LOCUS       sequence-471909-        5693 bp    DNA     circular SYN 14-JUL-2025
DEFINITION  Express SmBiT-VPS4B 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 5693)
  TITLE     Promega Early Access Collection
REFERENCE   2  (bases 1 to 5693)
  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: SmBiT-VPS4B Fusion Vector
FEATURES             Location/Qualifiers
     source          1..5693
                     /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             2102..2113
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     polyA_signal    2756..2877
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2767..2786)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(2992..3025)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3035..3082
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3114..3908
                     /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(3168..3187)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3778..3797
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(4066..4654)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4146..4165)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4780..4828
                     /note="synthetic polyadenylation signal"
     CDS             complement(4852..5250)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      5247..5256
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5281..5638)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5295..5430
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        5447..5638
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5595..5615
                     /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 cgctcaccat ggtgaccggc
     1201 taccggctgt tcgaggagat tctcgggagc tccggtggtg gcgggagcgg aggtggaggc
     1261 tcgagcggtg cgatcgccat gtcatccact tcgcccaacc tccagaaagc gatagatctg
     1321 gctagcaaag cagcgcaaga agacaaggct gggaactacg aagaagccct tcagctctat
     1381 cagcatgctg tgcagtattt tcttcatgtc gttaaatatg aagcacaggg tgataaagcc
     1441 aagcaaagta tcagggcaaa gtgtacagaa tatcttgata gagcagaaaa actaaaggag
     1501 tacctgaaaa ataaagagaa aaaagcacag aagccagtga aagaaggaca gccgagtcca
     1561 gcagatgaga aggggaatga cagtgatggg gaaggagaat ctgatgatcc tgaaaaaaag
     1621 aaactacaga atcaacttca aggtgccatt gttatagaac gaccaaatgt gaaatggagt
     1681 gacgttgctg gacttgaagg agccaaagaa gcactgaaag aggctgtgat actgcctatt
     1741 aaatttcctc atctttttac aggcaagaga acaccttgga ggggaatcct attatttggg
     1801 ccgcctggaa caggaaagtc ctacttagcc aaagctgtag caacagaagc caacaactca
     1861 acattttttt caatatcttc ctctgatctt gtttctaagt ggctaggtga aagtgaaaaa
     1921 ctggttaaga atttattcca acttgccaga gagaacaagc cctccattat cttcattgat
     1981 gaaattgatt ctctctgtgg ttcaagaagt gaaaatgaaa gtgaagccgc acgtagaatt
     2041 aagacggagt tcctagtgca aatgcaaggg gttggtgtag acaatgatgg aattttggtt
     2101 ctgggagcta caaatatacc ctgggttctg gattctgcca ttaggcgaag atttgagaaa
     2161 cgaatttata ttcccttgcc ggaaccccat gcccgagcag caatgttcaa actgcaccta
     2221 gggaccactc agaacagtct cacggaagca gactttcggg aacttgggag gaaaacagat
     2281 ggttattcag gggcagatat aagtatcatt gtacgtgatg cccttatgca gcctgttagg
     2341 aaagtacagt cagctactca ttttaaaaag gttcgcggac cttcccgagc tgatcctaac
     2401 catcttgtag atgatctgct aacaccttgc tctccaggtg accctggtgc cattgaaatg
     2461 acatggatgg atgtccctgg agataaactt ttggagccag ttgtttccat gtcggatatg
     2521 ttgcggtcac tatctaacac aaaacctaca gtcaatgaac atgacttgtt gaaattaaag
     2581 aagtttacag aagattttgg tcaagaaggc gtttaaacga attcgggctc ggtacccggg
     2641 gatcctctag agtcgacctg caggcatgca agctgatccg gctgctaaca aagcccgaaa
     2701 ggaagctgag ttggctgctg ccaccgctga gcaaggccgc ttcgagcaga catgataaga
     2761 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
     2821 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac
     2881 aacaacaatt gcattcattt tatgtttcag gttcaggggg agatgtggga ggttttttta
     2941 agcaagtaaa acctctacaa atgtggtaaa atcttaatta agaattctaa tataacttcg
     3001 tatagcatac attatacgaa gttatggatc cactgttgac aattaatcat cggcatagta
     3061 tatcggcata gtataatacg acaaggtgag gaactaaacc caggaggcag atcatgattg
     3121 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     3181 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     3241 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     3301 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     3361 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     3421 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     3481 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     3541 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     3601 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     3661 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     3721 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     3781 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     3841 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     3901 tcttctgatt cgaaatgacc gaccaagcga cgcccaaccg gtatcagctc actcaaaggc
     3961 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     4021 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4081 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4141 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4201 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     4261 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     4321 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     4381 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     4441 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     4501 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     4561 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     4621 gcagcagatt acgcgcagaa aaaaaggatt tcaagaagat cctttgatct tttctacggg
     4681 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     4741 aaggatcttc acctagatcc ttttatagtc cggaatcgtc acacaaaaaa ccaacacaca
     4801 gatgtaatga aaataaagat attttattgc ggccatcgtg atggctagag tttagccctc
     4861 ccacacataa ccagagggca gcaattcacg aatcccaact gccgtcggct gtccatcact
     4921 gtccttcact atggctttga tcccaggatg cagatcgaga agcacctgtc ggcaccgtcc
     4981 gcaggggctc aagatgcccc tgttctcatt tccgatcgcg acgatacaag tcaggttgcc
     5041 agctgccgca gcagcagcag tgcccagcac cacgagttct gcacaaggtc ccccagtaaa
     5101 atgatataca ttgacaccag tgaagatgcg gccgtcgcta gagagagctg cgctggcgac
     5161 gctgtagtct tcagagatgg ggatgctgtt gattgtagcc gttgctcttt caatgagggt
     5221 ggattcttct tgagacaaag gcttggccat ggtggcgcta gtgtcagaag aatcgagctt
     5281 tttgcaaaag cctaggcctc caaaaaagcc tcctcactac ttctggaata gctcagaggc
     5341 cgaggcggcc tcggcctctg cataaataaa aaaaattagt cagccatggg gcggagaatg
     5401 ggcggaactg ggcggagtta ggggcgggat gggcggagtt aggggcggga ctatggttgc
     5461 tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc
     5521 acacctggtt gctgactaat tgagatgcat gctttgcata cttctgcctg ctggggagcc
     5581 tggggacttt ccacacccta actgacacac attccacagc tggttctttc cgcctcagaa
     5641 ggtacctaac caagttcctc tttcagaggt tatttcaggc catggtgctg cgc
//
