LOCUS       sequence-201276-        5842 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses tagged 4EBP1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5842)
  AUTHORS   Grabiner BC, Nardi V, Birsoy K, Possemato R, Shen K, Sinha S, Jordan
            A, Beck AH, Sabatini DM
  TITLE     A diverse array of cancer-associated MTOR mutations are 
            hyperactivating and can predict rapamycin sensitivity.
  JOURNAL   Cancer Discov. 2014 May;4(5):554-63. doi: 
            10.1158/2159-8290.CD-13-0929. Epub 2014 Mar 14.
  PUBMED    24631838
REFERENCE   2  (bases 1 to 5842)
  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"; doi: 
            "10.1158/2159-8290.CD-13-0929"; journalName: "Cancer Discov"; date: 
            "2014-05"; volume: "4"; issue: "5"; pages: "554-63"
COMMENT     Sequence Label: pRK5-HA-GST-4EBP1-Low GC
FEATURES             Location/Qualifiers
     source          1..5842
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      12..147
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        164..355
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     312..332
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    424..558
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(448..467)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     502..521
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     regulatory      562..571
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(1028..1681)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     complement(1048..1070)
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             complement(1712..1738)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      1741..1750
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1835..1854)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        complement(1867..1885)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1868..1885)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2095..2119)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(2116..2319)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2145..2165)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2320..2699
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(2728..2744)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2728..2744)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2741..2763)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2752..2768
                     /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)."
     promoter        complement(join(2776..2782,2783..2800,2801..2806))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2821..2842
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2959..2976)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3130..3718)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3210..3229)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3889..4680,4681..4749))
                     /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     4512..4531
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4750..4854)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4922..4940
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4978..5000)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5100..5119
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(5136..5591)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5273..5294)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5485..5504
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5789..5811
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5803..5820
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5804..5820
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(join(5840..5842,1..355))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
ORIGIN
        1 gcaaaagcct aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggccga
       61 ggcggcctcg gcctctgcat aaataaaaaa aattagtcag ccatggggcg gagaatgggc
      121 ggaactgggc ggagttaggg gcgggatggg cggagttagg ggcgggacta tggttgctga
      181 ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca
      241 cctggttgct gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg
      301 ggactttcca caccctaact gacacacatt ccacagctgg ttctttccgc ctcagaaggt
      361 acctaaccaa gttcctcttt cagaggttat ttcaggccat ggtgctgcgc cgaattaatt
      421 cccgatccag acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag
      481 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata
      541 agctgcaata aacaagttgg gccatggcgg ccaagctggg gatccttact tacttagcgg
      601 ccgcttaaat atccatttca aactgagatt cttcaccacc tgcacgttta tcttctggag
      661 agttacgtaa atgagactga gatgcttcca ttggtggttc atcagaagat ggagaagtaa
      721 cacctggaat agttggtaaa tcacgtggtg gagttttagt aactggagag ttacgacatt
      781 ccattaaaaa tttacgatcg tagatgatac gagtaccacc tggagtagta gaaaataaag
      841 taccacctgg agtagtagaa taatcacctg gtggtaactg aacaccatca cctaaaacaa
      901 cacgacgagt tgctggaatt gcacgagatg gagtctgaga acaagaagaa ccaccagaca
      961 tcgtcgaccc agatccacct gaacctcctg atccaccaga acctcccctc gacccaccac
     1021 ctccaccttt tggaggatgg tcgccaccac caaacgtggc ttgccagccc tgcaaaggcc
     1081 atgctatata cttgctggat ttcaagtact tatcaatttg tgggatagct tcaatacgtt
     1141 ttttaaaaca aactaatttt gggaacgcat ccaggcacat tgggtccatg tataaaacaa
     1201 catcaagagc gtcatacaac atgaagtcag gatgggttac atgatcacca tttaaatatg
     1261 ttttatgaca taaacgatct tcgaacattt tcagcatttc aggtagcttg ctaagaaaat
     1321 caactttgag agtttcaaag tctttactat atgcaattct cgaaacaccg tatctaatat
     1381 ccaaaaccgc tccttcaagc attgaaatct ctgcacgctc ttttggacaa ccacccaaca
     1441 tgttgtgctt gtcagctata taacgtatga tggccataga ctgtgttaat ttaacatcac
     1501 catcaatata ataaggaaga ttgggaaact ccaaacccaa ttcaaacttt ttgtttcgcc
     1561 atttatcacc ttcatcgcgc tcatacaaat gctcttcata tttttcttca agatattcca
     1621 aaagaagtcg agtgggttgc acaaggccct taattttcca ataacctagt ataggggaca
     1681 ttctcgaccc accaccacca ccattgagat ctgcgtagtc aggcacatca taggggtagc
     1741 ccatggtggc gcgaatggcc ttggtgaaga gaccgactgt cggttcggga attcaatcga
     1801 tagaaccgag gtgcagttgg acctgggagt ggacacctgt ggagagaaag gcaaagtgga
     1861 tgttattcta tagtgtcacc taaatcgtat gtgtatgata cataaggtta tgtattaatt
     1921 gtagccgcgt tctaacgaag ccaagggggt gggcctatag actctatagg cggtacttac
     1981 gtcactcttg gcacggggaa tccgcgttcc aatgcaccgt tcccggccgc ggaggctgga
     2041 tcggtcccgg tgtcttctat ggaggtcaaa acagcgtgga tggcgtctcc aggcgatctg
     2101 acggttcact aaacgagctc tgcttatata gacctcccac cgtacacgcc taccgcccat
     2161 ttgcgtcaat ggggcggagt tgttacgaca ttttggaaag tcccgttgat tttggtgcca
     2221 aaacaaactc ccattgacgt caatggggtg gagacttgga aatccccgtg agtcaaaccg
     2281 ctatccacgc ccattgatgt actgccaaaa ccgcatcacc atggtaatag cgatgactaa
     2341 tacgtagatg tactgccaag taggaaagtc ccataaggtc atgtactggg cataatgcca
     2401 ggcgggccat ttaccgtcat tgacgtcaat agggggcgta cttggcatat gatacacttg
     2461 atgtactgcc aagtgggcag tttaccgtaa atactccacc cattgacgtc aatggaaagt
     2521 ccctattggc gttactatgg gaacatacgt cattattgac gtcaatgggc gggggtcgtt
     2581 gggcggtcag ccaggcgggc catttaccgt aagttatgta acgcggaact ccatatatgg
     2641 gctatgaact aatgaccccg taattgatta ctattaataa ctagtcaata atcaatgtcg
     2701 ggcgagctcg aattaattcg taatcatgtc atagctgttt cctgtgtgaa attgttatcc
     2761 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     2821 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     2881 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     2941 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     3001 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     3061 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     3121 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     3181 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     3241 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     3301 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     3361 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     3421 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     3481 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     3541 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     3601 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     3661 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     3721 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     3781 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     3841 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     3901 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     3961 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     4021 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     4081 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     4141 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     4201 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     4261 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     4321 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     4381 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     4441 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     4501 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     4561 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     4621 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     4681 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     4741 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     4801 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     4861 tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa
     4921 aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct
     4981 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     5041 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc
     5101 ggcatcagag cagattgtac tgagagtgca ccataaaatt gtaaacgtta atattttgtt
     5161 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     5221 caaaatccct tataaatcaa aagaatagcc cgagataggg ttgagtgttg ttccagtttg
     5281 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     5341 tcagggcgat ggcccactac gtgaaccatc acccaaatca agttttttgg ggtcgaggtg
     5401 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     5461 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     5521 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     5581 acagggcgcg tactatggtt gctttgacgt atgcggtgtg aaataccgca cagatgcgta
     5641 aggagaaaat accgcatcag gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg
     5701 cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg
     5761 cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt
     5821 gccaagctgt taacagcttt tt
//
