LOCUS       sequence-328085-        9406 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral vector for NPC1 expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9406)
  AUTHORS   Davis OB, Shin HR, Lim CY, Wu EY, Kukurugya M, Maher CF, Perera RM, 
            Ordonez MP, Zoncu R
  TITLE     NPC1-mTORC1 Signaling Couples Cholesterol Sensing to Organelle 
            Homeostasis and Is a Targetable Pathway in Niemann-Pick Type C.
  JOURNAL   Dev Cell. 2020 Dec 7. pii: S1534-5807(20)30925-4. doi: 
            10.1016/j.devcel.2020.11.016.
  PUBMED    33308480
REFERENCE   2  (bases 1 to 9406)
  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: "Dev Cell. 
            2020 Dec 7. pii: S1534-5807(20)30925-4. doi: 
            10.1016/j.devcel.2020.11.016."
COMMENT     Sequence Label: pLVX-NPC1(P691S)-FLAG
FEATURES             Location/Qualifiers
     source          1..9406
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(14..33)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     409..432
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     CDS             complement(496..519)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(4425..5607)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          4434..5376
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(4457..4477)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     misc_feature    5801..5916
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
     CDS             complement(6179..6223)
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    6408..6641
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    7138..7263
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             7310..7943
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    8150..8738
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             8256..8267
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     8665..8685
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8752..9351)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(8835..8855)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     9332..9351
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(join(9372..9406,1..467))
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
ORIGIN
        1 cggcagacgt gcgcttttga agcgtgcaga atgccgggcc tccggaggac cttcgggcgc
       61 ccgccccgcc cctgangncy ckcccctgag cccgcccccg gacccacccc ttcccagcct
      121 ctgagcccag aaagcgaagg agcaaagctg ctattggccg ctgccccaaa ggcctacccg
      181 cttccattgc tcagcggtgc tgtccatctg cacgagacta gtgagacgtg ctacttccat
      241 ttgtcacgtc ctgcacgacg cgagctgcgg ggcggggggg aacttcctga ctaggggagg
      301 agtagaaggt ggcgcgaagg ggccaccaaa gaacggagcc ggttggcgcc taccggtgga
      361 tgtggaatgt gtgcgaggcc agaggccact tgtgtagcgc caagtgccca gcggggctgc
      421 taaagcgcat gctccagact gccttgggaa aagcgcctcc cctacccggt agaattatct
      481 agatgagccg ggttatttat catcgtcatc cttgtagtca aagttcagca gtctctctcg
      541 ctcagtcccc ttgtatcttt cctcagtggc gcagcttttt gccttgttca cgctagggcc
      601 gatatatgac agcaggacgg gcaggaaaat cagcccgtgt gttgcgccca gcagcaccat
      661 agccaggtac atccggaaat aaaagatctg gaagatctgg ctcttagcaa aggccaggac
      721 cacgattccc ccgaactttg tcagggtaat gccactaaac actgaacttc ccatgtgtgc
      781 cagagcttcc tctgctcttt ccacccggct tcccttcatt gagacggtga aagctctagt
      841 gatatggcta caaaactcca cactaatccc gcaagacatg accagattca ccagactgac
      901 ggcgttcaga gagatccccc acagccacat cacgccgaac atattcacca ggaccatagc
      961 gatggtggcg cacataatca cggcagacca cagctcgcag cccagcagca ccatagtgac
     1021 caggaagata gctcccaggg acacccccag gttgaagatg gtatcgtcaa tgatagtcag
     1081 gtactgttca tagaacacgt aaaagacaga gtagggaaac actcgatagg cggatccatt
     1141 gatccccata gtctctgtca cgttgctagc gatcaggcgg gctttcttca gtgcatcaat
     1201 gaagtcagct gaggtctgca gcacagtgtg gtatgtcatg aagtatgtgg ctcccactcg
     1261 ggtcccatgt cccagcagga tgttgactgc agaggagtat gcagcgtggc ctcctttgcc
     1321 acacttggga tttgggttgt cggacaggaa cataggcagg aatctcatga agtcgcctcc
     1381 ctgtggtcgc tgtttgccct ctggagtcag tggcctacat ctcacgcagg cagggtcgac
     1441 cacgcttgca ttgcaaaact gatctgtgat gttgtccacg cgacagcagc ttgactgagg
     1501 cttgacccaa tcgaagtaat cgtcgatcca actagagggg gcaaagccaa tccttgtata
     1561 attgtccagc tgggctgcgt tgaagatctg ctgcaccagg gaatcattgt tacatcccat
     1621 tcccccgcac accatattct gccccttgga gctagtgtag tcatggcctt cctccagcac
     1681 gaagtagaca ggggggccgg cgtgcagata ctgactgata gatttaaagt aatccaccat
     1741 gtaagaatcg tcgggcatgg acaggctctg atccaggccg atgtcgacct tgttcagcac
     1801 ggcaattgag aaactcagca ctccgacaaa gattgcaatc acgatggggc gcatccagtc
     1861 tttcagcagc agtggagaat agctgttctt gaaaaacctg aacagacagc tttctgaagc
     1921 ctgcacggat gtgccgtcct ctgctccccg cacgcagcag aagatatcca ggcggttttt
     1981 ctcctgcctc ttaatgtcca ggcccagcag gctcacaaag caggtgatct gcagcaggaa
     2041 gtcaataaac actgccagcc cagcgaacag tgaaaatgtg tggactgctg gcatcacact
     2101 cagggcgccc aggaaaaatg ccacggtttc actgaatgaa ctcagaaaca tagatggagc
     2161 gacctctccc agcacccttc ccagctgctg atccagtgtt tcgccctgca gtcgctcgtc
     2221 gcgctggtat gcctgcacca gaatgaagat attgtccact ccgacagcca ggaccagaaa
     2281 tgagatcact tcaatgacga tcagggtcag aggcagtcca atataggaga acacccccag
     2341 gctacaagcg acagaggaca gcacaatcag gatgccggca attcccaggc tgacttttga
     2401 atccaccagc aggcgcctgc agctcttcat atggcccagt gccagtgaga tgtacaggaa
     2461 catgatagca tagctaatga ccacggtgaa cacgtcactg tcagattctc ggttcagttc
     2521 gtcctcgatg ctccgctcgg ctgtgaaact aatggtcaga tttgggtttt tgtaattctt
     2581 cacgaagttg atgaactctt tctcccaggc ctgtgctctc tgcagcttct cggtatcatt
     2641 atagtaattg ttgacaggga aggtgatgac cagggcggtg gcattgttgt agttctgatc
     2701 gtcatagcct cccaggacca gccaagggaa cacgggtccg ccaaatgtcc ccagacatgg
     2761 atcgtgcagc agtgaggtgt cgttcagact tgcaggagcc cgcacgcagt acaggaaatg
     2821 ggtgtgatag tcggcgtaca cgaagaagtc gtcgcctttc ttatgatcca gcacggagtg
     2881 gctattctga aagtagttca ggacggacag aatggtacaa ttagtgttat atgggctcag
     2941 aggagccagg cagatatcct gcagggtcac agtttcattg tcatagctag cggtaatgtt
     3001 ctcaatggcg atctgcaggt ccagcacctg atgcagaatc tggatatcca gtggagggcc
     3061 gaaaggcacg tcggctccag aaggataggg ctggtagatg tgtttatcag tcaggggtgc
     3121 cctaatgatc agctgctctg ttctgaaaaa tggtccgaaa tgctggtcaa agtattcctt
     3181 ctccagtcga gcctggcttg atggggcgct ccacagatcc actggatttg tggtcactcg
     3241 gacgaacacc agcccactag aacaggcggt gataaacacc aggctgaaga agatcacgca
     3301 tccggggttc cgcacgcaga aagaccccca tcgagtaaac agtctccgca ggcacccttc
     3361 gaaagcggcg ctcacgggat cacagcagga tgcctcgcct ttgtcggatg cattcacgct
     3421 gaaagcgatg ttggagtcaa tgggggtgta ctcgctcaca aagtaccgct ttcgataaca
     3481 ccacacggcg aaaaatgcgc cgaaaaagac cagcagaaag gccatgtaag tgatccacat
     3541 aatcacatac atggcgtcca gtcccagaat tgtccatggt gctggtggag gtggtggctg
     3601 tggtttaggc ccgcacacga tagagcaatc ctgacaggag caaggagcgg tcacttcatc
     3661 gacggactcg tcgcatccct tagtggcatt gttcataggc tccatcccgt ggacggggaa
     3721 gtcgctaaac actggtgtaa tggtgaaagg ggcctggcca ttatccttgt taaacatgta
     3781 ttcgatccaa ttggtagcgt tacaggcatc tgcgtctttt ccgcacagca gccccagggc
     3841 cttatcattg gagcttggtg cctccacgtc tcggcaagca ttatacatag cgttggcgaa
     3901 ggactggccc acatagtact gcagttcttt gacattagtc tttgtctggt ttgtcacggg
     3961 atcgacgtag tcctcggtgg cagtcacgtt caggaactgt gactgccttg gactacaggt
     4021 cagctcgcag aacagattca gcaggttata aaaacaagat gggcacctgg acagaaactg
     4081 cagaggcagc tgcagattat ccttcagggt ctgcagctgc cgcacgtcac agcacaggct
     4141 cacgttgccg aaaaagaatc ctgggcacag ttcctgcacc agatcgtagc cgtctttggg
     4201 cagtggctta gggggtccag agtattcaca gttatacctc ttgtccccat aggcgatgcc
     4261 gcactcgcca taccacacgc aagactgact gaacacctga gcggggcaca gcagcagcag
     4321 cagcagcccc agagccagcc ctctagcggt catggtggcg aattccacgt ggatccaatt
     4381 cgaagcttga gctcgagatc tgagtccggt agcgctagcg atcctcacga cacctgaaat
     4441 ggaagaaaaa aactttgaac cactgtctga ggcttgagaa tgaaccaaga tccaaactca
     4501 aaaagggcaa attccaagga gaattacatc aagtgccaag ctggcctaac ttcagtctcc
     4561 acccactcag tgtggggaaa ctccatcgca taaaacccct ccccccaacc taaagacgac
     4621 gtactccaaa agctcgagaa ctaatcgagg tgcctggacg gcgcccggta ctccgtggag
     4681 tcacatgaag cgacggctga ggacggaaag gcccttttcc tttgtgtggg tgactcaccc
     4741 gcccgctctc ccgagcgccg cgtcctccat tttgagctcc ctgcagcagg gccgggaagc
     4801 ggccatcttt ccgctcacgc aactggtgcc gaccgggcca gccttgccgc ccagggcggg
     4861 gcgatacacg gcggcgcgag gccaggcacc agagcaggcc ggccagcttg agactacccc
     4921 cgtccgattc tcggtggccg cgctcgcagg ccccgcctcg ccgaacatgt gcgctgggac
     4981 gcacgggccc cgtcgccgcc cgcggcccca aaaaccgaaa taccagtgtg cagatcttgg
     5041 cccgcattta caagactatc ttgccagaaa aaaagcgtcg cagcaggtca tcaaaaattt
     5101 taaatggcta gagacttatc gaaagcagcg agacaggcgc gaaggtgcca ccagattcgc
     5161 acgcggcggc cccagcgccc aagccaggcc tcaactcaag cacgaggcga aggggctcct
     5221 taagcgcaag gcctcgaact ctcccaccca cttccaaccc gaagctcggg atcaagaatc
     5281 acgtactgca gccaggggcg tggaagtaat tcaaggcacg caagggccat aacccgtaaa
     5341 gaggccaggc ccgcgggaac cacacacggc acttacctgt gttctggcgg caaacccgtt
     5401 gcgaaaaaga acgttcacgg cgactactgc acttatatac ggttctcccc caccctcggg
     5461 aaaaaggcgg agccagtaca cgacatcact ttcccagttt accccgcgcc accttctcta
     5521 ggcaccggtt caattgccga cccctccccc caacttctcg gggactgtgg gcgatgtgcg
     5581 ctctgcccac tgacgggcac cggagcctca cgcatgctct tctccacctc agtgatgacg
     5641 agagcgggcg ggtgaggggg cgggaacgca gcgatctctg ggttctacgt tagtgggagt
     5701 ttaacgacgg tccctgggat tccccaaggc aggggcgagt ccttttgtat gaattactca
     5761 tcgataaact ggatctctgc tgtccctgta ataaacccga aaattttgaa tttttgtaat
     5821 ttgtttttgt aattctttag tttgtatgtc tgttgctatt atgtctacta ttctttcccc
     5881 tgcactgtac cccccaatcc ccccttttct tttaaaggcg ataccgtcga gatccgttca
     5941 ctaatcgaat ggatctgtct ctgtctctct ctccaccttc ttcttctatt ccttcgggcc
     6001 tgtcgggtcc cctcggggtt gggaggtggg tctgaaacga taatggtgaa tatccctgcc
     6061 taactctatt cactatagaa agtacagcaa aaactattct taaacctacc aagcctccta
     6121 ctatcattat gaataatttt atataccaca gccaatttgt tatgttaaac caattccaca
     6181 aacttgccca tttatctaat tccaataatt cttgttcatt cttttcttgc tggttttgcg
     6241 attcttcaat taaggagtgt attaagcttg tgtaattgtt aatttctctg tcccactcca
     6301 tccaggtcgt gtgattccaa atctgttcca gagatttatt actccaacta gcattccaag
     6361 gcacagcagt ggtgcaaatg agttttccag agcaacccca aatccccagg agctgttgat
     6421 cctttaggta tctttccaca gccaggattc ttgcctggag ctgcttgatg ccccagactg
     6481 tgagttgcaa cagatgctgt tgcgcctcaa tagccctcag caaattgttc tgctgctgca
     6541 ctataccaga caataattgt ctggcctgta ccgtcagcgt cattgacgct gcgcccatag
     6601 tgcttcctgc tgctcccaag aacccaagga acaaagctcc tattcccact gctctttttt
     6661 ctctctgcac cactcttctc tttgccttgg tgggtgctac tcctaatggt tcaattttta
     6721 ctactttata tttatataat tcacttctcc aattgtccct catatctcct cctccaggtc
     6781 tgaagatcag cggccggccg cttgctgtgc ggtggtctta cttttgtttt gctcttcctc
     6841 tatcttgtct aaagcttcct tggtgtcttt tatctctatc ctttgatgca cacaatagag
     6901 ggttgctact gtattatata atgatctaag ttcttctgat cctgtctgaa gggatggttg
     6961 tagctgtccc agtatttgtc tacagccttc tgatgtttct aacaggccag gattaactgc
     7021 gaatcgttct agctccctgc ttgcccatac tatatgtttt aatttatatt ttttctttcc
     7081 ccctggcctt aaccgaattt tttcccatcg cgatctaatt ctcccccgct taatactgac
     7141 gctctcgcac ccatctctct ccttctagcc tccgctagtc aaaatttttg gcgtactcac
     7201 cagtcgccgc ccctcgcctc ttgccgtgcg cgcttcagca agccgagtcc tgcgtcgaga
     7261 gagctcctct ggtttccctt tcgctttcaa gtccctgttc gggcgccact gctagagatt
     7321 ttccacactg actaaaaggg tctgagggat ctctagttac cagagtcaca caacagacgg
     7381 gcacacacta cttgaagcac tcaaggcaag ctttattgag gcttaagcag tgggttccct
     7441 agttagccag agagctccca ggctcagatc tggtctaacc agagagaccc agtacaggca
     7501 aaaagcagct gcttatatgc aggatctgag ggctcgccac tccccagtcc cgcccaggcc
     7561 acgcctccct ggaaagtccc cagcggaaag tcccttgtag caagctcgat atcagcagtt
     7621 cttgaagtac tccggatgca gctctcgggc cacgtgatga aatgctaggc ggctgtcaaa
     7681 cctccactct aacacttctc tctccgggtc atccatccca tgcaggctca cagggtgtaa
     7741 caagctggtg ttctctcctt tattggcctc ttctacctta tctggctcaa ctggtactag
     7801 cttgtagcac catccaaagg tcagtggata tctgacccct ggccctggtg tgtagttctg
     7861 ctaatcaggg aagtagcctt gtgtgtggta gatccacaga tcaaggatat cttgtcttcg
     7921 ttgggagtga attagccctt ccagtcccct cttttctttt aaaaagtggc taagatctac
     7981 agctgccttg taagtcattg gtcttaaagg tacctgaggt gtgactggaa aacccacctc
     8041 ctcctcctct tgtgcttcta gccaggcaca atcagcattg gtagctgctg tattgctact
     8101 tgtgattgct ccatgttttt ctaggtctcg actgcagaat taattccagg cggggaggcg
     8161 gcccaaaggg agatccgact cgtctgaggg cgaaggcgaa gacgcggaag aggccgcaga
     8221 gccggcagca ggccgcggga aggaaggtcc gctggattga gggccgaagg gacgtagcag
     8281 aaggacgtcc cgcgcagaat ccaggtggca acacaggcga gcagccatgg aaaggacgtc
     8341 agcttccccg acaacaccac ggaattgtca gtgcccaaca gccgagcccc tgtccagcag
     8401 cgggcaaggc aggcggcgat gagttccgcc gtggcaatag ggagggggaa agcgaaagtc
     8461 ccggaaagga gctgacaggt ggtggcaatg ccccaaccag tgggggttgc gtcagcaaac
     8521 acagtgcaca ccacgccacg ttgcctgaca acgggccaca actcctcata aagagacagc
     8581 aaccaggatt tatacaagga ggagaaaatg aaagccatac gggaagcaat agcatgatac
     8641 aaaggcatta aagcagcgta tccacatagc gtaaaaggag caacatagtt aagaatacca
     8701 gtcaatcttt cacaaatttt gtaatccaga ggttgattgt tccagacgcg gtcaggcacc
     8761 gggcttgcgg gtcatgcacc aggtgcgcgg tccttcgggc acctcgacgt cggcggtgac
     8821 ggtgaagccg agccgctcgt agaaggggag gttgcggggc gcggaggtct ccaggaaggc
     8881 gggcaccccg gcgcgctcgg ccgcctccac tccggggagc acgacggcgc tgcccagacc
     8941 cttgccctgg tggtcgggcg agacgccgac ggtggccagg aaccacgcgg gctccttggg
     9001 ccggtgcggc gccaggaggc cttccatctg ttgctgcgcg gccagccggg aaccgctcaa
     9061 ctcggccatg cgcgggccga tctcggcgaa caccgccccc gcttcgacgc tctccggcgt
     9121 ggtccagacc gccaccgcgg cgccgtcgtc cgcgacccac accttgccga tgtcgagccc
     9181 gacgcgcgtg aggaagagtt cttgcagctc ggtgacccgc tcgatgtggc ggtccggatc
     9241 gacggtgtgg cgcgtggcgg ggtagtcggc gaacgcggcg gcgagggtgc gtacggccct
     9301 ggggacgtcg tcgcgggtgg cgaggcgcac cgtgggcttg tactcggtca tggtaagctt
     9361 gggctgcagg tcgaaaggcc cggagatgag gaagaggaga acagcg
//
