LOCUS       sequence-471908-        5634 bp    DNA     circular SYN 14-JUL-2025
DEFINITION  Express VPS4B-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 5634)
  TITLE     Promega Early Access Collection
REFERENCE   2  (bases 1 to 5634)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: VPS4B-SmBiT Fusion Vector
FEATURES             Location/Qualifiers
     source          1..5634
                     /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"
     CDS             2012..2023
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     polyA_signal    2697..2818
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2708..2727)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(2933..2966)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2976..3023
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3055..3849
                     /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(3109..3128)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3719..3738
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(4007..4595)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4087..4106)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4721..4769
                     /note="synthetic polyadenylation signal"
     CDS             complement(4793..5191)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      5188..5197
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5222..5579)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5236..5371
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        5388..5579
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5536..5556
                     /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 gtcatccact
     1201 tcgcccaacc tccagaaagc gatagatctg gctagcaaag cagcgcaaga agacaaggct
     1261 gggaactacg aagaagccct tcagctctat cagcatgctg tgcagtattt tcttcatgtc
     1321 gttaaatatg aagcacaggg tgataaagcc aagcaaagta tcagggcaaa gtgtacagaa
     1381 tatcttgata gagcagaaaa actaaaggag tacctgaaaa ataaagagaa aaaagcacag
     1441 aagccagtga aagaaggaca gccgagtcca gcagatgaga aggggaatga cagtgatggg
     1501 gaaggagaat ctgatgatcc tgaaaaaaag aaactacaga atcaacttca aggtgccatt
     1561 gttatagaac gaccaaatgt gaaatggagt gacgttgctg gacttgaagg agccaaagaa
     1621 gcactgaaag aggctgtgat actgcctatt aaatttcctc atctttttac aggcaagaga
     1681 acaccttgga ggggaatcct attatttggg ccgcctggaa caggaaagtc ctacttagcc
     1741 aaagctgtag caacagaagc caacaactca acattttttt caatatcttc ctctgatctt
     1801 gtttctaagt ggctaggtga aagtgaaaaa ctggttaaga atttattcca acttgccaga
     1861 gagaacaagc cctccattat cttcattgat gaaattgatt ctctctgtgg ttcaagaagt
     1921 gaaaatgaaa gtgaagccgc acgtagaatt aagacggagt tcctagtgca aatgcaaggg
     1981 gttggtgtag acaatgatgg aattttggtt ctgggagcta caaatatacc ctgggttctg
     2041 gattctgcca ttaggcgaag atttgagaaa cgaatttata ttcccttgcc ggaaccccat
     2101 gcccgagcag caatgttcaa actgcaccta gggaccactc agaacagtct cacggaagca
     2161 gactttcggg aacttgggag gaaaacagat ggttattcag gggcagatat aagtatcatt
     2221 gtacgtgatg cccttatgca gcctgttagg aaagtacagt cagctactca ttttaaaaag
     2281 gttcgcggac cttcccgagc tgatcctaac catcttgtag atgatctgct aacaccttgc
     2341 tctccaggtg accctggtgc cattgaaatg acatggatgg atgtccctgg agataaactt
     2401 ttggagccag ttgtttccat gtcggatatg ttgcggtcac tatctaacac aaaacctaca
     2461 gtcaatgaac atgacttgtt gaaattaaag aagtttacag aagattttgg tcaagaaggc
     2521 gtttctcaag gcagttcagg tggtggcggg agcggaggtg gaggctcgag cggtgtgacc
     2581 ggctaccggc tgttcgagga gattctgtaa ggccgcgact ctagagtcga cctgcaggca
     2641 tgcaagctga tttggctgct gccaccgctg agcaaggccg cttcgagcag acatgataag
     2701 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     2761 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
     2821 caacaacaat tgcattcatt ttatgtttca ggttcagggg gagatgtggg aggttttttt
     2881 aagcaagtaa aacctctaca aatgtggtaa aatcttaatt aagaattcta atataacttc
     2941 gtatagcata cattatacga agttatggat ccactgttga caattaatca tcggcatagt
     3001 atatcggcat agtataatac gacaaggtga ggaactaaac ccaggaggca gatcatgatt
     3061 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     3121 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     3181 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     3241 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     3301 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     3361 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     3421 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     3481 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     3541 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     3601 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     3661 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     3721 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     3781 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     3841 ttcttctgat tcgaaatgac cgaccaagcg acgcccaacc ggtatcagct cactcaaagg
     3901 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     3961 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4021 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4081 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4141 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4201 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4261 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4321 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4381 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4441 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     4501 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     4561 agcagcagat tacgcgcaga aaaaaaggat ttcaagaaga tcctttgatc ttttctacgg
     4621 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     4681 aaaggatctt cacctagatc cttttatagt ccggaatcgt cacacaaaaa accaacacac
     4741 agatgtaatg aaaataaaga tattttattg cggccatcgt gatggctaga gtttagccct
     4801 cccacacata accagagggc agcaattcac gaatcccaac tgccgtcggc tgtccatcac
     4861 tgtccttcac tatggctttg atcccaggat gcagatcgag aagcacctgt cggcaccgtc
     4921 cgcaggggct caagatgccc ctgttctcat ttccgatcgc gacgatacaa gtcaggttgc
     4981 cagctgccgc agcagcagca gtgcccagca ccacgagttc tgcacaaggt cccccagtaa
     5041 aatgatatac attgacacca gtgaagatgc ggccgtcgct agagagagct gcgctggcga
     5101 cgctgtagtc ttcagagatg gggatgctgt tgattgtagc cgttgctctt tcaatgaggg
     5161 tggattcttc ttgagacaaa ggcttggcca tggtggcgct agtgtcagaa gaatcgagct
     5221 ttttgcaaaa gcctaggcct ccaaaaaagc ctcctcacta cttctggaat agctcagagg
     5281 ccgaggcggc ctcggcctct gcataaataa aaaaaattag tcagccatgg ggcggagaat
     5341 gggcggaact gggcggagtt aggggcggga tgggcggagt taggggcggg actatggttg
     5401 ctgactaatt gagatgcatg ctttgcatac ttctgcctgc tggggagcct ggggactttc
     5461 cacacctggt tgctgactaa ttgagatgca tgctttgcat acttctgcct gctggggagc
     5521 ctggggactt tccacaccct aactgacaca cattccacag ctggttcttt ccgcctcaga
     5581 aggtacctaa ccaagttcct ctttcagagg ttatttcagg ccatggtgct gcgc
//
