LOCUS       sequence-476792-        5613 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express VPS4A-SmBiT Fusion Protein in Mammalian Cells under a CMV 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5613)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5613)
  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: 
            "Unpublished"
COMMENT     Sequence Label: VPS4A-SmBiT Fusion Vector
FEATURES             Location/Qualifiers
     source          1..5613
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    24..105
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        338..641
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        642..845
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     796..816
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          981..1113
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1096..1115
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1157..1176
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1157..1175
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    2676..2797
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2687..2706)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(2912..2945)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2955..3002
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3034..3828
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3088..3107)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3698..3717
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(3986..4574)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4066..4085)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4700..4748
                     /note="synthetic polyadenylation signal"
     CDS             complement(4772..5170)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      5167..5176
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5201..5558)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5215..5350
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        5367..5558
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5515..5535
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
ORIGIN
        1 aagacgcgtt aattaagacg tcggcagtga aaaaaatgct ttatttgtga aatttgtgat
       61 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
      121 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
      181 ctctacaaat gtggtatggc tgattatgat ccgcggccgc aatacgtcga cgttatcagc
      241 tgacttcgta cgagagccta ggattatggc gcgccactag ttattaatag taatcaatta
      301 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      361 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      421 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      481 ctgcccactt ggcagtacat caagtgtatc atatgccaag tccgccccct attgacgtca
      541 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttacgg gactttccta
      601 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      661 acaccaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
      721 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaata
      781 accccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      841 gagctggttt agtgaaccgt cagatcacta gaagctttat tgcggtagtt tatcacagtt
      901 aaattgctaa cgcagtcagt gcttctgaca caacagtctc gaacttaagc tgcagaagtt
      961 ggtcgtgagg cactgggcag gtaagtatca aggttacaag acaggtttaa ggagaccaat
     1021 agaaactggg cttgtcgaga cagagaagac tcttgcgttt ctgataggca cctattggtc
     1081 ttactgacat ccactttgcc tttctctcca caggtgtcca ctcccagttc aattacagct
     1141 cttaaggcta gagtattaat acgactcact atagggctag cgatcgccat gacaacgtca
     1201 accctccaga aagccattga tctggtgacg aaagccacag aggaggacaa agccaagaac
     1261 tacgaggagg cgctgcggct gtaccagcat gcggtggagt acttcctcca cgctatcaag
     1321 tatgaggccc acagcgacaa ggccaaggag agcattcgag ccaagtgcgt gcagtaccta
     1381 gaccgggccg agaagctgaa ggattattta cgaagcaaag agaaacacgg caagaagcca
     1441 gtcaaagaga accagagtga gggcaagggc agtgacagtg acagtgaagg ggataatccg
     1501 gagaaaaaga aactgcaaga acagctgatg ggtgccgtcg tgatggagaa gcccaacata
     1561 cggtggaacg acgtggccgg gctggagggg gccaaggagg ccctcaaaga agctgtcatt
     1621 ttgccaatca aattcccaca cttgttcaca ggcaagcgca ccccctggcg ggggattctg
     1681 ctgttcggac cccctggcac agggaaatcc tacctggcca aagccgtggc aacagaggcc
     1741 aacaactcca ccttcttctc tgtgtcctcc tcagatctga tgtccaagtg gctgggggag
     1801 agtgaaaagc tggtcaagaa cctgtttgag ctggccaggc agcacaagcc ctccatcatc
     1861 ttcatcgatg aggtggattc cctctgcggg tcccgaaatg aaaatgagag tgaggccgcc
     1921 cggaggatca aaacggagtt cttggtccag atgcaggggg tggggaataa caatgatggg
     1981 actctggttc ttggagccac aaacatccca tgggtgttgg attcggccat caggaggagg
     2041 tttgaaaaac gaatttatat ccccttgccg gaggaagctg cccgcgccca gatgttccgg
     2101 ttgcatctcg ggagcactcc ccacaacctc acggatgcaa acatccacga gctggcccgg
     2161 aagacggaag gctactcggg cgcggacatc agcatcatcg tgcgggactc tctcatgcag
     2221 cccgtgagga aggtgcagtc ggccacacac ttcaaaaagg tctgtggccc ctctcgcacc
     2281 aaccccagca tgatgattga tgacctcctg actccatgct caccagggga cccaggagcc
     2341 atggagatga cttggatgga tgtccctggg gacaaactct tagagcctgt ggtttgcatg
     2401 tcggacatgc tgcggtctct ggccaccacc cggcccacgg tgaatgcaga cgacctcctg
     2461 aaagtgaaga aattctcaga ggactttggg caagagagtg tttctcaagg cagttcaggt
     2521 ggtggcggga gcggaggtgg aggctcgagc ggtgtgaccg gctaccggct gttcgaggag
     2581 attctgtaag gccgcgactc tagagtcgac ctgcaggcat gcaagctgat ttggctgctg
     2641 ccaccgctga gcaaggccgc ttcgagcaga catgataaga tacattgatg agtttggaca
     2701 aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc
     2761 tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt
     2821 tatgtttcag gttcaggggg agatgtggga ggttttttta agcaagtaaa acctctacaa
     2881 atgtggtaaa atcttaatta agaattctaa tataacttcg tatagcatac attatacgaa
     2941 gttatggatc cactgttgac aattaatcat cggcatagta tatcggcata gtataatacg
     3001 acaaggtgag gaactaaacc caggaggcag atcatgattg aacaagatgg attgcacgca
     3061 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     3121 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     3181 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
     3241 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     3301 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     3361 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     3421 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     3481 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     3541 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     3601 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     3661 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     3721 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     3781 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgatt cgaaatgacc
     3841 gaccaagcga cgcccaaccg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     3901 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     3961 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4021 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4081 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4141 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4201 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4261 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4321 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     4381 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     4441 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4501 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     4561 aaaaaggatt tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     4621 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     4681 ttttatagtc cggaatcgtc acacaaaaaa ccaacacaca gatgtaatga aaataaagat
     4741 attttattgc ggccatcgtg atggctagag tttagccctc ccacacataa ccagagggca
     4801 gcaattcacg aatcccaact gccgtcggct gtccatcact gtccttcact atggctttga
     4861 tcccaggatg cagatcgaga agcacctgtc ggcaccgtcc gcaggggctc aagatgcccc
     4921 tgttctcatt tccgatcgcg acgatacaag tcaggttgcc agctgccgca gcagcagcag
     4981 tgcccagcac cacgagttct gcacaaggtc ccccagtaaa atgatataca ttgacaccag
     5041 tgaagatgcg gccgtcgcta gagagagctg cgctggcgac gctgtagtct tcagagatgg
     5101 ggatgctgtt gattgtagcc gttgctcttt caatgagggt ggattcttct tgagacaaag
     5161 gcttggccat ggtggcgcta gtgtcagaag aatcgagctt tttgcaaaag cctaggcctc
     5221 caaaaaagcc tcctcactac ttctggaata gctcagaggc cgaggcggcc tcggcctctg
     5281 cataaataaa aaaaattagt cagccatggg gcggagaatg ggcggaactg ggcggagtta
     5341 ggggcgggat gggcggagtt aggggcggga ctatggttgc tgactaattg agatgcatgc
     5401 tttgcatact tctgcctgct ggggagcctg gggactttcc acacctggtt gctgactaat
     5461 tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacccta
     5521 actgacacac attccacagc tggttctttc cgcctcagaa ggtacctaac caagttcctc
     5581 tttcagaggt tatttcaggc catggtgctg cgc
//
