LOCUS       sequence-315444-        8408 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral vector for stable expression of FLAG-TK-GAPDH.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8408)
  AUTHORS   Orozco JM, Krawczyk PA, Scaria SM, Cangelosi AL, Chan SH, Kunchok T,
            Lewis CA, Sabatini DM
  TITLE     Dihydroxyacetone phosphate signals glucose availability to mTORC1.
  JOURNAL   Nat Metab. 2020 Sep;2(9):893-901. doi: 10.1038/s42255-020-0250-5. 
            Epub 2020 Jul 27.
  PUBMED    32719541
REFERENCE   2  (bases 1 to 8408)
  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/s42255-020-0250-5"; journalName: "Nat Metab"; date: 
            "2020-09"; volume: "2"; issue: "9"; pages: "893-901"
COMMENT     Sequence Label: FLAG-TKGAPDH in pLJM60
FEATURES             Location/Qualifiers
     source          1..8408
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        231..249
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     231..248
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     262..281
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             340..363
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    1453..1570
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1619..2129
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2048..2066
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2151..2750
                     /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(2151..2170)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2647..2667
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             2878..3111
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3183..3304
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3220..3239)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3274..3293
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3344..3479
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3499..3518)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3500..3518)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3528..3545)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3528..3544)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3537..3559)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3686..4141
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3773..3792)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3983..4004
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4167..4271
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4272..4340,4341..5132)
                     /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(4490..4509)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5303..5891
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5792..5811
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6045..6062
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6179..6200
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6215..6220,6221..6238,6239..6245)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6253..6269
                     /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     6258..6280
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6277..6293
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6277..6293
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6312..6332
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6314..6332
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6360..6586
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6587..6767
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6814..6939
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7436..7669
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7854..7898
                     /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        8205..8408
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     8359..8379
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     join(8405..8408,1..21)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
ORIGIN
        1 cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga
       61 agacaccggg accgatccag cctccgcggc cgggaacggt gcattggaac gcggattccc
      121 cgtgccaaga gtgacgtaag taccgcctat agagtctata ggcccacccc cttggcttcg
      181 ttagaacgcg gctacaatta atacataacc ttatgtatca tacacatacg atttaggtga
      241 cactatagaa taacatccac tttgcctttc tctccacagg tgtccactcc caggtccaac
      301 tgcacctcgg ttctatcgat tgaattcacc atggaaatgg actacaagga tgacgatgac
      361 aagggtggtg gtggtggtgc gtcgacaatg gtcccaaaag ttggtataaa cgggttcggc
      421 cgcataggaa gggttgtgct gcggaacgca ctcgaaacag gcgcagtcga agtagtggca
      481 ctcaatgatc cgtttataga accccactat gctgagtaca tgttcaagta tgattccact
      541 catgggcgat tcaagggtga cattaaggtc gatggaaaag atcttgtgat cgacggaaaa
      601 aggataaagt tttaccaaga gcgcgatccc gcgaacattc cttggaaaga ttctggggcc
      661 gagtacatcg tagaaagtac tggagtattt acgacaaccg agaaggcatc tgctcatttc
      721 aagggagggg cgaaaaaagt aatcattagt gcaccatcag ctgacgcgcc gatgtatgtc
      781 atgggcgtaa acgaggatac ctacgcgggg gctaacgtaa tttctaacgc atcctgcaca
      841 acgaactgtc tggctccctt ggcgaaaaca ttgaacgata agttcacaat agttgaaggt
      901 ctcatgacag ccattcacgc gtacacggct tcccaaaaaa ccgttgatgg accgtcaagc
      961 aaagactggc ggggtggccg cgcagctgcc cagaacttga ttccctctag cactggagct
     1021 gctaaggccg tcggcaaggt tattccagag ctggcgggta aagtgacggg catgagcgtc
     1081 cgggtgccga ctgtgaacgt aagcctggtg gattttacag tccgcttcgc taaggacgtt
     1141 acatacgatg aggtgaaggc cgccattaag gaggcctctg agggtccctt gaagggcata
     1201 ctcgcataca ctgaggacga tattgtatct actgatattc tcaccgaccc tcattcttca
     1261 acctttgatg cgaaagcagg aatcgcattg aataaaaact ttgtcaaagt aatgtcatgg
     1321 tatgacaacg aatatgggta tagccgacgg gtagtggacc tcattgttta tgtcagtaaa
     1381 aaagacgccg gacaataagc ggccgcagtg agaattctcg acctcgagac aaatggcagt
     1441 attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat
     1501 agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat
     1561 tcaaaatttt cgggtttatt acagggacag cagagatcca ctttggccgc ggctcgaggg
     1621 ggttggggtt gcgccttttc caaggcagcc ctgggtttgc gcagggacgc ggctgctctg
     1681 ggcgtggttc cgggaaacgc agcggcgccg accctgggtc tcgcacattc ttcacgtccg
     1741 ttcgcagcgt cacccggatc ttcgccgcta cccttgtggg ccccccggcg acgcttcctg
     1801 ctccgcccct aagtcgggaa ggttccttgc ggttcgcggc gtgccggacg tgacaaacgg
     1861 aagccgcacg tctcactagt accctcgcag acggacagcg ccagggagca atggcagcgc
     1921 gccgaccgcg atgggctgtg gccaatagcg gctgctcagc agggcgcgcc gagagcagcg
     1981 gccgggaagg ggcggtgcgg gaggcggggt gtggggcggt agtgtgggcc ctgttcctgc
     2041 ccgcgcggtg ttccgcattc tgcaagcctc cggagcgcac gtcggcagtc ggctccctcg
     2101 ttgaccgaat caccgacctc tctccccagg gggatccacc ggagcttacc atgaccgagt
     2161 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg
     2221 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg
     2281 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg
     2341 tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag
     2401 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg
     2461 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt
     2521 tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg
     2581 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg
     2641 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg
     2701 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga cgcccgcccc
     2761 acgacccgca gcgcccgacc gaaaggagcg cacgacccca tgcatcggta cctttaagac
     2821 caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg
     2881 aagggctaat tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct
     2941 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
     3001 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
     3061 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtagtagtt
     3121 catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga
     3181 ggaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     3241 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     3301 cttatcatgt ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc
     3361 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     3421 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     3481 agggacgtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt
     3541 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     3601 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     3661 ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     3721 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     3781 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     3841 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     3901 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     3961 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     4021 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     4081 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     4141 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     4201 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     4261 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     4321 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     4381 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     4441 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     4501 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     4561 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     4621 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     4681 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     4741 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     4801 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     4861 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     4921 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     4981 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     5041 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     5101 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     5161 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     5221 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     5281 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     5341 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     5401 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     5461 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     5521 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     5581 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     5641 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     5701 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     5761 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     5821 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     5881 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     5941 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     6001 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     6061 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     6121 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     6181 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
     6241 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
     6301 acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagcttaat
     6361 gtagtcttat gcaatactct tgtagtcttg caacatggta acgatgagtt agcaacatgc
     6421 cttacaagga gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg
     6481 tgccttatta ggaaggcaac agacgggtct gacatggatt ggacgaacca ctgaattgcc
     6541 gcattgcaga gatattgtat ttaagtgcct agctcgatac ataaacgggt ctctctggtt
     6601 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
     6661 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
     6721 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac
     6781 agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct
     6841 gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact
     6901 agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt
     6961 agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa
     7021 catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa
     7081 acatcagaag gctgtagaca aatactggga cagctacaac catcccttca gacaggatca
     7141 gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata
     7201 gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag
     7261 accaccgcac agcaagcggc cggccgctga tcttcagacc tggaggagga gatatgaggg
     7321 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag
     7381 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag
     7441 ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc
     7501 tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga
     7561 gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc
     7621 aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg
     7681 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata
     7741 aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca
     7801 attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg
     7861 aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa
     7921 attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa
     7981 tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt
     8041 ttcagaccca cctcccaacc ccgaggggac cccatatgcc aagtacgccc cctattgacg
     8101 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     8161 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
     8221 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
     8281 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
     8341 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
     8401 gcagagct
//
