LOCUS       sequence-214529-        8612 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 2XFLAG-TMEM192 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8612)
  AUTHORS   Wyant GA, Abu-Remaileh M, Wolfson RL, Chen WW, Freinkman E, Danai 
            LV, Vander Heiden MG, Sabatini DM
  TITLE     mTORC1 Activator SLC38A9 Is Required to Efflux Essential Amino Acids
            from Lysosomes and Use Protein as a Nutrient.
  JOURNAL   Cell. 2017 Oct 19;171(3):642-654.e12. doi: 
            10.1016/j.cell.2017.09.046.
  PUBMED    29053970
REFERENCE   2  (bases 1 to 8612)
  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: 
            "10.1016/j.cell.2017.09"; journalName: "Cell"; date: "2017-10-19- 
            19"; volume: "171"; issue: "3"; pages: "642-654.e12"
COMMENT     Sequence Label: pLJC6-2XFLAG-TMEM192
FEATURES             Location/Qualifiers
     source          1..8612
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..227
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             228..408
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    455..580
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1073..1306
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1491..1535
                     /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"
     promoter        1824..3035
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     2872..2891
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     regulatory      3057..3066
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3887..3934
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     misc_feature    3979..4096
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4145..4655
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4574..4592
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4664..5059
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MVPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             5131..5364
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5436..5557
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5473..5492)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5527..5546
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5597..5732
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5752..5771)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5753..5771)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5781..5798)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5781..5797)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5790..5812)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5939..6394
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6026..6045)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6236..6257
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6420..6524
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6525..6593,6594..7385)
                     /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(6743..6762)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7556..8144
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8045..8064
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8298..8315
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8432..8453
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8468..8473,8474..8491,8492..8498)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8506..8522
                     /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     8511..8533
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8530..8546
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8530..8546
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     8565..8585
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        8567..8585
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg
       61 ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc
      121 gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc
      181 cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt
      241 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
      301 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
      361 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa
      421 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc
      481 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac
      541 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat
      601 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa
      661 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga
      721 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc
      781 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat
      841 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa
      901 gaccaccgca cagcaagcag cagctgatct tcagacctgg aggaggagat atgagggaca
      961 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac
     1021 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt
     1081 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga
     1141 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg
     1201 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg
     1261 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt
     1321 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat
     1381 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt
     1441 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac
     1501 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
     1561 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
     1621 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc
     1681 agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg
     1741 gagagagaga cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgatcac
     1801 gagactagcc tcgagcggga tctggcctcc gcgccgggtt ttggcgcctc ccgcgggcgc
     1861 ccccctcctc acggcgagcg ctgccacgtc agacgaaggg cgcagcgagc gtcctgatcc
     1921 ttccgcccgg acgctcagga cagcggcccg ctgctcataa gactcggcct tagaacccca
     1981 gtatcagcag aaggacattt taggacggga cttgggtgac tctagggcac tggttttctt
     2041 tccagagagc ggaacaggcg aggaaaagta gtcccttctc ggcgattctg cggagggatc
     2101 tccgtggggc ggtgaacgcc gatgattata taaggacgcg ccgggtgtgg cacagctagt
     2161 tccgtcgcag ccgggatttg ggtcgcggtt cttgtttgtg gatcgctgtg atcgtcactt
     2221 ggtgagtagc gggctgctgg gctggccggg gctttcgtgg ccgccgggcc gctcggtggg
     2281 acggaagcgt gtggagagac cgccaagggc tgtagtctgg gtccgcgagc aaggttgccc
     2341 tgaactgggg gttgggggga gcgcagcaaa atggcggctg ttcccgagtc ttgaatggaa
     2401 gacgcttgtg aggcgggctg tgaggtcgtt gaaacaaggt ggggggcatg gtgggcggca
     2461 agaacccaag gtcttgaggc cttcgctaat gcgggaaagc tcttattcgg gtgagatggg
     2521 ctggggcacc atctggggac cctgacgtga agtttgtcac tgactggaga actcggtttg
     2581 tcgtctgttg cgggggcggc agttatggcg gtgccgttgg gcagtgcacc cgtacctttg
     2641 ggagcgcgcg ccctcgtcgt gtcgtgacgt cacccgttct gttggcttat aatgcagggt
     2701 ggggccacct gccggtaggt gtgcggtagg cttttctccg tcgcaggacg cagggttcgg
     2761 gcctagggta ggctctcctg aatcgacagg cgccggacct ctggtgaggg gagggataag
     2821 tgaggcgtca gtttctttgg tcggttttat gtacctatct tcttaagtag ctgaagctcc
     2881 ggttttgaac tatgcgctcg gggttggcga gtgtgttttg tgaagttttt taggcacctt
     2941 ttgaaatgta atcatttggg tcaatatgta attttcagtg ttagactagt aaattgtccg
     3001 ctaaattctg gccgtttttg gcttttttgt tagacgaagc gctagcgcta ccggtaccat
     3061 ggcggcgggg ggcaggatgg aggacggttc cttggatatc acccagagta ttgaagacga
     3121 cccacttctg gatgcccagc ttctcccaca ccactcatta caagctcact ttagaccccg
     3181 attccatcct cttcctacag tcatcatagt gaatcttctg tggtttattc atctcgtgtt
     3241 tgttgtttta gcatttttaa caggtgtgct ttgttcttat cctaatccaa atgaggacaa
     3301 gtgcccagga aattacacaa acccattgaa agttcagacg gttataatcc ttgggaaagt
     3361 tattttgtgg attctccatt tactccttga atgctacatc cagtatcacc acagcaaaat
     3421 cagaaaccga ggctataact tgatctaccg atcaacaagg catctcaaga gacttgcgtt
     3481 gatgatacag tcctctggca acacagtgct tctcctcata ctgtgcatgc agcactcctt
     3541 cccagagcct ggcagattgt atcttgacct cattctggcc atcttggcac tggaactcat
     3601 ctgttccctg atatgtctcc tcatttacac agtgaaaatc cggagattta ataaagctaa
     3661 accagagcct gatatacttg aagaagaaaa aatctatgct taccccagca atattacctc
     3721 ggagactgga ttcagaacta tttcaagcct agaagaaatt gttgaaaagc aaggagacac
     3781 cattgaatac ctgaagcgac acaatgcgct gctgagtaag cgattgttgg ctctcacttc
     3841 ctcagacctg ggctgtcagc caagtagaac gggaggtgga ggtagtgact acaaagacga
     3901 tgacgacaag gactacaaag acgatgacga caagtaagaa ttctcgacct cgagacaaat
     3961 ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga
     4021 aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac
     4081 aaaaattcaa aattttcggg tttattacag ggacagcaga gatccacttt ggccgcggct
     4141 cgagggggtt ggggttgcgc cttttccaag gcagccctgg gtttgcgcag ggacgcggct
     4201 gctctgggcg tggttccggg aaacgcagcg gcgccgaccc tgggtctcgc acattcttca
     4261 cgtccgttcg cagcgtcacc cggatcttcg ccgctaccct tgtgggcccc ccggcgacgc
     4321 ttcctgctcc gcccctaagt cgggaaggtt ccttgcggtt cgcggcgtgc cggacgtgac
     4381 aaacggaagc cgcacgtctc actagtaccc tcgcagacgg acagcgccag ggagcaatgg
     4441 cagcgcgccg accgcgatgg gctgtggcca atagcggctg ctcagcaggg cgcgccgaga
     4501 gcagcggccg ggaaggggcg gtgcgggagg cggggtgtgg ggcggtagtg tgggccctgt
     4561 tcctgcccgc gcggtgttcc gcattctgca agcctccgga gcgcacgtcg gcagtcggct
     4621 ccctcgttga ccgaatcacc gacctctctc cccaggggga tccatggttc ctttgtctca
     4681 agaagaatcc accctcattg aaagagcaac ggctacaatc aacagcatcc ccatctctga
     4741 agactacagc gtcgccagcg cagctctctc tagcgacggc cgcatcttca ctggtgtcaa
     4801 tgtatatcat tttactgggg gaccttgtgc agaactcgtg gtgctgggca ctgctgctgc
     4861 tgcggcagct ggcaacctga cttgtatcgt cgcgatcgga aatgagaaca ggggcatctt
     4921 gagcccctgc ggacggtgcc gacaggtgct tctcgatctg catcctggga tcaaagccat
     4981 agtgaaggac agtgatggac agccgacggc agttgggatt cgtgaattgc tgccctctgg
     5041 ttatgtgtgg gagggctaag gtacctttaa gaccaatgac ttacaaggca gctgtagatc
     5101 ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac
     5161 aagatctgct ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc
     5221 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc
     5281 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt
     5341 agtcagtgtg gaaaatctct agcagtagta gttcatgtca tcttattatt cagtatttat
     5401 aacttgcaaa gaaatgaata tcagagagtg agaggaactt gtttattgca gcttataatg
     5461 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     5521 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggctc tagctatccc
     5581 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     5641 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
     5701 ccagaagtag tgaggaggct tttttggagg cctagggacg tacccaattc gccctatagt
     5761 gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     5821 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     5881 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac
     5941 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     6001 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     6061 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     6121 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     6181 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     6241 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     6301 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     6361 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     6421 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     6481 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     6541 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     6601 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     6661 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     6721 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     6781 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     6841 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     6901 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     6961 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     7021 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     7081 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     7141 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     7201 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     7261 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     7321 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     7381 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     7441 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     7501 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     7561 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     7621 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     7681 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     7741 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     7801 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     7861 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     7921 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     7981 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     8041 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     8101 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     8161 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     8221 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     8281 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     8341 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     8401 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
     8461 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
     8521 aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact
     8581 aaagggaaca aaagctggag ctgcaagctt aa
//
