LOCUS       sequence-224952-        6887 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express HA-GST-FRB-MetaP2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6887)
  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 6887)
  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: pRK5 GST MetaP2
FEATURES             Location/Qualifiers
     source          1..6887
                     /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"
     regulatory      925..934
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             937..963
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             994..1647
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1605..1627
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     regulatory      3187..3196
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3200..3334
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3237..3256)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3291..3310
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3403..3760
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3403..3594
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3426..3446)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3611..3746
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3780..3797)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3780..3796)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3789..3811)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4009..4464
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4096..4115)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4306..4327
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4481..4500)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4600..4622
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4660..4678)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4746..4850
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4851..4919,4920..5711)
                     /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(5069..5088)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5882..6470
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6371..6390
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6624..6641
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6758..6779
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6794..6799,6800..6817,6818..6824)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6832..6848
                     /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     6837..6859
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6856..6872
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6856..6872
                     /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 attcgccacc atgggctacc cctatgatgt gcctgactac
      961 gcagatctca atggtggtgg tggtgggtcg agaatgtccc ctatactagg ttattggaaa
     1021 attaagggcc ttgtgcaacc cactcgactt cttttggaat atcttgaaga aaaatatgaa
     1081 gagcatttgt atgagcgcga tgaaggtgat aaatggcgaa acaaaaagtt tgaattgggt
     1141 ttggagtttc ccaatcttcc ttattatatt gatggtgatg ttaaattaac acagtctatg
     1201 gccatcatac gttatatagc tgacaagcac aacatgttgg gtggttgtcc aaaagagcgt
     1261 gcagagattt caatgcttga aggagcggtt ttggatatta gatacggtgt ttcgagaatt
     1321 gcatatagta aagactttga aactctcaaa gttgattttc ttagcaagct acctgaaatg
     1381 ctgaaaatgt tcgaagatcg tttatgtcat aaaacatatt taaatggtga tcatgtaacc
     1441 catcctgact tcatgttgta tgacgctctt gatgttgttt tatacatgga cccaatgtgc
     1501 ctggatgcgt tcccaaaatt agtttgtttt aaaaaacgta ttgaagctat cccacaaatt
     1561 gataagtact tgaaatccag caagtatata gcatggcctt tgcagggctg gcaagccacg
     1621 tttggtggtg gcgaccatcc tccaaaaggt ggaggtggtg ggtcgggcgg aggtggatct
     1681 ggcggaggtg gatcgggcgg aggtggatca gcgtcgacga tggcgggtgt ggaggaggta
     1741 gcggcctccg ggagccacct gaatggcgac ctggatccag acgacaggga agaaggagct
     1801 gcctctacgg ctgaggaagc agccaagaaa aaaagacgaa agaagaagaa gagcaaaggg
     1861 ccttctgcag gggaacagga acctgataaa gaatcaggag cctcagtgga tgaagtagca
     1921 agacagttgg aaagatcagc attggaagat aaagaaagag atgaagatga tgaagatgga
     1981 gatggcgatg gagatggagc aactggaaag aagaagaaaa agaagaagaa gaagagagga
     2041 ccaaaagttc aaacagaccc tccctcagtt ccaatatgtg acctgtatcc taatggtgta
     2101 tttcccaaag gacaagaatg cgaataccca cccacacaag atgggcgaac agctgcttgg
     2161 agaactacaa gtgaagaaaa gaaagcatta gatcaggcaa gtgaagagat ttggaatgat
     2221 tttcgagaag ctgcagaagc acatcgacaa gttagaaaat acgtaatgag ctggatcaag
     2281 cctgggatga caatgataga aatctgtgaa aagttggaag actgttcacg caagttaata
     2341 aaagagaatg gattaaatgc aggcctggca tttcctactg gatgttctct caataattgt
     2401 gctgcccatt atactcccaa tgccggtgac acaacagtat tacagtatga tgacatctgt
     2461 aaaatagact ttggaacaca tataagtggt aggattattg actgtgcttt tactgtcact
     2521 tttaatccca aatatgatac gttattaaaa gctgtaaaag atgctactaa cactggaata
     2581 aagtgtgctg gaattgatgt tcgtctgtgt gatgttggtg aggccatcca agaagttatg
     2641 gagtcctatg aagttgaaat agatgggaag acatatcaag tgaaaccaat ccgtaatcta
     2701 aatggacatt caattgggca atatagaata catgctggaa aaacagtgcc gattgtgaaa
     2761 ggaggggagg caacaagaat ggaggaagga gaagtatatg caattgaaac ctttggtagt
     2821 acaggaaaag gtgttgttca tgatgatatg gaatgttcac attacatgaa aaattttgat
     2881 gttggacatg tgccaataag gcttccaaga acaaaacact tgttaaatgt catcaatgaa
     2941 aactttggaa cccttgcctt ctgccgcaga tggctggatc gcttgggaga aagtaaatac
     3001 ttgatggctc tgaagaatct gtgtgacttg ggcattgtag atccatatcc accattatgt
     3061 gacattaaag gatcatatac agcgcaattt gaacatacca tcctgttgcg tccaacatgt
     3121 aaagaagttg tcagcagggg agatgactat taagcggccg ctaagtaagt aaggatcccc
     3181 agcttggccg ccatggccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     3241 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     3301 caaactcatc aatgtatctt atcatgtctg gatcgggaat taattcggcg cagcaccatg
     3361 gcctgaaata acctctgaaa gaggaacttg gttaggtacc ttctgaggcg gaaagaacca
     3421 gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     3481 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     3541 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     3601 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     3661 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     3721 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctgttaa cagcttggca
     3781 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     3841 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     3901 ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt
     3961 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     4021 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     4081 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     4141 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     4201 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     4261 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     4321 aaactggaac aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc
     4381 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     4441 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     4501 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     4561 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     4621 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     4681 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     4741 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     4801 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     4861 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     4921 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     4981 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     5041 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     5101 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     5161 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     5221 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     5281 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     5341 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     5401 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     5461 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     5521 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     5581 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     5641 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     5701 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     5761 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     5821 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     5881 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     5941 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     6001 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     6061 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     6121 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     6181 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     6241 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     6301 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     6361 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     6421 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     6481 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     6541 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     6601 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     6661 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     6721 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
     6781 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
     6841 gataacaatt tcacacagga aacagctatg acatgattac gaattaa
//
