LOCUS       sequence-219892-        6526 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 6526)
  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 6526)
  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 RagB wt
FEATURES             Location/Qualifiers
     source          1..6526
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        7..386
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        387..590
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     541..561
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     587..611
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        821..839
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     821..838
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     852..871
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      956..965
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             968..994
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1025..1678
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1636..1658
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     regulatory      2819..2828
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2832..2966
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2869..2888)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2923..2942
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3035..3392
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3035..3226
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3058..3078)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3243..3378
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3412..3429)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3412..3428)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3421..3443)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3641..4096
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3728..3747)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3938..3959
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4113..4132)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4232..4254
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4292..4310)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4378..4482
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4483..4551,4552..5343)
                     /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(4701..4720)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5514..6102
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6003..6022
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6256..6273
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6390..6411
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6426..6431,6432..6449,6450..6456)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6464..6480
                     /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     6469..6491
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6488..6504
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6488..6504
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 tcgcccgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
       61 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      121 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      181 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      241 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      301 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      361 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      421 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      481 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      541 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct cgtttagtga
      601 accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg
      661 accgatccag cctccgcggc cgggaacggt gcattggaac gcggattccc cgtgccaaga
      721 gtgacgtaag taccgcctat agagtctata ggcccacccc cttggcttcg ttagaacgcg
      781 gctacaatta atacataacc ttatgtatca tacacatacg atttaggtga cactatagaa
      841 taacatccac tttgcctttc tctccacagg tgtccactcc caggtccaac tgcacctcgg
      901 ttctatcgat tgaattcccg aaccgacagt cggtctcttc accaaggcca ttcgcgccac
      961 catgggctac ccctatgatg tgcctgacta cgcagatctc aatggtggtg gtggtgggtc
     1021 gagaatgtcc cctatactag gttattggaa aattaagggc cttgtgcaac ccactcgact
     1081 tcttttggaa tatcttgaag aaaaatatga agagcatttg tatgagcgcg atgaaggtga
     1141 taaatggcga aacaaaaagt ttgaattggg tttggagttt cccaatcttc cttattatat
     1201 tgatggtgat gttaaattaa cacagtctat ggccatcata cgttatatag ctgacaagca
     1261 caacatgttg ggtggttgtc caaaagagcg tgcagagatt tcaatgcttg aaggagcggt
     1321 tttggatatt agatacggtg tttcgagaat tgcatatagt aaagactttg aaactctcaa
     1381 agttgatttt cttagcaagc tacctgaaat gctgaaaatg ttcgaagatc gtttatgtca
     1441 taaaacatat ttaaatggtg atcatgtaac ccatcctgac ttcatgttgt atgacgctct
     1501 tgatgttgtt ttatacatgg acccaatgtg cctggatgcg ttcccaaaat tagtttgttt
     1561 taaaaaacgt attgaagcta tcccacaaat tgataagtac ttgaaatcca gcaagtatat
     1621 agcatggcct ttgcagggct ggcaagccac gtttggtggt ggcgaccatc ctccaaaagg
     1681 tggaggtggt gggtcgaggg gaggttctgg tggatcagga ggttcaggtg gatctgggtc
     1741 gacgatggaa gaatctgact ctgagaaaac gacggagaaa gaaaatctgg ggccgagaat
     1801 ggatccacca ctaggggaac cggaaggatc gcttgggtgg gtgctaccaa atacagccat
     1861 gaagaaaaag gtgctgttga tgggtaaaag tgggtctggt aagaccagca tgaggtctat
     1921 tatctttgca aattatattg ccagagacac acgtcgcctt ggcgcaacaa ttgatgtaga
     1981 acattctcat gttcgatttc tgggaaacct ggtattgaac ctgtgggatt gtggtgggca
     2041 agacaccttc atggaaaatt atttcactag ccaacgggac aacatcttcc gaaatgtgga
     2101 ggttctgatt tatgtctttg atgtggagag ccgcgaactg gaaaaggaca tgcactatta
     2161 ccaatcatgc ctggaggcca ttctgcagaa ttctccagat gccaaaatat tttgcttggt
     2221 acacaaaatg gatctggtac aggaggatca acgggacctg atttttaaag agcgagaaga
     2281 agatttgagg cgtttgtctc gcccattgga atgttcttgt ttccgaacat ctatctggga
     2341 tgaaaccctc tataaggctt ggtccagcat agtttatcag ctgattccca atgttcagca
     2401 gctggaaatg aacctaagga attttgctga aattatcgaa gctgatgaag tacttctttt
     2461 tgagagagct acttttctgg taatttctca ctatcagtgt aaagagcagc gtgatgccca
     2521 tagatttgag aaaataagca acattattaa gcagttcaag ctgagctgca gcaagctggc
     2581 tgcctctttc cagagtatgg aagtcaggaa ctctaacttc gctgctttca ttgacatctt
     2641 tacatccaac acttatgtga tggttgtgat gtctgatccg tccattcctt ctgcagctac
     2701 tctgatcaac atccgcaatg ccaggaaaca ctttgaaaag ctggaaagag tggatggacc
     2761 aaagcagtgt cttctcatgc gctaagcggc cgctaagtaa gtaaggatcc ccagcttggc
     2821 cgccatggcc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     2881 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     2941 tcaatgtatc ttatcatgtc tggatcggga attaattcgg cgcagcacca tggcctgaaa
     3001 taacctctga aagaggaact tggttaggta ccttctgagg cggaaagaac cagctgtgga
     3061 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     3121 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     3181 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     3241 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     3301 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     3361 gaggcttttt tggaggccta ggcttttgca aaaagctgtt aacagcttgg cactggccgt
     3421 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
     3481 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     3541 acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg tattttctcc ttacgcatct
     3601 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     3661 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     3721 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     3781 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     3841 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     3901 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     3961 acaacactca accctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     4021 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     4081 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     4141 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     4201 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     4261 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     4321 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     4381 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     4441 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     4501 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     4561 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     4621 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     4681 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     4741 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     4801 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     4861 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     4921 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     4981 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     5041 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     5101 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     5161 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     5221 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     5281 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     5341 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     5401 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     5461 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     5521 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     5581 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     5641 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     5701 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     5761 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     5821 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     5881 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     5941 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     6001 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     6061 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     6121 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     6181 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     6241 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
     6301 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     6361 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
     6421 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
     6481 tttcacacag gaaacagcta tgacatgatt acgaattaat tcgagc
//
