LOCUS       sequence-335059-        5444 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of His-GFP-Rab3a..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5444)
  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 5444)
  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-Rab3a
FEATURES             Location/Qualifiers
     source          1..5444
                     /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"
     misc_feature    1366..1431
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2232..2353
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2269..2288)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2323..2342
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2360..2815)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2497..2518)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2709..2728
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2842..2946
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2948..3305
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2948..3139
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2971..2991)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3156..3291
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3340..4134
                     /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             3340..4134
                     /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(3394..3413)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4004..4023
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4322..4341)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4366..4413
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4742..5330
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5231..5250
                     /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 cgagctcaag cttcgaattc tgcagtcgac ggtaccgcgg gcccgggatc catggcttcc
     1441 gccacagact ctcgctatgg gcagaaggag tcctcagacc agaacttcga ctatatgttc
     1501 aagatcctga tcattgggaa cagcagcgtg ggcaaaacct cgttcctctt ccgctacgca
     1561 gatgactcct tcactccagc ctttgtcagc accgttggca tagacttcaa ggtcaaaacc
     1621 atctatcgca acgacaagag gatcaagctg cagatctggg acacagcagg gcaagagcgg
     1681 taccgcacca tcaccacagc ctattaccga ggcgccatgg gcttcatcct aatgtatgac
     1741 atcaccaatg aggagtcatt taatgcagtg caggactggt ccactcagat caaaacttac
     1801 tcgtgggaca atgcccaggt gctgctggtg ggaaacaagt gtgacatgga agatgagcga
     1861 gtggtgtcct cagagcgtgg ccggcagctg gctgaccacc tgggctttga gttctttgag
     1921 gccagcgcca aggacaacat taatgtcaag cagacgtttg aacgtctggt ggacgtgatc
     1981 tgtgagaaga tgtcagagtc cctggatact gcagaccctg cggtcaccgg tgccaagcag
     2041 ggcccgcagc tcaccgacca gcaggcgcca cctcatcagg attgtgcctg ctgaaatcac
     2101 tagtgaattc gcggccgcga ctctagatca taatcagcca taccacattt gtagaggttt
     2161 tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa
     2221 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     2281 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     2341 tcaatgtatc ttaacgcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     2401 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     2461 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     2521 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     2581 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     2641 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     2701 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     2761 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt
     2821 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     2881 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     2941 aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3001 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3061 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3121 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3181 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3241 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     3301 gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     3361 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     3421 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     3481 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta
     3541 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     3601 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     3661 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     3721 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     3781 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     3841 gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc
     3901 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     3961 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     4021 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     4081 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     4141 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     4201 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     4261 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa
     4321 ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac
     4381 agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct
     4441 ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct
     4501 tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg
     4561 cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc
     4621 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     4681 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4741 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4801 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4861 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     4921 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     4981 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     5041 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     5101 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5161 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5221 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     5281 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     5341 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     5401 cgttatcccc tgattctgtg gataaccgta ttaccgccat gcat
//
