LOCUS       sequence-313798-        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 66L
FEATURES             Location/Qualifiers
     source          1..6427
                     /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..1672
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1630..1652
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     regulatory      2714..2723
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2727..2861
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2764..2783)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2818..2837
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2930..3287
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2930..3121
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2953..2973)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3138..3273
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3307..3324)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3307..3323)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3316..3338)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3536..3991
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3623..3642)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3833..3854
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4008..4027)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4127..4149
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4187..4205)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4273..4377
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4378..4446,4447..5238)
                     /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(4596..4615)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5409..5997
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5898..5917
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6151..6168
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6285..6306
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6321..6326,6327..6344,6345..6351)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6359..6375
                     /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     6364..6386
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6383..6399
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6383..6399
                     /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 aaggtggagg
     1681 tggtgggtcg aggggaggtt ctggtggatc aggaggttca ggtggatctg ggtcgacgat
     1741 gccaaataca gccatgaaga aaaaggtgct gctgatgggg aagagcgggt cggggaagac
     1801 cagcatgagg tcgataatct tcgccaatta cattgctcgc gacacccggc gcctgggggc
     1861 caccattgac gtggaacact cccacgtccg attcctaggg aacctggtgc tgaacctgtg
     1921 ggactgtggc ggtctggaca ccttcatgga aaattacttc accagccagc gagacaatat
     1981 cttccgtaac gtggaagttt tgatttacgt gtttgacgtg gagagccgcg aactggaaaa
     2041 ggacatgcat tattaccagt cgtgtctgga ggccatcctc cagaactctc ctgacgccaa
     2101 aatcttctgc ctggtgcaca aaatggatct ggttcaggag gatcagcgtg acctgatttt
     2161 taaagagcga gaggaagacc tgaggcgtct gtctcgcccg ctggagtgtg cttgttttcg
     2221 aacgtccatc tgggatgaga cgctctacaa agcctggtcc agcatcgtct accagctgat
     2281 tcccaacgtt cagcagctgg agatgaacct caggaatttt gcccaaatca ttgaggccga
     2341 tgaagttctg ctgttcgaaa gagctacatt cttggttatt tcccactacc agtgcaaaga
     2401 gcagcgcgac gtccaccggt ttgagaagat cagcaacatc atcaaacagt tcaagctgag
     2461 ctgcagtaaa ttggccgctt ccttccagag catggaagtt aggaattcca acttcgctgc
     2521 tttcatcgac atcttcacct caaatacgta cgtgatggtg gtcatgtcag atccgtcgat
     2581 cccttctgcg gccactctga tcaacattcg caatgcccgg aaacactttg agaagctgga
     2641 gagagtggat ggccccaagc acagtctcct tatgcgttga gcggccgcta agtaagtaag
     2701 gatccccagc ttggccgcca tggcccaact tgtttattgc agcttataat ggttacaaat
     2761 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     2821 gtttgtccaa actcatcaat gtatcttatc atgtctggat cgggaattaa ttcggcgcag
     2881 caccatggcc tgaaataacc tctgaaagag gaacttggtt aggtaccttc tgaggcggaa
     2941 agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     3001 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     3061 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     3121 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     3181 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     3241 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctgttaacag
     3301 cttggcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     3361 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     3421 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt
     3481 tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg
     3541 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3601 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3661 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3721 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg
     3781 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     3841 ttgttccaaa ctggaacaac actcaaccct atctcgggct attcttttga tttataaggg
     3901 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     3961 aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct
     4021 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     4081 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     4141 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     4201 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     4261 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     4321 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     4381 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     4441 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     4501 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     4561 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     4621 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     4681 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     4741 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     4801 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     4861 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     4921 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     4981 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     5041 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     5101 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     5161 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     5221 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     5281 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     5341 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     5401 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     5461 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     5521 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     5581 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     5641 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     5701 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     5761 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     5821 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     5881 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     5941 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     6001 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     6061 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     6121 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     6181 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     6241 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
     6301 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
     6361 gtgagcggat aacaatttca cacaggaaac agctatgaca tgattacgaa ttaattcgag
     6421 ctcgccc
//
