LOCUS       sequence-392985-        6418 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Genetically encoded fluorescent probe for labelling endogenous 
            inhibitory synapses in vivo.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6418)
  AUTHORS   Okur Z, Schlauri N, Bitsikas V, Panopoulou M, Karmakar K, Schreiner 
            D, Scheiffele P
  TITLE     Control of neuronal excitation-inhibition balance by BMP-SMAD1 
            signaling
  JOURNAL   bioRxiv 2023
REFERENCE   2  (bases 1 to 6418)
  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: "bioRxiv 
            2023"
COMMENT     Sequence Label: pAAV-ZnF-hSyn-DIO-GPHNFingR-eGFP
FEATURES             Location/Qualifiers
     source          1..6418
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     protein_bind    148..159
                     /label=ZF binding site
                     /bound_moiety="CCR5TC zinc-finger domain"
                     /note="target sequence for the CCR5TC zinc-finger domain 
                     from the CCR5-224 (+) zinc-finger nuclease (Perez et al., 
                     2008; Gross et al., 2013)"
     promoter        189..636
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(674..707)
                     /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(758..791)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(806..931)
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the rat zinc finger protein Kid-1 
                     (Witzgall et al., 1994)"
                     /label=KRAB-A
                     /translation="VSVTFEDVAVLFTRDEWKKLDLSQRSLYREVMLENYSNLASM"
     CDS             complement(980..1321)
                     /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(1367..2074,2075..2077))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELY"
     CDS             complement(join(1367..2074,2075..2077))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELY"
     primer_bind     complement(1406..1427)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1755..1774
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2014..2035
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(2099..2122)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    2403..2436
                     /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    2487..2520
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2545..3133
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2598..2618)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3016..3027)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3165..3641
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3364..3383)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3681..3821
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3681..3810
                     /label=AAV2 ITR
     rep_origin      3896..4351
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3983..4002)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4193..4214
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4368..4387)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4487..4509
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4547..4565)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4633..4737
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4738..4806,4807..5598)
                     /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(4956..4975)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5769..6357
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6258..6277
                     /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 gcgtagagtc atcctcatcg tcctagactg cagagggccc
      181 tgcgtatgag tgcaagtggg ttttaggacc aggatgaggc ggggtggggg tgcctacctg
      241 acgaccgacc ccgacccact ggacaagcac ccaaccccca ttccccaaat tgcgcatccc
      301 ctatcagaga gggggagggg aaacaggatg cggcgaggcg cgtgcgcact gccagcttca
      361 gcaccgcgga cagtgccttc gcccccgcct ggcggcgcgc gccaccgccg cctcagcact
      421 gaaggcgcgc tgacgtcact cgccggtccc ccgcaaactc cccttcccgg ccaccttggt
      481 cgcgtccgcg ccgccgccgg cccagccgga ccgcaccacg cgaggcgcga gatagggggg
      541 cacgggcgcg accatctgcg ctgcggcgcc ggcgactcag cgctgcctca gtctgcggtg
      601 ggcagcggag gagtcgtgtc gtgcctgaga gcgcagtcga gaaggtaccg gatcctctag
      661 agtcgactcc ggaataactt cgtataggat actttatacg aagttatgca gaatggtagc
      721 tggattgtag ctgctattag caatatgaaa cctcttaata acttcgtata gcatacatta
      781 tacgaagtta tggcgcgcct taagccatag aagcaagatt agaataattt tcaagcataa
      841 cttcacgata aagagaacgt tgagaaagat caagtttttt ccattcatca cgagtaaaaa
      901 gaacagcaac atcttcaaaa gtaacagaaa cagaaacttg agaagcacct tcacgttgtt
      961 caggagcgtc gacaatcgat agctggctgc cccgcaggtg aatctttgtg tgccgggtca
     1021 gattgccgga ggtggcaaac tttctgccac agatatcaca ggcaaaaggt ttttcaccag
     1081 tatgtgtccg gatgtgtctg gccaggttgt cgcttctgct aaagtttctc atacaaattc
     1141 tgcactggaa aggtttctgg ctgccagtgt ggatcttggt gtggctgttc aggttgctgc
     1201 tgatggcgaa cttccggccg caaatgtcgc aggcgaaggg cttttctccg gtgtgggttc
     1261 tgatgtgcct gctcaggttg gaccggtcgc tgaagttccg catgcagatc cggcactgga
     1321 atctagagcc agcccctgca cccgctccag cgccagctcc cctagagtac agctcgtcca
     1381 tgccgagagt gatcccggcg gcggtcacga actccagcag gaccatgtga tcgcgcttct
     1441 cgttggggtc tttgctcagg gcggactggg tgctcaggta gtggttgtcg ggcagcagca
     1501 cggggccgtc gccgatgggg gtgttctgct ggtagtggtc ggcgagctgc acgctgccgt
     1561 cctcgatgtt gtggcggatc ttgaagttca ccttgatgcc gttcttctgc ttgtcggcca
     1621 tgatatagac gttgtggctg ttgtagttgt actccagctt gtgccccagg atgttgccgt
     1681 cctccttgaa gtcgatgccc ttcagctcga tgcggttcac cagggtgtcg ccctcgaact
     1741 tcacctcggc gcgggtcttg tagttgccgt cgtccttgaa gaagatggtg cgctcctgga
     1801 cgtagccttc gggcatggcg gacttgaaga agtcgtgctg cttcatgtgg tcggggtagc
     1861 ggctgaagca ctgcacgccg taggtcaggg tggtcacgag ggtgggccag ggcacgggca
     1921 gcttgccggt ggtgcagatg aacttcaggg tcagcttgcc gtaggtggca tcgccctcgc
     1981 cctcgccgga cacgctgaac ttgtggccgt ttacgtcgcc gtccagctcg accaggatgg
     2041 gcaccacccc ggtgaacagc tcctcgccct tgctcacccc agatctggag ccgctaccct
     2101 tatcgtcgtc atccttgtaa tcggatccgg tgcggtagtt gatggagatc gggatcgggt
     2161 cctggtagtt ccagtggtcg atcgtgacgg cgtacaccgt gatggtatag tcgacaccag
     2221 gtttcaggct gctgatggta gcagtggact tgctgccagg cacggtgaat tcctggacag
     2281 ggctattgcc accagtttca ccgtaggtga tgcggtagta gcgaaccatg accttgtact
     2341 tgccccagct gatctggatg ctggttggtg atgcttcctt gacttcgagc atggtggcta
     2401 gcataacttc gtataaagta tcctatacga agttatttgc cttaacccag aaattatcac
     2461 tgttattctt tagaatggtg caaagaataa cttcgtataa tgtatgctat acgaagttat
     2521 gaattcgata tcaagcttat cgataatcaa cctctggatt acaaaatttg tgaaagattg
     2581 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     2641 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
     2701 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact
     2761 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc
     2821 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc
     2881 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa
     2941 tcatcgtcct ttccttggct gctcgcctat gttgccacct ggattctgcg cgggacgtcc
     3001 ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg
     3061 gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg
     3121 gccgcctccc cgcatcgata ccgagcgctg ctcgagagat ctacgggtgg catccctgtg
     3181 acccctcccc agtgcctctc ctggccctgg aagttgccac tccagtgccc accagccttg
     3241 tcctaataaa attaagttgc atcattttgt ctgactaggt gtccttctat aatattatgg
     3301 ggtggagggg ggtggtatgg agcaaggggc aagttgggaa gacaacctgt agggcctgcg
     3361 gggtctattg ggaaccaagc tggagtgcag tggcacaatc ttggctcact gcaatctccg
     3421 cctcctgggt tcaagcgatt ctcctgcctc agcctcccga gttgttggga ttccaggcat
     3481 gcatgaccag gctcagctaa tttttgtttt tttggtagag acggggtttc accatattgg
     3541 ccaggctggt ctccaactcc taatctcagg tgatctaccc accttggcct cccaaattgc
     3601 tgggattaca ggcgtgaacc actgctccct tccctgtcct tctgattttg taggtaacca
     3661 cgtgcggacc gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg
     3721 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
     3781 ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt
     3841 tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg
     3901 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3961 cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     4021 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     4081 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg
     4141 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     4201 ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga tttataaggg
     4261 attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     4321 aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct
     4381 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     4441 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     4501 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     4561 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     4621 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     4681 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     4741 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     4801 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     4861 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     4921 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     4981 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     5041 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     5101 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     5161 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     5221 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     5281 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     5341 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     5401 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     5461 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     5521 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     5581 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     5641 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     5701 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     5761 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     5821 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     5881 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     5941 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     6001 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     6061 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     6121 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     6181 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     6241 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     6301 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     6361 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt
//
