LOCUS       sequence-369264-        7461 bp    DNA     circular SYN 14-APR-2025
DEFINITION  GABA sensor for localization to membrane contatct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7461)
  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 7461)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-TagBFP-P2A-Pre-SPHERE-iGABASnFR
FEATURES             Location/Qualifiers
     source          1..7461
                     /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"
     CDS             2661..3359
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             3366..3407
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3417..3473
                     /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(3591..3612)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4808..4825)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4814..5038
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5084..5512
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5171..5190)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5381..5402
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5521..5541)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5526..5855
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5706..5841
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5768..5787
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5922..6716
                     /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(5976..5995)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6586..6605
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6890..7011
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6927..6946)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6981..7000
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7060..7076)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7060..7076)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7073..7095)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7084..7100
                     /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(7108..7114,7115..7132,7133..7138))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7153..7174
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7291..7308)
                     /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 atgagcgagc tgattaagga gaacatgcac atgaagctgt
     2701 acatggaggg caccgtggac aaccatcact tcaagtgcac atccgagggc gaaggcaagc
     2761 cctacgaggg cacccagacc atgagaatca aggtggtcga gggcggccct ctccccttcg
     2821 ccttcgacat cctggctact agcttcctct acggcagcaa gaccttcatc aaccacaccc
     2881 agggcatccc cgacttcttc aagcagtcct tccctgaggg cttcacatgg gagagagtca
     2941 ccacatacga agacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct
     3001 gcctcatcta caacgtcaag atcagagggg tgaacttcac atccaacggc cctgtgatgc
     3061 agaagaaaac actcggctgg gaggccttca ccgagacgct gtaccccgct gacggcggcc
     3121 tggaaggcag aaacgacatg gccctgaagc tcgtgggcgg gagccatctg atcgcaaaca
     3181 tcaagaccac atatagatcc aagaaacccg ctaagaacct caagatgcct ggcgtctact
     3241 atgtggacta cagactggaa agaatcaagg aggccaacaa cgagacctac gtcgagcagc
     3301 acgaggtggc agtggccaga tactgcgacc tccctagcaa actggggcac aagcttaatg
     3361 gatccggcaa gcctatccct aatcctctgc tgggactgga ctctacagga agcggagcta
     3421 ctaacttcag cctgctgaag caggctggag acgtggagga gaaccctgga cctgagaccg
     3481 acaccctcct gctatgggtg ctgctgctct gggtgcccgg cagcaccggc gacgcaccgg
     3541 tcggaggcat gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     3601 tggacggcga cgtaaacggc cacaagttca gcgtgcgcgg cgagggcgag ggcgatgcca
     3661 ccaacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     3721 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     3781 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     3841 tcagcttcaa ggacgacggc acctacaaga cccgcgccga ggtgaagttc gagggcgaca
     3901 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     3961 ggcacaagct ggagtacaac tggaacgcca acctggcacc gaacctgccg accgcctacg
     4021 tcaaggatca gatcaccctc gatttcgcct actgggccaa gaacggtccg gccattgcga
     4081 cacggtggaa tgaatggcta gtcaaaaccg gtggatctgg aggcagtggc ggatctagag
     4141 ctcctaccta ctctccacca cctccacctg gaggaggttc cggaggcgga agcggtaccg
     4201 aagtgccttc ctctatgaca actgagtcga cagccactgc catgcagtcc gagatgtcca
     4261 cctcaatcat ggagaccacc acaaccctgg ctaccagcac agctcgacga ggaaagcccc
     4321 ccacaaagga gcctatcagc cagaccacgg atgatatcct tgtggcctcg gcagagtgtc
     4381 ccagcgacga tgaggacatt gacccctgtg agccgagctc aggtgggtta gccaacccca
     4441 cccgagtggg cggccgcgaa ccatacccag gctcggcaga ggtgatccgg gagtctagca
     4501 gtaccactgg catggtggtg gggattgtcg cagcagcagc tctgtgcatc ctcatcctcc
     4561 tctatgccat gtacaagtac agaaaccggg atgaaggctc atatcacgtg gatgagagtc
     4621 gaaactacat cagtaactca gcacagtcca atggggctgt ggtcaaggag aagcagccca
     4681 gcagtgcgaa aagcgccaac aaaaacaaga agaacaagga taaggagtat tatgtctgaa
     4741 ttctgcagat atccagcaca gtggcggccg ctcgagtcta gagggcccgt ttaaacccgc
     4801 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     4861 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     4921 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     4981 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     5041 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     5101 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     5161 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     5221 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     5281 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     5341 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     5401 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     5461 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     5521 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     5581 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     5641 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     5701 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     5761 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     5821 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     5881 ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     5941 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     6001 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     6061 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc
     6121 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     6181 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     6241 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     6301 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     6361 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     6421 cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga
     6481 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     6541 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     6601 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     6661 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     6721 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
     6781 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     6841 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     6901 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     6961 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     7021 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     7081 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     7141 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     7201 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     7261 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     7321 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     7381 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     7441 aaaggccgcg ttgctggcgt t
//
