LOCUS       sequence-274698-        7373 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a specific PSD-95 binder (Xph20) fused to SNAPf, CCR5TC zinc
            finger, and KRAB(A) transcriptional repressor domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7373)
  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 7373)
  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-SNAPf-CCR5TC
FEATURES             Location/Qualifiers
     source          1..7373
                     /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"
     intron          697..1713
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1637..1659
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1721..1740
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1785..1794
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2115..2657
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="DKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVEV
                     PAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKV
                     VKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGGLAV
                     KEWLLAHEGHRLGKPGLG"
     CDS             2727..3068
                     /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             3117..3242
                     /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          3291..3423
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     3406..3425
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    3432..4020
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3485..3505)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3903..3914)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4047..4064)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4053..4164
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4260..4280)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4260..4278)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4395..4414)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4460..4515
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4514..4533)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4876..4892)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4876..4892)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4889..4911)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4900..4916
                     /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(4924..4930,4931..4948,4949..4954))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4969..4990
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5049..5244
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5095..5230
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5250..5384
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5287..5306)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5341..5360
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5452..5469)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5623..6211)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5703..5722)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6382..7173,7174..7242))
                     /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     7005..7024
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7243..7347)
                     /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 ggcgcgccrg gggggggggg gggggggggg gggggggggg gggsgrggsg gagaggtgcg
      601 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
      661 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc
      721 cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta
      781 ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta gcgcttggtt
      841 taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct ccgggagggc
      901 cctttgtgcg gggggagcgg ctcggggggt gcgtgcgtgt gtgtgtgcgt ggggagcgcc
      961 gcgtgcggct ccgcgctgcc cggcggctgt gagcgctgcg ggcgcggcgc ggggctttgt
     1021 gcgctccgca gtgtgcgcga ggggagcgcg gccgggggcg gtgccccgcg gtgcgggggg
     1081 ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg tgggggggtg agcagggggt
     1141 gtgggcgcgt cggtcgggct gcaacccccc ctgcaccccc ctccccgagt tgctgagcac
     1201 ggcccggctt cgggtgcggg gctccgtacg gggcgtggcg cggggctcgc cgtgccgggc
     1261 ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc cgcctcgggc cggggrgggc
     1321 tcgggggagg ggcgcggcgg cccccggagc gccggcggct gtcgaggcgc ggcgagccgc
     1381 agccattgcc ttttatggta atcgtgcgag agggcgcagg gacttccttt gtcccaaatc
     1441 tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct ctagcgggcg cggggcgaag
     1501 cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc ttcgtgcgtc gccgcgccgc
     1561 cgtccccttc tccctctcca gcctcggggc tgtccgcggg gggacggctg ccttcggggg
     1621 ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag agcctctgct
     1681 aaccatgttc atgccttctt ctttttccta cagctcctgg gcaacgtgct ggttattgtg
     1741 ctgtctcatc attttggcaa agaattcggt accgcgggcc cggggccacc atggcgggca
     1801 gctctgtcag ttccgtgccg accaaactgg aagtggttgc ggccaccccg acgtctctgc
     1861 tgatcagctg ggatgcagtg gcgaagaatg tgaaggtcgg ttattaccgt atcacctatg
     1921 gcgaaacggg cggtaactcc ccggttcagg aattcaccgt cccgggtagt tcctcaaccg
     1981 caacgatttc cggcctgtca ccgggtgtcg actataccat tacggtttac gctaatgggg
     2041 tgctgagtaa gatggtgctt ccgatttcta tcaattaccg taccggtagc ggctccagta
     2101 gatctggtag cggcgacaaa gactgcgaaa tgaagcgcac caccctggat agccctctgg
     2161 gcaagctgga actgtctggg tgcgaacagg gcctgcaccg tatcatcttc ctgggcaaag
     2221 gaacatctgc cgccgacgcc gtggaagtgc ctgccccagc cgccgtgctg ggcggaccag
     2281 agccactgat gcaggccacc gcctggctca acgcctactt tcaccagcct gaggccatcg
     2341 aggagttccc tgtgccagcc ctgcaccacc cagtgttcca gcaggagagc tttacccgcc
     2401 aggtgctgtg gaaactgctg aaagtggtga agttcggaga ggtcatcagc tacagccacc
     2461 tggccgccct ggccggcaat cccgccgcca ccgccgccgt gaaaaccgcc ctgagcggaa
     2521 atcccgtgcc cattctgatc ccctgccacc gggtggtgca gggcgacctg gacgtggggg
     2581 gctacgaggg cgggctcgcc gtgaaagagt ggctgctggc ccacgagggc cacagactgg
     2641 gcaagcctgg gctgggtcct gcaggcggta gcggctcctc tgctagcgga gctggcgctg
     2701 gagcgggtgc aggggctggc tctagattcc agtgccggat ctgcatgcgg aacttcagcg
     2761 accggtccaa cctgagcagg cacatcagaa cccacaccgg agaaaagccc ttcgcctgcg
     2821 acatttgcgg ccggaagttc gccatcagca gcaacctgaa cagccacacc aagatccaca
     2881 ctggcagcca gaaacctttc cagtgcagaa tttgtatgag aaactttagc agaagcgaca
     2941 acctggccag acacatccgg acacatactg gtgaaaaacc ttttgcctgt gatatctgtg
     3001 gcagaaagtt tgccacctcc ggcaatctga cccggcacac aaagattcac ctgcggggca
     3061 gccagctatc gattgtcgac gctcctgaac aacgtgaagg tgcttctcaa gtttctgttt
     3121 ctgttacttt tgaagatgtt gctgttcttt ttactcgtga tgaatggaaa aaacttgatc
     3181 tttctcaacg ttctctttat cgtgaagtta tgcttgaaaa ttattctaat cttgcttcta
     3241 tggcttaaac gcgttgtaca agtaaagcgg ccccggactc gaggccgcag gtaagtatca
     3301 aggttacaag acaggtttaa ggagaccaat agaaactggg cttgtcgaga cagagaagac
     3361 tcttgcgttt ctgataggca cctattggtc ttactgacat ccactttgcc tttctctcca
     3421 caggtgtcga caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     3481 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     3541 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     3601 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     3661 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     3721 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     3781 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaagctg acgtcctttc
     3841 catggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     3901 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     3961 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     4021 ctggaattcg agctcggtac gatcagcctc gactgtgcct tctagttgcc agccatctgt
     4081 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     4141 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     4201 tggggtgggg caggacagca agggggagga ttgggaagac aatagcccag cttttgttcc
     4261 ctttagtgag ggttaattgc gcgcttggcg taatcatggt catmssykww tycwstsykm
     4321 arktgywrkc tgcctatcag aaggtggtgg ctggtgtggc caatgccctg gctcacaaat
     4381 accactgaga tcgatctttt tccctctgcc aaaaattatg gggacatcat gaagcccctt
     4441 gagcatctga cttctggcta ataaaggaaa tttattttca ttgcaatagt gtgttggaat
     4501 tttttgtgtc tctcactcgg aaggacatat gggagggcaa atcatttaaa acatcagaat
     4561 gagtatttgg tttagagttt ggcaacatat gcccatatgc tggctgccat gaacaaaggt
     4621 tggctataaa gaggtcatca gtatatgaaa cagccccctg ctgtccattc cttattccat
     4681 agaaaagcct tgacttgagg ttagattttt tttatatttt gttttgtgtt atttttttct
     4741 ttaacatccc taaaattttc cttacatgtt ttactagcca gatttttcct cctctcctga
     4801 ctactcccag tcatagctgt ccctcttctc ttatggagat ccctcgacct gcagcccaag
     4861 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     4921 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     4981 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     5041 gcggatccgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     5101 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     5161 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt
     5221 tttggaggcc taggcttttg caaaaagcta acttgtttat tgcagcttat aatggttaca
     5281 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     5341 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatccgctgc attaatgaat
     5401 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     5461 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     5521 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     5581 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5641 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5701 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5761 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     5821 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5881 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5941 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     6001 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     6061 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     6121 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6181 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     6241 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     6301 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     6361 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6421 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6481 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6541 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6601 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6661 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6721 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6781 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6841 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6901 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6961 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     7021 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     7081 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7141 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7201 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     7261 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     7321 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctg
//
