LOCUS       sequence-224990-        5852 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express GFP-RagB (C terminal domain only).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5852)
  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 5852)
  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  GFP RagB (CTD)
FEATURES             Location/Qualifiers
     source          1..5852
                     /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"
     regulatory      925..934
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(973..975,976..1686)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(973..975,976..1686)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1015..1036)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1276..1295)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1623..1644
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2152..2161
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2165..2299
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2202..2221)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2256..2275
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2368..2725
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2368..2559
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2391..2411)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2576..2711
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2745..2762)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2745..2761)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2754..2776)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2974..3429
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3061..3080)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3271..3292
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3446..3465)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3565..3587
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3625..3643)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3711..3815
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3816..3884,3885..4676)
                     /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(4034..4053)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4847..5435
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5336..5355
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5589..5606
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5723..5744
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5759..5764,5765..5782,5783..5789)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5797..5813
                     /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     5802..5824
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5821..5837
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5821..5837
                     /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 attcgccacc atggactaca aggatgccgc tgccaagggc
      961 ggaggtggat ctgtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     1021 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     1081 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     1141 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
     1201 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     1261 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     1321 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     1381 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     1441 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     1501 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     1561 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
     1621 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     1681 tacaagggag gcggtggatc gggcgggggt ggatcagtcg acaatgttca gcagctggaa
     1741 atgaacctaa ggaattttgc tgaaattatc gaagctgatg aagtacttct ttttgagaga
     1801 gctacttttc tggtaatttc tcactatcag tgtaaagagc agcgtgatgc ccatagattt
     1861 gagaaaataa gcaacattat taagcagttc aagctgagct gcagcaagct ggctgcctct
     1921 ttccagagta tggaagtcag gaactctaac ttcgctgctt tcattgacat ctttacatcc
     1981 aacacttatg tgatggttgt gatgtctgat ccgtccattc cttctgcagc tactctgatc
     2041 aacatccgca atgccaggaa acactttgaa aagctggaaa gagtggatgg accaaagcag
     2101 tgtcttctca tgcgcttagc ggccgctaag taagtaagga tccccagctt ggccgccatg
     2161 gcccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     2221 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     2281 atcttatcat gtctggatcg ggaattaatt cggcgcagca ccatggcctg aaataacctc
     2341 tgaaagagga acttggttag gtaccttctg aggcggaaag aaccagctgt ggaatgtgtg
     2401 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     2461 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     2521 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     2581 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     2641 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     2701 ttttggaggc ctaggctttt gcaaaaagct gttaacagct tggcactggc cgtcgtttta
     2761 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     2821 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     2881 cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     2941 atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg
     3001 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     3061 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     3121 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     3181 aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     3241 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     3301 tcaaccctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     3361 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     3421 ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     3481 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     3541 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     3601 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     3661 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     3721 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     3781 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     3841 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     3901 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     3961 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     4021 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     4081 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     4141 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     4201 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     4261 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     4321 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     4381 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     4441 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     4501 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     4561 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     4621 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     4681 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     4741 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     4801 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4861 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4921 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4981 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     5041 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     5101 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     5161 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     5221 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     5281 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     5341 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     5401 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     5461 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     5521 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     5581 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     5641 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     5701 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
     5761 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
     5821 caggaaacag ctatgacatg attacgaatt aa
//
