LOCUS       sequence-224267-        8176 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express pLJM1 TMEM192-Flag .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8176)
  AUTHORS   Lawrence RE, Cho KF, Rappold R, Thrun A, Tofaute M, Kim DJ, 
            Moldavski O, Hurley JH, Zoncu R
  TITLE     A nutrient-induced affinity switch controls mTORC1 activation by its
            Rag GTPase-Ragulator lysosomal scaffold.
  JOURNAL   Nat Cell Biol. 2018 Sep;20(9):1052-1063. doi: 
            10.1038/s41556-018-0148-6. Epub 2018 Jul 30.
  PUBMED    30061680
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"; doi: 
            "10.1038/s41556-018-0148-6"; journalName: "Nat Cell Biol"; date: 
            "2018-09"; volume: "20"; issue: "9"; pages: "1052-1063"
COMMENT     Sequence Label: pLJM1 TMEM192-Flag
FEATURES             Location/Qualifiers
     source          1..8176
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1010..1030
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1012..1030
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1058..1284
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             1285..1465
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1512..1637
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2130..2363
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2548..2592
                     /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     2890..2910
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     promoter        3199..3402
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3353..3373
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3440..3449
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      3444..3453
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4259..4282
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    4327..4444
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4493..5003
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4922..4940
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             5025..5624
                     /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(5025..5044)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5521..5541
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             5752..5985
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    6057..6178
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6094..6113)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6148..6167
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6218..6353
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6373..6392)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6374..6392)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6402..6419)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6402..6418)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6411..6433)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6560..7015
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6647..6666)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6857..6878
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        7041..7145
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7146..7214,7215..8006)
                     /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(7364..7383)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc
     1021 tcactaaagg gaacaaaagc tggagctgca agcttaatgt agtcttatgc aatactcttg
     1081 tagtcttgca acatggtaac gatgagttag caacatgcct tacaaggaga gaaaaagcac
     1141 cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg ccttattagg aaggcaacag
     1201 acgggtctga catggattgg acgaaccact gaattgccgc attgcagaga tattgtattt
     1261 aagtgcctag ctcgatacat aaacgggtct ctctggttag accagatctg agcctgggag
     1321 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     1381 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     1441 tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga
     1501 aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg
     1561 aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag
     1621 atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc
     1681 ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg
     1741 agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa
     1801 tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata
     1861 atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag
     1921 ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg
     1981 ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa
     2041 tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg
     2101 gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca
     2161 gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg
     2221 tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg
     2281 ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga
     2341 tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc
     2401 actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac
     2461 acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta
     2521 attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa
     2581 tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc
     2641 ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg
     2701 aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg
     2761 ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc
     2821 attcgattag tgaacggatc tcgacggtat cgatcacgag actagcctcg agcggccgcc
     2881 cccttcaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
     2941 gttagagaga taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg
     3001 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
     3061 gactatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
     3121 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
     3181 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
     3241 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
     3301 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
     3361 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag
     3421 atccgctagc gctaccggtg ccaccatggc ggcggggggc aggatggagg acggttcctt
     3481 ggatatcacc cagagtattg aagacgaccc acttctggat gcccagcttc tcccacacca
     3541 ctcattacaa gctcacttta gaccccgatt ccatcctctt cctacagtca tcatagtgaa
     3601 tcttctgtgg tttattcatc tcgtgtttgt tgttttagca tttttaacag gtgtgctttg
     3661 ttcttatcct aatccaaatg aggacaagtg cccaggaaat tacacaaacc cattgaaagt
     3721 tcagacggtt ataatccttg ggaaagttat tttgtggatt ctccatttac tccttgaatg
     3781 ctacatccag tatcaccaca gcaaaatcag aaaccgaggc tataacttga tctaccgatc
     3841 aacaaggcat ctcaagagac ttgcgttgat gatacagtcc tctggcaaca cagtgcttct
     3901 cctcatactg tgcatgcagc actccttccc agagcctggc agattgtatc ttgacctcat
     3961 tctggccatc ttggcactgg aactcatctg ttccctgata tgtctcctca tttacacagt
     4021 gaaaatccgg agatttaata aagctaaacc agagcctgat atacttgaag aagaaaaaat
     4081 ctatgcttac cccagcaata ttacctcgga gactggattc agaactattt caagcctaga
     4141 agaaattgtt gaaaagcaag gagacaccat tgaatacctg aagcgacaca atgcgctgct
     4201 gagtaagcga ttgttggctc tcacttcctc agacctgggc tgtcagccaa gtagaacgga
     4261 ttacaaggat gacgacgata agtaagaatt ctcgacctcg agacaaatgg cagtattcat
     4321 ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga
     4381 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa
     4441 ttttcgggtt tattacaggg acagcagaga tccactttgg ccgcggctcg agggggttgg
     4501 ggttgcgcct tttccaaggc agccctgggt ttgcgcaggg acgcggctgc tctgggcgtg
     4561 gttccgggaa acgcagcggc gccgaccctg ggtctcgcac attcttcacg tccgttcgca
     4621 gcgtcacccg gatcttcgcc gctacccttg tgggcccccc ggcgacgctt cctgctccgc
     4681 ccctaagtcg ggaaggttcc ttgcggttcg cggcgtgccg gacgtgacaa acggaagccg
     4741 cacgtctcac tagtaccctc gcagacggac agcgccaggg agcaatggca gcgcgccgac
     4801 cgcgatgggc tgtggccaat agcggctgct cagcagggcg cgccgagagc agcggccggg
     4861 aaggggcggt gcgggaggcg gggtgtgggg cggtagtgtg ggccctgttc ctgcccgcgc
     4921 ggtgttccgc attctgcaag cctccggagc gcacgtcggc agtcggctcc ctcgttgacc
     4981 gaatcaccga cctctctccc cagggggatc caccggagct taccatgacc gagtacaagc
     5041 ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg
     5101 cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg
     5161 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg
     5221 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg
     5281 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc
     5341 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg
     5401 ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc
     5461 ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc
     5521 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg
     5581 aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgacgcccg ccccacgacc
     5641 cgcagcgccc gaccgaaagg agcgcacgac cccatgcatc ggtaccttta agaccaatga
     5701 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc
     5761 taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag
     5821 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
     5881 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
     5941 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc
     6001 atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggaact
     6061 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     6121 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     6181 atgtctggct ctagctatcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     6241 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     6301 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctagggac
     6361 gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa
     6421 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     6481 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     6541 agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     6601 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     6661 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     6721 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     6781 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     6841 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     6901 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     6961 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
     7021 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     7081 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     7141 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     7201 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     7261 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     7321 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     7381 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     7441 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     7501 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     7561 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     7621 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     7681 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     7741 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     7801 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     7861 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     7921 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     7981 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     8041 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     8101 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     8161 agatcaaagg atcttc
//
