LOCUS       sequence-424509-        7245 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression, Bacterial Expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7245)
  AUTHORS   Giordano F, Saheki Y, Idevall-Hagren O, Colombo SF, Pirruccello M, 
            Milosevic I, Gracheva EO, Bagriantsev SN, Borgese N, De Camilli P
  TITLE     PI(4,5)P(2)-dependent and Ca(2+)-regulated ER-PM interactions 
            mediated by the extended synaptotagmins.
  JOURNAL   Cell. 2013 Jun 20;153(7):1494-509. doi: 10.1016/j.cell.2013.05.026.
  PUBMED    23791178
REFERENCE   2  (bases 1 to 7245)
  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: 
            "10.1016/j.cell.2013.05"; journalName: "Cell"; date: "2013-06-20- 
            20"; volume: "153"; issue: "7"; pages: "1494-509"
COMMENT     Sequence Label: EGFP-E-Syt2
FEATURES             Location/Qualifiers
     source          1..7245
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     135..154
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     394..415
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      457..466
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(505..708)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(534..554)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        709..1012
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(1187..1775)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1267..1286)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    2104..2151
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     2176..2195
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(2383..3177)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(2383..3177)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2494..2513)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3104..3123
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(3212..3569)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3226..3361
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        3378..3569
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     3526..3546
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(3571..3675)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3702..4157
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3789..3808)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3999..4020
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    4164..4285
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4175..4194)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4229..4248
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(6989..7245,1..454,455..457,458..460))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(6989..7245,1..454,455..457,458..460))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(7031..7052)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 tgatatagac gttgtggctg ttgtagttgt actccagctt gtgccccagg atgttgccgt
       61 cctccttgaa gtcgatgccc ttcagctcga tgcggttcac cagggtgtcg ccctcgaact
      121 tcacctcggc gcgggtcttg tagttgccgt cgtccttgaa gaagatggtg cgctcctgga
      181 cgtagccttc gggcatggcg gacttgaaga agtcgtgctg cttcatgtgg tcggggtagc
      241 ggctgaagca ctgcacgccg taggtcaggg tggtcacgag ggtgggccag ggcacgggca
      301 gcttgccggt ggtgcagatg aacttcaggg tcagcttgcc gtaggtggca tcgccctcgc
      361 cctcgccgga cacgctgaac ttgtggccgt ttacgtcgcc gtccagctcg accaggatgg
      421 gcaccacccc ggtgaacagc tcctcgccct tgctcaccat ggtggcgacc ggtagcgcta
      481 gcggatctga cggttcacta aaccagctct gcttatatag acctcccacc gtacacgcct
      541 accgcccatt tgcgtcaatg gggcggagtt gttacgacat tttggaaagt cccgttgatt
      601 ttggtgccaa aacaaactcc cattgacgtc aatggggtgg agacttggaa atccccgtga
      661 gtcaaaccgc tatccacgcc cattgatgta ctgccaaaac cgcatcacca tggtaatagc
      721 gatgactaat acgtagatgt actgccaagt aggaaagtcc cataaggtca tgtactgggc
      781 ataatgccag gcgggccatt taccgtcatt gacgtcaata gggggcgtac ttggcatatg
      841 atacacttga tgtactgcca agtgggcagt ttaccgtaaa tactccaccc attgacgtca
      901 atggaaagtc cctattggcg ttactatggg aacatacgtc attattgacg tcaatgggcg
      961 ggggtcgttg ggcggtcagc caggcgggcc atttaccgta agttatgtaa cgcggaactc
     1021 catatatggg ctatgaacta atgaccccgt aattgattac tattaataac taatgcatgg
     1081 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     1141 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     1201 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     1261 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     1321 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     1381 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     1441 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     1501 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     1561 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     1621 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     1681 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     1741 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     1801 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     1861 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     1921 tatatgagta acctgaggct atggcagggc ctgccgcccc gacgttggct gcgagccctg
     1981 ggccttcacc cgaacttggg gggtggggtg gggaaaagga agaaacgcgg gcgtattggc
     2041 cccaatgggg tctcggtggg gtatcgacag agtgccagcc ctgggaccga accccgcgtt
     2101 tatgaacaaa cgacccaaca ccgtgcgttt tattctgtct ttttattgcc gtcatagcgc
     2161 gggttccttc cggtattgtc tccttccgtg tttcagttag cctcccccta gggtgggcga
     2221 agaactccag catgagatcc ccgcgctgga ggatcatcca gccggcgtcc cggaaaacga
     2281 ttccgaagcc caacctttca tagaaggcgg cggtggaatc gaaatctcgt gatggcaggt
     2341 tgggcgtcgc ttggtcggtc atttcgaacc ccagagtccc gctcagaaga actcgtcaag
     2401 aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa
     2461 gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca acgctatgtc
     2521 ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa agcggccatt
     2581 ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat cctcgccgtc
     2641 gggcatgctc gccttgagcc tggcgaacag ttcggctggc gcgagcccct gatgctcttc
     2701 gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc gctcgatgcg
     2761 atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca gccgccgcat
     2821 tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca ggagatcctg
     2881 ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac
     2941 agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct cgtcttgcag
     3001 ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga
     3061 cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt catagccgaa
     3121 tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg
     3181 aaacgatcct catcctgtct cttgatcgat ctttgcaaaa gcctaggcct ccaaaaaagc
     3241 ctcctcacta cttctggaat agctcagagg ccgaggcggc ctcggcctct gcataaataa
     3301 aaaaaattag tcagccatgg ggcggagaat gggcggaact gggcggagtt aggggcggga
     3361 tgggcggagt taggggcggg actatggttg ctgactaatt gagatgcatg ctttgcatac
     3421 ttctgcctgc tggggagcct ggggactttc cacacctggt tgctgactaa ttgagatgca
     3481 tgctttgcat acttctgcct gctggggagc ctggggactt tccacaccct aactgacaca
     3541 cattccacag ctggttcttt ccgcctcagg actcttcctt tttcaatatt attgaagcat
     3601 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     3661 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgcgccct gtagcggcgc
     3721 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     3781 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     3841 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     3901 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     3961 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     4021 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     4081 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     4141 attaacgctt acaatttacg cgttaagata cattgatgag tttggacaaa ccacaactag
     4201 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     4261 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     4321 tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtatggc
     4381 tgattatgat cagttatcta gatccggtgg atccctatgt catcgcctga ggcctcgtcc
     4441 catcttccgt gaggtcatac cactgggtcc agcctttggc aagttcttca gatgccagag
     4501 caaccaatac tttgccaagg agccctttgt ctttggacag gaagccgccg ctgttcttca
     4561 cggcaacgtc gagcgttctc ctctgcactt ctggtaacga aacactgaaa tcaaagcttt
     4621 gatcaaacac tggatttaat gttttctttg acacgtgtgt tttcctcctt cctgaccgcc
     4681 tcttgtctgg taataaatac atgcggacat aggggtcaga gccgtcttca gagaaggcaa
     4741 tgaggtttct gcaggcatgc acgaccacga taagcttgtt tctctgcgag ctgtgccgga
     4801 tggtcagctg gatctgcccc agtggagact gtcccagggt cgtcccgttt tccagctgcc
     4861 tcagcctttg ccgcagctcc tgggtggcga tgggcagcga gatgtccgag gcgatgctgg
     4921 gggtcggctc cttgactgag atgtggcctg gggaggccag gaggctggag gagcttctgc
     4981 ccaggtcgtg cagcccctga gggccagcct caggcggctg ggccttttct tccataccag
     5041 gcttatcact gcccccaatg actggtgtgg atggagctgt gttgctgcca ccagggcctg
     5101 gagacccaga catatgagat ttgatggatg ttttcctccc ctctttggac acagagggac
     5161 gtttgacttg agctgagtgt tggtggtctg gaggcctttc tcgcttttcg agatggagca
     5221 cccgcagggc aatcttcatc ttgatggtgc tgtttggacc cgagttactg agctggaagc
     5281 gctggctcac agtcatgtcc tcactggtga gcagctggct gagggggacc ttcagattcc
     5341 ccagggaaca ctggtgctgc tcgtctctga cctcaacttc aaggtcctgg cgcttgggat
     5401 tgtgaatgaa gaaagtgaag ttttcctccc acacaggttc attggttttg tatcgaatct
     5461 tgctctcctg ggccttgtgc ccaactgaca tctggacaac aggatttggg ttgctgctta
     5521 ttttcttccc tgacggaagg ttccttgctg aatccaagta caagatcagc aatgcagagg
     5581 aaagaccatc gttggcttgg tctttgtcag ctttgatgtc tgttagcacc ttgtcgaggt
     5641 ttgacgcatt tggcattaac gtgagccact ccagtctcaa gtgtagcttc cccttgggaa
     5701 cctcgtccag agtgaaccat tcatctaaaa ggcgctcctt ttcaacttca atgaggtcaa
     5761 tcataagact tcctaaaaag tcatccttgt ctgggtcttc atcaaagagc tcaatctcta
     5821 attcttgtcc aggatgttca tacactaaag cctcatagac ttcattccac tttggactga
     5881 ggttctcctt gatgactctg ctttggaaga tttggttgcc aactctaatg attccatagg
     5941 ggtctgactt tcccttgaca agtcccttaa ggtaagtgtc tttcccctga agatcctgag
     6001 cttcaataaa atgtatcctt agaacaccct ttggtacagg aaaccgcaac tgagctattt
     6061 gaacttcact gacaagtgga acggtgattc gattgggaag caccagatag tttgatatta
     6121 tatccaaaat gatagtatct gataaaccat tcaatccagg gacatccaga agattcgtca
     6181 gtcctgtcca gttaatttct aaaagtggtt tcctaaggaa gaagatagac aaagctccaa
     6241 ctaagggcat atctccaatc aacggttcca ggatcacccg catggtacca tgaatctgga
     6301 tacttttcac accagctcta caaaaatatc gtttgatctc caaatcaatc tcacaatttc
     6361 ctacaaaact aatctgaagg tccaaaataa tttgcctttt gtctacattt tcagtgtata
     6421 ccttaacacc attgatcctg aggggctgct ggcccacgtc gaccttcgtg aaactaaagg
     6481 tgctaaggtg ggtgtttgct ccccgcacgg ctggttctat agtttctcga aacaacttct
     6541 ctataaattg gcaaatgaaa ggccacatgt gttttacagt cttatttagc cattctgctc
     6601 tttcagtgtc tggaaaatga acccaggcgg gcaggtcgca ggcgcgcacc cccaggcgca
     6661 cgacgcgctc ctcgtcttcc agcagcgcca gcgcgcggca caggcgcagg gccttgaggc
     6721 cgcggctgcg gcgacaccag gcgagcagcg cgagcgcgag gagaacccag ctgaagctga
     6781 gccccaggta gcccagcgcg tacacgggca gcagcagcgc gaagctccgc gccagctgcg
     6841 ccagcagccc gggcagctcc acgctcagca cgcccccggg gttctcaggc gccgcgcggc
     6901 ccccagcccc gccggcgccc gcctccgggc cctcgccccg ggcgccgctc atcgaattcg
     6961 aagcttgagc tcgagatctg agtccggact tgtacagctc gtccatgccg agagtgatcc
     7021 cggcggcggt cacgaactcc agcaggacca tgtgatcgcg cttctcgttg gggtctttgc
     7081 tcagggcgga ctgggtgctc aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga
     7141 tgggggtgtt ctgctggtag tggtcggcga gctgcacgct gccgtcctcg atgttgtggc
     7201 ggatcttgaa gttcaccttg atgccgttct tctgcttgtc ggcca
//
