LOCUS       sequence-274693-        7520 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a specific PSD-95 binder (Xph15) 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 7520)
  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 7520)
  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-mNeonGreen-CCR5TC
FEATURES             Location/Qualifiers
     source          1..7520
                     /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             2109..2813
                     /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      2361..2370
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2874..3215
                     /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             3264..3389
                     /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          3438..3570
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     3553..3572
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    3579..4167
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3632..3652)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4050..4061)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4194..4211)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4200..4311
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4407..4427)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4407..4425)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4542..4561)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4607..4662
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4661..4680)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5023..5039)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5023..5039)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5036..5058)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5047..5063
                     /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(5071..5077,5078..5095,5096..5101))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5116..5137
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5196..5391
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5242..5377
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5397..5531
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5434..5453)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5488..5507
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5599..5616)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5770..6358)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5850..5869)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6529..7320,7321..7389))
                     /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     7152..7171
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7390..7494)
                     /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 ggggggggsg 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 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 gtagcggcgt gagcaagggc gaggaggata acatggcctc tctcccagcg acacatgagt
     2161 tacacatctt tggctccatc aacggtgtgg actttgacat ggtgggtcag ggcaccggca
     2221 atccaaatga tggttatgag gagttaaacc tgaagtccac caagggtgac ctccagttct
     2281 ccccctggat tctggtccct catatcgggt atggcttcca tcagtacctg ccctaccctg
     2341 acgggatgtc gcctttccag gccgccatgg tagatggctc cggataccaa gtccatcgca
     2401 caatgcagtt tgaagatggt gcctccctta ctgttaacta ccgctacacc tacgagggaa
     2461 gccacatcaa aggagaggcc caggtgaagg ggactggttt ccctgctgac ggtcctgtga
     2521 tgaccaactc gctgaccgct gcggactggt gcaggtcgaa gaagacttac cccaacgaca
     2581 aaaccatcat cagtaccttt aagtggagtt acaccactgg aaatggcaag cgctaccgga
     2641 gcactgcgcg gaccacctac acctttgcca agccaatggc ggctaactat ctgaagaacc
     2701 agccgatgta cgtgttccgt aagacggagc tcaagcactc caagaccgag ctcaacttca
     2761 aggagtggca aaaggccttt accgatgtga tgggcatgga cgagctgtac aagggtagcg
     2821 gctcctctgc tagcggagct ggcgctggag cgggtgcagg ggctggctct agattccagt
     2881 gccggatctg catgcggaac ttcagcgacc ggtccaacct gagcaggcac atcagaaccc
     2941 acaccggaga aaagcccttc gcctgcgaca tttgcggccg gaagttcgcc atcagcagca
     3001 acctgaacag ccacaccaag atccacactg gcagccagaa acctttccag tgcagaattt
     3061 gtatgagaaa ctttagcaga agcgacaacc tggccagaca catccggaca catactggtg
     3121 aaaaaccttt tgcctgtgat atctgtggca gaaagtttgc cacctccggc aatctgaccc
     3181 ggcacacaaa gattcacctg cggggcagcc agctatcgat tgtcgacgct cctgaacaac
     3241 gtgaaggtgc ttctcaagtt tctgtttctg ttacttttga agatgttgct gttcttttta
     3301 ctcgtgatga atggaaaaaa cttgatcttt ctcaacgttc tctttatcgt gaagttatgc
     3361 ttgaaaatta ttctaatctt gcttctatgg cttaaacgcg ttgtacaagt aaagcggccc
     3421 cggactcgag gccgcaggta agtatcaagg ttacaagaca ggtttaagga gaccaataga
     3481 aactgggctt gtcgagacag agaagactct tgcgtttctg ataggcacct attggtctta
     3541 ctgacatcca ctttgccttt ctctccacag gtgtcgacaa tcaacctctg gattacaaaa
     3601 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     3661 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     3721 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
     3781 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
     3841 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
     3901 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     3961 tgttgtcggg gaagctgacg tcctttccat ggctgctcgc ctgtgttgcc acctggattc
     4021 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
     4081 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
     4141 ggatctccct ttgggccgcc tccccgcctg gaattcgagc tcggtacgat cagcctcgac
     4201 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     4261 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     4321 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     4381 ggaagacaat agcccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa
     4441 tcatggtcat mssykwwtyc wstsykmark tgcaggctgc ctatcagaag gtggtggctg
     4501 gtgtggccaa tgccctggct cacaaatacc actgagatcg atctttttcc ctctgccaaa
     4561 aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt
     4621 attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggaag gacatatggg
     4681 agggcaaatc atttaaaaca tcagaatgag tatttggttt agagtttggc aacatatgcc
     4741 catatgctgg ctgccatgaa caaaggttgg ctataaagag gtcatcagta tatgaaacag
     4801 ccccctgctg tccattcctt attccataga aaagccttga cttgaggtta gatttttttt
     4861 atattttgtt ttgtgttatt tttttcttta acatccctaa aattttcctt acatgtttta
     4921 ctagccagat ttttcctcct ctcctgacta ctcccagtca tagctgtccc tcttctctta
     4981 tggagatccc tcgacctgca gcccaagctt ggcgtaatca tggtcatagc tgtttcctgt
     5041 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     5101 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     5161 tttccagtcg ggaaacctgt cgtgccagcg gatccgcatc tcaattagtc agcaaccata
     5221 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     5281 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     5341 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctaact
     5401 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     5461 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     5521 atgtctggat ccgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     5581 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     5641 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     5701 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     5761 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     5821 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     5881 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     5941 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     6001 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     6061 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     6121 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     6181 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     6241 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     6301 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     6361 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     6421 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     6481 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     6541 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     6601 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     6661 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     6721 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     6781 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     6841 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     6901 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     6961 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     7021 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     7081 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     7141 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     7201 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     7261 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     7321 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     7381 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     7441 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     7501 tccccgaaaa gtgccacctg
//
