LOCUS       sequence-424398-        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 77L
FEATURES             Location/Qualifiers
     source          1..6688
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(37..54)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(37..53)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(46..68)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      266..721
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(353..372)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     563..584
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(738..757)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     857..879
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(917..935)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1003..1107
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1108..1176,1177..1968)
                     /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(1326..1345)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2139..2727
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2628..2647
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2881..2898
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3015..3036
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3051..3056,3057..3074,3075..3081)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3089..3105
                     /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     3094..3116
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3113..3129
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3113..3129
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        3158..3537
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3538..3741
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3692..3712
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3738..3762
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        3972..3990
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3972..3989
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     4003..4022
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      4107..4116
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4119..4145
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4176..4829
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     4787..4809
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     regulatory      6132..6141
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    6145..6279
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6182..6201)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6236..6255
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        join(6348..6688,1..17)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        6348..6539
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(6371..6391)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      join(6556..6688,1..3)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 gcctaggctt ttgcaaaaag ctgttaacag cttggcactg gccgtcgttt tacaacgtcg
       61 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
      121 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
      181 gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
      241 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
      301 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
      361 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
      421 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
      481 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
      541 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
      601 atctcgggct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
      661 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
      721 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
      781 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
      841 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
      901 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
      961 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     1021 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     1081 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     1141 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     1201 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     1261 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     1321 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     1381 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     1441 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     1501 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     1561 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     1621 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     1681 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     1741 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     1801 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     1861 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     1921 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     1981 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     2041 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     2101 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     2161 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     2221 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     2281 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     2341 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     2401 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     2461 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     2521 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     2581 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     2641 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     2701 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     2761 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     2821 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     2881 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     2941 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     3001 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
     3061 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
     3121 agctatgaca tgattacgaa ttaattcgag ctcgcccgac attgattatt gactagttat
     3181 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
     3241 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
     3301 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
     3361 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
     3421 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
     3481 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
     3541 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
     3601 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
     3661 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
     3721 gaggtctata taagcagagc tcgtttagtg aaccgtcaga tcgcctggag acgccatcca
     3781 cgctgttttg acctccatag aagacaccgg gaccgatcca gcctccgcgg ccgggaacgg
     3841 tgcattggaa cgcggattcc ccgtgccaag agtgacgtaa gtaccgccta tagagtctat
     3901 aggcccaccc ccttggcttc gttagaacgc ggctacaatt aatacataac cttatgtatc
     3961 atacacatac gatttaggtg acactataga ataacatcca ctttgccttt ctctccacag
     4021 gtgtccactc ccaggtccaa ctgcacctcg gttctatcga ttgaattccc gaaccgacag
     4081 tcggtctctt caccaaggcc attcgcgcca ccatgggcta cccctatgat gtgcctgact
     4141 acgcagatct caatggtggt ggtggtgggt cgagaatgtc ccctatacta ggttattgga
     4201 aaattaaggg ccttgtgcaa cccactcgac ttcttttgga atatcttgaa gaaaaatatg
     4261 aagagcattt gtatgagcgc gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg
     4321 gtttggagtt tcccaatctt ccttattata ttgatggtga tgttaaatta acacagtcta
     4381 tggccatcat acgttatata gctgacaagc acaacatgtt gggtggttgt ccaaaagagc
     4441 gtgcagagat ttcaatgctt gaaggagcgg ttttggatat tagatacggt gtttcgagaa
     4501 ttgcatatag taaagacttt gaaactctca aagttgattt tcttagcaag ctacctgaaa
     4561 tgctgaaaat gttcgaagat cgtttatgtc ataaaacata tttaaatggt gatcatgtaa
     4621 cccatcctga cttcatgttg tatgacgctc ttgatgttgt tttatacatg gacccaatgt
     4681 gcctggatgc gttcccaaaa ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa
     4741 ttgataagta cttgaaatcc agcaagtata tagcatggcc tttgcagggc tggcaagcca
     4801 cgtttggtgg tggcgaccat cctccaaaag gtggaggtgg tgggtcgagg ggaggttctg
     4861 gtggatcagg aggttcaggt ggatctgggt cgacgatgag ccaggtgctg gggaagccgc
     4921 agccgcagga cgaggacgac gcggaggagg aggaggagga ggatgagctg gtggggctag
     4981 cggactacgg agacgggccc gactcctccg acgccgatcc ggacagcggc acagaggagg
     5041 gagttctgga cttcagtgac cccttcagca ctgaagtgaa gccgagaatc ctgctcatgg
     5101 gcctgaggag aagcggcaag tcgcttattc agaaagttgt ctttcacaaa atgtctccca
     5161 acgaaactct gttcttggag agcactaata agatatgccg ggaagatgtt tccaacagct
     5221 cctttgtcaa ttttcagatt tgggacttcc caggacagat tgactttttt gaccctacat
     5281 ttgactatga gatgatcttc cggggaacag gagcactgat atttgtcatt gactcacagg
     5341 atgattacat ggaagccctg gccaggctcc acctcacggt gaccagggcc tacaaagtga
     5401 atactgacat caacttcgag gtgtttattc ataaagtgga tggtctgtca gatgaccaca
     5461 aaattgaaac ccaaagagat attcaccaga gggcaaacga tgaccttgca gatgctggat
     5521 tagaaaaaat tcacctcagc ttttatctga caagcatata tgatcattca atatttgaag
     5581 cttttagcaa agttgttcag aaactgattc cacaactccc aactctggag aatttgctga
     5641 acatctttat ctcaaattct ggaattgaaa aggcatttct atttgatgtg gtcagtaaaa
     5701 tttatattgc aactgatagt actccggtgg atatgcaaac ctatgagctc tgctgtgata
     5761 tgatagatgt ggttattgac atctcttgta tttatggtct caaagaagat ggagcaggaa
     5821 ccccctatga caaggaatcc acagccatca taaagcttaa taatacaacc gtgctttatt
     5881 taaaagaggt gacaaagttc ctggctctcg tttgctttgt cagagaggaa agctttgaaa
     5941 gaaaagggct aattgactat aattttcatt gcttccggaa ggccattcat gaagtttttg
     6001 aggtgagaat gaaagtagta aaatctcgaa aggttcagaa tcggctgcag aagaaaaaga
     6061 gagccacccc taatgggacc cctagagtgc tgctgtaggc ggccgctaag taagtaagga
     6121 tccccagctt ggccgccatg gcccaacttg tttattgcag cttataatgg ttacaaataa
     6181 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     6241 ttgtccaaac tcatcaatgt atcttatcat gtctggatcg ggaattaatt cggcgcagca
     6301 ccatggcctg aaataacctc tgaaagagga acttggttag gtaccttctg aggcggaaag
     6361 aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     6421 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     6481 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     6541 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     6601 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     6661 cagaagtagt gaggaggctt ttttggag
//
