LOCUS       sequence-376959-        7346 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV-driven expression of an N-terminally Flag-tagged MIOS (core 
            subunit of GATOR2) — native sequence for transient expression in 
            mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7346)
  AUTHORS   Valenstein ML, Rogala KB, Lalgudi PV, Brignole EJ, Gu X, Saxton RA, 
            Chantranupong L, Kolibius J, Quast JP, Sabatini DM
  TITLE     Structure of the nutrient-sensing hub GATOR2.
  JOURNAL   Nature. 2022 Jul;607(7919):610-616. doi: 10.1038/s41586-022-04939-z.
            Epub 2022 Jul 13.
  PUBMED    35831510
REFERENCE   2  (bases 1 to 7346)
  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/s41586-022-04939-z"; journalName: "Nature"; date: 
            "2022-07"; volume: "607"; issue: "7919"; pages: "610-616"
COMMENT     Sequence Label: Flag-MIOS(1-875) in pRK5
FEATURES             Location/Qualifiers
     source          1..7346
                     /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      3646..3655
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3659..3793
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3696..3715)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3750..3769
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3862..4219
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3862..4053
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3885..3905)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4070..4205
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4239..4256)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4239..4255)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4248..4270)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4468..4923
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4555..4574)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4765..4786
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4940..4959)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5059..5081
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5119..5137)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5205..5309
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5310..5378,5379..6170)
                     /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(5528..5547)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6341..6929
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6830..6849
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7083..7100
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7217..7238
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7253..7258,7259..7276,7277..7283)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7291..7307
                     /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     7296..7318
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7315..7331
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7315..7331
                     /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 gacgatgtcg ggcaccaaac cggatattct gtgggcaccg
     1021 catcatgttg accgcttcgt tgtctgtgac tcggaactgt ccctgtacca tgtcgaaagt
     1081 accgtgaaca gcgaactgaa agcaggcagc ctgcgtctgt cagaagactc ggcggccacc
     1141 ctgctgagca ttaactctga tacgccgtat atgaaatgtg tcgcgtggta tctgaactac
     1201 gatccggaat gcctgctggc cgttggccag gcaaatggtc gcgtggttct gaccagcctg
     1261 ggccaagacc acaactcaaa attcaaagat ctgatcggta aagaatttgt gccgaaacat
     1321 gcccgtcagt gtaacaccct ggcatggaat ccgctggatt ctaactggct ggcagctggc
     1381 ctggacaaac accgcgcgga tttttctgtg ctgatttggg acatctgcag taaatatacc
     1441 ccggatattg ttccgatgga aaaagtcaaa ctgtccgctg gcgaaaccga aaccacgctg
     1501 ctggttacga aaccgctgta cgaactgggt cagaatgacg cctgtctgtc actgtgctgg
     1561 ctgccgcgtg atcaaaaact gctgctggct ggtatgcacc gtaatctggc gatttttgat
     1621 ctgcgcaaca cctcgcagaa aatgttcgtg aataccaaag cggttcaagg cgtgacggtt
     1681 gacccgtatt ttcatgatcg tgtcgctagc ttctacgagg gtcaggtggc gatctgggat
     1741 ctgcgcaaat ttgaaaaacc agtgctgacc ctgacggaac aaccgaaacc gctgaccaaa
     1801 gtcgcatggt gtccgacccg tacgggcctg ctggcaaccc tgacgcgtga ctctaatatt
     1861 atccgcctgt atgatatgca gcacaccccg acgccgattg gtgacgaaac cgaaccgacg
     1921 attatcgaac gcagtgtgca gccgtgcgat aactacattg cttcgttcgc gtggcatccg
     1981 accagccaaa accgtatgat cgtcgtgacc ccgaatcgca cgatgagcga ttttaccgtt
     2041 ttcgaacgta tttcactggc ttggtcgccg atcacgagcc tgatgtgggc gtgtggtcgc
     2101 cacctgtatg aatgcaccga agaagaaaac gacaattcgc tggaaaaaga tattgccacg
     2161 aaaatgcgtc tgcgcgcact gagccgttac ggcctggaca ccgaacaagt gtggcgcaat
     2221 cacatcctgg ccggtaacga agatccgcaa ctgaaatccc tgtggtatac cctgcatttt
     2281 atgaaacagt acacggaaga tatggaccaa aaatcgccgg gcaataaagg tagcctggtt
     2341 tatgcgggca ttaaaagcat cgtgaaaagc tctctgggta tggttgaaag ttcccgtcat
     2401 aactggtctg gcctggataa acagagtgac attcaaaacc tgaatgaaga acgcatcctg
     2461 gccctgcagc tgtgtggctg gattaagaaa ggcaccgatg tggatgtggg cccgtttctg
     2521 aacagcctgg tgcaagaagg tgaatgggaa cgcgcggcgg cagttgcgct gttcaacctg
     2581 gatattcgtc gcgccattca gatcctgaat gaaggcgcat catcggaaaa aggtgatctg
     2641 aacctgaatg ttgtcgctat ggcgctgagt ggttataccg atgagaaaaa cagcctgtgg
     2701 cgtgaaatgt gtagtacgct gcgcctgcag ctgaacaatc cgtacctgtg cgtgatgttt
     2761 gctttcctga cctctgaaac gggcagttat gatggtgtcc tgtacgaaaa taaagttgcg
     2821 gtccgtgacc gcgtggcctt tgcatgtaaa ttcctgtcag atacccagct gaaccgttat
     2881 atcgaaaaac tgacgaacga aatgaaagaa gccggcaacc tggaaggtat cctgctgacc
     2941 ggcctgacga aagatggtgt tgacctgatg gaaagctatg tcgatcgcac cggcgacgtg
     3001 cagacggcat cctactgcat gctgcaaggt tcaccgctgg atgttctgaa agacgaacgt
     3061 gtccagtatt ggattgaaaa ctaccgcaat ctgctggatg cttggcgttt ttggcacaaa
     3121 cgcgcggaat ttgacatcca tcgttctaaa ctggatccga gctctaaacc gctggcccag
     3181 gtgtttgtta gttgtaactt ctgcggcaaa tcaatttcgt atagctgctc tgcagtgccg
     3241 catcagggtc gcggttttag ccaatacggc gttagtggtt ccccgaccaa atccaaagtc
     3301 acgtcatgtc cgggttgccg taaaccgctg ccgcgttgtg cactgtgcct gatcaatatg
     3361 ggtaccccgg tgtcttcctg tccgggcggt acgaaatcgg atgaaaaagt tgacctgagc
     3421 aaagataaaa aactggccca gtttaacaat tggttcacct ggtgtcacaa ctgccgtcat
     3481 ggcggtcacg caggtcatat gctgtcctgg tttcgcgatc atgctgaatg cccggtgtca
     3541 gcgtgcacct gtaaatgcat gcaactggat acgacgggta atctggtccc ggcggaaacc
     3601 gttcaaccgt aagcggccgc taagtaagta aggatcccca gcttggccgc catggcccaa
     3661 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     3721 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     3781 tcatgtctgg atcgggaatt aattcggcgc agcaccatgg cctgaaataa cctctgaaag
     3841 aggaacttgg ttaggtacct tctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt
     3901 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3961 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     4021 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     4081 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     4141 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     4201 aggcctaggc ttttgcaaaa agctgttaac agcttggcac tggccgtcgt tttacaacgt
     4261 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     4321 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     4381 ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     4441 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     4501 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     4561 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     4621 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     4681 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     4741 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     4801 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     4861 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     4921 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     4981 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     5041 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     5101 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     5161 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     5221 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     5281 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     5341 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     5401 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     5461 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     5521 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     5581 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     5641 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     5701 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     5761 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     5821 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     5881 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     5941 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     6001 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     6061 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     6121 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     6181 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     6241 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     6301 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     6361 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     6421 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     6481 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     6541 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     6601 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     6661 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     6721 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     6781 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     6841 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     6901 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     6961 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     7021 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     7081 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     7141 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     7201 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
     7261 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     7321 acagctatga catgattacg aattaa
//
