LOCUS       sequence-163519-        6427 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6427)
  AUTHORS   Sancak Y, Peterson TR, Shaul YD, Lindquist RA, Thoreen CC, Bar-Peled
            L, Sabatini DM
  TITLE     The Rag GTPases bind raptor and mediate amino acid signaling to 
            mTORC1.
  JOURNAL   Science. 2008 Jun 13. 320(5882):1496-501.
  PUBMED    18497260
REFERENCE   2  (bases 1 to 6427)
  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. 
            2008 Jun 13. 320(5882):1496-501."
COMMENT     Sequence Label: pRK5-HA GST RagA wt
FEATURES             Location/Qualifiers
     source          1..6427
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    55..76
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(91..96,97..114,115..121)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    129..145
                     /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     134..156
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     153..169
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     153..169
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        198..577
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        578..781
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     732..752
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     778..802
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        1012..1030
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1012..1029
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     1043..1062
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      1147..1156
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1159..1185
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1216..1869
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1827..1849
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     regulatory      2911..2920
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2924..3058
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2961..2980)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3015..3034
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3127..3484
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3127..3318
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3150..3170)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3335..3470
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3504..3521)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3504..3520)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3513..3535)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3733..4188
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3820..3839)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4030..4051
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4205..4224)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4324..4346
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4384..4402)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4470..4574
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4575..4643,4644..5435)
                     /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(4793..4812)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5606..6194
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6095..6114
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6348..6365
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
       61 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
      121 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgaca tgattacgaa
      181 ttaattcgag ctcgcccgac attgattatt gactagttat taatagtaat caattacggg
      241 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      301 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      361 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      421 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
      481 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
      541 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
      601 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      661 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
      721 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      781 tcgtttagtg aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag
      841 aagacaccgg gaccgatcca gcctccgcgg ccgggaacgg tgcattggaa cgcggattcc
      901 ccgtgccaag agtgacgtaa gtaccgccta tagagtctat aggcccaccc ccttggcttc
      961 gttagaacgc ggctacaatt aatacataac cttatgtatc atacacatac gatttaggtg
     1021 acactataga ataacatcca ctttgccttt ctctccacag gtgtccactc ccaggtccaa
     1081 ctgcacctcg gttctatcga ttgaattccc gaaccgacag tcggtctctt caccaaggcc
     1141 attcgcgcca ccatgggcta cccctatgat gtgcctgact acgcagatct caatggtggt
     1201 ggtggtgggt cgagaatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
     1261 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
     1321 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
     1381 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
     1441 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
     1501 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
     1561 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
     1621 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
     1681 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
     1741 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
     1801 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
     1861 cctccaaaag gtggaggtgg tgggtcgagg ggaggttctg gtggatcagg aggttcaggt
     1921 ggatctgggt cgacgatgcc aaatacagcc atgaagaaaa aggtgctgct gatggggaag
     1981 agcgggtcgg ggaagaccag catgaggtcg ataatcttcg ccaattacat tgctcgcgac
     2041 acccggcgcc tgggggccac cattgacgtg gaacactccc acgtccgatt cctagggaac
     2101 ctggtgctga acctgtggga ctgtggcggt caggacacct tcatggaaaa ttacttcacc
     2161 agccagcgag acaatatctt ccgtaacgtg gaagttttga tttacgtgtt tgacgtggag
     2221 agccgcgaac tggaaaagga catgcattat taccagtcgt gtctggaggc catcctccag
     2281 aactctcctg acgccaaaat cttctgcctg gtgcacaaaa tggatctggt tcaggaggat
     2341 cagcgtgacc tgatttttaa agagcgagag gaagacctga ggcgtctgtc tcgcccgctg
     2401 gagtgtgctt gttttcgaac gtccatctgg gatgagacgc tctacaaagc ctggtccagc
     2461 atcgtctacc agctgattcc caacgttcag cagctggaga tgaacctcag gaattttgcc
     2521 caaatcattg aggccgatga agttctgctg ttcgaaagag ctacattctt ggttatttcc
     2581 cactaccagt gcaaagagca gcgcgacgtc caccggtttg agaagatcag caacatcatc
     2641 aaacagttca agctgagctg cagtaaattg gccgcttcct tccagagcat ggaagttagg
     2701 aattccaact tcgctgcttt catcgacatc ttcacctcaa atacgtacgt gatggtggtc
     2761 atgtcagatc cgtcgatccc ttctgcggcc actctgatca acattcgcaa tgcccggaaa
     2821 cactttgaga agctggagag agtggatggc cccaagcaca gtctccttat gcgttgagcg
     2881 gccgctaagt aagtaaggat ccccagcttg gccgccatgg cccaacttgt ttattgcagc
     2941 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     3001 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatcgg
     3061 gaattaattc ggcgcagcac catggcctga aataacctct gaaagaggaa cttggttagg
     3121 taccttctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc
     3181 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     3241 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     3301 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     3361 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     3421 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     3481 caaaaagctg ttaacagctt ggcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     3541 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     3601 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     3661 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     3721 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3781 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     3841 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3901 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     3961 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     4021 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcgggctatt
     4081 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     4141 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     4201 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     4261 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4321 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     4381 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     4441 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     4501 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     4561 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     4621 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     4681 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     4741 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     4801 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     4861 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     4921 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     4981 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     5041 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     5101 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     5161 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     5221 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     5281 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     5341 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     5401 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     5461 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     5521 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     5581 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     5641 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     5701 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     5761 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     5821 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     5881 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     5941 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6001 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6061 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     6121 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     6181 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     6241 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     6301 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     6361 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     6421 attaatg
//
