LOCUS       sequence-424397-        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 wt
FEATURES             Location/Qualifiers
     source          1..6688
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    66..87
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(102..107,108..125,126..132)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    140..156
                     /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     145..167
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     164..180
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     164..180
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        209..588
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        589..792
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     743..763
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     789..813
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        1023..1041
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1023..1040
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     1054..1073
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      1158..1167
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1170..1196
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1227..1880
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1838..1860
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     regulatory      3183..3192
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3196..3330
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3233..3252)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3287..3306
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3399..3756
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3399..3590
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3422..3442)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3607..3742
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3776..3793)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3776..3792)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3785..3807)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4005..4460
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4092..4111)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4302..4323
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4477..4496)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4596..4618
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4656..4674)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4742..4846
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4847..4915,4916..5707)
                     /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(5065..5084)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5878..6466
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6367..6386
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6620..6637
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
       61 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
      121 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
      181 atgattacga attaattcga gctcgcccga cattgattat tgactagtta ttaatagtaa
      241 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
      301 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
      361 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
      421 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
      481 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
      541 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
      601 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
      661 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
      721 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
      781 ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt
      841 gacctccata gaagacaccg ggaccgatcc agcctccgcg gccgggaacg gtgcattgga
      901 acgcggattc cccgtgccaa gagtgacgta agtaccgcct atagagtcta taggcccacc
      961 cccttggctt cgttagaacg cggctacaat taatacataa ccttatgtat catacacata
     1021 cgatttaggt gacactatag aataacatcc actttgcctt tctctccaca ggtgtccact
     1081 cccaggtcca actgcacctc ggttctatcg attgaattcc cgaaccgaca gtcggtctct
     1141 tcaccaaggc cattcgcgcc accatgggct acccctatga tgtgcctgac tacgcagatc
     1201 tcaatggtgg tggtggtggg tcgagaatgt cccctatact aggttattgg aaaattaagg
     1261 gccttgtgca acccactcga cttcttttgg aatatcttga agaaaaatat gaagagcatt
     1321 tgtatgagcg cgatgaaggt gataaatggc gaaacaaaaa gtttgaattg ggtttggagt
     1381 ttcccaatct tccttattat attgatggtg atgttaaatt aacacagtct atggccatca
     1441 tacgttatat agctgacaag cacaacatgt tgggtggttg tccaaaagag cgtgcagaga
     1501 tttcaatgct tgaaggagcg gttttggata ttagatacgg tgtttcgaga attgcatata
     1561 gtaaagactt tgaaactctc aaagttgatt ttcttagcaa gctacctgaa atgctgaaaa
     1621 tgttcgaaga tcgtttatgt cataaaacat atttaaatgg tgatcatgta acccatcctg
     1681 acttcatgtt gtatgacgct cttgatgttg ttttatacat ggacccaatg tgcctggatg
     1741 cgttcccaaa attagtttgt tttaaaaaac gtattgaagc tatcccacaa attgataagt
     1801 acttgaaatc cagcaagtat atagcatggc ctttgcaggg ctggcaagcc acgtttggtg
     1861 gtggcgacca tcctccaaaa ggtggaggtg gtgggtcgag gggaggttct ggtggatcag
     1921 gaggttcagg tggatctggg tcgacgatga gccaggtgct ggggaagccg cagccgcagg
     1981 acgaggacga cgcggaggag gaggaggagg aggatgagct ggtggggcta gcggactacg
     2041 gagacgggcc cgactcctcc gacgccgatc cggacagcgg cacagaggag ggagttctgg
     2101 acttcagtga ccccttcagc actgaagtga agccgagaat cctgctcatg ggcctgagga
     2161 gaagcggcaa gtcgtctatt cagaaagttg tctttcacaa aatgtctccc aacgaaactc
     2221 tgttcttgga gagcactaat aagatatgcc gggaagatgt ttccaacagc tcctttgtca
     2281 attttcagat ttgggacttc ccaggacaga ttgacttttt tgaccctaca tttgactatg
     2341 agatgatctt ccggggaaca ggagcactga tatttgtcat tgactcacag gatgattaca
     2401 tggaagccct ggccaggctc cacctcacgg tgaccagggc ctacaaagtg aatactgaca
     2461 tcaacttcga ggtgtttatt cataaagtgg atggtctgtc agatgaccac aaaattgaaa
     2521 cccaaagaga tattcaccag agggcaaacg atgaccttgc agatgctgga ttagaaaaaa
     2581 ttcacctcag cttttatctg acaagcatat atgatcattc aatatttgaa gcttttagca
     2641 aagttgttca gaaactgatt ccacaactcc caactctgga gaatttgctg aacatcttta
     2701 tctcaaattc tggaattgaa aaggcatttc tatttgatgt ggtcagtaaa atttatattg
     2761 caactgatag tactccggtg gatatgcaaa cctatgagct ctgctgtgat atgatagatg
     2821 tggttattga catctcttgt atttatggtc tcaaagaaga tggagcagga accccctatg
     2881 acaaggaatc cacagccatc ataaagctta ataatacaac cgtgctttat ttaaaagagg
     2941 tgacaaagtt cctggctctc gtttgctttg tcagagagga aagctttgaa agaaaagggc
     3001 taattgacta taattttcat tgcttccgga aggccattca tgaagttttt gaggtgagaa
     3061 tgaaagtagt aaaatctcga aaggttcaga atcggctgca gaagaaaaag agagccaccc
     3121 ctaatgggac ccctagagtg ctgctgtagg cggccgctaa gtaagtaagg atccccagct
     3181 tggccgccat ggcccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     3241 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     3301 ctcatcaatg tatcttatca tgtctggatc gggaattaat tcggcgcagc accatggcct
     3361 gaaataacct ctgaaagagg aacttggtta ggtaccttct gaggcggaaa gaaccagctg
     3421 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3481 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     3541 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     3601 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     3661 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     3721 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tgttaacagc ttggcactgg
     3781 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     3841 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     3901 cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc
     3961 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg
     4021 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     4081 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     4141 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     4201 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
     4261 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     4321 tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat
     4381 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     4441 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata
     4501 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     4561 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     4621 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     4681 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     4741 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     4801 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     4861 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     4921 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     4981 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     5041 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     5101 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     5161 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     5221 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     5281 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     5341 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     5401 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     5461 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     5521 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     5581 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     5641 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     5701 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     5761 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     5821 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     5881 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     5941 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     6001 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     6061 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     6121 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     6181 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     6241 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     6301 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     6361 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     6421 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     6481 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     6541 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     6601 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
     6661 caaaccgcct ctccccgcgc gttggccg
//
