LOCUS       sequence-225014-        5759 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express Flag-RagB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5759)
  AUTHORS   Lawrence RE, Cho KF, Rappold R, Thrun A, Tofaute M, Kim DJ, 
            Moldavski O, Hurley JH, Zoncu R
  TITLE     A nutrient-induced affinity switch controls mTORC1 activation by its
            Rag GTPase-Ragulator lysosomal scaffold.
  JOURNAL   Nat Cell Biol. 2018 Sep;20(9):1052-1063. doi: 
            10.1038/s41556-018-0148-6. Epub 2018 Jul 30.
  PUBMED    30061680
REFERENCE   2  (bases 1 to 5759)
  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: 
            "10.1038/s41556-018-0148-6"; journalName: "Nat Cell Biol"; date: 
            "2018-09"; volume: "20"; issue: "9"; pages: "1052-1063"
COMMENT     Sequence Label: pRK5 Flag RagB
FEATURES             Location/Qualifiers
     source          1..5759
                     /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"
     CDS             937..960
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      2059..2068
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2072..2206
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2109..2128)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2163..2182
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2275..2632
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2275..2466
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2298..2318)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2483..2618
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2652..2669)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2652..2668)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2661..2683)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2881..3336
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2968..2987)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3178..3199
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3353..3372)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3472..3494
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3532..3550)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3618..3722
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3723..3791,3792..4583)
                     /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(3941..3960)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4754..5342
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5243..5262
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5496..5513
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5630..5651
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5666..5671,5672..5689,5690..5696)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5704..5720
                     /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     5709..5731
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5728..5744
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5728..5744
                     /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 attcaccatg gaaatggact acaaggatga cgatgacaag
      961 ggtggtggtg gtggtgcgtc gacgatggaa gaatctgact ctgagaaaac gacggagaaa
     1021 gaaaatctgg ggccgagaat ggatccacca ctaggggaac cggaaggatc gcttgggtgg
     1081 gtgctaccaa atacagccat gaagaaaaag gtgctgttga tgggtaaaag tgggtctggt
     1141 aagaccagca tgaggtctat tatctttgca aattatattg ccagagacac acgtcgcctt
     1201 ggcgcaacaa ttgatgtaga acattctcat gttcgatttc tgggaaacct ggtattgaac
     1261 ctgtgggatt gtggtgggca agacaccttc atggaaaatt atttcactag ccaacgggac
     1321 aacatcttcc gaaatgtgga ggttctgatt tatgtctttg atgtggagag ccgcgaactg
     1381 gaaaaggaca tgcactatta ccaatcatgc ctggaggcca ttctgcagaa ttctccagat
     1441 gccaaaatat tttgcttggt acacaaaatg gatctggtac aggaggatca acgggacctg
     1501 atttttaaag agcgagaaga agatttgagg cgtttgtctc gcccattgga atgttcttgt
     1561 ttccgaacat ctatctggga tgaaaccctc tataaggctt ggtccagcat agtttatcag
     1621 ctgattccca atgttcagca gctggaaatg aacctaagga attttgctga aattatcgaa
     1681 gctgatgaag tacttctttt tgagagagct acttttctgg taatttctca ctatcagtgt
     1741 aaagagcagc gtgatgccca tagatttgag aaaataagca acattattaa gcagttcaag
     1801 ctgagctgca gcaagctggc tgcctctttc cagagtatgg aagtcaggaa ctctaacttc
     1861 gctgctttca ttgacatctt tacatccaac acttatgtga tggttgtgat gtctgatccg
     1921 tccattcctt ctgcagctac tctgatcaac atccgcaatg ccaggaaaca ctttgaaaag
     1981 ctggaaagag tggatggacc aaagcagtgt cttctcatgc gctaagcggc cgctaagtaa
     2041 gtaaggatcc ccagcttggc cgccatggcc caacttgttt attgcagctt ataatggtta
     2101 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     2161 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatcggga attaattcgg
     2221 cgcagcacca tggcctgaaa taacctctga aagaggaact tggttaggta ccttctgagg
     2281 cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     2341 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     2401 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     2461 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     2521 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     2581 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctgtt
     2641 aacagcttgg cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     2701 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     2761 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg
     2821 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt
     2881 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     2941 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     3001 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     3061 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
     3121 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     3181 gactcttgtt ccaaactgga acaacactca accctatctc gggctattct tttgatttat
     3241 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     3301 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
     3361 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     3421 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     3481 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     3541 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     3601 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     3661 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     3721 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     3781 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     3841 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     3901 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     3961 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     4021 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     4081 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     4141 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     4201 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     4261 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     4321 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     4381 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     4441 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     4501 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     4561 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     4621 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     4681 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     4741 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     4801 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4861 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     4921 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4981 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     5041 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     5101 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     5161 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     5221 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     5281 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     5341 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     5401 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     5461 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     5521 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     5581 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     5641 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     5701 gaattgtgag cggataacaa tttcacacag gaaacagcta tgacatgatt acgaattaa
//
