LOCUS       sequence-391940-        6172 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of Synaptotagmin 4 with pH-sensitive 
            fluorescent protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6172)
  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 6172)
  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 Syt4
FEATURES             Location/Qualifiers
     source          1..6172
                     /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    3286..3407
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3297..3316)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3351..3370
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3588..4043
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3675..3694)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3885..3906
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4110..4129)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4229..4251
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4289..4307)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4375..4479
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4480..4548,4549..5340)
                     /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(4698..4717)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5511..6099
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6000..6019
                     /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 cggcggagct cctatcacca ccagccgcgt ggaattcgat gaaattccca cagtggtggg
     2041 catcttcagt gcttttggcc tggtcttcac tgtgtctctc tttgcctgga tctgctgcca
     2101 gagaagatcc gctaagtcca acaagactcc tccatacaag tttgtgcacg tgcttaaagg
     2161 agttgatatc tacccagaaa acctaagtag caaaaagaag tttggaggag atgacaagag
     2221 tgaagcaaag cgtaaagcag ctttgcctaa cctttccctg caccttgatc tcgagaagcg
     2281 agacctcaat ggcaatttcc ccaaaaccaa ccccaaagct ggcagctctt ctgatctgga
     2341 aaatgtcacc ccaaagctct ttccggagac ggaaaaggag gccgtctccc ctgagagctt
     2401 gaagtccagc acttccctca cttcagagga gaaacaagag aagctgggca ctctcttctt
     2461 gtctctagag tacaacttcg agaagaaagc atttgtggtg aacatcaagg aagcccaggg
     2521 cttaccagcc atggatgaac aatccatgac ctctgaccca tacatcaaga tgacaatctt
     2581 accagagaag aagcacaaag tgaaaaccag agtgctgagg aagacgctgg accccgtgtt
     2641 tgacgaaacc ttcacattct atggggtccc ttatccccac atccaagagc tgtccctgca
     2701 cttcaccgtc ctgagttttg acaggttctc aagagatgat gtcattggag aagtcctcgt
     2761 tcctctttca gggattgaat tgtcagacgg gaaaatgtta atgaccagag agatcatcaa
     2821 gagaaatgct aagaagtctt ctggacgggg tgaacttctg gtctccctct gttatcagtc
     2881 cactacaaac acgctcactg tggttgtctt aaaagcgcgg cacctaccga aatctgatgt
     2941 gtctggactt tcagatccct acgtcaaagt gaacctgtac catgccaaga agagaatctc
     3001 taaaaagaag actcacgtga aaaagtgcac tcccaacgca gtgttcaacg aactgtttgt
     3061 ctttgatatt ccttgtgaga gtcttgaaga aataagtgtt gaatttttag ttttggattc
     3121 tgaaagggga tcccgaaatg aggtgatcgg gcggttggtc ctgggtgcca cagcagaagg
     3181 aagtggtggg gggcactgga aggagatctg tgacttcccc aggagacaaa ttgctaagtg
     3241 gcatatgctc tgtgatggtt aggcggccgc ttcgagcaga catgataaga tacattgatg
     3301 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     3361 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     3421 gcattcattt tatgtttcag gttcaggggg agatgtggga ggttttttaa agcaagtaaa
     3481 acctctacaa atgtggtaaa atcgataagg atccgggctg gcgtaatagc gaagaggccc
     3541 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggacg cgccctgtag
     3601 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     3661 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     3721 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     3781 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     3841 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     3901 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     3961 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     4021 caaaatatta acgcttacaa tttcctgatg cggtattttc tccttacgca tctgtgcggt
     4081 atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     4141 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     4201 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     4261 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     4321 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     4381 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     4441 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     4501 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     4561 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     4621 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     4681 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     4741 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     4801 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     4861 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     4921 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     4981 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     5041 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     5101 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     5161 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     5221 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     5281 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     5341 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     5401 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     5461 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     5521 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     5581 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     5641 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     5701 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     5761 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     5821 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     5881 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     5941 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     6001 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     6061 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     6121 ttacggttcc tggccttttg ctggcctttt gctcacatgg ctcgacagat ct
//
