LOCUS       sequence-374405-        6707 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 6707)
  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 6707)
  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: pAAV.hSynapsin.TagBFP-P2A-Pre-SPHERE-SF-iGluSnFR
FEATURES             Location/Qualifiers
     source          1..6707
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        321..768
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             783..1481
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             1488..1529
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1539..1595
                     /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(1713..1734)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     misc_feature    2820..3408
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2873..2893)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3291..3302)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3428..3904
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3627..3646)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3950..4090
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3950..4079
                     /label=AAV2 ITR
     rep_origin      4165..4620
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4252..4271)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4462..4483
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4637..4656)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4756..4778
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4816..4834)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4902..5006
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5007..5075,5076..5867)
                     /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     complement(5225..5244)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6038..6626
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6527..6546
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(6688..6707,1..110)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg cctcagtgag
       61 cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct gcggccgcac
      121 gcgttgcaaa gatggataaa gttttaaaca gagaggaatc tttgcagcta atggaccttc
      181 taggtcttga aaggagtggg aattggctcc ggtgcccgtc agtgggcaga gcgcacatcg
      241 cccacagtcc ccgagaagtt ggggggaggg gtcggcagca aatggttaat taatctagac
      301 tgcagagggc cctgcgtatg agtgcaagtg ggttttagga ccaggatgag gcggggtggg
      361 ggtgcctacc tgacgaccga ccccgaccca ctggacaagc acccaacccc cattccccaa
      421 attgcgcatc ccctatcaga gagggggagg ggaaacagga tgcggcgagg cgcgtgcgca
      481 ctgccagctt cagcaccgcg gacagtgcct tcgcccccgc ctggcggcgc gcgccaccgc
      541 cgcctcagca ctgaaggcgc gctgacgtca ctcgccggtc ccccgcaaac tccccttccc
      601 ggccaccttg gtcgcgtccg cgccgccgcc ggcccagccg gaccgcacca cgcgaggcgc
      661 gagatagggg ggcacgggcg cgaccatctg cgctgcggcg ccggcgactc agcgctgcct
      721 cagtctgcgg tgggcagcgg aggagtcgtg tcgtgcctga gagcgcagtc gagaacgcca
      781 ccatgagcga gctgattaag gagaacatgc acatgaagct gtacatggag ggcaccgtgg
      841 acaaccatca cttcaagtgc acatccgagg gcgaaggcaa gccctacgag ggcacccaga
      901 ccatgagaat caaggtggtc gagggcggcc ctctcccctt cgccttcgac atcctggcta
      961 ctagcttcct ctacggcagc aagaccttca tcaaccacac ccagggcatc cccgacttct
     1021 tcaagcagtc cttccctgag ggcttcacat gggagagagt caccacatac gaagacgggg
     1081 gcgtgctgac cgctacccag gacaccagcc tccaggacgg ctgcctcatc tacaacgtca
     1141 agatcagagg ggtgaacttc acatccaacg gccctgtgat gcagaagaaa acactcggct
     1201 gggaggcctt caccgagacg ctgtaccccg ctgacggcgg cctggaaggc agaaacgaca
     1261 tggccctgaa gctcgtgggc gggagccatc tgatcgcaaa catcaagacc acatatagat
     1321 ccaagaaacc cgctaagaac ctcaagatgc ctggcgtcta ctatgtggac tacagactgg
     1381 aaagaatcaa ggaggccaac aacgagacct acgtcgagca gcacgaggtg gcagtggcca
     1441 gatactgcga cctccctagc aaactggggc acaagcttaa tggatccggc aagcctatcc
     1501 ctaatcctct gctgggactg gactctacag gaagcggagc tactaacttc agcctgctga
     1561 agcaggctgg agacgtggag gagaaccctg gacctgagac cgacaccctc ctgctatggg
     1621 tgctgctgct ctgggtgccc ggcagcaccg gcgacgcacc ggtcggaggc atgagcaagg
     1681 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     1741 gccacaagtt cagcgtgcgc ggcgagggcg agggcgatgc caccaacggc aagctgaccc
     1801 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     1861 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
     1921 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcagcttc aaggacgacg
     1981 gcacctacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     2041 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     2101 actttaacaa ccccctgaac atgaatttcg aactgtcaga cgaaatgaaa gcactgttca
     2161 aagaaccgaa tgacaaggca ctgaaaaccg gtggatctgg aggcagtggc ggatctagag
     2221 ctcctaccta ctctccacca cctccacctg gaggaggttc cggaggcgga agcggtaccg
     2281 aagtgccttc ctctatgaca actgagtcga cagccactgc catgcagtcc gagatgtcca
     2341 cctcaatcat ggagaccacc acaaccctgg ctaccagcac agctcgacga ggaaagcccc
     2401 ccacaaagga gcctatcagc cagaccacgg atgatatcct tgtggcctcg gcagagtgtc
     2461 ccagcgacga tgaggacatt gacccctgtg agccgagctc aggtgggtta gccaacccca
     2521 cccgagtggg cggccgcgaa ccatacccag gctcggcaga ggtgatccgg gagtctagca
     2581 gtaccactgg catggtggtg gggattgtcg cagcagcagc tctgtgcatc ctcatcctcc
     2641 tctatgccat gtacaagtac agaaaccggg atgaaggctc atatcacgtg gatgagagtc
     2701 gaaactacat cagtaactca gcacagtcca atggggctgt ggtcaaggag aagcagccca
     2761 gcagtgcgaa aagcgccaac aaaaacaaga agaacaagga taaggagtat tatgtctgaa
     2821 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc
     2881 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta
     2941 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt
     3001 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg
     3061 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta
     3121 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt
     3181 tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg
     3241 cctatgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca
     3301 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc
     3361 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccggc
     3421 tcgagacggg tggcatccct gtgacccctc cccagtgcct ctcctggccc tggaagttgc
     3481 cactccagtg cccaccagcc ttgtcctaat aaaattaagt tgcatcattt tgtctgacta
     3541 ggtgtccttc tataatatta tggggtggag gggggtggta tggagcaagg ggcaagttgg
     3601 gaagacaacc tgtagggcct gcggggtcta ttgggaacca agctggagtg cagtggcaca
     3661 atcttggctc actgcaatct ccgcctcctg ggttcaagcg attctcctgc ctcagcctcc
     3721 cgagttgttg ggattccagg catgcatgac caggctcagc taatttttgt ttttttggta
     3781 gagacggggt ttcaccatat tggccaggct ggtctccaac tcctaatctc aggtgatcta
     3841 cccaccttgg cctcccaaat tgctgggatt acaggcgtga accactgctc ccttccctgt
     3901 ccttctcgag ctgattttgt aggtaaccac gtgcggaccg agcggccgca ggaaccccta
     3961 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
     4021 aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc
     4081 tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     4141 cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     4201 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg
     4261 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     4321 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     4381 tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     4441 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta
     4501 tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa
     4561 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     4621 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     4681 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     4741 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     4801 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     4861 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     4921 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     4981 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     5041 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     5101 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     5161 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     5221 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     5281 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     5341 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     5401 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     5461 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     5521 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     5581 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     5641 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     5701 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     5761 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     5821 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     5881 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     5941 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     6001 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     6061 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     6121 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     6181 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     6241 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     6301 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     6361 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     6421 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     6481 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     6541 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     6601 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     6661 ccttttgctg gccttttgct cacatgtcct gcaggcagct gcgcgct
//
