LOCUS       sequence-177654-        5632 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-term HA-GST tagged overexpression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5632)
  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 5632)
  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..5632
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        14..393
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        394..597
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     548..568
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     594..618
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        828..846
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     828..845
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     859..878
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             1699..1725
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1756..2409
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2367..2389
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     primer_bind     complement(2525..2542)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2525..2541)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2534..2556)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2754..3209
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2841..2860)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3051..3072
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3226..3245)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3345..3367
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3405..3423)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3491..3595
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3596..3664,3665..4456)
                     /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(3814..3833)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4627..5215
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5116..5135
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5369..5386
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5503..5524
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5539..5544,5545..5562,5563..5569)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5577..5593
                     /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     5582..5604
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5601..5617
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5601..5617
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca
       61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
      601 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
      661 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt
      721 gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta
      781 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac
      841 tatagaataa catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc
      901 acctcggttc tatcgattga attcgtcgac gccaccatgt cgggcaaaga ccgaattgaa
      961 atctttccct cgcgaatggc acagaccatc atgaaggctc gtttaaaggg agcacagaca
     1021 ggtcgaaacc tcctgaagaa aaaatctgat gccttaactc ttcgatttcg acagatccta
     1081 aagaagataa tagagactaa aatgttgatg ggcgaagtga tgagagaagc tgccttttca
     1141 ctagctgaag ccaagttcac agcaggtgac ttcagcacta cagttatcca aaatgtcaat
     1201 aaagcgcaag tgaagattcg agcgaagaaa gataatgtag caggtgttac tttgccagta
     1261 tttgaacatt accatgaagg aactgacagt tatgaactga ctggtttagc cagaggtggg
     1321 gaacagttgg ctaaattaaa gaggaattat gccaaagcag tggaactact ggtggaacta
     1381 gcttctctgc agacttcttt tgttactttg gatgaagcta ttaagataac caacaggcgt
     1441 gtaaatgcca ttgaacatgt catcattccc cggattgaac gtactcttgc ttatatcatc
     1501 acagagctgg atgagagaga gcgagaagag ttctataggt taaagaaaat acaagagaag
     1561 aaaaagattc taaaggaaaa atctgagaag gacttggagc aaaggagagc agctggagag
     1621 gtgttggagc ctgctaatct tctggctgaa gagaaggacg aggatcttct atttgaagcg
     1681 gccgcaggag gtggaggtta cccctatgat gtgcctgact acgcagatct caatggtggt
     1741 ggtggtgggt cgagaatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
     1801 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
     1861 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
     1921 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
     1981 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
     2041 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
     2101 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
     2161 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
     2221 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
     2281 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
     2341 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
     2401 cctccaaaag gtggatgata atatgcagag gccgaggccg cctcggcctc tgagctattc
     2461 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct gttaacagct
     2521 tggcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     2581 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     2641 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc
     2701 tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc
     2761 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2821 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2881 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2941 acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc
     3001 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     3061 gttccaaact ggaacaacac tcaaccctat ctcgggctat tcttttgatt tataagggat
     3121 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     3181 ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga
     3241 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     3301 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     3361 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     3421 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     3481 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     3541 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     3601 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     3661 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     3721 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     3781 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     3841 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     3901 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     3961 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     4021 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     4081 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     4141 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     4201 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     4261 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     4321 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     4381 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     4441 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     4501 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     4561 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     4621 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     4681 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     4741 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     4801 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     4861 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     4921 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     4981 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     5041 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     5101 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     5161 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     5221 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     5281 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     5341 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     5401 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     5461 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
     5521 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     5581 gagcggataa caatttcaca caggaaacag ctatgacatg attacgaatt aa
//
