LOCUS       sequence-393122-        6160 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of Synaptotagmin 1 with pH-sensitive 
            fluorescent protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6160)
  TITLE     Rothman lab pHluorin Syt1 plasmid
REFERENCE   2  (bases 1 to 6160)
  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"
COMMENT     Sequence Label: pCI pHluorin Syt1
FEATURES             Location/Qualifiers
     source          1..6160
                     /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    3274..3395
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3285..3304)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3339..3358
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3576..4031
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3663..3682)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3873..3894
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4098..4117)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4217..4239
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4277..4295)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4363..4467
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4468..4536,4537..5328)
                     /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(4686..4705)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5499..6087
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5988..6007
                     /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 cggcggagtg agtgccagtc atcctgaggc cctggccgcc cctgtcacca ctgttgcgac
     2041 ccttgtccca cacaatgcca ctgagccagc cagtcctggg gaagggaagg aagatgcctt
     2101 ttccaagctg aagcagaagt ttatgaatga gctgcataaa attccattgc caccgtgggc
     2161 cttaatagcc atagccatag ttgcggtcct tttagtcgta acctgctgct tttgtgtctg
     2221 taagaaatgt ttgttcaaaa agaaaaacaa gaagaagggg aaggaaaagg gaggaaagaa
     2281 cgccattaac atgaaagacg tgaaagactt agggaagacc atgaaggatc aggcccttaa
     2341 ggatgacgat gctgaaaccg gactgactga tggagaagaa aaggaagagc ccaaggaaga
     2401 ggagaaactg ggaaagctcc aatattcact ggactatgac ttccagaata accagctgtt
     2461 ggtgggaatc atccaggctg ctgaactgcc cgccctggac atggggggta catccgatcc
     2521 atacgtcaaa gtcttcctgc tgcctgacaa aaagaagaaa tttgagacta aagtccaccg
     2581 gaaaaccctc aatccagtct tcaatgaaca atttactttc aaggtaccct actcggaatt
     2641 aggtggcaaa accctggtga tggctgtgta tgactttgat cgcttctcca agcacgacat
     2701 catcggagag ttcaaagttc ctatgaacac cgtggatttt ggccatgtga ccgaggagtg
     2761 gcgcgatctc cagagcgctg agaaagaaga gcaagagaaa ctgggtgaca tctgcttctc
     2821 cctccgctac gtccctactg ccggcaaact gactgttgtc attctggaag ccaagaacct
     2881 gaagaagatg gatgtgggtg gcttatctga tccctacgtg aagattcacc tgatgcagaa
     2941 cggtaagagg ctgaagaaga aaaagacgac gattaagaag aacacactca acccctacta
     3001 caacgagtcc ttcagctttg aagttccgtt cgagcaaatc cagaaagtgc aagtggtggt
     3061 aactgttttg gactatgaca agattggcaa gaacgacgcc atcggcaaag tcttcgttgg
     3121 ttacaacagc actggggcgg agctgcgaca ctggtcagac atgctggcca acccccggcg
     3181 acccatcgca cagtggcaca ctctgcaggt agaggaggag gtcgatgcca tgctggctgt
     3241 caagaagtaa gcggccgctt cgagcagaca tgataagata cattgatgag tttggacaaa
     3301 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
     3361 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta
     3421 tgtttcaggt tcagggggag atgtgggagg ttttttaaag caagtaaaac ctctacaaat
     3481 gtggtaaaat cgataaggat ccgggctggc gtaatagcga agaggcccgc accgatcgcc
     3541 cttcccaaca gttgcgcagc ctgaatggcg aatggacgcg ccctgtagcg gcgcattaag
     3601 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     3661 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     3721 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     3781 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     3841 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     3901 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     3961 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     4021 gcttacaatt tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     4081 atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     4141 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     4201 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     4261 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     4321 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     4381 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     4441 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     4501 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     4561 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     4621 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     4681 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     4741 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     4801 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     4861 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     4921 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     4981 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     5041 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     5101 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     5161 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     5221 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     5281 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     5341 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     5401 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5461 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5521 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5581 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     5641 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     5701 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5761 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     5821 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     5881 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     5941 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6001 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6061 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     6121 gccttttgct ggccttttgc tcacatggct cgacagatct
//
