LOCUS       sequence-335076-        5332 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of His-GFP SNAP25..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5332)
  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 5332)
  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 SNAP25
FEATURES             Location/Qualifiers
     source          1..5332
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        152..355
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     306..326
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      394..403
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             412..429
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(439..441,442..1152)
                     /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(481..502)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(742..761)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1089..1110
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1907..2028
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1944..1963)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1998..2017
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2035..2490)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2172..2193)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2384..2403
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2517..2621
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2623..2980
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2623..2814
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2646..2666)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2831..2966
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3015..3809
                     /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             3015..3809
                     /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(3069..3088)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3679..3698
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3997..4016)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4041..4088
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4417..5005
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4906..4925
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        join(5180..5332,1..151)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
       61 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
      121 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
      181 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
      241 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
      301 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctggttt
      361 agtgaaccgt cagatccgct agcgctaccg gtcgccacca tgggcagcag ccatcatcat
      421 catcatcaca gcagcggcgt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
      481 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
      541 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
      601 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
      661 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
      721 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
      781 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
      841 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
      901 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
      961 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     1021 ctgcccgaca accactacct gagcacccag tccaagctga gcaaagaccc caacgagaag
     1081 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     1141 gagctgtaca aagcttctat ggccgaagac gcagacatgc gcaatgagct ggaggagatg
     1201 cagaggaggg ctgaccagct ggctgatgag tccctggaaa gcacccgtcg catgctgcag
     1261 ctggttgaag agagtaaaga tgctggcatc aggactttgg ttatgttgga tgagcaaggc
     1321 gaacaactgg aacgcattga ggaagggatg gaccaaatca ataaggacat gaaagaagca
     1381 gaaaagaatt tgacggacct aggaaaattc tgcgggcttt gtgtgtgtcc ctgtaacaag
     1441 cttaaatcca gtgatgctta caaaaaagcc tggggcaata atcaggatgg agtagtggcc
     1501 agccagcctg cccgtgtggt ggatgaacgg gagcagatgg ccatcagtgg tggcttcatc
     1561 cgcagggtaa caaatgatgc ccgggaaaat gagatggatg agaacctgga gcaggtgagc
     1621 ggcatcatcg gaaacctccg ccatatggct ctagacatgg gcaatgagat tgacacccag
     1681 aatcgccaga tcgacaggat catggagaag gctgattcca acaaaaccag aattgatgaa
     1741 gccaaccaac gtgcaacaaa gatgctggga agtggttaag gtaccgcggc cgcgactcta
     1801 gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca
     1861 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc
     1921 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     1981 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaag gcgtaaattg
     2041 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     2101 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     2161 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     2221 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     2281 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     2341 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     2401 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     2461 ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg
     2521 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     2581 aaccctgata aatgcttcaa taatattgaa aaaggaagag tcctgaggcg gaaagaacca
     2641 gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     2701 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     2761 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     2821 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     2881 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     2941 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa gagacaggat
     3001 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     3061 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     3121 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     3181 ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     3241 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     3301 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     3361 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     3421 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     3481 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     3541 cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     3601 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     3661 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     3721 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     3781 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
     3841 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     3901 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     3961 gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc
     4021 ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc
     4081 gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag
     4141 accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc
     4201 gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagg
     4261 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     4321 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     4381 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     4441 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     4501 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     4561 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     4621 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     4681 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     4741 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     4801 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     4861 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     4921 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     4981 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     5041 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     5101 ccgtattacc gccatgcatt agttattaat agtaatcaat tacggggtca ttagttcata
     5161 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     5221 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     5281 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac tt
//
