LOCUS       sequence-194374-        5707 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Mammalian expression of HA-RagA(Q66L).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5707)
  AUTHORS   Shen K, Choe A, Sabatini DM
  TITLE     Intersubunit Crosstalk in the Rag GTPase Heterodimer Enables mTORC1 
            to Respond Rapidly to Amino Acid Availability.
  JOURNAL   Mol Cell. 2017 Oct 17. pii: S1097-2765(17)30704-9. doi: 
            10.1016/j.molcel.2017.09.026.
  PUBMED    29056322
REFERENCE   2  (bases 1 to 5707)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Cell. 
            2017 Oct 17. pii: S1097-2765(17)30704-9. doi: 
            10.1016/j.molcel.2017.09.026."
COMMENT     Sequence Label: pRK5 HA-RagA(Q66L)
FEATURES             Location/Qualifiers
     source          1..5707
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     581..605
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        815..833
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     815..832
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     846..865
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      950..959
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             962..988
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      1994..2003
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2007..2141
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2044..2063)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2098..2117
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2210..2567
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2210..2401
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2233..2253)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2418..2553
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2587..2604)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2587..2603)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2596..2618)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2816..3271
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2903..2922)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3113..3134
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3288..3307)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3407..3429
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3467..3485)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3553..3657
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3658..3726,3727..4518)
                     /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(3876..3895)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4689..5277
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5178..5197
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5431..5448
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5565..5586
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5601..5606,5607..5624,5625..5631)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5639..5655
                     /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     5644..5666
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5663..5679
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5663..5679
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc
      601 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
      661 ccagcctccg cggccgggaa cggtgcattg gaacgcggat tccccgtgcc aagagtgacg
      721 taagtaccgc ctatagagtc tataggccca cccccttggc ttcgttagaa cgcggctaca
      781 attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat agaataacat
      841 ccactttgcc tttctctcca caggtgtcca ctcccaggtc caactgcacc tcggttctat
      901 cgattgaatt cccgaaccga cagtcggtct cttcaccaag gccattcgcg ccaccatggg
      961 ctacccctat gatgtgcctg actacgcaga tctcaatggt ggtggtggtg ggtcgacgat
     1021 gccaaataca gccatgaaga aaaaggtgct gctgatgggg aagagcgggt cggggaagac
     1081 cagcatgagg tcgataatct tcgccaatta cattgctcgc gacacccggc gcctgggggc
     1141 caccattgac gtggaacact cccacgtccg attcctaggg aacctggtgc tgaacctgtg
     1201 ggactgtggc ggtctggaca ccttcatgga aaattacttc accagccagc gagacaatat
     1261 cttccgtaac gtggaagttt tgatttacgt gtttgacgtg gagagccgcg aactggaaaa
     1321 ggacatgcat tattaccagt cgtgtctgga ggccatcctc cagaactctc ctgacgccaa
     1381 aatcttctgc ctggtgcaca aaatggatct ggttcaggag gatcagcgtg acctgatttt
     1441 taaagagcga gaggaagacc tgaggcgtct gtctcgcccg ctggagtgtg cttgttttcg
     1501 aacgtccatc tgggatgaga cgctctacaa agcctggtcc agcatcgtct accagctgat
     1561 tcccaacgtt cagcagctgg agatgaacct caggaatttt gcccaaatca ttgaggccga
     1621 tgaagttctg ctgttcgaaa gagctacatt cttggttatt tcccactacc agtgcaaaga
     1681 gcagcgcgac gtccaccggt ttgagaagat cagcaacatc atcaaacagt tcaagctgag
     1741 ctgcagtaaa ttggccgctt ccttccagag catggaagtt aggaattcca acttcgctgc
     1801 tttcatcgac atcttcacct caaatacgta cgtgatggtg gtcatgtcag atccgtcgat
     1861 cccttctgcg gccactctga tcaacattcg caatgcccgg aaacactttg agaagctgga
     1921 gagagtggat ggccccaagc acagtctcct tatgcgttga gcggccgcta agtaagtaag
     1981 gatccccagc ttggccgcca tggcccaact tgtttattgc agcttataat ggttacaaat
     2041 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     2101 gtttgtccaa actcatcaat gtatcttatc atgtctggat cgggaattaa ttcggcgcag
     2161 caccatggcc tgaaataacc tctgaaagag gaacttggtt aggtaccttc tgaggcggaa
     2221 agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     2281 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     2341 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     2401 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     2461 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     2521 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctgttaacag
     2581 cttggcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     2641 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     2701 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt
     2761 tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg
     2821 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     2881 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     2941 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3001 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg
     3061 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     3121 ttgttccaaa ctggaacaac actcaaccct atctcgggct attcttttga tttataaggg
     3181 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     3241 aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct
     3301 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     3361 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     3421 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     3481 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     3541 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     3601 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     3661 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     3721 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     3781 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     3841 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     3901 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     3961 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     4021 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     4081 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     4141 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     4201 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     4261 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     4321 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     4381 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     4441 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     4501 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     4561 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     4621 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     4681 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     4741 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4801 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     4861 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4921 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4981 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     5041 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     5101 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     5161 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     5221 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     5281 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     5341 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     5401 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     5461 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     5521 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
     5581 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
     5641 gtgagcggat aacaatttca cacaggaaac agctatgaca tgattacgaa ttaattcgag
     5701 ctcgccc
//
