LOCUS       sequence-313825-        6685 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 6685)
  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 6685)
  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 RagC 75L
FEATURES             Location/Qualifiers
     source          1..6685
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     581..605
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        815..833
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     815..832
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     846..865
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      950..959
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             962..988
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1019..1669
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPP"
     primer_bind     1630..1652
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     regulatory      2972..2981
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2985..3119
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3022..3041)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3076..3095
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3188..3545
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3188..3379
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3211..3231)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3396..3531
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3565..3582)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3565..3581)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3574..3596)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3794..4249
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3881..3900)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4091..4112
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4266..4285)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4385..4407
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4445..4463)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4531..4635
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4636..4704,4705..5496)
                     /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(4854..4873)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5667..6255
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6156..6175
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6409..6426
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6543..6564
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6579..6584,6585..6602,6603..6609)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6617..6633
                     /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     6622..6644
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6641..6657
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6641..6657
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc
      601 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
      661 ccagcctccg cggccgggaa cggtgcattg gaacgcggat tccccgtgcc aagagtgacg
      721 taagtaccgc ctatagagtc tataggccca cccccttggc ttcgttagaa cgcggctaca
      781 attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat agaataacat
      841 ccactttgcc tttctctcca caggtgtcca ctcccaggtc caactgcacc tcggttctat
      901 cgattgaatt cccgaaccga cagtcggtct cttcaccaag gccattcgcg ccaccatggg
      961 ctacccctat gatgtgcctg actacgcaga tctcaatggt ggtggtggtg ggtcgagaat
     1021 gtcccctata ctaggttatt ggaaaattaa gggccttgtg caacccactc gacttctttt
     1081 ggaatatctt gaagaaaaat atgaagagca tttgtatgag cgcgatgaag gtgataaatg
     1141 gcgaaacaaa aagtttgaat tgggtttgga gtttcccaat cttccttatt atattgatgg
     1201 tgatgttaaa ttaacacagt ctatggccat catacgttat atagctgaca agcacaacat
     1261 gttgggtggt tgtccaaaag agcgtgcaga gatttcaatg cttgaaggag cggttttgga
     1321 tattagatac ggtgtttcga gaattgcata tagtaaagac tttgaaactc tcaaagttga
     1381 ttttcttagc aagctacctg aaatgctgaa aatgttcgaa gatcgtttat gtcataaaac
     1441 atatttaaat ggtgatcatg taacccatcc tgacttcatg ttgtatgacg ctcttgatgt
     1501 tgttttatac atggacccaa tgtgcctgga tgcgttccca aaattagttt gttttaaaaa
     1561 acgtattgaa gctatcccac aaattgataa gtacttgaaa tccagcaagt atatagcatg
     1621 gcctttgcag ggctggcaag ccacgtttgg tggtggcgac catcctccaa acggtggagg
     1681 tggtgggtcg aggggaggtt ctggtggatc aggaggttca ggtggatctg ggtcgacgat
     1741 gtccctgcag tacggggcgg aggagacgcc cctcgccggc agttacggcg cggccgattc
     1801 gtttccaaag gacttcggct acggcgtgga ggaggaggaa gaggaggcgg cggcggcggg
     1861 cggaggggtt ggggcagggg caggcggtgg ctgtggtccg gggggcgctg acagctccaa
     1921 gccgaggatt ctgctcatgg gactccggcg cagcggcaag ctctccatcc agaaggtggt
     1981 gtttcataag atgtcaccca acgagaccct ctttttggaa agtaccaaca agatttataa
     2041 ggatgacatt tccaatagct cctttgtgaa tttccagata tgggattttc ctgggcaaat
     2101 ggactttttt gacccaacct ttgactatga gatgatcttc aggggaacag gagcattgat
     2161 atacgtcatt gacgcacagg atgactacat ggaggcttta acaagacttc acattactgt
     2221 ttctaaagcc tacaaagtta acccagacat gaattttgag gtttttattc acaaagttga
     2281 tggtctgtct gatgatcaca aaatagaaac acagagggac attcatcaaa gggccaatga
     2341 tgaccttgca gatgctgggc tagaaaaact ccatcttagc ttttatctga ctagtatcta
     2401 tgaccattca atatttgaag cctttagtaa ggtggtgcag aaactcattc cacaactgcc
     2461 gaccttggaa aacctattaa atatctttat atcaaattca ggtattgaaa aagcttttct
     2521 ctttgatgtt gtcagcaaaa tctacattgc aacagacagt tcccctgtgg atatgcaatc
     2581 ttatgaactt tgctgtgaca tgatcgatgt tgtaattgat gtgtcttgta tatatgggtt
     2641 aaaggaagat ggaagtggaa gtgcttatga caaagaatct atggcaatta tcaagctgaa
     2701 taatacaact gtcctttatt taaaggaggt gactaaattt ttggcactgg tctgcattct
     2761 aagggaagaa agctttgaaa gaaaaggttt aatagactac aacttccact gtttccgaaa
     2821 agctattcat gaggtttttg aggtgggtgt gacttctcac aggagctgtg gtcaccagac
     2881 tagtgcctcc agtctgaaag cgctgacaca caatggcacg ccacgaaacg ccatctaggc
     2941 ggccgctaag taagtaagga tccccagctt ggccgccatg gcccaacttg tttattgcag
     3001 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     3061 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatcg
     3121 ggaattaatt cggcgcagca ccatggcctg aaataacctc tgaaagagga acttggttag
     3181 gtaccttctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3241 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3301 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3361 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3421 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3481 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     3541 gcaaaaagct gttaacagct tggcactggc cgtcgtttta caacgtcgtg actgggaaaa
     3601 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     3661 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     3721 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca
     3781 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3841 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3901 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3961 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt
     4021 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     4081 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcgggctat
     4141 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     4201 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact
     4261 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     4321 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     4381 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     4441 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     4501 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     4561 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     4621 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     4681 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     4741 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     4801 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     4861 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     4921 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     4981 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     5041 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     5101 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     5161 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     5221 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     5281 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     5341 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     5401 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     5461 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     5521 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     5581 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     5641 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     5701 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     5761 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     5821 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     5881 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     5941 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     6001 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     6061 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     6121 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     6181 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     6241 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     6301 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     6361 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     6421 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     6481 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     6541 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     6601 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgacatg
     6661 attacgaatt aattcgagct cgccc
//
