LOCUS       sequence-423180-        6412 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  transcriptionally regulated, double floxed, postsynaptic marker of 
            PSD95 fused to Citrine.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6412)
  AUTHORS   Willig KI, Wegner W, Muller A, Calvet-Fournier V, Steffens H
  TITLE     Multi-label in vivo STED microscopy by parallelized switching of 
            reversibly switchable fluorescent proteins.
  JOURNAL   Cell Rep. 2021 Jun 1;35(9):109192. doi: 
            10.1016/j.celrep.2021.109192.
  PUBMED    34077731
REFERENCE   2  (bases 1 to 6412)
  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: "Cell Rep.";
            date: "2021-06-1"; volume: "35(9):109192. doi"; pages: " 
            10.1016/j.celrep.2021.109192"
COMMENT     Sequence Label: pAAV-ZFN-hSyn-DIO-PSD95-FingR-Citrine-CCR5TC
FEATURES             Location/Qualifiers
     source          1..6412
                     /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    145..156
                     /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        187..634
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(666..699)
                     /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(750..783)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(798..923)
                     /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(972..1313)
                     /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(1359..2069,2070..2072,2073..2075))
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1401..1422)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1750..1769
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2009..2030
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(2097..2120)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    2397..2430
                     /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    2481..2514
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2539..3127
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2592..2612)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3010..3021)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3159..3635
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3358..3377)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3675..3815
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3675..3804
                     /label=AAV2 ITR
     rep_origin      3890..4345
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3977..3996)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4187..4208
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4362..4381)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4481..4503
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4541..4559)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4627..4731
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4732..4800,4801..5592)
                     /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(4950..4969)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5763..6351
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6252..6271
                     /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 gcgtgtcatc ctcatcgtgt ctagactgca gagggccctg
      181 cgtatgagtg caagtgggtt ttaggaccag gatgaggcgg ggtgggggtg cctacctgac
      241 gaccgacccc gacccactgg acaagcaccc aacccccatt ccccaaattg cgcatcccct
      301 atcagagagg gggaggggaa acaggatgcg gcgaggcgcg tgcgcactgc cagcttcagc
      361 accgcggaca gtgccttcgc ccccgcctgg cggcgcgcgc caccgccgcc tcagcactga
      421 aggcgcgctg acgtcactcg ccggtccccc gcaaactccc cttcccggcc accttggtcg
      481 cgtccgcgcc gccgccggcc cagccggacc gcaccacgcg aggcgcgaga taggggggca
      541 cgggcgcgac catctgcgct gcggcgccgg cgactcagcg ctgcctcagt ctgcggtggg
      601 cagcggagga gtcgtgtcgt gcctgagagc gcagtcgaga aggtaccgga tccgtcgact
      661 ccggaataac ttcgtatagg atactttata cgaagttatg cagaatggta gctggattgt
      721 agctgctatt agcaatatga aacctcttaa taacttcgta tagcatacat tatacgaagt
      781 tatggcgcgc cttaagccat agaagcaaga ttagaataat tttcaagcat aacttcacga
      841 taaagagaac gttgagaaag atcaagtttt ttccattcat cacgagtaaa aagaacagca
      901 acatcttcaa aagtaacaga aacagaaact tgagaagcac cttcacgttg ttcaggagcg
      961 tcgacaatcg atagctggct gccccgcagg tgaatctttg tgtgccgggt cagattgccg
     1021 gaggtggcaa actttctgcc acagatatca caggcaaaag gtttttcacc agtatgtgtc
     1081 cggatgtgtc tggccaggtt gtcgcttctg ctaaagtttc tcatacaaat tctgcactgg
     1141 aaaggtttct ggctgccagt gtggatcttg gtgtggctgt tcaggttgct gctgatggcg
     1201 aacttccggc cgcaaatgtc gcaggcgaag ggcttttctc cggtgtgggt tctgatgtgc
     1261 ctgctcaggt tggaccggtc gctgaagttc cgcatgcaga tccggcactg gaatctagag
     1321 ccagcccctg cacccgctcc ggcgccagct cccctagact tgtacagctc gtccatgccg
     1381 agagtgatcc cggcggcggt cacgaactcc agcaggacca tgtgatcgcg cttctcgttg
     1441 gggtctttgc tcagggcgga ctggtagctc aggtagtggt tgtcgggcag cagcacgggg
     1501 ccgtcgccga tgggggtgtt ctgctggtag tggtcggcga gctgcacgct gccgtcctcg
     1561 atgttgtggc ggatcttgaa gttcaccttg atgccgttct tctgcttgtc ggccatgata
     1621 tagacgttgt ggctgttgta gttgtactcc agcttgtgcc ccaggatgtt gccgtcctcc
     1681 ttgaagtcga tgcccttcag ctcgatgcgg ttcaccaggg tgtcgccctc gaacttcacc
     1741 tcggcgcggg tcttgtagtt gccgtcgtcc ttgaagaaga tggtgcgctc ctggacgtag
     1801 ccttcgggca tggcggactt gaagaagtcg tgctgcttca tgtggtcggg gtagcgggcg
     1861 aagcacatca ggccgtagcc gaaggtggtc acgagggtgg gccagggcac gggcagcttg
     1921 ccggtggtgc agatgaactt cagggtcagc ttgccgtagg tggcatcgcc ctcgccctcg
     1981 ccggacacgc tgaacttgtg gccgtttacg tcgccgtcca gctcgaccag gatgggcacc
     2041 accccggtga acagctcctc gcccttgctc accatggatc tactggagcc gctaccctta
     2101 tcgtcgtcat ccttgtaatc ggttccggtg cggtagttga tggagatcgg cggccaggcg
     2161 gagcggtagg cgctgaagat cgtgacggcg tacaccgtga tggtatagtc gacaccaggt
     2221 ttcaggccgc tgatggtagc agtggacttg ctgccaggca cggtgaattc ctggacaggg
     2281 ctattgccac cagtttcacc gtaggtgatg cggtagtagc gaacgtggcg caagtggagc
     2341 acccagctga tctggatgct ggttggtgat gcttccttga cttcgagcat gctagcataa
     2401 cttcgtataa agtatcctat acgaagttat ttgccttaac ccagaaatta tcactgttat
     2461 tctttagaat ggtgcaaaga ataacttcgt ataatgtatg ctatacgaag ttatgaattc
     2521 gatatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     2581 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     2641 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
     2701 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
     2761 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
     2821 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     2881 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg
     2941 tcctttcctt ggctgctcgc ctatgttgcc acctggattc tgcgcgggac gtccttctgc
     3001 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
     3061 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
     3121 tccccgcatc gataccgagc gctgctcgag agatctacgg gtggcatccc tgtgacccct
     3181 ccccagtgcc tctcctggcc ctggaagttg ccactccagt gcccaccagc cttgtcctaa
     3241 taaaattaag ttgcatcatt ttgtctgact aggtgtcctt ctataatatt atggggtgga
     3301 ggggggtggt atggagcaag gggcaagttg ggaagacaac ctgtagggcc tgcggggtct
     3361 attgggaacc aagctggagt gcagtggcac aatcttggct cactgcaatc tccgcctcct
     3421 gggttcaagc gattctcctg cctcagcctc ccgagttgtt gggattccag gcatgcatga
     3481 ccaggctcag ctaatttttg tttttttggt agagacgggg tttcaccata ttggccaggc
     3541 tggtctccaa ctcctaatct caggtgatct acccaccttg gcctcccaaa ttgctgggat
     3601 tacaggcgtg aaccactgct cccttccctg tccttctgat tttgtaggta accacgtgcg
     3661 gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
     3721 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc
     3781 ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct
     3841 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt
     3901 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     3961 agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     4021 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     4081 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga
     4141 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     4201 caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg
     4261 ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     4321 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc
     4381 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     4441 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     4501 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     4561 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     4621 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     4681 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     4741 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     4801 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     4861 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     4921 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     4981 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     5041 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     5101 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     5161 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     5221 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     5281 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     5341 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     5401 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     5461 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     5521 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     5581 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     5641 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     5701 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     5761 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     5821 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     5881 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     5941 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6001 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6061 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6121 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6181 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcatgagg
     6241 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     6301 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     6361 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gt
//
