LOCUS       sequence-274692-        7476 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a specific PSD-95 binder (Xph20) fused to mEos3.2, CCR5 
            left-handed zinc finger, and KRAB(A) transcriptional repressor 
            domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7476)
  AUTHORS   Rimbault C, Breillat C, Compans B, Toulme E, Nunes Vicente F, 
            Fernandez-Monreal M, Mascalchi P, Genuer C, Puente-Munoz V, 
            Gauthereau I, Hosy E, Claverol S, Giannone G, Chamma I, Mackereth 
            CD, Poujol C, Choquet D, Sainlos M
  TITLE     Engineering paralog-specific PSD-95 recombinant binders as minimally
            interfering multimodal probes for advanced imaging techniques.
  JOURNAL   Elife. 2024 Jan 3;13:e69620. doi: 10.7554/eLife.69620.
  PUBMED    38167295
REFERENCE   2  (bases 1 to 7476)
  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: "2024-01-3"; volume: "13:e69620. doi"; pages: " 
            10.7554/eLife.69620"
COMMENT     Sequence Label: pCAG_Xph20-mEos3.2-CCR5TC
FEATURES             Location/Qualifiers
     source          1..7476
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    25..36
                     /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)"
     enhancer        40..419
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        421..697
                     /label=chicken beta-actin promoter
     intron          698..1714
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1638..1660
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1722..1741
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1786..1795
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2110..2784
                     /codon_start=1
                     /product="improved monomeric variant of green-to-red 
                     photoswitchable fluorescent protein EosFP (Zhang et al., 
                     2012)"
                     /label=mEos3.2
                     /translation="SAIKPDMKIKLRMEGNVNGHHFVIDGDGTGKPFEGKQSMDLEVKE
                     GGPLPFAFDILTTAFHYGNRVFAKYPDNIQDYFKQSFPKGYSWERSLTFEDGGICNARN
                     DITMEGDTFYNKVRFYGTNFPANGPVMQKKTLKWEPSTEKMYVRDGVLTGDIEMALLLE
                     GNAHYRCDFRTTYKAKEKGVKLPGAHFVDHCIEILSHDKDYNKVKLYEHAVAHSGLPDN
                     ARR"
     CDS             2830..3171
                     /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             3220..3345
                     /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"
     intron          3394..3526
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     3509..3528
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    3535..4123
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3588..3608)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4006..4017)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4150..4167)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4156..4267
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4363..4383)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4363..4381)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4498..4517)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4563..4618
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4617..4636)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4979..4995)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4979..4995)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4992..5014)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5003..5019
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5027..5033,5034..5051,5052..5057))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5072..5093
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5152..5347
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5198..5333
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5353..5487
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5390..5409)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5444..5463
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5555..5572)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5726..6314)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5806..5825)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6485..7276,7277..7345))
                     /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     7108..7127
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7346..7450)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagagtcatc ctcatcgtcg acattgatta ttgactagtt
       61 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
      121 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
      181 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
      241 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
      301 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
      361 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
      421 tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc ccacccccaa
      481 ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg gggggggggg
      541 ggcgcgcnca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc
      601 ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg
      661 gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg
      721 ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt
      781 actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agcgcttggt
      841 ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg
      901 ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc
      961 cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg cggggctttg
     1021 tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg
     1081 gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg
     1141 tgtgggcgcg tcggtcgggc tgcaaccccc cctgcacccc cctccccgag ttgctgagca
     1201 cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc gcggggctcg ccgtgccggg
     1261 cggggggtgg cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg ccggggaggg
     1321 ctcgggggag gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg cggcgagccg
     1381 cagccattgc cttttatggt aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat
     1441 ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc gcggggcgaa
     1501 gcggtgcggc gccggcagga aggaaatggg cggggagggc cttcgtgcgt cgccgcgccg
     1561 ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg ggggacggct gccttcgggg
     1621 gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta gagcctctgc
     1681 taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc tggttattgt
     1741 gctgtctcat cattttggca aagaattcgg taccgcgggc ccggggccac catggcgggc
     1801 agctctgtca gttccgtgcc gaccaaactg gaagtggttg cggccacccc gacgtctctg
     1861 ctgatcagct gggatgcagt ggcgaagaat gtgaaggtcg gttattaccg tatcacctat
     1921 ggcgaaacgg gcggtaactc cccggttcag gaattcaccg tcccgggtag ttcctcaacc
     1981 gcaacgattt ccggcctgtc accgggtgtc gactatacca ttacggttta cgctaatggg
     2041 gtgctgagta agatggtgct tccgatttct atcaattacc gtaccggtag cggctccagt
     2101 agatctggga gtgcgattaa gccagacatg aagatcaaac tccgtatgga aggcaacgta
     2161 aacgggcacc actttgtgat cgacggagat ggtacaggca agccttttga gggaaaacag
     2221 agtatggatc ttgaagtcaa agagggcgga cctctgcctt ttgcctttga tatcctgacc
     2281 actgcattcc attacggcaa cagggtattc gccaaatatc cagacaacat acaagactat
     2341 tttaagcagt cgtttcctaa ggggtattcg tgggaacgaa gcttgacttt cgaagacggg
     2401 ggcatttgca acgccagaaa cgacataaca atggaagggg acactttcta taataaagtt
     2461 cgattttatg gtaccaactt tcccgccaat ggtccagtta tgcagaagaa gacgctgaaa
     2521 tgggagccct ccactgagaa aatgtatgtg cgtgatggag tgctgacggg tgatattgag
     2581 atggctttgt tgcttgaagg aaatgcccat taccgatgtg acttcagaac tacttacaaa
     2641 gctaaggaga agggtgtcaa gttaccaggc gcccactttg tggaccactg cattgagatt
     2701 ttaagccatg acaaagatta caacaaggtt aagctgtatg agcatgctgt tgctcattct
     2761 ggattgcctg acaatgccag acgagctagc ggagctggcg ctggagcggg tgcaggggct
     2821 ggctctagat tccagtgccg gatctgcatg cggaacttca gcgaccggtc caacctgagc
     2881 aggcacatca gaacccacac cggagaaaag cccttcgcct gcgacatttg cggccggaag
     2941 ttcgccatca gcagcaacct gaacagccac accaagatcc acactggcag ccagaaacct
     3001 ttccagtgca gaatttgtat gagaaacttt agcagaagcg acaacctggc cagacacatc
     3061 cggacacata ctggtgaaaa accttttgcc tgtgatatct gtggcagaaa gtttgccacc
     3121 tccggcaatc tgacccggca cacaaagatt cacctgcggg gcagccagct atcgattgtc
     3181 gacgctcctg aacaacgtga aggtgcttct caagtttctg tttctgttac ttttgaagat
     3241 gttgctgttc tttttactcg tgatgaatgg aaaaaacttg atctttctca acgttctctt
     3301 tatcgtgaag ttatgcttga aaattattct aatcttgctt ctatggctta aacgcgttgt
     3361 acaagtaaag cggccccgga ctcgaggccg caggtaagta tcaaggttac aagacaggtt
     3421 taaggagacc aatagaaact gggcttgtcg agacagagaa gactcttgcg tttctgatag
     3481 gcacctattg gtcttactga catccacttt gcctttctct ccacaggtgt cgacaatcaa
     3541 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     3601 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     3661 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc
     3721 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg
     3781 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc
     3841 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
     3901 actgacaatt ccgtggtgtt gtcggggaag ctgacgtcct ttccatggct gctcgcctgt
     3961 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca
     4021 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt
     4081 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcctggaat tcgagctcgg
     4141 tacgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     4201 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     4261 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     4321 gcaaggggga ggattgggaa gacaatagcc cagcttttgt tccctttagt gagggttaat
     4381 tgcgcgcttg gcgtaatcat ggtcatagsy kwwtycwsts ykmarktgca ggctgcctat
     4441 cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca aataccactg agatcgatct
     4501 ttttccctct gccaaaaatt atggggacat catgaagccc cttgagcatc tgacttctgg
     4561 ctaataaagg aaatttattt tcattgcaat agtgtgttgg aattttttgt gtctctcact
     4621 cggaaggaca tatgggaggg caaatcattt aaaacatcag aatgagtatt tggtttagag
     4681 tttggcaaca tatgcccata tgctggctgc catgaacaaa ggttggctat aaagaggtca
     4741 tcagtatatg aaacagcccc ctgctgtcca ttccttattc catagaaaag ccttgacttg
     4801 aggttagatt ttttttatat tttgttttgt gttatttttt tctttaacat ccctaaaatt
     4861 ttccttacat gttttactag ccagattttt cctcctctcc tgactactcc cagtcatagc
     4921 tgtccctctt ctcttatgga gatccctcga cctgcagccc aagcttggcg taatcatggt
     4981 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     5041 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     5101 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagcggatc cgcatctcaa
     5161 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     5221 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     5281 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     5341 ttgcaaaaag ctaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     5401 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     5461 atcaatgtat cttatcatgt ctggatccgc tgcattaatg aatcggccaa cgcgcgggga
     5521 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     5581 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     5641 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     5701 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     5761 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     5821 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     5881 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     5941 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     6001 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     6061 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     6121 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     6181 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     6241 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     6301 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     6361 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     6421 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     6481 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     6541 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     6601 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     6661 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     6721 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     6781 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     6841 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     6901 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     6961 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     7021 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     7081 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     7141 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     7201 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     7261 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     7321 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     7381 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     7441 gggttccgcg cacatttccc cgaaaagtgc cacctg
//
