LOCUS       sequence-274694-        7526 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a specific PSD-95 binder (Xph20) fused to mNeonGreen, 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 7526)
  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 7526)
  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-mNeonGreen-CCR5TC
FEATURES             Location/Qualifiers
     source          1..7526
                     /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..2819
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="VSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEE
                     LNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDG
                     ASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIST
                     FKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQK
                     AFTDVMGMDELYK"
     regulatory      2367..2376
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2880..3221
                     /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             3270..3395
                     /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          3444..3576
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     3559..3578
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    3585..4173
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3638..3658)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4056..4067)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4200..4217)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4206..4317
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4413..4433)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4413..4431)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4548..4567)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4613..4668
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4667..4686)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5029..5045)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5029..5045)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5042..5064)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5053..5069
                     /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(5077..5083,5084..5101,5102..5107))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5122..5143
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5202..5397
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5248..5383
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5403..5537
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5440..5459)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5494..5513
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5605..5622)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5776..6364)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5856..5875)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6535..7326,7327..7395))
                     /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     7158..7177
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7396..7500)
                     /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 gggggggggg gggggggggg ggggggggsg gggsgrggcg grgaggtgcg
      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 cggggagggc
     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 cggcgtgagc aagggcgagg aggataacat ggcctctctc ccagcgacac
     2161 atgagttaca catctttggc tccatcaacg gtgtggactt tgacatggtg ggtcagggca
     2221 ccggcaatcc aaatgatggt tatgaggagt taaacctgaa gtccaccaag ggtgacctcc
     2281 agttctcccc ctggattctg gtccctcata tcgggtatgg cttccatcag tacctgccct
     2341 accctgacgg gatgtcgcct ttccaggccg ccatggtaga tggctccgga taccaagtcc
     2401 atcgcacaat gcagtttgaa gatggtgcct cccttactgt taactaccgc tacacctacg
     2461 agggaagcca catcaaagga gaggcccagg tgaaggggac tggtttccct gctgacggtc
     2521 ctgtgatgac caactcgctg accgctgcgg actggtgcag gtcgaagaag acttacccca
     2581 acgacaaaac catcatcagt acctttaagt ggagttacac cactggaaat ggcaagcgct
     2641 accggagcac tgcgcggacc acctacacct ttgccaagcc aatggcggct aactatctga
     2701 agaaccagcc gatgtacgtg ttccgtaaga cggagctcaa gcactccaag accgagctca
     2761 acttcaagga gtggcaaaag gcctttaccg atgtgatggg catggacgag ctgtacaagg
     2821 gtagcggctc ctctgctagc ggagctggcg ctggagcggg tgcaggggct ggctctagat
     2881 tccagtgccg gatctgcatg cggaacttca gcgaccggtc caacctgagc aggcacatca
     2941 gaacccacac cggagaaaag cccttcgcct gcgacatttg cggccggaag ttcgccatca
     3001 gcagcaacct gaacagccac accaagatcc acactggcag ccagaaacct ttccagtgca
     3061 gaatttgtat gagaaacttt agcagaagcg acaacctggc cagacacatc cggacacata
     3121 ctggtgaaaa accttttgcc tgtgatatct gtggcagaaa gtttgccacc tccggcaatc
     3181 tgacccggca cacaaagatt cacctgcggg gcagccagct atcgattgtc gacgctcctg
     3241 aacaacgtga aggtgcttct caagtttctg tttctgttac ttttgaagat gttgctgttc
     3301 tttttactcg tgatgaatgg aaaaaacttg atctttctca acgttctctt tatcgtgaag
     3361 ttatgcttga aaattattct aatcttgctt ctatggctta aacgcgttgt acaagtaaag
     3421 cggccccgga ctcgaggccg caggtaagta tcaaggttac aagacaggtt taaggagacc
     3481 aatagaaact gggcttgtcg agacagagaa gactcttgcg tttctgatag gcacctattg
     3541 gtcttactga catccacttt gcctttctct ccacaggtgt cgacaatcaa cctctggatt
     3601 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     3661 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     3721 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
     3781 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
     3841 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
     3901 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
     3961 ccgtggtgtt gtcggggaag ctgacgtcct ttccatggct gctcgcctgt gttgccacct
     4021 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
     4081 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
     4141 cgagtcggat ctccctttgg gccgcctccc cgcctggaat tcgagctcgg tacgatcagc
     4201 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     4261 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     4321 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     4381 ggattgggaa gacaatagcc cagcttttgt tccctttagt gagggttaat tgcgcgcttg
     4441 gcgtaatcat ggtyatmssy kwwtycwsts ykcaggtgca ggctgcctat cagaaggtgg
     4501 tggctggtgt ggccaatgcc ctggctcaca aataccactg agatcgatct ttttccctct
     4561 gccaaaaatt atggggacat catgaagccc cttgagcatc tgacttctgg ctaataaagg
     4621 aaatttattt tcattgcaat agtgtgttgg aattttttgt gtctctcact cggaaggaca
     4681 tatgggaggg caaatcattt aaaacatcag aatgagtatt tggtttagag tttggcaaca
     4741 tatgcccata tgctggctgc catgaacaaa ggttggctat aaagaggtca tcagtatatg
     4801 aaacagcccc ctgctgtcca ttccttattc catagaaaag ccttgacttg aggttagatt
     4861 ttttttatat tttgttttgt gttatttttt tctttaacat ccctaaaatt ttccttacat
     4921 gttttactag ccagattttt cctcctctcc tgactactcc cagtcatagc tgtccctctt
     4981 ctcttatgga gatccctcga cctgcagccc aagcttggcg taatcatggt catagctgtt
     5041 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     5101 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     5161 gcccgctttc cagtcgggaa acctgtcgtg ccagcggatc cgcatctcaa ttagtcagca
     5221 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     5281 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     5341 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     5401 ctaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     5461 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     5521 cttatcatgt ctggatccgc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     5581 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     5641 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     5701 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     5761 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     5821 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5881 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5941 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     6001 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     6061 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     6121 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     6181 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     6241 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     6301 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     6361 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     6421 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     6481 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     6541 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     6601 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     6661 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     6721 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     6781 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     6841 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     6901 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     6961 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     7021 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     7081 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     7141 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     7201 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     7261 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     7321 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     7381 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     7441 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     7501 cacatttccc cgaaaagtgc cacctg
//
