LOCUS       sequence-287544-        6688 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 6688)
  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 6688)
  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 RagD 121L
FEATURES             Location/Qualifiers
     source          1..6688
                     /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      963..972
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             975..1001
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1032..1685
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1643..1665
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     regulatory      2988..2997
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3001..3135
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3038..3057)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3092..3111
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3204..3561
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3204..3395
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3227..3247)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3412..3547
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3581..3598)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3581..3597)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3590..3612)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3810..4265
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3897..3916)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4107..4128
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4282..4301)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4401..4423
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4461..4479)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4547..4651
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4652..4720,4721..5512)
                     /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(4870..4889)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5683..6271
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6172..6191
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6425..6442
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6559..6580
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6595..6600,6601..6618,6619..6625)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6633..6649
                     /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     6638..6660
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6657..6673
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6657..6673
                     /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 attcccgaac cgacagtcgg tctcttcacc aaggccattc
      961 gcgccaccat gggctacccc tatgatgtgc ctgactacgc agatctcaat ggtggtggtg
     1021 gtgggtcgag aatgtcccct atactaggtt attggaaaat taagggcctt gtgcaaccca
     1081 ctcgacttct tttggaatat cttgaagaaa aatatgaaga gcatttgtat gagcgcgatg
     1141 aaggtgataa atggcgaaac aaaaagtttg aattgggttt ggagtttccc aatcttcctt
     1201 attatattga tggtgatgtt aaattaacac agtctatggc catcatacgt tatatagctg
     1261 acaagcacaa catgttgggt ggttgtccaa aagagcgtgc agagatttca atgcttgaag
     1321 gagcggtttt ggatattaga tacggtgttt cgagaattgc atatagtaaa gactttgaaa
     1381 ctctcaaagt tgattttctt agcaagctac ctgaaatgct gaaaatgttc gaagatcgtt
     1441 tatgtcataa aacatattta aatggtgatc atgtaaccca tcctgacttc atgttgtatg
     1501 acgctcttga tgttgtttta tacatggacc caatgtgcct ggatgcgttc ccaaaattag
     1561 tttgttttaa aaaacgtatt gaagctatcc cacaaattga taagtacttg aaatccagca
     1621 agtatatagc atggcctttg cagggctggc aagccacgtt tggtggtggc gaccatcctc
     1681 caaaaggtgg aggtggtggg tcgaggggag gttctggtgg atcaggaggt tcaggtggat
     1741 ctgggtcgac gatgagccag gtgctgggga agccgcagcc gcaggacgag gacgacgcgg
     1801 aggaggagga ggaggaggat gagctggtgg ggctagcgga ctacggagac gggcccgact
     1861 cctccgacgc cgatccggac agcggcacag aggagggagt tctggacttc agtgacccct
     1921 tcagcactga agtgaagccg agaatcctgc tcatgggcct gaggagaagc ggcaagtcgt
     1981 ctattcagaa agttgtcttt cacaaaatgt ctcccaacga aactctgttc ttggagagca
     2041 ctaataagat atgccgggaa gatgtttcca acagctcctt tgtcaatttt cagatttggg
     2101 acttcccagg actgattgac ttttttgacc ctacatttga ctatgagatg atcttccggg
     2161 gaacaggagc actgatattt gtcattgact cacaggatga ttacatggaa gccctggcca
     2221 ggctccacct cacggtgacc agggcctaca aagtgaatac tgacatcaac ttcgaggtgt
     2281 ttattcataa agtggatggt ctgtcagatg accacaaaat tgaaacccaa agagatattc
     2341 accagagggc aaacgatgac cttgcagatg ctggattaga aaaaattcac ctcagctttt
     2401 atctgacaag catatatgat cattcaatat ttgaagcttt tagcaaagtt gttcagaaac
     2461 tgattccaca actcccaact ctggagaatt tgctgaacat ctttatctca aattctggaa
     2521 ttgaaaaggc atttctattt gatgtggtca gtaaaattta tattgcaact gatagtactc
     2581 cggtggatat gcaaacctat gagctctgct gtgatatgat agatgtggtt attgacatct
     2641 cttgtattta tggtctcaaa gaagatggag caggaacccc ctatgacaag gaatccacag
     2701 ccatcataaa gcttaataat acaaccgtgc tttatttaaa agaggtgaca aagttcctgg
     2761 ctctcgtttg ctttgtcaga gaggaaagct ttgaaagaaa agggctaatt gactataatt
     2821 ttcattgctt ccggaaggcc attcatgaag tttttgaggt gagaatgaaa gtagtaaaat
     2881 ctcgaaaggt tcagaatcgg ctgcagaaga aaaagagagc cacccctaat gggaccccta
     2941 gagtgctgct gtaggcggcc gctaagtaag taaggatccc cagcttggcc gccatggccc
     3001 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     3061 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     3121 tatcatgtct ggatcgggaa ttaattcggc gcagcaccat ggcctgaaat aacctctgaa
     3181 agaggaactt ggttaggtac cttctgaggc ggaaagaacc agctgtggaa tgtgtgtcag
     3241 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3301 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3361 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     3421 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     3481 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     3541 ggaggcctag gcttttgcaa aaagctgtta acagcttggc actggccgtc gttttacaac
     3601 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     3661 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     3721 gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     3781 cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc
     3841 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     3901 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3961 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     4021 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     4081 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     4141 ccctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     4201 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
     4261 aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     4321 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     4381 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     4441 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     4501 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     4561 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     4621 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     4681 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     4741 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     4801 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     4861 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     4921 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     4981 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     5041 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     5101 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     5161 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     5221 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     5281 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     5341 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     5401 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     5461 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     5521 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     5581 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     5641 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     5701 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     5761 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     5821 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     5881 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     5941 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     6001 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     6061 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     6121 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     6181 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     6241 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     6301 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     6361 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     6421 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
     6481 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     6541 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
     6601 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
     6661 aaacagctat gacatgatta cgaattaa
//
