LOCUS       sequence-471885-        5433 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express NCK1-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 5433)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5433)
  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            "Unpublished"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: NCK1-SmBiT Fusion Vector
FEATURES             Location/Qualifiers
     source          1..5433
                     /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    2496..2617
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2507..2526)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(2732..2765)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2775..2822
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2854..3648
                     /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(2908..2927)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3518..3537
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(3806..4394)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3886..3905)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4520..4568
                     /note="synthetic polyadenylation signal"
     CDS             complement(4592..4990)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      4987..4996
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5021..5378)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5035..5170
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        5187..5378
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5335..5355
                     /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 ggcagaagaa
     1201 gtggtggtag tagccaaatt tgattatgtg gcccaacaag aacaagagtt ggacatcaag
     1261 aagaatgaga gattatggct tctggatgat tctaagtcct ggtggcgagt tcgaaattcc
     1321 atgaataaaa caggttttgt gccttctaac tatgtggaaa ggaaaaacag tgctcggaaa
     1381 gcatctattg tgaaaaacct aaaggatacc ttaggcattg gaaaagtgaa aagaaaacct
     1441 agtgtgccag attctgcatc tcctgctgat gatagttttg ttgacccagg ggaacgtctc
     1501 tatgacctca acatgcccgc ttatgtgaaa tttaactaca tggctgagag agaggatgaa
     1561 ttatcattga taaaggggac aaaggtgatc gtcatggaga aatgcagtga tgggtggtgg
     1621 cgtggtagct acaatggaca agttggatgg ttcccttcaa actatgtaac tgaagaaggt
     1681 gacagtcctt tgggtgacca tgtgggttct ctgtcagaga aattagcagc agtcgtcaat
     1741 aacctaaata ctgggcaagt gttgcatgtg gtacaggctc tttacccatt cagctcatct
     1801 aatgatgaag aacttaattt cgagaaagga gatgtaatgg atgttattga aaaacctgaa
     1861 aatgacccag agtggtggaa atgcaggaag atcaatggta tggttggtct agtaccaaaa
     1921 aactatgtta ccgttatgca gaataatcca ttaacttcag gtttggaacc atcacctcca
     1981 cagtgtgatt acattaggcc ttcactcact ggaaagtttg ctggcaatcc ttggtattat
     2041 ggcaaagtca ccaggcatca agcagaaatg gcattaaatg aaagaggaca tgaaggggat
     2101 ttcctcattc gtgatagtga atcttcgcca aatgatttct cagtatcact aaaagcacaa
     2161 gggaaaaaca agcattttaa agtccaacta aaagagactg tctactgcat tgggcagcgt
     2221 aaattcagca ccatggaaga acttgtagaa cattacaaaa aggcaccaat ttttacaagt
     2281 gaacaaggag aaaaattata tcttgtcaag catttatcag tttctcaagg cagttcaggt
     2341 ggtggcggga gcggaggtgg aggctcgagc ggtgtgaccg gctaccggct gttcgaggag
     2401 attctgtaag gccgcgactc tagagtcgac ctgcaggcat gcaagctgat ttggctgctg
     2461 ccaccgctga gcaaggccgc ttcgagcaga catgataaga tacattgatg agtttggaca
     2521 aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc
     2581 tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt
     2641 tatgtttcag gttcaggggg agatgtggga ggttttttta agcaagtaaa acctctacaa
     2701 atgtggtaaa atcttaatta agaattctaa tataacttcg tatagcatac attatacgaa
     2761 gttatggatc cactgttgac aattaatcat cggcatagta tatcggcata gtataatacg
     2821 acaaggtgag gaactaaacc caggaggcag atcatgattg aacaagatgg attgcacgca
     2881 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     2941 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     3001 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
     3061 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     3121 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     3181 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     3241 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     3301 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     3361 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     3421 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     3481 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     3541 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     3601 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgatt cgaaatgacc
     3661 gaccaagcga cgcccaaccg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     3721 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     3781 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     3841 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     3901 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     3961 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4021 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4081 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4141 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     4201 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     4261 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4321 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     4381 aaaaaggatt tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     4441 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     4501 ttttatagtc cggaatcgtc acacaaaaaa ccaacacaca gatgtaatga aaataaagat
     4561 attttattgc ggccatcgtg atggctagag tttagccctc ccacacataa ccagagggca
     4621 gcaattcacg aatcccaact gccgtcggct gtccatcact gtccttcact atggctttga
     4681 tcccaggatg cagatcgaga agcacctgtc ggcaccgtcc gcaggggctc aagatgcccc
     4741 tgttctcatt tccgatcgcg acgatacaag tcaggttgcc agctgccgca gcagcagcag
     4801 tgcccagcac cacgagttct gcacaaggtc ccccagtaaa atgatataca ttgacaccag
     4861 tgaagatgcg gccgtcgcta gagagagctg cgctggcgac gctgtagtct tcagagatgg
     4921 ggatgctgtt gattgtagcc gttgctcttt caatgagggt ggattcttct tgagacaaag
     4981 gcttggccat ggtggcgcta gtgtcagaag aatcgagctt tttgcaaaag cctaggcctc
     5041 caaaaaagcc tcctcactac ttctggaata gctcagaggc cgaggcggcc tcggcctctg
     5101 cataaataaa aaaaattagt cagccatggg gcggagaatg ggcggaactg ggcggagtta
     5161 ggggcgggat gggcggagtt aggggcggga ctatggttgc tgactaattg agatgcatgc
     5221 tttgcatact tctgcctgct ggggagcctg gggactttcc acacctggtt gctgactaat
     5281 tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacccta
     5341 actgacacac attccacagc tggttctttc cgcctcagaa ggtacctaac caagttcctc
     5401 tttcagaggt tatttcaggc catggtgctg cgc
//
