LOCUS       sequence-369260-        7428 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Glutamate sensor for localization to membrane contatct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7428)
  AUTHORS   Shindo Y, Ashida K, Masamoto K, Takuwa H, Takahashi M, Higuchi M, 
            Ide R, Hotta K, Oka K
  TITLE     Genetically encoded sensors for analysing neurotransmission among 
            synaptically-connected neurons
  JOURNAL   bioRxiv 2022
REFERENCE   2  (bases 1 to 7428)
  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: "bioRxiv 
            2022"
COMMENT     Sequence Label: 
            pcDNA3.1-mCherry-P2A-Pre-SPHERE-sfmTurquoise2-iGluSnFR
FEATURES             Location/Qualifiers
     source          1..7428
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(1..589)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(81..100)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(760..1551,1552..1620))
                     /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     1383..1402
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1621..1725)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1800..1819)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1991..2370
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2371..2574
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2525..2545
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2619..2638
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2619..2637
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2655..2664
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2661..3368
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2806..2824)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3074..3093
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3375..3416
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3426..3482
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(3600..3621)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4775..4792)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4781..5005
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5051..5479
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5138..5157)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5348..5369
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5488..5508)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5493..5822
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5673..5808
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5735..5754
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5889..6683
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5943..5962)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6553..6572
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6857..6978
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6894..6913)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6948..6967
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7027..7043)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7027..7043)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7040..7062)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7051..7067
                     /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(7075..7081,7082..7099,7100..7105))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7120..7141
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7258..7275)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
       61 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      121 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      181 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      241 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
      301 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
      361 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
      421 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
      481 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
      541 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
      601 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
      661 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
      721 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
      781 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
      841 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
      901 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
      961 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     1021 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     1081 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     1141 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     1201 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     1261 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     1321 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     1381 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     1441 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     1501 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     1561 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     1621 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     1681 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     1741 agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt
     1801 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag
     1861 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat
     1921 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
     1981 tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
     2041 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
     2101 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
     2161 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
     2221 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
     2281 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
     2341 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
     2401 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
     2461 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
     2521 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg
     2581 ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag
     2641 acccaagctg gctagccacc atggtgagca agggcgagga ggataacatg gccatcatca
     2701 aggagttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga
     2761 tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga
     2821 ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct
     2881 ccaaggccta cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg
     2941 agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc
     3001 aggactcctc cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact
     3061 tcccctccga cggccccgta atgcagaaga agaccatggg ctgggaggcc tcctccgagc
     3121 ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg
     3181 acggcggcca ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc
     3241 tgcccggcgc ctacaacgtc aacatcaagt tggacatcac ctcccacaac gaggactaca
     3301 ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc
     3361 tgtacaaggg atccggcaag cctatcccta atcctctgct gggactggac tctacaggaa
     3421 gcggagctac taacttcagc ctgctgaagc aggctggaga cgtggaggag aaccctggac
     3481 ctgagaccga caccctcctg ctatgggtgc tgctgctctg ggtgcccggc agcaccggcg
     3541 acgcaccggt cggaggcatg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     3601 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgcgcggc gagggcgagg
     3661 gcgatgccac caacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     3721 tgccctggcc caccctcgtg accaccctga gctggggcgt gcagtgcttc gcccgctacc
     3781 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     3841 agcgcaccat cagcttcaag gacgacggca cctacaagac ccgcgccgag gtgaagttcg
     3901 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     3961 acatcctggg gcacaagctg gagtacaact ttgccatccg cctgaacatg aatttcgaac
     4021 tgtcagacga aatgaaagca ctgttcaaag aaccgaatga caaggcactg aaaaccggtg
     4081 gatctggagg cagtggcgga tctagagctc ctacctactc tccaccacct ccacctggag
     4141 gaggttccgg aggcggaagc ggtaccgaag tgccttcctc tatgacaact gagtcgacag
     4201 ccactgccat gcagtccgag atgtccacct caatcatgga gaccaccaca accctggcta
     4261 ccagcacagc tcgacgagga aagcccccca caaaggagcc tatcagccag accacggatg
     4321 atatccttgt ggcctcggca gagtgtccca gcgacgatga ggacattgac ccctgtgagc
     4381 cgagctcagg tgggttagcc aaccccaccc gagtgggcgg ccgcgaacca tacccaggct
     4441 cggcagaggt gatccgggag tctagcagta ccactggcat ggtggtgggg attgtcgcag
     4501 cagcagctct gtgcatcctc atcctcctct atgccatgta caagtacaga aaccgggatg
     4561 aaggctcata tcacgtggat gagagtcgaa actacatcag taactcagca cagtccaatg
     4621 gggctgtggt caaggagaag cagcccagca gtgcgaaaag cgccaacaaa aacaagaaga
     4681 acaaggataa ggagtattat gtctgaattc tgcagatatc cagcacagtg gcggccgctc
     4741 gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     4801 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     4861 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     4921 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     4981 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     5041 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     5101 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     5161 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     5221 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     5281 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     5341 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     5401 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     5461 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     5521 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     5581 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     5641 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     5701 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     5761 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     5821 aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat
     5881 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     5941 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     6001 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     6061 atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     6121 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     6181 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     6241 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     6301 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     6361 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
     6421 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     6481 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     6541 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     6601 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     6661 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     6721 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     6781 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     6841 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     6901 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     6961 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     7021 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     7081 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     7141 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     7201 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     7261 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     7321 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     7381 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtt
//
