LOCUS       sequence-335139-        6775 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Mammalian expression of His-GFP-Granuphilin..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6775)
  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 6775)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-Granuphilin
FEATURES             Location/Qualifiers
     source          1..6775
                     /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    3563..3684
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3600..3619)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3654..3673
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3691..4146)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3828..3849)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4040..4059
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4173..4277
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4279..4636
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4279..4470
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4302..4322)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4487..4622
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4671..5465
                     /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             4671..5465
                     /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(4725..4744)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5335..5354
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5653..5672)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5697..5744
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6073..6661
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6562..6581
                     /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 cgagaaatgt cggagttact ggacctttct tttctgtctg aggaggaaaa ggatttgatt
     1441 ctcagtgttc tacagcgaga tgaagaggtc cggaaagcag atgagaaaag gattaggcga
     1501 ctaaagaatg agttactgga gataaaaagg aaaggggcca agaggggcag ccaacactac
     1561 agtgatcgga cctgtgcccg gtgccaggag agcctgggcc gtttgagtcc caaaaccaat
     1621 acttgtcggg gttgtaatca cctggtgtgt cgggactgcc gcatacagga aagcaatggt
     1681 acctggaggt gcaaggtgtg cgccaaggaa atagagttga agaaagcaac tggggactgg
     1741 ttttatgacc agaaagtgaa tcgctttgct taccgcacag gtagtgagat aatcaggatg
     1801 tccctgcgcc acaaacctgc agtgagtaaa agagagacag tgggacagtc cctccttcat
     1861 cagacacaga tgggtgacat ctggccagga agaaagatca ttcaggagcg gcagaaggag
     1921 cccagtgtgc tatttgaagt gccaaagctg aaaagtggaa agagtgcatt ggaagctgag
     1981 agtgagagtc tggatagctt cacagctgac tcggatagca cctccaggag agactctctg
     2041 gataaatctg gcctctttcc agaatggaag aagatgtctg ctcccaaatc tcaagtagaa
     2101 aaggaaactc agcctggagg tcaaaatgtg gtatttgtgg atgagggtga gatgatattt
     2161 aagaagaaca ccagaaaaat cctcaggcct tcagagtaca ctaaatctgt gatagatctt
     2221 cgcccagaag atgtggtaca tgaaagtggc tccttgggag acagaagcaa atccgtccca
     2281 ggcctcaatg tggatatgga agaggaagaa gaagaagaag acattgacca cctagtgaag
     2341 ttacatcgcc agaagctagc cagaagcagc atgcaaagtg gctcctccat gagtacgatc
     2401 ggcagcatga tgagcatcta cagtgaagct ggtgatttcg ggaacatctt tgtgactggc
     2461 aggattgcct tttccctgaa gtatgagcag caaacccaga gtctggttgt ccatgtgaag
     2521 gagtgccatc agctggccta tgctgatgaa gccaagaagc gctctaaccc atatgtgaag
     2581 acttaccttc tgcctgacaa gtcccgccaa ggaaaaagaa aaaccagcat caagcgggac
     2641 actgttaatc cactatatga tgagacgctg aggtatgaga tcccagaatc tctcctggcc
     2701 cagaggaccc tgcagttctc agtttggcat catggtcgtt ttggcagaaa cactttcctt
     2761 ggagaggcag agatccagat ggattcctgg aagcttgata agaaactgga tcattgcctc
     2821 cctttacatg gaaagatcag tgctgagtcc ccgactggct tgccatcaca caaaggcgag
     2881 ttggtggttt cattgaaata catcccagcc tccaaaaccc ctgttggagg tgaccggaaa
     2941 aagagtaaag gtggggaagg gggagagctc caggtgtgga tcaaagaagc caagaacttg
     3001 acggctgcca aagcaggagg gacttcagac agctttgtca agggatacct ccttcccatg
     3061 aggaacaagg ccagtaaacg taaaactcct gtgatgaaga agaccctgaa tcctcactac
     3121 aaccatacat ttgtctacaa tggtgtgagg ctggaagatc tacagcatat gtgcctggaa
     3181 ctgactgtgt gggaccggga gcccctggcc agcaatgact tcctgggagg ggtcaggctg
     3241 ggtgttggca ctgggatcag taatggggaa gtggtggact ggatggactc gactggggaa
     3301 gaagtgagcc tgtggcagaa gatgcgacag tacccagggt cttgggcaga agggactctg
     3361 cagctccgtt cctcaatggc caagcagaag ctgggtttat gagaattctg cagtcgacgg
     3421 taccgcgggc ccgggatcca ccggatctag ataactgatc ataatcagcc ataccacatt
     3481 tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa
     3541 aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag
     3601 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     3661 gtccaaactc atcaatgtat cttaacgcgt aaattgtaag cgttaatatt ttgttaaaat
     3721 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     3781 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     3841 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     3901 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     3961 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     4021 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     4081 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     4141 gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     4201 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     4261 attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg
     4321 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     4381 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     4441 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     4501 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     4561 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     4621 cctaggcttt tgcaaagatc gatcaagaga caggatgagg atcgtttcgc atgattgaac
     4681 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     4741 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     4801 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg
     4861 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     4921 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     4981 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     5041 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     5101 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     5161 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc
     5221 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     5281 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     5341 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     5401 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     5461 tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg
     5521 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     5581 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccctag
     5641 ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc
     5701 aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttcataaac gcggggttcg
     5761 gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg
     5821 cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc
     5881 caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac tttagattga
     5941 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     6001 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6061 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6121 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6181 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     6241 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6301 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     6361 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     6421 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     6481 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     6541 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     6601 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     6661 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     6721 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcca tgcat
//
