LOCUS       sequence-177057-        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 99L
FEATURES             Location/Qualifiers
     source          1..6526
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        11..390
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        391..594
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     545..565
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     591..615
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        825..843
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     825..842
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     856..875
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      960..969
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             972..998
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1029..1682
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1640..1662
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     regulatory      2823..2832
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2836..2970
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2873..2892)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2927..2946
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3039..3396
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3039..3230
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3062..3082)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3247..3382
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3416..3433)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3416..3432)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3425..3447)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3645..4100
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3732..3751)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3942..3963
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4117..4136)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4236..4258
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4296..4314)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4382..4486
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4487..4555,4556..5347)
                     /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(4705..4724)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5518..6106
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6007..6026
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6260..6277
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6394..6415
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6430..6435,6436..6453,6454..6460)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6468..6484
                     /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     6473..6495
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6492..6508
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6492..6508
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gagctcgccc gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
       61 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
      121 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
      181 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
      241 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
      301 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
      361 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
      421 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
      481 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
      541 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta
      601 gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac
      661 cgggaccgat ccagcctccg cggccgggaa cggtgcattg gaacgcggat tccccgtgcc
      721 aagagtgacg taagtaccgc ctatagagtc tataggccca cccccttggc ttcgttagaa
      781 cgcggctaca attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat
      841 agaataacat ccactttgcc tttctctcca caggtgtcca ctcccaggtc caactgcacc
      901 tcggttctat cgattgaatt cccgaaccga cagtcggtct cttcaccaag gccattcgcg
      961 ccaccatggg ctacccctat gatgtgcctg actacgcaga tctcaatggt ggtggtggtg
     1021 ggtcgagaat gtcccctata ctaggttatt ggaaaattaa gggccttgtg caacccactc
     1081 gacttctttt ggaatatctt gaagaaaaat atgaagagca tttgtatgag cgcgatgaag
     1141 gtgataaatg gcgaaacaaa aagtttgaat tgggtttgga gtttcccaat cttccttatt
     1201 atattgatgg tgatgttaaa ttaacacagt ctatggccat catacgttat atagctgaca
     1261 agcacaacat gttgggtggt tgtccaaaag agcgtgcaga gatttcaatg cttgaaggag
     1321 cggttttgga tattagatac ggtgtttcga gaattgcata tagtaaagac tttgaaactc
     1381 tcaaagttga ttttcttagc aagctacctg aaatgctgaa aatgttcgaa gatcgtttat
     1441 gtcataaaac atatttaaat ggtgatcatg taacccatcc tgacttcatg ttgtatgacg
     1501 ctcttgatgt tgttttatac atggacccaa tgtgcctgga tgcgttccca aaattagttt
     1561 gttttaaaaa acgtattgaa gctatcccac aaattgataa gtacttgaaa tccagcaagt
     1621 atatagcatg gcctttgcag ggctggcaag ccacgtttgg tggtggcgac catcctccaa
     1681 aaggtggagg tggtgggtcg aggggaggtt ctggtggatc aggaggttca ggtggatctg
     1741 ggtcgacgat ggaagaatct gactctgaga aaacgacgga gaaagaaaat ctggggccga
     1801 gaatggatcc accactaggg gaaccggaag gatcgcttgg gtgggtgcta ccaaatacag
     1861 ccatgaagaa aaaggtgctg ttgatgggta aaagtgggtc tggtaagacc agcatgaggt
     1921 ctattatctt tgcaaattat attgccagag acacacgtcg ccttggcgca acaattgatg
     1981 tagaacattc tcatgttcga tttctgggaa acctggtatt gaacctgtgg gattgtggtg
     2041 ggctagacac cttcatggaa aattatttca ctagccaacg ggacaacatc ttccgaaatg
     2101 tggaggttct gatttatgtc tttgatgtgg agagccgcga actggaaaag gacatgcact
     2161 attaccaatc atgcctggag gccattctgc agaattctcc agatgccaaa atattttgct
     2221 tggtacacaa aatggatctg gtacaggagg atcaacggga cctgattttt aaagagcgag
     2281 aagaagattt gaggcgtttg tctcgcccat tggaatgttc ttgtttccga acatctatct
     2341 gggatgaaac cctctataag gcttggtcca gcatagttta tcagctgatt cccaatgttc
     2401 agcagctgga aatgaaccta aggaattttg ctgaaattat cgaagctgat gaagtacttc
     2461 tttttgagag agctactttt ctggtaattt ctcactatca gtgtaaagag cagcgtgatg
     2521 cccatagatt tgagaaaata agcaacatta ttaagcagtt caagctgagc tgcagcaagc
     2581 tggctgcctc tttccagagt atggaagtca ggaactctaa cttcgctgct ttcattgaca
     2641 tctttacatc caacacttat gtgatggttg tgatgtctga tccgtccatt ccttctgcag
     2701 ctactctgat caacatccgc aatgccagga aacactttga aaagctggaa agagtggatg
     2761 gaccaaagca gtgtcttctc atgcgctaag cggccgctaa gtaagtaagg atccccagct
     2821 tggccgccat ggcccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     2881 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     2941 ctcatcaatg tatcttatca tgtctggatc gggaattaat tcggcgcagc accatggcct
     3001 gaaataacct ctgaaagagg aacttggtta ggtaccttct gaggcggaaa gaaccagctg
     3061 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3121 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     3181 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     3241 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     3301 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     3361 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tgttaacagc ttggcactgg
     3421 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     3481 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     3541 cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc
     3601 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg
     3661 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3721 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3781 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3841 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3901 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3961 tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat
     4021 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     4081 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata
     4141 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     4201 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     4261 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     4321 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     4381 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     4441 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     4501 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     4561 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     4621 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     4681 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     4741 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     4801 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     4861 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     4921 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     4981 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     5041 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     5101 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     5161 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     5221 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     5281 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     5341 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     5401 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     5461 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     5521 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     5581 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     5641 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     5701 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     5761 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     5821 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     5881 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     5941 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     6001 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     6061 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     6121 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     6181 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     6241 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
     6301 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     6361 cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc
     6421 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
     6481 acaatttcac acaggaaaca gctatgacat gattacgaat taattc
//
