LOCUS       sequence-335131-        6739 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of dGFP-Granuphilin..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6739)
  AUTHORS   Prasai B, Haber GJ, Strub MP, Ahn R, Ciemniecki JA, Sochacki KA, 
            Taraska JW
  TITLE     The nanoscale molecular morphology of docked exocytic dense-core 
            vesicles in neuroendocrine cells.
  JOURNAL   Nat Commun. 2021 Jun 25;12(1):3970. doi: 10.1038/s41467-021-24167-9.
  PUBMED    34172739
REFERENCE   2  (bases 1 to 6739)
  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"; journalName: "Nat 
            Commun."; date: "2021-06-25"; volume: "12(1):3970. doi"; pages: " 
            10.1038/s41467-021-24167-9"
COMMENT     Sequence Label: dGFP-Granuphilin
FEATURES             Location/Qualifiers
     source          1..6739
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLGGGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3527..3648
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3564..3583)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3618..3637
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3655..4110)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3792..3813)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4004..4023
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4137..4241
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4243..4600
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4243..4434
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4266..4286)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4451..4586
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4635..5429
                     /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             4635..5429
                     /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(4689..4708)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5299..5318
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5617..5636)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5661..5708
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6037..6625
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6526..6545
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctggga ggtggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc aagctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt ccggactcag atctcgagaa atgtcggagt tactggacct ttcttttctg
     1381 tctgaggagg aaaaggattt gattctcagt gttctacagc gagatgaaga ggtccggaaa
     1441 gcagatgaga aaaggattag gcgactaaag aatgagttac tggagataaa aaggaaaggg
     1501 gccaagaggg gcagccaaca ctacagtgat cggacctgtg cccggtgcca ggagagcctg
     1561 ggccgtttga gtcccaaaac caatacttgt cggggttgta atcacctggt gtgtcgggac
     1621 tgccgcatac aggaaagcaa tggtacctgg aggtgcaagg tgtgcgccaa ggaaatagag
     1681 ttgaagaaag caactgggga ctggttttat gaccagaaag tgaatcgctt tgcttaccgc
     1741 acaggtagtg agataatcag gatgtccctg cgccacaaac ctgcagtgag taaaagagag
     1801 acagtgggac agtccctcct tcatcagaca cagatgggtg acatctggcc aggaagaaag
     1861 atcattcagg agcggcagaa ggagcccagt gtgctatttg aagtgccaaa gctgaaaagt
     1921 ggaaagagtg cattggaagc tgagagtgag agtctggata gcttcacagc tgactcggat
     1981 agcacctcca ggagagactc tctggataaa tctggcctct ttccagaatg gaagaagatg
     2041 tctgctccca aatctcaagt agaaaaggaa actcagcctg gaggtcaaaa tgtggtattt
     2101 gtggatgagg gtgagatgat atttaagaag aacaccagaa aaatcctcag gccttcagag
     2161 tacactaaat ctgtgataga tcttcgccca gaagatgtgg tacatgaaag tggctccttg
     2221 ggagacagaa gcaaatccgt cccaggcctc aatgtggata tggaagagga agaagaagaa
     2281 gaagacattg accacctagt gaagttacat cgccagaagc tagccagaag cagcatgcaa
     2341 agtggctcct ccatgagtac gatcggcagc atgatgagca tctacagtga agctggtgat
     2401 ttcgggaaca tctttgtgac tggcaggatt gccttttccc tgaagtatga gcagcaaacc
     2461 cagagtctgg ttgtccatgt gaaggagtgc catcagctgg cctatgctga tgaagccaag
     2521 aagcgctcta acccatatgt gaagacttac cttctgcctg acaagtcccg ccaaggaaaa
     2581 agaaaaacca gcatcaagcg ggacactgtt aatccactat atgatgagac gctgaggtat
     2641 gagatcccag aatctctcct ggcccagagg accctgcagt tctcagtttg gcatcatggt
     2701 cgttttggca gaaacacttt ccttggagag gcagagatcc agatggattc ctggaagctt
     2761 gataagaaac tggatcattg cctcccttta catggaaaga tcagtgctga gtccccgact
     2821 ggcttgccat cacacaaagg cgagttggtg gtttcattga aatacatccc agcctccaaa
     2881 acccctgttg gaggtgaccg gaaaaagagt aaaggtgggg aagggggaga gctccaggtg
     2941 tggatcaaag aagccaagaa cttgacggct gccaaagcag gagggacttc agacagcttt
     3001 gtcaagggat acctccttcc catgaggaac aaggccagta aacgtaaaac tcctgtgatg
     3061 aagaagaccc tgaatcctca ctacaaccat acatttgtct acaatggtgt gaggctggaa
     3121 gatctacagc atatgtgcct ggaactgact gtgtgggacc gggagcccct ggccagcaat
     3181 gacttcctgg gaggggtcag gctgggtgtt ggcactggga tcagtaatgg ggaagtggtg
     3241 gactggatgg actcgactgg ggaagaagtg agcctgtggc agaagatgcg acagtaccca
     3301 gggtcttggg cagaagggac tctgcagctc cgttcctcaa tggccaagca gaagctgggt
     3361 ttatgagaat tctgcagtcg acggtaccgc gggcccggga tccaccggat ctagataact
     3421 gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca
     3481 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc
     3541 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     3601 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaac gcgtaaattg
     3661 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     3721 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     3781 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     3841 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     3901 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     3961 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     4021 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     4081 ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg
     4141 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     4201 aaccctgata aatgcttcaa taatattgaa aaaggaagag tcctgaggcg gaaagaacca
     4261 gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     4321 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     4381 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     4441 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     4501 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     4561 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa gagacaggat
     4621 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     4681 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     4741 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     4801 ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     4861 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     4921 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     4981 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     5041 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     5101 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     5161 cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     5221 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     5281 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     5341 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     5401 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
     5461 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     5521 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     5581 gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc
     5641 ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc
     5701 gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag
     5761 accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc
     5821 gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagg
     5881 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     5941 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     6001 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6061 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6121 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6181 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6241 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6301 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6361 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6421 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     6481 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     6541 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     6601 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     6661 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     6721 ccgtattacc gccatgcat
//
