LOCUS       sequence-335057-        6885 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of His-GFP-Rabphilin..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6885)
  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 6885)
  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: His-GFP-Rabphilin
FEATURES             Location/Qualifiers
     source          1..6885
                     /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             625..642
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(652..654,655..1365)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(694..715)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(955..974)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1302..1323
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3673..3794
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3710..3729)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3764..3783
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3801..4256)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3938..3959)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4150..4169
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4283..4387
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4389..4746
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4389..4580
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4412..4432)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4597..4732
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4781..5575
                     /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             4781..5575
                     /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(4835..4854)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5445..5464
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5763..5782)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5807..5854
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6183..6771
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6672..6691
                     /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 ccatgggcag cagccatcat catcatcatc acagcagcgg cgtgagcaag
      661 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
      721 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
      781 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
      841 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
      901 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
      961 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     1021 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     1081 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     1141 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     1201 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     1261 cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     1321 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtccgg actcagatct
     1381 atgactgaca ccgtgttcag cagcagctca agccgttgga tgtgtcccag tgaccgaccc
     1441 ctgcagtcaa atgataaaga gcagctccag acaggatggt ctgtccaccc cagcggtcag
     1501 ccggacagac agaggaaaca ggaagagctg acagatgagg agaaggaaat catcaacagg
     1561 gtgatcgctc gcgctgagaa gatggaagag atggaacagg agcgaatcgg gcgcctggtg
     1621 gaccgcctag agaacatgag gaagaacgtg gccggggacg gggtgaaccg ctgcatcctg
     1681 tgtggagaac agctggggat gctgggctct gcctgtgtgg tgtgtgagga ctgtaagaag
     1741 aatgtctgca ccaagtgtgg ggtggagacc tccaacaacc gcccacaccc tgtgtggctc
     1801 tgcaaaatct gcatcgagca gagagaggtg tggaagcgct ctggagcatg gttcttcaag
     1861 ggcttcccca aacaggtcct cccacagcct atgcccataa agaagaacaa gccccagcag
     1921 ccagtcagtg agcctgtgcc cgctgcccct gagcagccca ctcctgagcc caagcaccct
     1981 gcccgggctc caactcgagt tttttctctc tctgcaggtg acactgaaga caggaggggc
     2041 ccaggtcaga agacaggccc tgacatgact tctgctcctg ggcgaggaag ctacgggcct
     2101 cctgtgcgca gggcctctga ggcacgaatg agctcctctg gccgggactc agacagctgg
     2161 gaccagggcc acggcatggc tgctggggac cccagccaga gcccagcagg tttgagacgg
     2221 gccaactcag tccaggcttc tagacctgcc ccagcctcaa tgcagagccc ggcacctccc
     2281 caaccggggc aaccaggacc cccagaaggc agcagaccca gtccggggcc gacgggacgc
     2341 tttccagatc agagaccaga ggtggctccc agtgaccccg actatacagg ggccgccgcc
     2401 caaccccggg aggagagaac cgggggaatc ggaggctact cggcagctgg aaccagggag
     2461 gaccgagcgg gccacccccc gggctcctat acccaagcct ctgcggctgc tccccagccg
     2521 gtggtggcct cggcccgtca gcccccaccc cctgaggagg acgaggagga agctaacagc
     2581 tacgattcgg atgaagcaac caccctgggt gccctggagt tcagccttct ctatgaccag
     2641 gacaacagct ccctgcactg caccatcata aaggccaagg gactgaagcc catggattcg
     2701 aatggcttgg ccgatcccta cgttaagctg cacctcctgc cgggagccag caagtccaac
     2761 aagcttcgta caaaaaccct gcggaacacc agaaacccca tctggaacga aactctggtc
     2821 taccatggca tcacagacga ggacatgcaa aggaagaccc tcagaatctc tgtctgtgat
     2881 gaggacaaat tcggccataa tgagtttatt ggtgagacca gattctccct caagaaactg
     2941 aagcccaatc agaggaagaa cttcaacatc tgtctggagc gggtgatccc gatgaagcgt
     3001 gccggaacca ctgggtcagc ccgaggcatg gccctctatg aggaggagca ggtggaacgt
     3061 attggtgaca tagaggaacg cggcaagatc ctggtgtccc tcatgtacag cacacagcag
     3121 ggaggcctca tcgtgggcat catacgctgc gtgcacctgg cggccatgga tgccaatggc
     3181 tactcagatc catttgtcaa gctctggctg aaaccagaca tgggaaagaa agccaaacac
     3241 aagactcaaa ttaagaagaa aaccttgaat cctgaattta atgaggaatt tttctatgac
     3301 atcaaacaca gtgacctggc gaagaagtca ctggacatct cagtctggga ctatgacatc
     3361 ggcaagtcca atgattacat tggaggctgc cagctgggga tctcggccaa gggagagcgc
     3421 ttaaaacact ggtacgagtg tctgaaaaac aaggacaaga agatcgaacg ctggcaccag
     3481 ctacagaacg agaaccacgt gtcgagcgat tagaattctg cagtcgacgg taccgcgggc
     3541 ccgggatcca ccggatctag ataactgatc ataatcagcc ataccacatt tgtagaggtt
     3601 ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca
     3661 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     3721 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     3781 atcaatgtat cttaacgcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     3841 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     3901 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     3961 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     4021 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     4081 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     4141 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     4201 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg
     4261 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     4321 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     4381 gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag
     4441 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     4501 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     4561 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     4621 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     4681 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     4741 tgcaaagatc gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     4801 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     4861 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     4921 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct
     4981 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     5041 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     5101 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     5161 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     5221 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     5281 agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac
     5341 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     5401 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     5461 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     5521 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     5581 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     5641 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     5701 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta
     5761 actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga
     5821 cagaataaaa cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc
     5881 tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc
     5941 ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg
     6001 gcggcaggcc ctgccatagc ctcaggttac tcatatatac tttagattga tttaaaactt
     6061 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     6121 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6181 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6241 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6301 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     6361 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6421 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6481 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6541 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6601 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     6661 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     6721 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     6781 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     6841 gcgttatccc ctgattctgt ggataaccgt attaccgcca tgcat
//
