LOCUS       sequence-274678-        7514 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a specific PSD-95 binder (Xph18) fused to eGFP, 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 7514)
  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 7514)
  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_Xph18-eGFP-CCR5TC
FEATURES             Location/Qualifiers
     source          1..7514
                     /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             join(2112..2114,2115..2822)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELY"
     CDS             join(2112..2114,2115..2822)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELY"
     primer_bind     complement(2154..2175)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2415..2434)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2762..2783
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2868..3209
                     /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             3258..3383
                     /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          3432..3564
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     3547..3566
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    3573..4161
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3626..3646)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4044..4055)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4188..4205)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4194..4305
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4401..4421)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4401..4419)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4536..4555)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4601..4656
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4655..4674)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5017..5033)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5017..5033)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5030..5052)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5041..5057
                     /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(5065..5071,5072..5089,5090..5095))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5110..5131
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5190..5385
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5236..5371
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5391..5525
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5428..5447)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5482..5501
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5593..5610)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5764..6352)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5844..5863)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6523..7314,7315..7383))
                     /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     7146..7165
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7384..7488)
                     /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 gggsgrggcg 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 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 ggatgcacat tcgcggatgg cgtgggtcaa ttattaccgt atcacctatg
     1921 gcgaaacggg cggtaactcc ccggttcagg aattcaccgt cccgggtagt tcctcaaccg
     1981 caacgatttc cggcctgtca ccgggtgtcg actataccat tacggtttac gctaatgggg
     2041 gggggtttcg tcgggggatg gcgccgattt ctatcaatta ccgtaccggt agcggctcca
     2101 gtagatctgg ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     2161 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     2221 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     2281 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     2341 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     2401 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     2461 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     2521 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     2581 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     2641 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     2701 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
     2761 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     2821 actctagggg agctggcgct ggagcgggtg caggggctgg ctctagattc cagtgccgga
     2881 tctgcatgcg gaacttcagc gaccggtcca acctgagcag gcacatcaga acccacaccg
     2941 gagaaaagcc cttcgcctgc gacatttgcg gccggaagtt cgccatcagc agcaacctga
     3001 acagccacac caagatccac actggcagcc agaaaccttt ccagtgcaga atttgtatga
     3061 gaaactttag cagaagcgac aacctggcca gacacatccg gacacatact ggtgaaaaac
     3121 cttttgcctg tgatatctgt ggcagaaagt ttgccacctc cggcaatctg acccggcaca
     3181 caaagattca cctgcggggc agccagctat cgattgtcga cgctcctgaa caacgtgaag
     3241 gtgcttctca agtttctgtt tctgttactt ttgaagatgt tgctgttctt tttactcgtg
     3301 atgaatggaa aaaacttgat ctttctcaac gttctcttta tcgtgaagtt atgcttgaaa
     3361 attattctaa tcttgcttct atggcttaaa cgcgttgtac aagtaaagcg gccccggact
     3421 cgaggccgca ggtaagtatc aaggttacaa gacaggttta aggagaccaa tagaaactgg
     3481 gcttgtcgag acagagaaga ctcttgcgtt tctgataggc acctattggt cttactgaca
     3541 tccactttgc ctttctctcc acaggtgtcg acaatcaacc tctggattac aaaatttgtg
     3601 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt
     3661 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata
     3721 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg
     3781 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc
     3841 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct
     3901 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt
     3961 cggggaagct gacgtccttt ccatggctgc tcgcctgtgt tgccacctgg attctgcgcg
     4021 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc
     4081 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct
     4141 ccctttgggc cgcctccccg cctggaattc gagctcggta cgatcagcct cgactgtgcc
     4201 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     4261 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     4321 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     4381 caatagccca gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg
     4441 tcatassykw wtycwstsyk marktgywrk ctgcctatca gaaggtggtg gctggtgtgg
     4501 ccaatgccct ggctcacaaa taccactgag atcgatcttt ttccctctgc caaaaattat
     4561 ggggacatca tgaagcccct tgagcatctg acttctggct aataaaggaa atttattttc
     4621 attgcaatag tgtgttggaa ttttttgtgt ctctcactcg gaaggacata tgggagggca
     4681 aatcatttaa aacatcagaa tgagtatttg gtttagagtt tggcaacata tgcccatatg
     4741 ctggctgcca tgaacaaagg ttggctataa agaggtcatc agtatatgaa acagccccct
     4801 gctgtccatt ccttattcca tagaaaagcc ttgacttgag gttagatttt ttttatattt
     4861 tgttttgtgt tatttttttc tttaacatcc ctaaaatttt ccttacatgt tttactagcc
     4921 agatttttcc tcctctcctg actactccca gtcatagctg tccctcttct cttatggaga
     4981 tccctcgacc tgcagcccaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     5041 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     5101 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     5161 gtcgggaaac ctgtcgtgcc agcggatccg catctcaatt agtcagcaac catagtcccg
     5221 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     5281 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     5341 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct aacttgttta
     5401 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     5461 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     5521 ggatccgctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5581 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     5641 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     5701 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     5761 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     5821 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     5881 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     5941 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     6001 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     6061 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     6121 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     6181 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     6241 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     6301 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     6361 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     6421 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     6481 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     6541 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     6601 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     6661 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     6721 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     6781 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     6841 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     6901 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     6961 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     7021 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     7081 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     7141 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     7201 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     7261 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     7321 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     7381 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     7441 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     7501 aaaagtgcca cctg
//
