LOCUS       sequence-471581-        7150 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Anterograde transsynaptic tracer protein to express in presynaptic 
            glutamatergic neurons that express Cre, Flp payload. Must use with 
            AAV8-fDIO-Reporter viruses infected in postsynaptic cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7150)
  AUTHORS   Rivera JF, Huang H, Weng W, Sohn H, Girasole AE, Li S, Albanese MA, 
            Qin M, Tao C, Klug ME, Rao S, Paletzki R, Herring BE, Kanoski SE, 
            Zhang LI, Gerfen CR, Sabatini BL, Arnold DB
  TITLE     ATLAS: a rationally designed anterograde transsynaptic tracer.
  JOURNAL   Nat Methods. 2025 May;22(5):1101-1111. doi: 
            10.1038/s41592-025-02670-x. Epub 2025 May 1.
  PUBMED    40312509
REFERENCE   2  (bases 1 to 7150)
  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/s41592-025-02670-x"; journalName: "Nat Methods"; date: 
            "2025-05"; volume: "22"; issue: "5"; pages: "1101-1111"
COMMENT     Sequence Label: pAAV-DIO-ATLASsnFlp
FEATURES             Location/Qualifiers
     source          1..7150
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        175..622
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(660..693)
                     /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)."
     protein_bind    complement(744..777)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(789..1130)
                     /codon_start=1
                     /product="zinc-finger domain from the CCR5-224 (+) 
                     zinc-finger nuclease that recognizes the left half-site 
                     target sequence in the human CCR5 gene (Perez et al., 2008;
                     Gross et al., 2013)"
                     /label=CCR5TC
                     /translation="FQCRICMRNFSDRSNLSRHIRTHTGEKPFACDICGRKFAISSNLN
                     SHTKIHTGSQKPFQCRICMRNFSRSDNLARHIRTHTGEKPFACDICGRKFATSGNLTRH
                     TKIHLRGSQL"
     CDS             complement(join(1146..2414,2415..2435,2436..2441))
                     /codon_start=1
                     /product="nuclear-targeted site-specific recombinase"
                     /label=FLPo
                     /note="FLPo is a mouse codon-optimized fusion between the 
                     SV40 nuclear localization signal and a thermostable version
                     of the Saccharomyces cerevisiae site-specific recombinase 
                     FLP.  Recombination occurs at FRT sequences."
                     /translation="MAPKKKRKVMSQFDILCKTPPKVLVRQFVERFERPSGEKIASCAA
                     ELTYLCWMITHNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKK
                     LIPAWEFTIIPYNGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESI
                     WEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGV
                     IIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEY
                     QLLKDNLVRSYNKALKKNAPYPIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWS
                     DKRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQL
                     KGSAEGSIRYPAWNGIISQEVLDYLSSYINRRI"
     CDS             complement(2454..2483)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(2658..2681)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(3009..3047)
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     protein_bind    3510..3543
                     /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)."
     protein_bind    3594..3627
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3652..4240
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3705..3725)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4123..4134)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4306..4325)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4360..4379
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   4413..4553
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4413..4542
                     /label=AAV2 ITR
     rep_origin      4628..5083
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4715..4734)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4925..4946
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5100..5119)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5219..5241
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5279..5297)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5365..5469
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5470..5538,5539..6330)
                     /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(5688..5707)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6501..7089
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6990..7009
                     /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 gcgtgtgtct agactgcaga gggccctgcg tatgagtgca
      181 agtgggtttt aggaccagga tgaggcgggg tgggggtgcc tacctgacga ccgaccccga
      241 cccactggac aagcacccaa cccccattcc ccaaattgcg catcccctat cagagagggg
      301 gaggggaaac aggatgcggc gaggcgcgtg cgcactgcca gcttcagcac cgcggacagt
      361 gccttcgccc ccgcctggcg gcgcgcgcca ccgccgcctc agcactgaag gcgcgctgac
      421 gtcactcgcc ggtcccccgc aaactcccct tcccggccac cttggtcgcg tccgcgccgc
      481 cgccggccca gccggaccgc accacgcgag gcgcgagata ggggggcacg ggcgcgacca
      541 tctgcgctgc ggcgccggcg actcagcgct gcctcagtct gcggtgggca gcggaggagt
      601 cgtgtcgtgc ctgagagcgc agtcgagaag gtaccggatc ctctagagtc gactccggaa
      661 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc
      721 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc
      781 gcgccctata gctggctgcc ccgcaggtga atctttgtgt gccgggtcag attgccggag
      841 gtggcaaact ttctgccaca gatatcacag gcaaaaggtt tttcaccagt atgtgtccgg
      901 atgtgtctgg ccaggttgtc gcttctgcta aagtttctca tacaaattct gcactggaaa
      961 ggtttctggc tgccagtgtg gatcttggtg tggctgttca ggttgctgct gatggcgaac
     1021 ttccggccgc aaatgtcgca ggcgaagggc ttttctccgg tgtgggttct gatgtgcctg
     1081 ctcaggttgg accggtcgct gaagttccgc atgcagatcc ggcactggaa gctaccgggc
     1141 gcgccgatcc gcctgttgat gtagctgctc aggtagtcca gcacctcctg gctgatgatg
     1201 ccgttccagg cggggtatct gatgctgccc tcggcgctgc ccttcagctg ctcgatgtgc
     1261 tgccactcct cgatggggtt ggtctcgtcc ttcagggcga tcatctcctt gctgatgggg
     1321 tcgtaggcgt agtacctgga caccagggcg aagtagtggt cggggatggc ggtgatctgg
     1381 tgggtgtagg tggtcctggc cacggcggag gccctcttgt cgctccagtt gcccaccacg
     1441 tttgtcagct cggtcaggcc cttcatgctc agaaagctgg tcatcaggtg cctgccgatg
     1501 tggctcttag ggccgttctt gatagcgaag atggggtagg gggcgttctt cttcagggcc
     1561 ttgttgtagc tgcgcaccag gttgtccttc agcagctggt actcctgctt gttgctgctg
     1621 ctgttgccgg tcctgttcac tctcttcagc acgggctcgc tgttcctcag gaactcgtcc
     1681 aggtacacca gggggtcgat cctgcctctg gcgctgaaaa agtagatgtg cctggacacg
     1741 cttgtcttgg tctcggtcac caggcactga atgatcacgc ccaggtactt gttctgcacc
     1801 agcttgaagc tcttggggtc cacgttcttg atgtcgctga acctgccgca gttgatgaat
     1861 gtggccagga acaggaactg gtacagggtc ttggtcttgg tgaacctgct ggtgtactcg
     1921 aagctgttca ggatcttctc ggtgatctcc cagatgctct cgccctcgga cagcagggcc
     1981 ttcagcatct tcttgctgtg gctgttgccc ttgtcggcct cctcgctgct ctcgaactgc
     2041 agctgcaggc tggacacgat gtcggtgatg tcgctctggt gcttctggcc gttgtaaggg
     2101 atgatggtga actcccaggc ggggatcagc ttcttcaggc tggcctccag gatggtggcc
     2161 ttctgggtct tgtacttgaa ctgcaggctc ttgttcacga tgtcgaagct caggctgttg
     2221 ctgatgatgg tgttgtagct catgaaggtg gccctcttga tggcggtgcc gttgtgggtg
     2281 atcatccagc acaggtaggt cagctcggcg gcacagctgg cgatcttctc gccgctgggc
     2341 ctctcgaatc tctccacgaa ctgccgcacc agcaccttgg ggggggtctt gcacaggatg
     2401 tcgaactggc tcatcacctt cctcttcttc ttaggagcca ttgtacagct acccagatcc
     2461 tcttctgaga tgagtttttg ttcactacca agcttgcatc cccctccggg atgacggctc
     2521 acataaccgc agcctcctcc cggatgtcga cttacataac cgcaaccacc gccggggtgc
     2581 cgtgaaacgt aaccgcaccc gcctccggga tgcctgctta catatccgct tccgcttcct
     2641 ctagaggagc cgctaccctt atcgtcgtca tccttgtagt cggatccggt gcggtagttg
     2701 atggagatcg ggccgtgcca gtcccagaag ttggcgaagg gcgtgacggc gtacaccgtg
     2761 atggtatagt caacaccagg tttcaggccg ctgatggtag cagtggattt gctgccaggc
     2821 acggtgaatt tttggacagg gctattgcca ccagtttcgc cgtaggtgat gcggtagtag
     2881 cgaatcacct ggtccctcct gctccagctg atctggatgc tggttggtga tgcttccttg
     2941 acctcgagca tgctagctct aaattcagcg tctaggttta cttcactaat ttcactagcg
     3001 agctccggtt cggtcaggcg gcggcgcagt tcttcttcca ggcggctcgg actagtagtg
     3061 ctgaagtaaa cgatgatgat gatgaggatg atggcgcaaa tcactcccaa gatgatcatc
     3121 atcaattggg accctccgcc cttgggttct gaggagacag tgacctgggt cccctgaccc
     3181 ctcgtcatat cccgggccca gctccctttt gcacagtaat agacggccgt gtcctccggt
     3241 ttcaggctgt tcatctgcag atacagcgtg ttcttggcgt tgtctctggc gatggtgaat
     3301 cggcccttca cggactctgt ataggtttca taattgccac tagcactaat agttgagacc
     3361 cattcgagcc cctttcctgg agcctggcgg acccagctca taaaatattt actgaagggg
     3421 aatccagagg ctgcacagga gagtcttaga gaccccccag gccgcaccaa gccgccccca
     3481 gactcctcca gctgcacctc catgctagca taacttcgta taaagtatcc tatacgaagt
     3541 tatttgcctt aacccagaaa ttatcactgt tattctttag aatggtgcaa agaataactt
     3601 cgtataatgt atgctatacg aagttatgaa ttcgatatca agcttatcga taatcaacct
     3661 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     3721 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
     3781 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
     3841 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
     3901 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
     3961 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
     4021 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctatgtt
     4081 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
     4141 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
     4201 cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg agcgctgctc
     4261 gagagatcta cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4321 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4381 atgtatctta cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg agttggccac
     4441 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     4501 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct
     4561 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa
     4621 ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     4681 gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt
     4741 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     4801 cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt
     4861 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     4921 aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt
     4981 gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa
     5041 aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt
     5101 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     5161 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     5221 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     5281 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     5341 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     5401 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     5461 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     5521 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     5581 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     5641 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     5701 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     5761 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     5821 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     5881 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     5941 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     6001 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     6061 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     6121 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     6181 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     6241 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     6301 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     6361 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     6421 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     6481 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     6541 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     6601 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     6661 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     6721 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     6781 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     6841 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     6901 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     6961 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     7021 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     7081 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     7141 gctcacatgt
//
