LOCUS       sequence-256359-        6471 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses constitutively GTP-bound RAGB in mammalian cells in a 
            G418-selectable plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6471)
  AUTHORS   Walton ZE, Patel CH, Brooks RC, Yu Y, Ibrahim-Hashim A, Riddle M, 
            Porcu A, Jiang T, Ecker BL, Tameire F, Koumenis C, Weeraratna AT, 
            Welsh DK, Gillies R, Alwine JC, Zhang L, Powell JD, Dang CV
  TITLE     Acid Suspends the Circadian Clock in Hypoxia through Inhibition of 
            mTOR.
  JOURNAL   Cell. 2018 Jun 28;174(1):72-87.e32. doi: 10.1016/j.cell.2018.05.009.
            Epub 2018 May 31.
  PUBMED    29861175
REFERENCE   2  (bases 1 to 6471)
  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: 
            "10.1016/j.cell.2018.05.009"; journalName: "Cell"; date: 
            "2018-06-28- 28"; volume: "174"; issue: "1"; pages: "72-87.e32"
COMMENT     Sequence Label: pcDNA3.1+ Flag-RAGB99L
FEATURES             Location/Qualifiers
     source          1..6471
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(32..51)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        223..602
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        603..806
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     757..777
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     851..870
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        851..869
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             901..924
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2050..2067)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2056..2280
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2326..2754
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2413..2432)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2623..2644
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2763..2783)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2768..3097
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2948..3083
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3010..3029
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3164..3958
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3218..3237)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3828..3847
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4132..4253
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4169..4188)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4223..4242
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4302..4318)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4302..4318)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4315..4337)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4326..4342
                     /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(4350..4356,4357..4374,4375..4380))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4395..4416
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4533..4550)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4704..5292)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4784..4803)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5463..6254,6255..6323))
                     /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     6086..6105
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6324..6428)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
       61 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
      121 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
      181 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta
      241 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
      301 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      361 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      421 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      481 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
      541 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
      601 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
      661 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
      721 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
      781 ggtgggaggt ctatataagc agagctctct ggctaactag agaacccact gcttactggc
      841 ttatcgaaat taatacgact cactataggg agacccaagc tggctagcgc taccggtatg
      901 gactacaagg atgacgatga caagggtggt ggtggtggtg cgtcgacgat ggaagaatct
      961 gactctgaga aaacgacgga gaaagaaaat ctggggccga gaatggatcc accactaggg
     1021 gaaccggaag gatcgcttgg gtgggtgcta ccaaatacag ccatgaagaa aaaggtgctg
     1081 ttgatgggta aaagtgggtc tggtaagacc agcatgaggt ctattatctt tgcaaattat
     1141 attgccagag acacacgtcg ccttggcgca acaattgatg tagaacattc tcatgttcga
     1201 tttctgggaa acctggtatt gaacctgtgg gattgtggtg ggctagacac cttcatggaa
     1261 aattatttca ctagccaacg ggacaacatc ttccgaaatg tggaggttct gatttatgtc
     1321 tttgatgtgg agagccgcga actggaaaag gacatgcact attaccaatc atgcctggag
     1381 gccattctgc agaattctcc agatgccaaa atattttgct tggtacacaa aatggatctg
     1441 gtacaggagg atcaacggga cctgattttt aaagagcgag aagaagattt gaggcgtttg
     1501 tctcgcccat tggaatgttc ttgtttccga acatctatct gggatgaaac cctctataag
     1561 gcttggtcca gcatagttta tcagctgatt cccaatgttc agcagctgga aatgaaccta
     1621 aggaattttg ctgaaattat cgaagctgat gaagtacttc tttttgagag agctactttt
     1681 ctggtaattt ctcactatca gtgtaaagag cagcgtgatg cccatagatt tgagaaaata
     1741 agcaacatta ttaagcagtt caagctgagc tgcagcaagc tggctgcctc tttccagagt
     1801 atggaagtca ggaactctaa cttcgctgct ttcattgaca tctttacatc caacacttat
     1861 gtgatggttg tgatgtctga tccgtccatt ccttctgcag ctactctgat caacatccgc
     1921 aatgccagga aacactttga aaagctggaa agagtggatg gaccaaagca gtgtcttctc
     1981 atgcgctaag atatccagca cagtggcggc cgctcgagtc tagagggccc gtttaaaccc
     2041 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     2101 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     2161 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     2221 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     2281 cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg
     2341 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     2401 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     2461 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     2521 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     2581 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     2641 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     2701 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct
     2761 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     2821 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     2881 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     2941 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     3001 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta
     3061 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc
     3121 cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
     3181 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     3241 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     3301 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg
     3361 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     3421 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     3481 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     3541 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     3601 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     3661 gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt
     3721 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     3781 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     3841 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     3901 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc
     3961 gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
     4021 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     4081 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
     4141 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     4201 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     4261 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     4321 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     4381 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     4441 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4501 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4561 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4621 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4681 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4741 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4801 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4861 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     4921 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4981 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5041 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5101 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     5161 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5221 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5281 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5341 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5401 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5461 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5521 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     5581 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     5641 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     5701 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     5761 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     5821 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     5881 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     5941 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     6001 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6061 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     6121 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     6181 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     6241 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     6301 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     6361 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6421 gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg a
//
