LOCUS       sequence-174472-        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 wt
FEATURES             Location/Qualifiers
     source          1..6685
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      20..608
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     509..528
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     762..779
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    896..917
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(932..937,938..955,956..962)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    970..986
                     /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     975..997
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     994..1010
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     994..1010
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        1039..1418
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1419..1622
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1573..1593
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1619..1643
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        1853..1871
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1853..1870
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     1884..1903
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      1988..1997
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2000..2026
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2057..2710
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2668..2690
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     regulatory      4010..4019
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    4023..4157
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4060..4079)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4114..4133
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        4226..4583
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4226..4417
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4249..4269)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4434..4569
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4603..4620)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4603..4619)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4612..4634)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4832..5287
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4919..4938)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5129..5150
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5304..5323)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5423..5445
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5483..5501)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5569..5673
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5674..5742,5743..6534)
                     /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(5892..5911)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
       61 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
      121 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
      181 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
      241 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
      301 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
      361 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
      421 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
      481 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
      541 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
      601 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
      661 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
      721 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
      781 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
      841 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
      901 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
      961 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac atgattacga
     1021 attaattcga gctcgcccga cattgattat tgactagtta ttaatagtaa tcaattacgg
     1081 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
     1141 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     1201 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     1261 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
     1321 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
     1381 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
     1441 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
     1501 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
     1561 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag
     1621 ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt gacctccata
     1681 gaagacaccg ggaccgatcc agcctccgcg gccgggaacg gtgcattgga acgcggattc
     1741 cccgtgccaa gagtgacgta agtaccgcct atagagtcta taggcccacc cccttggctt
     1801 cgttagaacg cggctacaat taatacataa ccttatgtat catacacata cgatttaggt
     1861 gacactatag aataacatcc actttgcctt tctctccaca ggtgtccact cccaggtcca
     1921 actgcacctc ggttctatcg attgaattcc cgaaccgaca gtcggtctct tcaccaaggc
     1981 cattcgcgcc accatgggct acccctatga tgtgcctgac tacgcagatc tcaatggtgg
     2041 tggtggtggg tcgagaatgt cccctatact aggttattgg aaaattaagg gccttgtgca
     2101 acccactcga cttcttttgg aatatcttga agaaaaatat gaagagcatt tgtatgagcg
     2161 cgatgaaggt gataaatggc gaaacaaaaa gtttgaattg ggtttggagt ttcccaatct
     2221 tccttattat attgatggtg atgttaaatt aacacagtct atggccatca tacgttatat
     2281 agctgacaag cacaacatgt tgggtggttg tccaaaagag cgtgcagaga tttcaatgct
     2341 tgaaggagcg gttttggata ttagatacgg tgtttcgaga attgcatata gtaaagactt
     2401 tgaaactctc aaagttgatt ttcttagcaa gctacctgaa atgctgaaaa tgttcgaaga
     2461 tcgtttatgt cataaaacat atttaaatgg tgatcatgta acccatcctg acttcatgtt
     2521 gtatgacgct cttgatgttg ttttatacat ggacccaatg tgcctggatg cgttcccaaa
     2581 attagtttgt tttaaaaaac gtattgaagc tatcccacaa attgataagt acttgaaatc
     2641 cagcaagtat atagcatggc ctttgcaggg ctggcaagcc acgtttggtg gtggcgacca
     2701 tcctccaaaa ggtggaggtg gtgggtcgag gggaggttct ggtggatcag gaggttcagg
     2761 tggatctggg tcgacgatgt ccctgcagta cggggcggag gagacgcccc tcgccggcag
     2821 ttacggcgcg gccgattcgt ttccaaagga cttcggctac ggcgtggagg aggaggaaga
     2881 ggaggcggcg gcggcgggcg gaggggttgg ggcaggggca ggcggtggct gtggtccggg
     2941 gggcgctgac agctccaagc cgaggattct gctcatggga ctccggcgca gcggcaagtc
     3001 ctccatccag aaggtggtgt ttcataagat gtcacccaac gagaccctct ttttggaaag
     3061 taccaacaag atttataagg atgacatttc caatagctcc tttgtgaatt tccagatatg
     3121 ggattttcct gggcaaatgg acttttttga cccaaccttt gactatgaga tgatcttcag
     3181 gggaacagga gcattgatat acgtcattga cgcacaggat gactacatgg aggctttaac
     3241 aagacttcac attactgttt ctaaagccta caaagttaac ccagacatga attttgaggt
     3301 ttttattcac aaagttgatg gtctgtctga tgatcacaaa atagaaacac agagggacat
     3361 tcatcaaagg gccaatgatg accttgcaga tgctgggcta gaaaaactcc atcttagctt
     3421 ttatctgact agtatctatg accattcaat atttgaagcc tttagtaagg tggtgcagaa
     3481 actcattcca caactgccga ccttggaaaa cctattaaat atctttatat caaattcagg
     3541 tattgaaaaa gcttttctct ttgatgttgt cagcaaaatc tacattgcaa cagacagttc
     3601 ccctgtggat atgcaatctt atgaactttg ctgtgacatg atcgatgttg taattgatgt
     3661 gtcttgtata tatgggttaa aggaagatgg aagtggaagt gcttatgaca aagaatctat
     3721 ggcaattatc aagctgaata atacaactgt cctttattta aaggaggtga ctaaattttt
     3781 ggcactggtc tgcattctaa gggaagaaag ctttgaaaga aaaggtttaa tagactacaa
     3841 cttccactgt ttccgaaaag ctattcatga ggtttttgag gtgggtgtga cttctcacag
     3901 gagctgtggt caccagacta gtgcctccag tctgaaagcg ctgacacaca atggcacgcc
     3961 acgaaacgcc atctaggcgg ccgctaagta agtaaggatc cccagcttgg ccgccatggc
     4021 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4081 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4141 cttatcatgt ctggatcggg aattaattcg gcgcagcacc atggcctgaa ataacctctg
     4201 aaagaggaac ttggttaggt accttctgag gcggaaagaa ccagctgtgg aatgtgtgtc
     4261 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     4321 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     4381 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     4441 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     4501 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     4561 ttggaggcct aggcttttgc aaaaagctgt taacagcttg gcactggccg tcgttttaca
     4621 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     4681 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     4741 cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     4801 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     4861 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     4921 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     4981 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     5041 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     5101 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     5161 aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     5221 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     5281 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     5341 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     5401 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     5461 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     5521 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     5581 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     5641 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     5701 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     5761 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     5821 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     5881 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     5941 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     6001 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     6061 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     6121 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     6181 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     6241 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     6301 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     6361 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     6421 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     6481 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     6541 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     6601 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     6661 ttccactgag cgtcagaccc cgtag
//
