LOCUS       sequence-177208-        5589 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  C-term HA-GST tagged overexpression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5589)
  AUTHORS   Zoncu R, Bar-Peled L, Efeyan A, Wang S, Sancak Y, Sabatini DM
  TITLE     mTORC1 senses lysosomal amino acids through an inside-out mechanism 
            that requires the vacuolar H-ATPase.
  JOURNAL   Science. 2011 Nov 4;334(6056):678-83.
  PUBMED    22053050
REFERENCE   2  (bases 1 to 5589)
  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: "Science."; 
            date: "2011-11-4"; volume: "334(6056)"; pages: "678-83"
COMMENT     Sequence Label: pRK5-ATP6V1D-HAGST
FEATURES             Location/Qualifiers
     source          1..5589
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     complement(59..76)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(59..75)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(68..90)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      288..743
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(375..394)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..606
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(760..779)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     879..901
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(939..957)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1025..1129
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1130..1198,1199..1990)
                     /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(1348..1367)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2161..2749
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2650..2669
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2903..2920
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3037..3058
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3073..3078,3079..3096,3097..3103)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3111..3127
                     /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     3116..3138
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3135..3151
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3135..3151
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        3180..3559
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3560..3763
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3714..3734
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3760..3784
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        3994..4012
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3994..4011
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     4025..4044
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             4865..4891
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4922..5575
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     5533..5555
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
ORIGIN
        1 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctgttaac agcttggcac
       61 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc
      121 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc
      181 cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta
      241 cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag
      301 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
      361 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
      421 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
      481 cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata
      541 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
      601 aactggaaca acactcaacc ctatctcggg ctattctttt gatttataag ggattttgcc
      661 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
      721 caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc
      781 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
      841 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
      901 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
      961 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     1021 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     1081 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     1141 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     1201 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     1261 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     1321 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     1381 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     1441 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     1501 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     1561 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     1621 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     1681 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     1741 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     1801 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     1861 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     1921 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     1981 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     2041 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     2101 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     2161 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     2221 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     2281 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     2341 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     2401 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     2461 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     2521 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     2581 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     2641 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     2701 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     2761 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     2821 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     2881 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
     2941 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     3001 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
     3061 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
     3121 ataacaattt cacacaggaa acagctatga catgattacg aattaattcg agctcgcccg
     3181 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
     3241 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
     3301 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     3361 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     3421 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     3481 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     3541 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
     3601 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
     3661 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
     3721 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca
     3781 gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc
     3841 cagcctccgc ggccgggaac ggtgcattgg aacgcggatt ccccgtgcca agagtgacgt
     3901 aagtaccgcc tatagagtct ataggcccac ccccttggct tcgttagaac gcggctacaa
     3961 ttaatacata accttatgta tcatacacat acgatttagg tgacactata gaataacatc
     4021 cactttgcct ttctctccac aggtgtccac tcccaggtcc aactgcacct cggttctatc
     4081 gattgaattc gtcgacgcca ccatgtcggg caaagaccga attgaaatct ttccctcgcg
     4141 aatggcacag accatcatga aggctcgttt aaagggagca cagacaggtc gaaacctcct
     4201 gaagaaaaaa tctgatgcct taactcttcg atttcgacag atcctaaaga agataataga
     4261 gactaaaatg ttgatgggcg aagtgatgag agaagctgcc ttttcactag ctgaagccaa
     4321 gttcacagca ggtgacttca gcactacagt tatccaaaat gtcaataaag cgcaagtgaa
     4381 gattcgagcg aagaaagata atgtagcagg tgttactttg ccagtatttg aacattacca
     4441 tgaaggaact gacagttatg aactgactgg tttagccaga ggtggggaac agttggctaa
     4501 attaaagagg aattatgcca aagcagtgga actactggtg gaactagctt ctctgcagac
     4561 ttcttttgtt actttggatg aagctattaa gataaccaac aggcgtgtaa atgccattga
     4621 acatgtcatc attccccgga ttgaacgtac tcttgcttat atcatcacag agctggatga
     4681 gagagagcga gaagagttct ataggttaaa gaaaatacaa gagaagaaaa agattctaaa
     4741 ggaaaaatct gagaaggact tggagcaaag gagagcagct ggagaggtgt tggagcctgc
     4801 taatcttctg gctgaagaga aggacgagga tcttctattt gaagcggccg caggaggtgg
     4861 aggttacccc tatgatgtgc ctgactacgc agatctcaat ggtggtggtg gtgggtcgag
     4921 aatgtcccct atactaggtt attggaaaat taagggcctt gtgcaaccca ctcgacttct
     4981 tttggaatat cttgaagaaa aatatgaaga gcatttgtat gagcgcgatg aaggtgataa
     5041 atggcgaaac aaaaagtttg aattgggttt ggagtttccc aatcttcctt attatattga
     5101 tggtgatgtt aaattaacac agtctatggc catcatacgt tatatagctg acaagcacaa
     5161 catgttgggt ggttgtccaa aagagcgtgc agagatttca atgcttgaag gagcggtttt
     5221 ggatattaga tacggtgttt cgagaattgc atatagtaaa gactttgaaa ctctcaaagt
     5281 tgattttctt agcaagctac ctgaaatgct gaaaatgttc gaagatcgtt tatgtcataa
     5341 aacatattta aatggtgatc atgtaaccca tcctgacttc atgttgtatg acgctcttga
     5401 tgttgtttta tacatggacc caatgtgcct ggatgcgttc ccaaaattag tttgttttaa
     5461 aaaacgtatt gaagctatcc cacaaattga taagtacttg aaatccagca agtatatagc
     5521 atggcctttg cagggctggc aagccacgtt tggtggtggc gaccatcctc caaaaggtgg
     5581 atgataagg
//
