LOCUS       sequence-456521-        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 120L
FEATURES             Location/Qualifiers
     source          1..6685
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        14..393
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        394..597
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     548..568
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     594..618
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        828..846
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     828..845
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     859..878
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      963..972
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             975..1001
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1032..1685
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1643..1665
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     regulatory      2985..2994
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2998..3132
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3035..3054)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3089..3108
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3201..3558
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3201..3392
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3224..3244)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3409..3544
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3578..3595)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3578..3594)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3587..3609)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3807..4262
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3894..3913)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4104..4125
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4279..4298)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4398..4420
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4458..4476)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4544..4648
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4649..4717,4718..5509)
                     /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(4867..4886)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5680..6268
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6169..6188
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6422..6439
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6556..6577
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6592..6597,6598..6615,6616..6622)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6630..6646
                     /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     6635..6657
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6654..6670
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6654..6670
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca
       61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
      601 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
      661 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt
      721 gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta
      781 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac
      841 tatagaataa catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc
      901 acctcggttc tatcgattga attcccgaac cgacagtcgg tctcttcacc aaggccattc
      961 gcgccaccat gggctacccc tatgatgtgc ctgactacgc agatctcaat ggtggtggtg
     1021 gtgggtcgag aatgtcccct atactaggtt attggaaaat taagggcctt gtgcaaccca
     1081 ctcgacttct tttggaatat cttgaagaaa aatatgaaga gcatttgtat gagcgcgatg
     1141 aaggtgataa atggcgaaac aaaaagtttg aattgggttt ggagtttccc aatcttcctt
     1201 attatattga tggtgatgtt aaattaacac agtctatggc catcatacgt tatatagctg
     1261 acaagcacaa catgttgggt ggttgtccaa aagagcgtgc agagatttca atgcttgaag
     1321 gagcggtttt ggatattaga tacggtgttt cgagaattgc atatagtaaa gactttgaaa
     1381 ctctcaaagt tgattttctt agcaagctac ctgaaatgct gaaaatgttc gaagatcgtt
     1441 tatgtcataa aacatattta aatggtgatc atgtaaccca tcctgacttc atgttgtatg
     1501 acgctcttga tgttgtttta tacatggacc caatgtgcct ggatgcgttc ccaaaattag
     1561 tttgttttaa aaaacgtatt gaagctatcc cacaaattga taagtacttg aaatccagca
     1621 agtatatagc atggcctttg cagggctggc aagccacgtt tggtggtggc gaccatcctc
     1681 caaaaggtgg aggtggtggg tcgaggggag gttctggtgg atcaggaggt tcaggtggat
     1741 ctgggtcgac gatgtccctg cagtacgggg cggaggagac gcccctcgcc ggcagttacg
     1801 gcgcggccga ttcgtttcca aaggacttcg gctacggcgt ggaggaggag gaagaggagg
     1861 cggcggcggc gggcggaggg gttggggcag gggcaggcgg tggctgtggt ccggggggcg
     1921 ctgacagctc caagccgagg attctgctca tgggactccg gcgcagcggc aagtcctcca
     1981 tccagaaggt ggtgtttcat aagatgtcac ccaacgagac cctctttttg gaaagtacca
     2041 acaagattta taaggatgac atttccaata gctcctttgt gaatttccag atatgggatt
     2101 ttcctgggct aatggacttt tttgacccaa cctttgacta tgagatgatc ttcaggggaa
     2161 caggagcatt gatatacgtc attgacgcac aggatgacta catggaggct ttaacaagac
     2221 ttcacattac tgtttctaaa gcctacaaag ttaacccaga catgaatttt gaggttttta
     2281 ttcacaaagt tgatggtctg tctgatgatc acaaaataga aacacagagg gacattcatc
     2341 aaagggccaa tgatgacctt gcagatgctg ggctagaaaa actccatctt agcttttatc
     2401 tgactagtat ctatgaccat tcaatatttg aagcctttag taaggtggtg cagaaactca
     2461 ttccacaact gccgaccttg gaaaacctat taaatatctt tatatcaaat tcaggtattg
     2521 aaaaagcttt tctctttgat gttgtcagca aaatctacat tgcaacagac agttcccctg
     2581 tggatatgca atcttatgaa ctttgctgtg acatgatcga tgttgtaatt gatgtgtctt
     2641 gtatatatgg gttaaaggaa gatggaagtg gaagtgctta tgacaaagaa tctatggcaa
     2701 ttatcaagct gaataataca actgtccttt atttaaagga ggtgactaaa tttttggcac
     2761 tggtctgcat tctaagggaa gaaagctttg aaagaaaagg tttaatagac tacaacttcc
     2821 actgtttccg aaaagctatt catgaggttt ttgaggtggg tgtgacttct cacaggagct
     2881 gtggtcacca gactagtgcc tccagtctga aagcgctgac acacaatggc acgccacgaa
     2941 acgccatcta ggcggccgct aagtaagtaa ggatccccag cttggccgcc atggcccaac
     3001 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     3061 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     3121 catgtctgga tcgggaatta attcggcgca gcaccatggc ctgaaataac ctctgaaaga
     3181 ggaacttggt taggtacctt ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta
     3241 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3301 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3361 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     3421 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     3481 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     3541 ggcctaggct tttgcaaaaa gctgttaaca gcttggcact ggccgtcgtt ttacaacgtc
     3601 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     3661 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     3721 tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     3781 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg
     3841 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3901 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3961 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     4021 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     4081 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     4141 tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     4201 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     4261 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     4321 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     4381 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     4441 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     4501 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     4561 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     4621 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     4681 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     4741 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     4801 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     4861 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     4921 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     4981 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     5041 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     5101 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     5161 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     5221 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     5281 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     5341 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     5401 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     5461 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     5521 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     5581 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     5641 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     5701 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     5761 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     5821 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     5881 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     5941 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     6001 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     6061 acagcgtgag ctatgagaaa gcgccacgtt tcccgaaggg agaaaggcgg acaggtatcc
     6121 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     6181 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     6241 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     6301 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     6361 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     6421 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     6481 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     6541 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     6601 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     6661 cagctatgac atgattacga attaa
//
