LOCUS       sequence-391948-        6064 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of Synaptotagmin 8 with pH-sensitive 
            fluorescent protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6064)
  AUTHORS   Dean C, Dunning FM, Liu H, Bomba-Warczak E, Martens H, Bharat V, 
            Ahmed S, Chapman ER
  TITLE     Axonal and dendritic synaptotagmin isoforms revealed by a 
            pHluorin-syt functional screen.
  JOURNAL   Mol Biol Cell. 2012 May;23(9):1715-27. doi: 10.1091/mbc.E11-08-0707.
            Epub 2012 Mar 7.
  PUBMED    22398727
REFERENCE   2  (bases 1 to 6064)
  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: 
            "10.1091/mbc.E11-08-0707"; journalName: "Mol Biol Cell"; date: 
            "2012-05"; volume: "23"; issue: "9"; pages: "1715-27"
COMMENT     Sequence Label: pCI pHluorin Syt8
FEATURES             Location/Qualifiers
     source          1..6064
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..721
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     672..692
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     718..742
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          857..989
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     972..991
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1034..1052
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     5'UTR           1076..1118
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     CDS             1127..1216
                     /codon_start=1
                     /product="signal sequence from bovine prolactin"
                     /label=prolactin signal sequence
                     /translation="MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS"
     CDS             1235..1945
                     /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(1262..1290)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1885..1905
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     polyA_signal    3178..3299
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3189..3208)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3243..3262
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3480..3935
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3567..3586)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3777..3798
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4002..4021)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4121..4143
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4181..4199)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4267..4371
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4372..4440,4441..5232)
                     /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(4590..4609)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5403..5991
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5892..5911
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      661 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      721 tcgtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc acagttaaat
      781 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc
      841 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa
      901 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac
      961 tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta
     1021 aggctagagt acttaatacg actcactata ggctagcctc gagaattcga tatcgcttgt
     1081 tctttttgca gaagctcaga ataaacgctc aactttggca gataccatgg acagcaaagg
     1141 ttcgtcgcag aaagggtccc gcctgctcct gctgctggtg gtgtcaaatc tactcttgtg
     1201 ccagggtgtg gtctccgata tcggtaccac gcgtagtaaa ggcgaagaac ttttcactgg
     1261 agttgtccca attcttgttg aattagatgg tgatgttaat gggcacaaat tttctgtcag
     1321 tggagagggt gaaggtgatg caacatacgg aaaacttacc cttaaattta tttgcactac
     1381 tggaaaacta cctgttcctt ggccaacact tgtcactact ttaacttatg gtgttcaatg
     1441 cttttcaaga tacccagatc atatgaaacg gcatgacttt ttcaagagtg ccatgcccga
     1501 aggttatgta caggaaagaa ctatattttt caaagatgac gggaactaca agacacgtgc
     1561 tgaagtcaag tttgaaggtg atacccttgt taatagaatc gagttaaaag gtattgattt
     1621 taaagaagat ggaaacattc ttggacacaa attggaatac aactataacg atcaccaggt
     1681 gtacatcatg gcagacaaac aaaagaatgg aatcaaagct aacttcaaaa ttagacacaa
     1741 cattgaagat ggaggcgttc aactagcaga ccattatcaa caaaatactc caattggcga
     1801 tgggcccgtc cttttaccag acaaccatta cctgtttaca acttctactc tttcgaaaga
     1861 tcccaacgaa aagagagacc acatggtcct tcttgagttt gtaacagctg ctgggattac
     1921 acatggcatg gatgaactat acaaagaatt ctcaggcgga agcggaggct ccggcgggag
     1981 cggcggaaag atggggcacg ccttaaaccc cttcagcacc tctgccccac ttgatgccac
     2041 agctgggccc agtctcatcc cagacctcat caccaggatc ccctggcctc gctggaccct
     2101 ctttatcgcc atccttgctg ctggagtcct cttagtctcc tgtttgctct gtgtcatctg
     2161 ttgctattgc caccgccatc gccacaggaa gcaacccaag gataaagaaa ctgtgggcct
     2221 gggcagtgcc cgtaatagca ccactactca cctggttcaa ccagatgtgg actgcctaga
     2281 accctgctct ggaggggacc aacagtgggg tcgactcctg ctgtcactag agtatgattt
     2341 tggaagccag gagattagag tgggcctgag gcaggctgga aacctgaagg ctgagggcac
     2401 agcagacccc tacgcctggg tcagtgtgtc cacccagtct gggagaagac atgagacaaa
     2461 ggtgcaccga gggacgctct ctcccatgtt tgaagagaca tgctgctttc tggtcccacc
     2521 agcagagctg cccaaagcca ctctgaaggt gcagctgtgg gacttcaagc ggttttcaga
     2581 acacgagcca ctgggtgagc tccaactacc cctgggcacc gtggaccttc aacatgtcct
     2641 ggagagctgg taccagctag gccctccagg gaccactgag cctgagcaga tgggggagct
     2701 gtgcttctca ctacgctacg tgcccagctc aggcagcctg actgtggttg tactggaggc
     2761 tcggggtctg aatccggggc ttgcagaggc ctacgtgaag atccagctta tgttaaacca
     2821 gagaaaatgg aagaagagca aaacatcctc caagaaaggc acgactaccc cctacttcaa
     2881 cgaggccttc gtcttcctgg ttcccgttag ccagctccag agtgtggacc tggtgctggc
     2941 tgtctgggcc cgtggtctgc agctccggac tgagcctgtg ggcaaagtgc tgctgggttc
     3001 ccgggcttct ggccaacccc tacagcactg ggcagacatg ctggcccatg ctcggcggcc
     3061 catcgcccag tggcaccacc tgcggtcccc cagggaagtg gatcgtgttc tggccctgca
     3121 gcctcgcctt cccctgctta ggcctcgctc ctgagcggcc gcttcgagca gacatgataa
     3181 gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt
     3241 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta
     3301 acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggagatgtgg gaggtttttt
     3361 aaagcaagta aaacctctac aaatgtggta aaatcgataa ggatccgggc tggcgtaata
     3421 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga
     3481 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3541 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3601 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     3661 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     3721 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     3781 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     3841 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     3901 cgcgaatttt aacaaaatat taacgcttac aatttcctga tgcggtattt tctccttacg
     3961 catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc
     4021 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     4081 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     4141 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     4201 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     4261 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     4321 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     4381 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     4441 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     4501 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     4561 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     4621 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     4681 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     4741 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     4801 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     4861 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     4921 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     4981 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     5041 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     5101 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     5161 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     5221 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     5281 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     5341 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     5401 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     5461 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     5521 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     5581 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     5641 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     5701 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     5761 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     5821 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     5881 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     5941 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     6001 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat ggctcgacag
     6061 atct
//
