LOCUS       sequence-173045-        7065 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BDNF with pH sensitive fluorophore for visualization of release.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7065)
  AUTHORS   Harward SC, Hedrick NG, Hall CE, Parra-Bueno P, Milner TA, Pan E, 
            Laviv T, Hempstead BL, Yasuda R, McNamara JO
  TITLE     Autocrine BDNF-TrkB signalling within a single dendritic spine.
  JOURNAL   Nature. 2016 Sep 28;538(7623):99-103. doi: 10.1038/nature19766.
  PUBMED    27680698
REFERENCE   2  (bases 1 to 7065)
  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"; doi: "10"; journalName: 
            "Nature"; date: "2016-09-28- 28"; volume: "538"; issue: "7623"; 
            pages: "99-103"
COMMENT     Sequence Label: BDNF-SEP
FEATURES             Location/Qualifiers
     source          1..7065
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     9..30
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(149..169)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        154..483
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      334..469
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     396..415
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             532..1557
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             532..1557
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    1687..1808
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1724..1743)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1778..1797
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1857..1873)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1857..1873)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1870..1892)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1881..1897
                     /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(1905..1911,1912..1929,1930..1935))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1950..1971
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2088..2105)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2259..2847)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2339..2358)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3018..3809,3810..3878))
                     /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     3641..3660
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3879..3983)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4058..4077)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4249..4628
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4629..4832
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4783..4803
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4877..4896
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4877..4895
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             5003..5029
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             5003..5029
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             5726..5749
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5750..6463
                     /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(5777..5805)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     6400..6420
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(6501..6518)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6507..6731
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(6777..7065,1..140)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6864..6883)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
       61 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      121 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc
      181 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
      241 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
      301 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
      361 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
      421 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
      481 aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcagcacgt gatgaaaaag
      541 cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc
      601 gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg
      661 cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga tcgttatgtt
      721 tatcggcact ttgcatcggc cgcgctcccg attccggaag tgcttgacat tggggaattc
      781 agcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg
      841 cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct
      901 gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa
      961 tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta tcactggcaa
     1021 actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt
     1081 tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg ctccaacaat
     1141 gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc gatgttcggg
     1201 gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc ttgtatggag
     1261 cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc gcggctccgg
     1321 gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga cggcaatttc
     1381 gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg agccgggact
     1441 gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa
     1501 gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa ggaatagcac
     1561 gtgctacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     1621 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     1681 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     1741 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     1801 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     1861 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     1921 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     1981 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     2041 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     2101 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     2161 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     2221 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     2281 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     2341 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     2401 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     2461 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     2521 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     2581 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     2641 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     2701 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     2761 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     2821 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     2881 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     2941 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     3001 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     3061 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     3121 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     3181 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     3241 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     3301 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     3361 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     3421 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     3481 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     3541 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     3601 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     3661 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     3721 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     3781 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     3841 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     3901 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     3961 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
     4021 tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat
     4081 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca
     4141 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg
     4201 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat
     4261 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
     4321 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     4381 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
     4441 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
     4501 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
     4561 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
     4621 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
     4681 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
     4741 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
     4801 gtgtacggtg ggaggtctat ataagcagag ctctctggct aactagagaa cccactgctt
     4861 actggcttat cgaaattaat acgactcact atagggagac ccaagctggc tagcgtttaa
     4921 acttaagctt gccaccatga ccatcctttt ccttactatg gttatttcat actttggttg
     4981 catgaaggcg gccgccggag gctaccccta cgacgtgccc gactacgccg gcgcgcccat
     5041 gaaagaagca aacatccgag gacaaggtgg cttggcctac ccaggtgtgc ggacccatgg
     5101 gactctggag agcgtgaatg ggcccaaggc aggttcaaga ggcttgacat cattggctga
     5161 cactttcgaa cacgtgatag aagagctgtt ggatgaggac cagaaagttc ggcccaatga
     5221 agaaaacaat aaggacgcag acttgtacac gtccagggtg atgctcagta gtcaagtgcc
     5281 tttggagcct cctcttctct ttctgctgga ggaatacaaa aattacctag atgctgcaaa
     5341 catgtccatg agggtccggc gccactctga ccctgcccgc cgaggggagc tgagcgtgtg
     5401 tgacagtatt agtgagtggg taacggcggc agacaaaaag actgcagtgg acatgtcggg
     5461 cgggacggtc acagtccttg aaaaggtccc tgtatcaaaa ggccaactga agcaatactt
     5521 ctacgagacc aagtgcaatc ccatgggtta cacaaaagaa ggctgcaggg gcatagacaa
     5581 aaggcattgg aactcccagt gccgaactac ccagtcgtac gtgcgggccc ttaccatgga
     5641 tagcaaaaag agaattggct ggcgattcat aaggatagac acttcttgtg tatgtacatt
     5701 gaccattaaa aggggaagag gatccgacta caaggatgac gatgacaaga gtaaaggaga
     5761 agaacttttc actggagttg tcccaattct tgttgaatta gatggtgatg ttaatgggca
     5821 caaattttct gtcagtggag agggtgaagg tgatgcaaca tacggaaaac ttacccttaa
     5881 atttatttgc actactggaa aactacctgt tccttggcca acacttgtca ctactttaac
     5941 ttatggtgtt caatgctttt caagataccc agatcatatg aaacggcatg actttttcaa
     6001 gagtgccatg cccgaaggtt atgtacagga aagaactata tttttcaaag atgacgggaa
     6061 ctacaagaca cgtgctgaag tcaagtttga aggtgatacc cttgttaata gaatcgagtt
     6121 aaaaggtatt gattttaaag aagatggaaa cattcttgga cacaaattgg aatacaacta
     6181 taacgatcac caggtgtaca tcatggcaga caaacaaaag aatggaatca aagctaactt
     6241 caaaattaga cacaacattg aagatggagg cgttcaacta gcagaccatt atcaacaaaa
     6301 tactccaatt ggcgatgggc ccgtcctttt accagacaac cattacctgt ttacaacttc
     6361 tactctttcg aaagatccca acgaaaagag agaccacatg gtccttcttg agtttgtaac
     6421 agctgctggg attacacatg gcatggatga actatacaaa tagctcgagt ctagagggcc
     6481 cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     6541 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     6601 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     6661 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     6721 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     6781 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     6841 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     6901 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     6961 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     7021 gccctgatag acggtttttc gccctttgac gttggagtcc acgtt
//
