LOCUS       sequence-160461-        7062 bp    DNA     circular SYN 14-APR-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 7062)
  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 7062)
  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"; 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..7062
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             78..104
                     /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             78..104
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             801..824
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             825..1538
                     /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(852..880)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1475..1495
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(1576..1593)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1582..1806
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1852..2280
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1939..1958)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2149..2170
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2289..2309)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2294..2623
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2474..2609
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2536..2555
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2672..3697
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2672..3697
                     /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    3827..3948
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3864..3883)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3918..3937
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3997..4013)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3997..4013)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4010..4032)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4021..4037
                     /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(4045..4051,4052..4069,4070..4075))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4090..4111
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4228..4245)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4399..4984)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4479..4498)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5155..5946,5947..6015))
                     /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     5778..5797
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6016..6120)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6195..6214)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6386..6765
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6766..6969
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6920..6940
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     7014..7033
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7014..7032
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 agcttgccac catgaccatc cttttcctta ctatggttat ttcatacttt ggttgcatga
       61 aggcggccgc cggaggctac ccctacgacg tgcccgacta cgccggcgcg cccatgaaag
      121 aagcaaacat ccgaggacaa ggtggcttgg cctacccagg tgtgcggacc catgggactc
      181 tggagagcgt gaatgggccc aaggcaggtt caagaggctt gacatcattg gctgacactt
      241 tcgaacacgt gatagaagag ctgttggatg aggaccagaa agttcggccc aatgaagaaa
      301 acaataagga cgcagacttg tacacgtcca gggtgatgct cagtagtcaa gtgcctttgg
      361 agcctcctct tctctttctg ctggaggaat acaaaaatta cctagatgct gcaaacatgt
      421 ccatgagggt ccggcgccac tctgaccctg cccgccgagg ggagctgagc gtgtgtgaca
      481 gtattagtga gtgggtaacg gcggcagaca aaaagactgc agtggacatg tcgggcggga
      541 cggtcacagt ccttgaaaag gtccctgtat caaaaggcca actgaagcaa tacttctacg
      601 agaccaagtg caatcccatg ggttacacaa aagaaggctg caggggcata gacaaaaggc
      661 attggaactc ccagtgccga actacccagt cgtacgtgcg ggcccttacc atggatagca
      721 aaaagagaat tggctggcga ttcataagga tagacacttc ttgtgtatgt acattgacca
      781 ttaaaagggg aagaggatcc gactacaagg atgacgatga caagagtaaa ggagaagaac
      841 ttttcactgg agttgtccca attcttgttg aattagatgg tgatgttaat gggcacaaat
      901 tttctgtcag tggagagggt gaaggtgatg caacatacgg aaaacttacc cttaaattta
      961 tttgcactac tggaaaacta cctgttcctt ggccaacact tgtcactact ttaacttatg
     1021 gtgttcaatg cttttcaaga tacccagatc atatgaaacg gcatgacttt ttcaagagtg
     1081 ccatgcccga aggttatgta caggaaagaa ctatattttt caaagatgac gggaactaca
     1141 agacacgtgc tgaagtcaag tttgaaggtg atacccttgt taatagaatc gagttaaaag
     1201 gtattgattt taaagaagat ggaaacattc ttggacacaa attggaatac aactataacg
     1261 atcaccaggt gtacatcatg gcagacaaac aaaagaatgg aatcaaagct aacttcaaaa
     1321 ttagacacaa cattgaagat ggaggcgttc aactagcaga ccattatcaa caaaatactc
     1381 caattggcga tgggcccgtc cttttaccag acaaccatta cctgtttaca acttctactc
     1441 tttcgaaaga tcccaacgaa aagagagacc acatggtcct tcttgagttt gtaacagctg
     1501 ctgggattac acatggcatg gatgaactat acaaatagct cgagtctaga gggcccgttt
     1561 aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     1621 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     1681 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     1741 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     1801 ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
     1861 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     1921 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     1981 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     2041 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     2101 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     2161 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     2221 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     2281 aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     2341 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     2401 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     2461 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     2521 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca
     2581 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg
     2641 tatatccatt ttcggatctg atcagcacgt gatgaaaaag cctgaactca ccgcgacgtc
     2701 tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga
     2761 gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt
     2821 aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc
     2881 cgcgctcccg attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg
     2941 catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc
     3001 tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac
     3061 gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt
     3121 catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt
     3181 cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga
     3241 agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg
     3301 cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc
     3361 caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga
     3421 gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg
     3481 tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca
     3541 gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc
     3601 ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa
     3661 ccgacgcccc agcactcgtc cgagggcaaa ggaatagcac gtgctacgag atttcgattc
     3721 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     3781 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     3841 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     3901 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     3961 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     4021 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     4081 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     4141 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4201 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     4261 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     4321 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     4381 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     4441 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4501 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     4561 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     4621 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     4681 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     4741 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     4801 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     4861 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     4921 caccgctggt agcggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     4981 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     5041 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     5101 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     5161 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     5221 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     5281 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     5341 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     5401 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     5461 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     5521 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     5581 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     5641 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     5701 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     5761 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     5821 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     5881 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     5941 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6001 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     6061 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6121 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta
     6181 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
     6241 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa
     6301 ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc
     6361 gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca
     6421 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     6481 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     6541 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     6601 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     6661 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     6721 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
     6781 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
     6841 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
     6901 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
     6961 agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga aattaatacg
     7021 actcactata gggagaccca agctggctag cgtttaaact ta
//
