LOCUS       sequence-122772-        8042 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 8042)
  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 8042)
  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-Syt1
FEATURES             Location/Qualifiers
     source          1..8042
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        68..371
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        372..575
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     526..546
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      614..623
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(620..622,623..625,626..1336)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(620..622,623..625,626..1336)
                     /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(665..686)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(926..945)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1273..1294
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4837..4958
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4874..4893)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4928..4947
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4965..5420)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5102..5123)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5314..5333
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5447..5551
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5553..5910
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5553..5744
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5576..5596)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5761..5896
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5945..6739
                     /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             5945..6739
                     /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(5999..6018)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6609..6628
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6927..6946)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6971..7018
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7347..7935
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7836..7855
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgcattag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
       61 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
      121 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
      181 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
      241 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
      301 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      361 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      421 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      481 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      541 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      601 agcgctaccg gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
      661 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
      721 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
      781 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
      841 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
      901 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
      961 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     1021 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     1081 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     1141 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     1201 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     1261 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     1321 ggacgagctg tacaagtccg gactcagatc tcgagctcaa gcttcgaatt ctgcagtcga
     1381 catggagcga tctccaggag agggccccag ccccagcccc atggaccagc cctctgctcc
     1441 ctccgacccc actgaccagc cccccgctgc tcacgcaaag ccagacccag gttctggggg
     1501 ccaacctgct ggccctggcg cggcgggtga ggccctggcg gtgctgactt cattcgggag
     1561 gcggttgctg gtgctgatac ctgtgtattt ggccggggca gtgggactca gcgtgggttt
     1621 cgtgctcttc ggcctcgccc tctacctggg ctggcgccgg gtccgcgacg agaaagaacg
     1681 gagccttcga gcagcgaggc agctactgga cgacgaggag cagctcactg cgaaaactct
     1741 ctatatgagt catcgagagc tacctgcctg ggtcagcttc ccagacgtgg aaaaggctga
     1801 atggctcaat aagattgtgg cccaggtctg gcccttcctg ggccagtata tggagaagct
     1861 tctggctgaa actgtggctc cggctgttag gggatctaac ccccatctgc aaacatttac
     1921 atttacacga gtggaactgg gtgaaaagcc attgcgcatc attggagtca aggttcaccc
     1981 aggtcagaga aaagagcaga tcctgctgga cttgaacatc agctatgtag gtgatgtgca
     2041 gattgatgtg gaagtgaaga aatatttttg caaagcagga gtcaagggca tgcagctaca
     2101 tggcgttttg cgggtgatac tggagccact cattggggac cttcccttcg tgggggctgt
     2161 gtcaatgttc ttcatccgac gcccgaccct agacatcaac tggacaggga tgaccaacct
     2221 gctggatatc ccaggactta gctcactctc tgacaccatg atcatggact ccattgctgc
     2281 cttcctcgtg ttgcccaacc gattactggt gccccttgtg cctgaccttc aagatgtggc
     2341 tcagttgcgt tcccctctgc ccaggggcat tattcgaatt cacctgctgg ctgctcgagg
     2401 gctgagttcc aaggacaaat atgtgaaggg cctgattgag ggcaagtcag acccatatgc
     2461 acttgtgcgt ttgggtaccc agacattctg cagtcgtgtc attgatgaag aactcaaccc
     2521 acagtgggga gagacttatg aggtgatggt acacgaggtc ccagggcagg agattgaagt
     2581 ggaggtgttc gacaaggatc cagataaaga tgactttctg ggcagaatga agctggatgt
     2641 agggaaggtg ttacaggcta gcgttctgga tgattggttc cctctacaag gtgggcaagg
     2701 ccaagttcac ttgaggctag aatggctgtc acttttgtca gatgcagaga aactggagca
     2761 ggttctacag tggaattggg gagtctcctc tcgaccagat cccccgtcag ctgccatctt
     2821 agttgtctac ctggatcggg cccaggatct tcctctgaag aaggggaaca aggaacccaa
     2881 ccctatggta caactgtcaa ttcaggatgt gactcaggag agcaaggctg tctacagtac
     2941 caactgccca gtgtgggagg aagcgttccg gttcttccta caagaccctc aaagccagga
     3001 gctcgatgtg caagtgaagg atgattccag ggccctgact ttaggagcac tgacgctgcc
     3061 tctggcccgc ctgctgactg ccccagaact catcctggac cagtggttcc agctcagcag
     3121 ctctggtcca aactccagac tctatatgaa actagtcatg aggatcctgt acttggattc
     3181 atcagaaata tgcttcccca cggtgcctgg ttgtcctggt gcttgggacg tggacagtga
     3241 gaatccccag agaggcagca gtgtggatgc cccacctcga ccctgtcaca cgactcctga
     3301 tagccagttt gggactgagc atgtgcttcg gatccatgta ttagaggccc aggacctgat
     3361 tgccaaagac cgtttcttgg ggggactggt gaagggcaag tcagacccct atgtcaaact
     3421 aaagttggca ggacgaagct tccggagcca tgttgttcgg gaagatctca atccccgctg
     3481 gaatgaggtt tttgaggtga tcgtcacatc agttccaggc caagagctag aggttgaagt
     3541 ctttgacaag gacttggaca aggatgattt tctgggcagg tgtaaagtgc gtctcaccac
     3601 agtcttaaac agtggcttcc ttgatgagtg gctgaccctg gaggatgtcc catctggccg
     3661 cctgcacttg cgcctggagc gtctcacccc ccgtcccact gctgctgagt tagaggaggt
     3721 gctgcaggtg aatagtttga tccagactca gaagagtgcg gagctggctg cggccctgct
     3781 atccatctat atggagcggg cagaggacct cccgctgcga aaaggcacca agcacctcag
     3841 cccttatgct actctcactg tgggagatag ttctcataaa accaagacta tttcgcaaac
     3901 ttcagcccct gtctgggatg agagtgcctc ctttctcatc aggaaaccac acactgagag
     3961 cctagagttg caggttcggg gtgagggcac tggcgtgctg ggctcattat ccctgcccct
     4021 ctcagagctc ctcgtggctg accagctctg cttggaccgc tggtttacac tcagcagtgg
     4081 tcaggggcag gtgctactga gagcacagct agggatcctg gtgtcccagc actcgggagt
     4141 ggaagctcat agccacagct acagccacag ctcctcatcg ctgagtgaag aaccagagct
     4201 ctcgggggga ccccctcaca tcacctcctc agccccagag ctccggcagc gcctaacaca
     4261 tgttgacagt ccccttgagg ctccagccgg gcctctgggc caggtgaaac tgactctgtg
     4321 gtactacagt gaagaacgaa agctggtcag cattgttcat ggttgccggt cccttcgaca
     4381 gaatggacgt gatcctcctg atccctatgt gtcactgttg ctactgccag acaagaaccg
     4441 aggcaccaag aggaggacct cacagaagaa gaggaccctg agtcctgaat ttaatgaacg
     4501 gtttgagtgg gaactccccc tggatgaggc ccagagacga aagctggatg tctctgtcaa
     4561 gtctaattcc tccttcatgt caagagagcg tgagctgctg gggaaggtgc agctggacct
     4621 agctgagaca gacctttccc agggtgtagc ccggtggtat gacctgatgg acaacaagga
     4681 caagggcagc tcctagccgc gggcccggga tccaccggat ctagataact gatcataatc
     4741 agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg
     4801 aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat
     4861 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     4921 tctagttgtg gtttgtccaa actcatcaat gtatcttaac gcgtaaattg taagcgttaa
     4981 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     5041 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     5101 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     5161 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     5221 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     5281 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     5341 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     5401 tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     5461 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     5521 aatgcttcaa taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat
     5581 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     5641 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     5701 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     5761 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     5821 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     5881 ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt
     5941 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     6001 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     6061 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     6121 actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     6181 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     6241 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     6301 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     6361 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     6421 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc
     6481 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     6541 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     6601 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     6661 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     6721 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     6781 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     6841 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     6901 ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc
     6961 cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc gtttgttcat
     7021 aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg
     7081 ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc gggtgaaggc
     7141 ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagg ttactcatat
     7201 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     7261 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     7321 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     7381 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     7441 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     7501 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     7561 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     7621 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     7681 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     7741 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     7801 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     7861 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     7921 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     7981 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     8041 gc
//
