LOCUS       sequence-335125-        5421 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of His-GFP-Clathrin..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5421)
  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 5421)
  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-Clathrin
FEATURES             Location/Qualifiers
     source          1..5421
                     /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"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(652..654,655..1365)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     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    2209..2330
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2246..2265)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2300..2319
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2337..2792)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2474..2495)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2686..2705
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2819..2923
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2925..3282
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2925..3116
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2948..2968)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3133..3268
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3317..4111
                     /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             3317..4111
                     /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(3371..3390)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3981..4000
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4299..4318)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4343..4390
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4719..5307
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5208..5227
                     /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 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     1321 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtccgg actcagatct
     1381 cgagctcaag cttcgaattc tatggccgag ttggatccat tcggcgatcc cgccggtgcg
     1441 cccggtggcc ccgctctagg aaacggggtg gcgggcgccg gcgaagaaga cccggccgcg
     1501 gccttcttgg cgcagcaaga gagtgagatt gcgggcatcg agaacgatga ggccttcgcc
     1561 atcctggacg gcggcgcccc cgggccgcag ccacacggcg agccgccggg aggtccggat
     1621 gctgttgacg gagtgatgaa tggtgaatac taccaggaga gcaatggtcc aacagacagt
     1681 tacgcagcca tttcagaagt ggatcgattg cagtcagagc ctgaaagtat ccgtaagtgg
     1741 agagaagagc agacagagcg cctggaagcc ctcgatgcca attctcggaa gcaagaagcg
     1801 gagtggaaag aaaaggcgat aaaggagctg gaagagtggt atgcgaggca ggacgagcag
     1861 ctacagaaga caaaggcaaa caacagggca gcagaagaag cctttgtaaa cgacattgac
     1921 gagtcatccc caggcactga gtgggagcgg gtggcccgcc tgtgtgactt taaccccaag
     1981 tccagcaaac aggccaaaga tgtctctcgc atgcgctcag tcctcatctc cctgaagcag
     2041 gcccccctgg tgcactgagt cgacggtacc gcgggcccgg gatccaccgg atctagataa
     2101 ctgatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca
     2161 cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt
     2221 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     2281 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta acgcgtaaat
     2341 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     2401 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     2461 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     2521 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     2581 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     2641 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     2701 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     2761 cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg
     2821 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     2881 ataaccctga taaatgcttc aataatattg aaaaaggaag agtcctgagg cggaaagaac
     2941 cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     3001 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     3061 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     3121 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     3181 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     3241 aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg
     3301 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     3361 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     3421 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     3481 tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     3541 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     3601 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     3661 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     3721 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     3781 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     3841 ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     3901 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     3961 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     4021 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     4081 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     4141 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     4201 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     4261 atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata
     4321 ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg
     4381 tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg
     4441 agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt
     4501 tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca
     4561 ggttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     4621 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     4681 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     4741 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     4801 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     4861 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     4921 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     4981 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     5041 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     5101 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     5161 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     5221 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     5281 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     5341 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     5401 aaccgtatta ccgccatgca t
//
