LOCUS       sequence-274687-        7529 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a specific PSD-95 binder (Xph20) fused to mRuby2, 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 7529)
  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 7529)
  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-mRuby2-CCR5TC
FEATURES             Location/Qualifiers
     source          1..7529
                     /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..2822
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="VSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTMR
                     IKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGGV
                     VTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTHM
                     ALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAVA
                     KFAGLGGGMDELYK"
     CDS             2883..3224
                     /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             3273..3398
                     /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          3447..3579
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     3562..3581
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    3588..4176
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3641..3661)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4059..4070)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4203..4220)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4209..4320
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4416..4436)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4416..4434)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4551..4570)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4616..4671
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4670..4689)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5032..5048)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5032..5048)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5045..5067)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5056..5072
                     /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(5080..5086,5087..5104,5105..5110))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5125..5146
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5205..5400
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5251..5386
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5406..5540
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5443..5462)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5497..5516
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5608..5625)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5779..6367)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5859..5878)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6538..7329,7330..7398))
                     /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     7161..7180
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7399..7503)
                     /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 sgcgcgccrg gsgggggggg gggggggggg ggggggggsg gggsgaggcg 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 cggcgtgtct aagggcgaag agctgatcaa ggaaaatatg cgtatgaagg
     2161 tggtcatgga aggttcggtc aacggccacc aattcaaatg cacaggtgaa ggagaaggca
     2221 atccgtacat gggaactcaa accatgagga tcaaagtcat cgagggagga cccctgccat
     2281 ttgcctttga cattcttgcc acgtcgttca tgtatggcag ccgtactttt atcaagtacc
     2341 cgaaaggcat tcctgatttc tttaaacagt cctttcctga gggttttact tgggaaagag
     2401 ttacgagata cgaagatggt ggagtcgtca ccgtcatgca ggacaccagc cttgaggatg
     2461 gctgtctcgt ttaccacgtc caagtcagag gggtaaactt tccctccaat ggtcccgtga
     2521 tgcagaagaa gaccaagggt tgggagccta atacagagat gatgtatcca gcagatggtg
     2581 gtctgagggg atacactcat atggcactga aagttgatgg tggtggccat ctgtcttgct
     2641 ctttcgtaac aacttacagg tcaaaaaaga ccgtcgggaa catcaagatg cccggtatcc
     2701 atgccgttga tcaccgcctg gaaaggttag aggaaagtga caatgaaatg ttcgtagtac
     2761 aacgcgaaca cgcagttgcc aagttcgccg ggcttggtgg tgggatggac gagctgtaca
     2821 agggtagcgg ctcctctgct agcggagctg gcgctggagc gggtgcaggg gctggctcta
     2881 gattccagtg ccggatctgc atgcggaact tcagcgaccg gtccaacctg agcaggcaca
     2941 tcagaaccca caccggagaa aagcccttcg cctgcgacat ttgcggccgg aagttcgcca
     3001 tcagcagcaa cctgaacagc cacaccaaga tccacactgg cagccagaaa cctttccagt
     3061 gcagaatttg tatgagaaac tttagcagaa gcgacaacct ggccagacac atccggacac
     3121 atactggtga aaaacctttt gcctgtgata tctgtggcag aaagtttgcc acctccggca
     3181 atctgacccg gcacacaaag attcacctgc ggggcagcca gctatcgatt gtcgacgctc
     3241 ctgaacaacg tgaaggtgct tctcaagttt ctgtttctgt tacttttgaa gatgttgctg
     3301 ttctttttac tcgtgatgaa tggaaaaaac ttgatctttc tcaacgttct ctttatcgtg
     3361 aagttatgct tgaaaattat tctaatcttg cttctatggc ttaaacgcgt tgtacaagta
     3421 aagcggcccc ggactcgagg ccgcaggtaa gtatcaaggt tacaagacag gtttaaggag
     3481 accaatagaa actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta
     3541 ttggtcttac tgacatccac tttgcctttc tctccacagg tgtcgacaat caacctctgg
     3601 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
     3661 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
     3721 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca
     3781 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg
     3841 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg
     3901 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
     3961 attccgtggt gttgtcgggg aagctgacgt cctttccatg gctgctcgcc tgtgttgcca
     4021 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc
     4081 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc
     4141 agacgagtcg gatctccctt tgggccgcct ccccgcctgg aattcgagct cggtacgatc
     4201 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     4261 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     4321 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     4381 ggaggattgg gaagacaata gcccagcttt tgttcccttt agtgagggtt aattgcgcgc
     4441 ttggcgtaat catkgtyatm ssykwwtycw stsykcaggt gcaggctgcc tatcagaagg
     4501 tggtggctgg tgtggccaat gccctggctc acaaatacca ctgagatcga tctttttccc
     4561 tctgccaaaa attatgggga catcatgaag ccccttgagc atctgacttc tggctaataa
     4621 aggaaattta ttttcattgc aatagtgtgt tggaattttt tgtgtctctc actcggaagg
     4681 acatatggga gggcaaatca tttaaaacat cagaatgagt atttggttta gagtttggca
     4741 acatatgccc atatgctggc tgccatgaac aaaggttggc tataaagagg tcatcagtat
     4801 atgaaacagc cccctgctgt ccattcctta ttccatagaa aagccttgac ttgaggttag
     4861 atttttttta tattttgttt tgtgttattt ttttctttaa catccctaaa attttcctta
     4921 catgttttac tagccagatt tttcctcctc tcctgactac tcccagtcat agctgtccct
     4981 cttctcttat ggagatccct cgacctgcag cccaagcttg gcgtaatcat ggtcatagct
     5041 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     5101 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     5161 actgcccgct ttccagtcgg gaaacctgtc gtgccagcgg atccgcatct caattagtca
     5221 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     5281 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     5341 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     5401 aagctaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     5461 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     5521 tatcttatca tgtctggatc cgctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5581 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     5641 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     5701 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5761 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     5821 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     5881 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5941 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     6001 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     6061 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     6121 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     6181 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     6241 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     6301 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     6361 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     6421 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     6481 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     6541 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     6601 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     6661 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     6721 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     6781 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     6841 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     6901 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     6961 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     7021 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     7081 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     7141 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     7201 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     7261 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     7321 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     7381 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     7441 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     7501 gcgcacattt ccccgaaaag tgccacctg
//
