LOCUS       sequence-477543-        6319 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV version of Tet inducible PFE3 for ablating excitatory synapses..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6319)
  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 6319)
  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-TRE-PFE3-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..6319
                     /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        148..462
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    156..174
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    192..210
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    227..245
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    263..281
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    299..317
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    334..352
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    370..388
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     435..459
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             749..775
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1667..1723
                     /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(1736..1738,1739..2449)
                     /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(1778..1799)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2039..2058)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2386..2407
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2459..3047
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2512..2532)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2930..2941)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3066..3542
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3265..3284)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3582..3722
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3582..3711
                     /label=AAV2 ITR
     rep_origin      3797..4252
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3884..3903)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4094..4115
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4269..4288)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4388..4410
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4448..4466)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4534..4638
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4639..4707,4708..5499)
                     /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(4857..4876)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5670..6258
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6159..6178
                     /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 gcgtctcgag tttactccct atcagtgata gagaacgtat
      181 gtcgagttta ctccctatca gtgatagaga acgatgtcga gtttactccc tatcagtgat
      241 agagaacgta tgtcgagttt actccctatc agtgatagag aacgtatgtc gagtttactc
      301 cctatcagtg atagagaacg tatgtcgagt ttatccctat cagtgataga gaacgtatgt
      361 cgagtttact ccctatcagt gatagagaac gtatgtcgag gtaggcgtgt acggtgggag
      421 gcctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagaat tctctagaat
      481 gctcgaagtc aaggaagcat caccaaccag catccagatc agctgggtgc tccacttgcg
      541 ccacgttcgc tactaccgca tcacctacgg tgaaactggt ggcaatagcc ctgtccagga
      601 attcaccgtg cctggcagca agtccactgc taccatcagc ggcctgaaac ctggtgtcga
      661 ctataccatc acggtgtacg ccgtcacgat cttcagcgcc taccgctccg cctggccgcc
      721 gatctccatc aactaccgca ccaccggtta cccatacgat gttccagatt acgctggtgg
      781 aagcacgcgt tgtgtgattt gccaggggcg gcctaaaaat ggttgcattg ttcacggcaa
      841 aaccggacac ctgatgtcat gtttcacgtg tgcaaaggca ctagctgcgg cgaatgcgcc
      901 ctgccccgtg tgcagagcta gcaagctctc aggtggcgga gggagcggag gaggtggctc
      961 tggaggtggt ggctcagctg aagcatggta caatctggga aacgcttatt acaagcaggg
     1021 cgactatcaa aaagccattg agtattacca aaaggctctt gagctagacc ctaataacgc
     1081 cgaagcgtgg tataacctag gcaacgcata ttacaagcag ggagactacc aaaaggcaat
     1141 cgaatattac cagaaggcac tcgaactgga cccaaataac gcagaggctt ggtataatct
     1201 gggaaacgcc tactataaac agggtgacta ccaaaaggcg atagaggact atcagaaggc
     1261 cttggagctc gatccgaaca atctgcaacg tagtgctggt ggcggagggt ctggtggagg
     1321 aggctcagga ggaggtggcg catcaggtac caagaagaaa ctcagcaagt cagacatcat
     1381 gttggtgcag agcgccaatg tccctagcaa cccgacccag gtgccggtag aggagtccgg
     1441 aggcttcggt tcccatcacc acaatcagaa ctattgctat caggtctgcc tcaccccaga
     1501 gtccgccaag accgacctga tgttcttgaa gccctgcagc ccttcccgga gtacagacgc
     1561 tgagcacaat ccctgtggag ccatcgtcac cgggtacagc gaccagcaac cagacatcat
     1621 ttccaacgga agcattttgt ccaacgagaa tggtggatca tgtacagcta ctaacttcag
     1681 cctgctgaag caggctggag acgtggagga gaaccctgga cctggaggta ctggggtgag
     1741 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     1801 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     1861 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     1921 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     1981 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     2041 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     2101 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     2161 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     2221 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     2281 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     2341 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     2401 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtaaaagc ttatcgataa
     2461 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     2521 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     2581 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     2641 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     2701 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
     2761 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     2821 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc
     2881 ctatgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
     2941 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     3001 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgaga
     3061 tctacgggtg gcatccctgt gacccctccc cagtgcctct cctggccctg gaagttgcca
     3121 ctccagtgcc caccagcctt gtcctaataa aattaagttg catcattttg tctgactagg
     3181 tgtccttcta taatattatg gggtggaggg gggtggtatg gagcaagggg caagttggga
     3241 agacaacctg tagggcctgc ggggtctatt gggaaccaag ctggagtgca gtggcacaat
     3301 cttggctcac tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct cagcctcccg
     3361 agttgttggg attccaggca tgcatgacca ggctcagcta atttttgttt ttttggtaga
     3421 gacggggttt caccatattg gccaggctgg tctccaactc ctaatctcag gtgatctacc
     3481 caccttggcc tcccaaattg ctgggattac aggcgtgaac cactgctccc ttccctgtcc
     3541 ttctgatttt gtaggtaacc acgtgcggac cgagcggccg caggaacccc tagtgatgga
     3601 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc
     3661 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca
     3721 ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg
     3781 tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     3841 acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc
     3901 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     3961 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat
     4021 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     4081 acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc
     4141 tattcttttg atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg
     4201 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc
     4261 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     4321 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     4381 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     4441 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     4501 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     4561 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     4621 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     4681 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     4741 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     4801 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     4861 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     4921 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     4981 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     5041 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     5101 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     5161 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     5221 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     5281 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     5341 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     5401 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     5461 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     5521 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     5581 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     5641 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     5701 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     5761 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     5821 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     5881 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     5941 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     6001 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     6061 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     6121 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     6181 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     6241 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     6301 tggccttttg ctcacatgt
//
