccatcattgg ccctttgttg
     7501 ttctctcggt acttgttctg caggaagatg taggtggcca gggcccccat ggagtagagg
     7561 aaggcaaaca cagccacggt gacaaagaat tcagccgagg aggagtagtc accaactagg
     7621 aag
//
