LOCUS       sequence-274686-        7511 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a specific PSD-95 binder (Xph20) 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 7511)
  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 7511)
  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-eGFP-CCR5TC
FEATURES             Location/Qualifiers
     source          1..7511
                     /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(2109..2111,2112..2819)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELY"
     CDS             join(2109..2111,2112..2819)
                     /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(2151..2172)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2412..2431)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2759..2780
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2865..3206
                     /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             3255..3380
                     /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          3429..3561
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     3544..3563
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    3570..4158
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3623..3643)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4041..4052)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4185..4202)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4191..4302
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4398..4418)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4398..4416)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4533..4552)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4598..4653
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4652..4671)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5014..5030)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5014..5030)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5027..5049)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5038..5054
                     /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(5062..5068,5069..5086,5087..5092))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5107..5128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5187..5382
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5233..5368
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5388..5522
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5425..5444)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5479..5498
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5590..5607)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5761..6349)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5841..5860)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6520..7311,7312..7380))
                     /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     7143..7162
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7381..7485)
                     /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 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 gatctggggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     2161 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     2221 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     2281 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     2341 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     2401 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     2461 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     2521 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     2581 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     2641 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     2701 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     2761 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtact
     2821 ctaggggagc tggcgctgga gcgggtgcag gggctggctc tagattccag tgccggatct
     2881 gcatgcggaa cttcagcgac cggtccaacc tgagcaggca catcagaacc cacaccggag
     2941 aaaagccctt cgcctgcgac atttgcggcc ggaagttcgc catcagcagc aacctgaaca
     3001 gccacaccaa gatccacact ggcagccaga aacctttcca gtgcagaatt tgtatgagaa
     3061 actttagcag aagcgacaac ctggccagac acatccggac acatactggt gaaaaacctt
     3121 ttgcctgtga tatctgtggc agaaagtttg ccacctccgg caatctgacc cggcacacaa
     3181 agattcacct gcggggcagc cagctatcga ttgtcgacgc tcctgaacaa cgtgaaggtg
     3241 cttctcaagt ttctgtttct gttacttttg aagatgttgc tgttcttttt actcgtgatg
     3301 aatggaaaaa acttgatctt tctcaacgtt ctctttatcg tgaagttatg cttgaaaatt
     3361 attctaatct tgcttctatg gcttaaacgc gttgtacaag taaagcggcc ccggactcga
     3421 ggccgcaggt aagtatcaag gttacaagac aggtttaagg agaccaatag aaactgggct
     3481 tgtcgagaca gagaagactc ttgcgtttct gataggcacc tattggtctt actgacatcc
     3541 actttgcctt tctctccaca ggtgtcgaca atcaacctct ggattacaaa atttgtgaaa
     3601 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     3661 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     3721 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     3781 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     3841 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     3901 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     3961 ggaagctgac gtcctttcca tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     4021 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     4081 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     4141 tttgggccgc ctccccgcct ggaattcgag ctcggtacga tcagcctcga ctgtgccttc
     4201 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     4261 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     4321 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     4381 tagcccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca
     4441 tmssykwwty cwstsykcag gtgcaggctg cctatcagaa ggtggtggct ggtgtggcca
     4501 atgccctggc tcacaaatac cactgagatc gatctttttc cctctgccaa aaattatggg
     4561 gacatcatga agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt
     4621 gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat
     4681 catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgctg
     4741 gctgccatga acaaaggttg gctataaaga ggtcatcagt atatgaaaca gccccctgct
     4801 gtccattcct tattccatag aaaagccttg acttgaggtt agattttttt tatattttgt
     4861 tttgtgttat ttttttcttt aacatcccta aaattttcct tacatgtttt actagccaga
     4921 tttttcctcc tctcctgact actcccagtc atagctgtcc ctcttctctt atggagatcc
     4981 ctcgacctgc agcccaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     5041 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     5101 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     5161 gggaaacctg tcgtgccagc ggatccgcat ctcaattagt cagcaaccat agtcccgccc
     5221 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     5281 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     5341 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctaac ttgtttattg
     5401 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     5461 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga
     5521 tccgctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     5581 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     5641 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     5701 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     5761 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     5821 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     5881 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     5941 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     6001 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     6061 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     6121 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     6181 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     6241 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     6301 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     6361 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     6421 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     6481 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     6541 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     6601 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     6661 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     6721 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     6781 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     6841 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     6901 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     6961 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     7021 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     7081 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     7141 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     7201 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     7261 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     7321 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     7381 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     7441 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     7501 agtgccacct g
//
