LOCUS       sequence-328072-        8176 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral vector for TMEM192 expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8176)
  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 8176)
  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: pLJM1-TMEM192-FLAG
FEATURES             Location/Qualifiers
     source          1..8176
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    12..137
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    630..863
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1048..1092
                     /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"
     primer_bind     1390..1410
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     promoter        1699..1902
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1853..1873
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1940..1949
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1944..1953
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2759..2782
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    2827..2944
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2993..3503
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3422..3440
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3525..4124
                     /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(3525..3544)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4021..4041
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4252..4485
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4557..4678
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4594..4613)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4648..4667
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4718..4853
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4873..4892)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4874..4892)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4902..4919)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4902..4918)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4911..4933)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5060..5515
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5147..5166)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5357..5378
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5541..5645
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5646..5714,5715..6506)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5864..5883)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6677..7265
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7166..7185
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7419..7436
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7553..7574
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7589..7594,7595..7612,7613..7619)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7627..7643
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     7632..7654
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7651..7667
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7651..7667
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7686..7706
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7688..7706
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        7734..7960
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             7961..8141
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
ORIGIN
        1 aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg
       61 aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag
      121 atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc
      181 ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg
      241 agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa
      301 tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata
      361 atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag
      421 ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg
      481 ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa
      541 tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg
      601 gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca
      661 gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg
      721 tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg
      781 ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga
      841 tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc
      901 actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac
      961 acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta
     1021 attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa
     1081 tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc
     1141 ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg
     1201 aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg
     1261 ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc
     1321 attcgattag tgaacggatc tcgacggtat cgatcacgag actagcctcg agcggccgcc
     1381 cccttcaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
     1441 gttagagaga taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg
     1501 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
     1561 gactatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
     1621 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
     1681 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
     1741 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
     1801 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
     1861 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag
     1921 atccgctagc gctaccggtg ccaccatggc ggcggggggc aggatggagg acggttcctt
     1981 ggatatcacc cagagtattg aagacgaccc acttctggat gcccagcttc tcccacacca
     2041 ctcattacaa gctcacttta gaccccgatt ccatcctctt cctacagtca tcatagtgaa
     2101 tcttctgtgg tttattcatc tcgtgtttgt tgttttagca tttttaacag gtgtgctttg
     2161 ttcttatcct aatccaaatg aggacaagtg cccaggaaat tacacaaacc cattgaaagt
     2221 tcagacggtt ataatccttg ggaaagttat tttgtggatt ctccatttac tccttgaatg
     2281 ctacatccag tatcaccaca gcaaaatcag aaaccgaggc tataacttga tctaccgatc
     2341 aacaaggcat ctcaagagac ttgcgttgat gatacagtcc tctggcaaca cagtgcttct
     2401 cctcatactg tgcatgcagc actccttccc agagcctggc agattgtatc ttgacctcat
     2461 tctggccatc ttggcactgg aactcatctg ttccctgata tgtctcctca tttacacagt
     2521 gaaaatccgg agatttaata aagctaaacc agagcctgat atacttgaag aagaaaaaat
     2581 ctatgcttac cccagcaata ttacctcgga gactggattc agaactattt caagcctaga
     2641 agaaattgtt gaaaagcaag gagacaccat tgaatacctg aagcgacaca atgcgctgct
     2701 gagtaagcga ttgttggctc tcacttcctc agacctgggc tgtcagccaa gtagaacgga
     2761 ttacaaggat gacgacgata agtaagaatt ctcgacctcg agacaaatgg cagtattcat
     2821 ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga
     2881 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa
     2941 ttttcgggtt tattacaggg acagcagaga tccactttgg ccgcggctcg agggggttgg
     3001 ggttgcgcct tttccaaggc agccctgggt ttgcgcaggg acgcggctgc tctgggcgtg
     3061 gttccgggaa acgcagcggc gccgaccctg ggtctcgcac attcttcacg tccgttcgca
     3121 gcgtcacccg gatcttcgcc gctacccttg tgggcccccc ggcgacgctt cctgctccgc
     3181 ccctaagtcg ggaaggttcc ttgcggttcg cggcgtgccg gacgtgacaa acggaagccg
     3241 cacgtctcac tagtaccctc gcagacggac agcgccaggg agcaatggca gcgcgccgac
     3301 cgcgatgggc tgtggccaat agcggctgct cagcagggcg cgccgagagc agcggccggg
     3361 aaggggcggt gcgggaggcg gggtgtgggg cggtagtgtg ggccctgttc ctgcccgcgc
     3421 ggtgttccgc attctgcaag cctccggagc gcacgtcggc agtcggctcc ctcgttgacc
     3481 gaatcaccga cctctctccc cagggggatc caccggagct taccatgacc gagtacaagc
     3541 ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg
     3601 cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg
     3661 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg
     3721 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg
     3781 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc
     3841 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg
     3901 ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc
     3961 ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc
     4021 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg
     4081 aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgacgcccg ccccacgacc
     4141 cgcagcgccc gaccgaaagg agcgcacgac cccatgcatc ggtaccttta agaccaatga
     4201 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc
     4261 taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag
     4321 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
     4381 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
     4441 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc
     4501 atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggaact
     4561 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     4621 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     4681 atgtctggct ctagctatcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     4741 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     4801 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctagggac
     4861 gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa
     4921 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     4981 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     5041 agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     5101 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     5161 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     5221 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     5281 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     5341 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     5401 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     5461 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
     5521 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     5581 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     5641 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     5701 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     5761 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     5821 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     5881 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     5941 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     6001 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     6061 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     6121 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     6181 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     6241 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     6301 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     6361 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     6421 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     6481 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     6541 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     6601 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     6661 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     6721 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     6781 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     6841 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     6901 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     6961 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     7021 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     7081 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     7141 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     7201 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     7261 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     7321 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     7381 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     7441 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     7501 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
     7561 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
     7621 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
     7681 agcgcgcaat taaccctcac taaagggaac aaaagctgga gctgcaagct taatgtagtc
     7741 ttatgcaata ctcttgtagt cttgcaacat ggtaacgatg agttagcaac atgccttaca
     7801 aggagagaaa aagcaccgtg catgccgatt ggtggaagta aggtggtacg atcgtgcctt
     7861 attaggaagg caacagacgg gtctgacatg gattggacga accactgaat tgccgcattg
     7921 cagagatatt gtatttaagt gcctagctcg atacataaac gggtctctct ggttagacca
     7981 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
     8041 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
     8101 atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc gaacagggac
     8161 ttgaaagcga aaggga
//
