LOCUS       sequence-274671-        7505 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a specific PSD-95 binder (Xph15) 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 7505)
  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 7505)
  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_Xph15-eGFP-CCR5TC
FEATURES             Location/Qualifiers
     source          1..7505
                     /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"
     promoter        421..696
                     /label=chicken beta-actin promoter
     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(2103..2105,2106..2813)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELY"
     CDS             join(2103..2105,2106..2813)
                     /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(2145..2166)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2406..2425)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2753..2774
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2859..3200
                     /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             3249..3374
                     /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          3423..3555
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     3538..3557
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    3564..4152
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3617..3637)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4035..4046)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4179..4196)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4185..4296
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4392..4412)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4392..4410)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4527..4546)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4592..4647
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4646..4665)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5008..5024)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5008..5024)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5021..5043)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5032..5048
                     /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(5056..5062,5063..5080,5081..5086))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5101..5122
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5181..5376
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5227..5362
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5382..5516
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5419..5438)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5473..5492
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5584..5601)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5755..6343)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5835..5854)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6514..7305,7306..7374))
                     /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     7137..7156
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7375..7479)
                     /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 ggggggsgrg gggsggggsg gggcgaggcg 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 ggatgcaggg cctcgtaatg tcagttatta ccgtatcacc tatggcgaaa
     1921 cgggcggtaa ctccccggtt caggaattca ccgtcccggg tagttcctca accgcaacga
     1981 tttccggcct gtcaccgggt gtcgactata ccattacggt ttacgcttct ggtcatgtta
     2041 gtacgctgat gacgccgatt tctatcaatt accgtaccgg tagcggctcc agtagatctg
     2101 gggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     2161 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     2221 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     2281 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     2341 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     2401 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     2461 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     2521 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     2581 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     2641 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     2701 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     2761 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tactctaggg
     2821 gagctggcgc tggagcgggt gcaggggctg gctctagatt ccagtgccgg atctgcatgc
     2881 ggaacttcag cgaccggtcc aacctgagca ggcacatcag aacccacacc ggagaaaagc
     2941 ccttcgcctg cgacatttgc ggccggaagt tcgccatcag cagcaacctg aacagccaca
     3001 ccaagatcca cactggcagc cagaaacctt tccagtgcag aatttgtatg agaaacttta
     3061 gcagaagcga caacctggcc agacacatcc ggacacatac tggtgaaaaa ccttttgcct
     3121 gtgatatctg tggcagaaag tttgccacct ccggcaatct gacccggcac acaaagattc
     3181 acctgcgggg cagccagcta tcgattgtcg acgctcctga acaacgtgaa ggtgcttctc
     3241 aagtttctgt ttctgttact tttgaagatg ttgctgttct ttttactcgt gatgaatgga
     3301 aaaaacttga tctttctcaa cgttctcttt atcgtgaagt tatgcttgaa aattattcta
     3361 atcttgcttc tatggcttaa acgcgttgta caagtaaagc ggccccggac tcgaggccgc
     3421 aggtaagtat caaggttaca agacaggttt aaggagacca atagaaactg ggcttgtcga
     3481 gacagagaag actcttgcgt ttctgatagg cacctattgg tcttactgac atccactttg
     3541 cctttctctc cacaggtgtc gacaatcaac ctctggatta caaaatttgt gaaagattga
     3601 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     3661 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     3721 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     3781 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     3841 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     3901 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaagc
     3961 tgacgtcctt tccatggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
     4021 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     4081 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
     4141 ccgcctcccc gcctggaatt cgagctcggt acgatcagcc tcgactgtgc cttctagttg
     4201 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     4261 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     4321 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagccc
     4381 agcttttgtt ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg gtcatmssyk
     4441 wwtycwstsy kmarktgcar gctgcctatc agaaggtggt ggctggtgtg gccaatgccc
     4501 tggctcacaa ataccactga gatcgatctt tttccctctg ccaaaaatta tggggacatc
     4561 atgaagcccc ttgagcatct gacttctggc taataaagga aatttatttt cattgcaata
     4621 gtgtgttgga attttttgtg tctctcactc ggaaggacat atgggagggc aaatcattta
     4681 aaacatcaga atgagtattt ggtttagagt ttggcaacat atgcccatat gctggctgcc
     4741 atgaacaaag gttggctata aagaggtcat cagtatatga aacagccccc tgctgtccat
     4801 tccttattcc atagaaaagc cttgacttga ggttagattt tttttatatt ttgttttgtg
     4861 ttattttttt ctttaacatc cctaaaattt tccttacatg ttttactagc cagatttttc
     4921 ctcctctcct gactactccc agtcatagct gtccctcttc tcttatggag atccctcgac
     4981 ctgcagccca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     5041 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     5101 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     5161 cctgtcgtgc cagcggatcc gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     5221 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     5281 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     5341 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc taacttgttt attgcagctt
     5401 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     5461 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatccgct
     5521 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     5581 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     5641 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     5701 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     5761 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     5821 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     5881 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     5941 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     6001 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     6061 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     6121 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     6181 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     6241 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     6301 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     6361 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     6421 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     6481 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     6541 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     6601 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     6661 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     6721 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     6781 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     6841 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     6901 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     6961 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     7021 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     7081 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     7141 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     7201 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     7261 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     7321 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     7381 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     7441 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     7501 acctg
//
