LOCUS       sequence-477492-        6669 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV version of constitutive PFE3 for ablating excitatory synapses..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6669)
  AUTHORS   Bareghamyan A, Deng C, Daoudi S, Yadav SC, Lu X, Zhang W, Campbell 
            RE, Kramer RH, Chenoweth DM, Arnold DB
  TITLE     A toolbox for ablating excitatory and inhibitory synapses.
  JOURNAL   eLife. 2025 Apr 29;13:RP103757. doi: 10.7554/eLife.103757.
  PUBMED    40298577
REFERENCE   2  (bases 1 to 6669)
  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"; journalName: "eLife."; 
            date: "2025-04-29"; volume: "13:RP103757. doi"; pages: " 
            10.7554/eLife.103757"
COMMENT     Sequence Label: AAV-CAG-PFE3-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..6669
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     promoter        441..718
                     /label=chicken beta-actin promoter
     primer_bind     997..1019
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1081..1100
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1403..1429
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2321..2377
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(2390..2392,2393..3103)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDEL*K"
     primer_bind     complement(2432..2453)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2693..2712)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3040..3061
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3113..3701
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3166..3186)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3584..3595)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3744..3865
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3755..3774)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3809..3828
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4027..4156
                     /label=AAV2 ITR
     rep_origin      complement(4393..4981)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4473..4492)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5152..5943,5944..6012))
                     /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     5775..5794
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6013..6117)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6143..6598)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6280..6301)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6492..6511
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct
      181 aggaagagta ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      241 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      301 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      361 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      421 agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc
      481 ccccccctcc ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat
      541 gggggcgggg gggggggggg ggcgcgcgcc gggggggggg gggsggggsg rggggsgggg
      601 sggggsgrgg cggagaggkg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc
      661 ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg
      721 agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc
      781 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      841 gggctgtaat tagcgcttgg tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag
      901 ccttgagggg ctccgggagg gccctttgtg cggggggagc ggctcggggc tgtccgcggg
      961 gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg
     1021 gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg
     1081 gcaacgtgct ggttattgtg ctgtctcatc attttggcaa agaattggat ccatgctcga
     1141 agtcaaggaa gcatcaccaa ccagcatcca gatcagctgg gtgctccact tgcgccacgt
     1201 tcgctactac cgcatcacct acggtgaaac tggtggcaat agccctgtcc aggaattcac
     1261 cgtgcctggc agcaagtcca ctgctaccat cagcggcctg aaacctggtg tcgactatac
     1321 catcacggtg tacgccgtca cgatcttcag cgcctaccgc tccgcctggc cgccgatctc
     1381 catcaactac cgcaccaccg gttacccata cgatgttcca gattacgctg gtggaagcac
     1441 gcgttgtgtg atttgccagg ggcggcctaa aaatggttgc attgttcacg gcaaaaccgg
     1501 acacctgatg tcatgtttca cgtgtgcaaa ggcactagct gcggcgaatg cgccctgccc
     1561 cgtgtgcaga gctagcaagc tctcaggtgg cggagggagc ggaggaggtg gctctggagg
     1621 tggtggctca gctgaagcat ggtacaatct gggaaacgct tattacaagc agggcgacta
     1681 tcaaaaagcc attgagtatt accaaaaggc tcttgagcta gaccctaata acgccgaagc
     1741 gtggtataac ctaggcaacg catattacaa gcagggagac taccaaaagg caatcgaata
     1801 ttaccagaag gcactcgaac tggacccaaa taacgcagag gcttggtata atctgggaaa
     1861 cgcctactat aaacagggtg actaccaaaa ggcgatagag gactatcaga aggccttgga
     1921 gctcgatccg aacaatctgc aacgtagtgc tggtggcgga gggtctggtg gaggaggctc
     1981 aggaggaggt ggcgcatcag gtaccaagaa gaaactcagc aagtcagaca tcatgttggt
     2041 gcagagcgcc aatgtcccta gcaacccgac ccaggtgccg gtagaggagt ccggaggctt
     2101 cggttcccat caccacaatc agaactattg ctatcaggtc tgcctcaccc cagagtccgc
     2161 caagaccgac ctgatgttct tgaagccctg cagcccttcc cggagtacag acgctgagca
     2221 caatccctgt ggagccatcg tcaccgggta cagcgaccag caaccagaca tcatttccaa
     2281 cggaagcatt ttgtccaacg agaatggtgg atcatgtaca gctactaact tcagcctgct
     2341 gaagcaggct ggagacgtgg aggagaaccc tggacctgga ggtactgggg tgagcaaggg
     2401 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     2461 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     2521 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     2581 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     2641 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     2701 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     2761 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     2821 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     2881 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     2941 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     3001 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     3061 gaccgccgcc gggatcactc tcggcatgga cgagctgtaa aagcttatcg ataatcaacc
     3121 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     3181 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     3241 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     3301 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     3361 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     3421 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     3481 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt
     3541 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     3601 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg
     3661 ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc gtcgacccgg
     3721 gcggccgctt cgagcagaca tgataagata cattgatgag tttggacaaa ccacaactag
     3781 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     3841 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     3901 tcagggggag atgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtaaaat
     3961 cgataaggat cttcctagag catggctacg tagataagta gcatggcggg ttaatcatta
     4021 actacaagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
     4081 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
     4141 gcgagcgagc gcgcagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     4201 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4261 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4321 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4381 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4441 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4501 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4561 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4621 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4681 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4741 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4801 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     4861 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4921 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4981 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     5041 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     5101 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     5161 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     5221 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     5281 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     5341 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     5401 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     5461 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     5521 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5581 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     5641 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     5701 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     5761 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     5821 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     5881 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     5941 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     6001 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     6061 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     6121 atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta
     6181 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     6241 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     6301 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     6361 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     6421 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     6481 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     6541 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc
     6601 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     6661 ttacgccag
//
