LOCUS       sequence-369455-        6714 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses surface-localized pHluorin fused to intracellular 
            expression of mRuby under CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6714)
  AUTHORS   Cuhadar U, Calzado-Reyes L, Pascual-Caro C, Aberra AS, Ritzau-Jost 
            A, Aggarwal A, Ibata K, Podgorski K, Yuzaki M, Geis C, Hallerman S, 
            Hoppa MB, de Juan-Sanz J
  TITLE     Activity-driven synaptic translocation of LGI1 controls excitatory 
            neurotransmission.
  JOURNAL   Cell Rep. 2024 May 28;43(5):114186. doi: 
            10.1016/j.celrep.2024.114186. Epub 2024 May 2.
  PUBMED    38700985
REFERENCE   2  (bases 1 to 6714)
  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: "Cell Rep.";
            date: "2024-05-28"; volume: "43(5):114186. doi"; pages: " 
            10.1016/j.celrep.2024.114186. Epub 2024 May 2"
COMMENT     Sequence Label: CMV-IgK-pHluorin-TM-mRuby
FEATURES             Location/Qualifiers
     source          1..6714
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        25..404
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        405..608
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     559..579
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     653..672
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        653..671
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     sig_peptide     752..811
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     CDS             824..1534
                     /codon_start=1
                     /product="pH-sensitive mutant of green fluorescent protein 
                     (Ng et al., 2002)"
                     /label=superecliptic pHluorin
                     /note="excited strongly by blue light at neutral pH but 
                     minimally fluorescent at acidic pH"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIKAN
                     FKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(851..879)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1474..1494
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             1541..1570
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1574..1720
                     /codon_start=1
                     /product="transmembrane domain from platelet derived growth
                     factor receptor beta"
                     /label=PDGFR-beta TM domain
                     /translation="AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ
                     KKPR"
     CDS             1721..2431
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     primer_bind     complement(2480..2497)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2486..2710
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(2842..3430)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2922..2941)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3759..3806
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     3831..3850
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(4038..4832)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASKPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4149..4168)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4759..4778
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(4931..4950)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     enhancer        5029..5220
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5177..5197
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             complement(join(5280..6071,6072..6140))
                     /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     5903..5922
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6141..6245)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6272..6700
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6359..6378)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6569..6590
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 acgcttacaa tttacgcgcg cgttgacatt gattattgac tagttattaa tagtaatcaa
       61 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
      121 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
      181 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
      241 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
      301 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
      361 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
      421 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
      481 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
      541 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
      601 gcagaactct ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga
      661 ctcactatag ggagacccaa gcttggtacc gaactcggat ccactagtaa cggccgccag
      721 tgtgctggaa ttcggcttgg ggatatccac catggagaca gacacactcc tgctatgggt
      781 actgctgctc tgggttccag gttccactgg tgacagatct ggaagtaaag gagaagaact
      841 tttcactgga gttgtcccaa ttcttgttga attagatggt gatgttaatg ggcacaaatt
      901 ttctgtcagt ggagagggtg aaggtgatgc aacatacgga aaacttaccc ttaaatttat
      961 ttgcactact ggaaaactac ctgttccttg gccaacactt gtcactactt taacttatgg
     1021 tgttcaatgc ttttcaagat acccagatca tatgaaacgg catgactttt tcaagagtgc
     1081 catgcccgaa ggttatgtac aggaaagaac tatatttttc aaagatgacg ggaactacaa
     1141 gacacgtgct gaagtcaagt ttgaaggtga tacccttgtt aatagaatcg agttaaaagg
     1201 tattgatttt aaagaagatg gaaacattct tggacacaaa ttggaataca actataacga
     1261 tcaccaggtg tacatcatgg cagacaaaca aaagaatgga atcaaagcta acttcaaaat
     1321 tagacacaac attgaagatg gaggcgttca actagcagac cattatcaac aaaatactcc
     1381 aattggcgat gggcccgtcc ttttaccaga caaccattac ctgtttacaa cttctactct
     1441 ttcgaaagat cccaacgaaa agagagacca catggtcctt cttgagtttg taacagctgc
     1501 tgggattaca catggcatgg atgaactata caaagtcgac gaacaaaaac tcatctcaga
     1561 agaggatctg aatgctgtgg gccaggacac gcaggaggtc atcgtggtgc cacactcctt
     1621 gccctttaag gtggtggtga tctcagccat cctggccctg gtggtgctca ccatcatctc
     1681 ccttatcatc ctcatcatgc tttggcagaa gaagccacgt atggtgtcta agggcgaaga
     1741 gctgatcaag gaaaatatgc gtatgaaggt ggtcatggaa ggttcggtca acggccacca
     1801 attcaaatgc acaggtgaag gagaaggcaa tccgtacatg ggaactcaaa ccatgaggat
     1861 caaagtcatc gagggaggac ccctgccatt tgcctttgac attcttgcca cgtcgttcat
     1921 gtatggcagc cgtactttta tcaagtaccc gaaaggcatt cctgatttct ttaaacagtc
     1981 ctttcctgag ggttttactt gggaaagagt tacgagatac gaagatggtg gagtcgtcac
     2041 cgtcatgcag gacaccagcc ttgaggatgg ctgtctcgtt taccacgtcc aagtcagagg
     2101 ggtaaacttt ccctccaatg gtcccgtgat gcagaagaag accaagggtt gggagcctaa
     2161 tacagagatg atgtatccag cagatggtgg tctgagggga tacactcata tggcactgaa
     2221 agttgatggt ggtggccatc tgtcttgctc tttcgtaaca acttacaggt caaaaaagac
     2281 cgtcgggaac atcaagatgc ccggtatcca tgccgttgat caccgcctgg aaaggttaga
     2341 ggaaagtgac aatgaaatgt tcgtagtaca acgcgaacac gcagttgcca agttcgccgg
     2401 gcttggtggt gggatggacg agctgtacaa gttctactgc gagaacgagg tgggcagctg
     2461 agcggccgct cgagatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     2521 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     2581 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     2641 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcgtgctgg ggatgcggtg
     2701 ggctctatgg cttctgaggc ggaaagaacc agtggcggta atacggttat ccacagaatc
     2761 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     2821 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     2881 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     2941 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     3001 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     3061 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     3121 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     3181 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     3241 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     3301 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     3361 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     3421 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     3481 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     3541 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaacctg aggctatggc
     3601 agggcctgcc gccccgacgt tggctgcgag ccctgggcct tcacccgaac ttggggggtg
     3661 gggtggggaa aaggaagaaa cgcgggcgta ttggccccaa tggggtctcg gtggggtatc
     3721 gacagagtgc cagccctggg accgaacccc gcgtttatga acaaacgacc caacaccgtg
     3781 cgttttattc tgtcttttta ttgccgtcat agcgcgggtt ccttccggta ttgtctcctt
     3841 ccgtgtttca gttagcctcc ccctagggtg ggcgaagaac tccagcatga gatccccgcg
     3901 ctggaggatc atccagccgg cgtcccggaa aacgattccg aagcccaacc tttcatagaa
     3961 ggcggcggtg gaatcgaaat ctcgtgatgg caggttgggc gtcgcttggt cggtcatttc
     4021 gaaccccaga gtcccactca gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc
     4081 gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc gccgccaagc
     4141 tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc cacacccagc
     4201 cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt cggcaagcag
     4261 gcatcgccat gggtcacgac gagatcctcg ccgtcgggct tgctcgcctt gagcctggcg
     4321 aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg atcgacaaga
     4381 ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg
     4441 caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat ggatactttc
     4501 tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc caatagcagc
     4561 cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg
     4621 gccagccacg atagccgcgc tgcctcgtct tgcagttcat tcagggcacc ggacaggtcg
     4681 gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc ggcatcagag
     4741 cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca agcggccgga
     4801 gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc tgtctcttga
     4861 tcgatctttg caaaagccta ggcctccaaa aaagcctcct cactacttct ggaatagctc
     4921 agaggccggg cctcggcctc tgcataaata aaaaaaatta gtcagccatg gggcggagaa
     4981 tgggcggaac tgggcggagt taggggcggg atgggcggag ttaggggcgg gactatggtt
     5041 gctgactaat tgagatgctt gctttgcata cttctgcctg ctggggagcc tggggacttt
     5101 ccacacctgg ttgctgacta attgagatgc ttgctttgca tacttctgcc tgctggggag
     5161 cctggggact ttccacaccc taactgacac acattccaca gctggttctt tccgcctcag
     5221 gactcttcct ttttcaataa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5281 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5341 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5401 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5461 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5521 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     5581 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     5641 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     5701 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     5761 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     5821 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     5881 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     5941 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6001 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6061 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6121 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6181 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6241 ccgcgcacat ttccccgaaa agtgccacct gacgcgccct gtagcggcgc attaagcgcg
     6301 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     6361 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     6421 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     6481 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     6541 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     6601 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     6661 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat atta
//
