LOCUS       sequence-474950-        8639 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient expression of N-terminally FLAG-tagged human WDR59-SEC13 
            ""single chain"" in the GATOR2 complex.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8639)
  AUTHORS   Valenstein ML, Wranik M, Lalgudi PV, Linde-Garelli KY, Choi Y, 
            Chivukula RR, Sabatini DM, Rogala KB
  TITLE     Structural basis for the dynamic regulation of mTORC1 by amino 
            acids.
  JOURNAL   Nature. 2025 Aug 20. doi: 10.1038/s41586-025-09428-7.
  PUBMED    40836086
REFERENCE   2  (bases 1 to 8639)
  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"; journalName: "Nature. 
            2025 Aug 20. doi: 10.1038/s41586-025-09428-7."
COMMENT     Sequence Label: FLAG-sc-WDR59-SEC13
FEATURES             Location/Qualifiers
     source          1..8639
                     /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      4939..4948
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    4952..5086
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4989..5008)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5043..5062
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        5155..5512
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5155..5346
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5178..5198)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5363..5498
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5532..5548)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5541..5563)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5761..6216
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5848..5867)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6058..6079
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6233..6252)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6352..6374
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6412..6430)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6498..6602
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6603..6671,6672..7463)
                     /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(6821..6840)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7634..8222
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8123..8142
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8376..8393
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8510..8531
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8546..8551,8552..8569,8570..8576)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8584..8600
                     /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     8589..8611
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8608..8624
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8608..8624
                     /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 gacgatggcg gcgcgatgga gcagcgaaaa cgtggttgta
     1021 gagttccgtg actcccaggc aactgcgatg tctgtggact gtcttgggca gcatgcagtg
     1081 ctttctggcc gcagattctt atacatcgtc aatctagatg cccctttcga aggtcaccga
     1141 aagatctctc gccagagcaa atgggacatt ggagctgtgc agtggaatcc tcatgacagc
     1201 tttgcacact attttgcggc ttcgagtaac caacgagtag acctttacaa gtggaaagac
     1261 ggcagtgggg aagttggcac aaccttacaa ggccacactc gtgtcatcag cgacttggac
     1321 tgggcggtgt ttgagcctga cctcctggtt accagctctg tggacaccta catctacatt
     1381 tgggatatca aagacacaag gaaacctact gttgcactgt ctgctgttgc gggtgcctcc
     1441 caggtcaaat ggaataaaaa aaatgctaac tgccttgcca ccagccatga cggcgatgtg
     1501 cggatatggg ataagaggaa acccagtaca gcagtggaat atctagccgc ccacctctcc
     1561 aaaatccatg gcctggactg gcacccagac agcgagcaca ttcttgctac ctccagtcaa
     1621 gacaattctg tgaagttctg ggattaccgc cagcctcgga aatacctcaa tattcttcct
     1681 tgccaggtgc ctgtctggaa ggccagatac acacctttca gcaatggatt ggtgactgtg
     1741 atggttcccc agctgcggag ggaaaacagc cttctcctgt ggaatgtctt tgacttgaac
     1801 accccagtcc acaccttcgt ggggcatgat gatgtggtcc tggagttcca gtggaggaag
     1861 cagaaggaag ggtccaagga ctatcaactg gtgacgtggt cccgggatca gaccttgaga
     1921 atgtggcggg tggattccca gatgcagagg ctttgtgcaa atgacatatt agatggtgtt
     1981 gatgagttca ttgagagtat ttcccttctg ccggaacctg agaagaccct gcacactgaa
     2041 gatacagatc accagcacac tgcaagccat ggggaggaag aagccctaaa agaagatccc
     2101 cctagaaatc tcctggaaga gaggaaatca gatcaactgg ggctgcctca gaccttgcag
     2161 caggaattct ccctgatcaa tgtgcaaatc cggaatgtca atgtggagat ggatgcggca
     2221 gacaggagct gcacagtgtc tgtgcactgc agcaaccatc gtgtcaagat gctggtgaag
     2281 ttccctgcac agtacccaaa caacgccgcc ccttccttcc agtttattaa ccccacaacc
     2341 atcacatcca ccatgaaagc taagctgctg aagatcctga aggacacagc cctgcagaaa
     2401 gtgaagcgtg gccagagctg cctggagccc tgcctgcgcc agctcgtctc ctgccttgag
     2461 tcctttgtga accaggaaga cagcgcttcc agcaacccgt ttgcactccc caactctgtc
     2521 actcccccct taccgacgtt tgcgcgggtg accacggctt acgggtcgta ccaggacgcc
     2581 aacattccct ttcctaggac ttctggggcc aggttctgcg gagcaggtta cctggtatat
     2641 ttcacaaggc ccatgacaat gcatcgggcg gtgtctccca cagagcctac tccgagatct
     2701 ctctcagcct tgtctgctta tcacactggc ttgatcgcgc ccatgaagat ccgcacagag
     2761 gcccctggga accttcgttt atacagtggg agccccactc gcagcgagaa agagcaggtc
     2821 tccatcagct ccttctacta caaggagcgg aaatcaagac gatggaaaag taagcgtgag
     2881 ggatcagact ctggcaatcg acagatcaag gctgctggga aagtcatcat ccaggatggc
     2941 agcggaagtg gtatggtgtc agtaattaac actgtggata cctcccatga ggacatgatt
     3001 cacgacgccc agatggacta ctatggcacc cgcctggcaa cctgctcatc agacaggtcc
     3061 gtcaaaatct ttgatgtgcg caatggaggg cagatcctta tcgccgacct caggggtcat
     3121 gagggtcctg tgtggcaagt ggcctgggct caccccatgt acggcaacat cctggcatcg
     3181 tgctcctatg accggaaagt cattatctgg agagaggaaa acggcacctg ggagaagagc
     3241 cacgagcatg cgggacacga ctcctcagtg aactcggtgt gctgggcccc ccatgactac
     3301 ggcctgatcc tggcctgtgg gagctcggat ggggccatct ccctgctgac ttacaccggg
     3361 gaaggccaat gggaagtaaa gaagatcaac aacgctcaca ccattggctg caatgccgtc
     3421 agctgggccc ctgctgttgt acctggaagc ctcatagacc acccatcggg gcagaaaccc
     3481 aattacatca agaggtttgc atcaggtggc tgtgacaacc tcatcaagct gtggaaggag
     3541 gaggaggacg gccagtggaa ggaggagcag aagctagaag cgcacagtga ctgggttcga
     3601 gatgtggcct gggccccctc catcggcctg cccaccagca ccatcgccag ctgctcccag
     3661 gatggtcgtg tgttcatttg gacctgtgat gatgcctcaa gcaatacgtg gtcccctaaa
     3721 ttgttgcaca agttcaacga tgtggtgtgg catgtgagct ggtccatcac agccaacatc
     3781 ctggctgtct ctggtggaga caataaggtg accctgtgga aggagtcagt tgatgggcag
     3841 tgggtgtgca tcagtgatgt caacaagggc cagggctccg tatcagcatc agtgacagag
     3901 ggccagcaga acgagcaggg tagcggcagt gggattgctt gcctcctgcc tgttcacaaa
     3961 tcgctgggag agctgtacat attgaatgtg aatgatattc aggaaacatg tcagaagaat
     4021 gccgcctctg ccttgctcgt tggaagaaag gatcttgtcc aggtttggtc gctggctacg
     4081 gtagctacag atctttgcct tggtccgaaa tctgacccag atttggaaac accctgggct
     4141 cgacatccat ttgggcggca gctgctggag tccctgttgg ctcactattg ccggctccgg
     4201 gatgttcaga cactggcgat gctctgtagc gtgtttgaag cccagtctcg gcctcagggg
     4261 ctaccaaacc cctttgggcc ttttcctaac cgttcttcta atcttgtggt gtcccatagt
     4321 cgatatccta gctttacctc ttctggttcc tgctccagta tgtcagaccc agggctcaac
     4381 actggcggct ggaacatagc gggaagagag gcagagcact tgtcctcccc ttggggagaa
     4441 tcctcaccag aagagctccg ctttgggagt ctgacctaca gtgatccccg tgagcgagaa
     4501 cgcgaccagc atgataaaaa taaaaggctc ctggaccccg ccaataccca gcaatttgat
     4561 gactttaaga aatgctatgg ggaaatcctc taccgttggg gtctgagaga gaagcgagct
     4621 gaagtgttga agtttgtctc ctgtcctcct gaccctcaca aagggatcga gttcggcgtg
     4681 tactgcagcc actgccggag tgaggtccgt ggcacgcagt gtgccatctg caaaggcttc
     4741 acgttccagt gtgccatctg tcacgtggct gtgcggggat cgtccaattt ctgcctgacc
     4801 tgtgggcacg gtggccacac cagccacatg atggagtggt ttcggaccca ggaggtgtgt
     4861 cccaccgggt gtgggtgcca ctgcctgctt gaaagcactt tctgagcggc cgctaagtaa
     4921 gtaaggatcc ccagcttggc cgccatggcc caacttgttt attgcagctt ataatggtta
     4981 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     5041 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatcggga attaattcgg
     5101 cgcagcacca tggcctgaaa taacctctga aagaggaact tggttaggta ccttctgagg
     5161 cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     5221 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     5281 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     5341 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     5401 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     5461 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctgtt
     5521 aacagcttgg cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     5581 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     5641 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg
     5701 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt
     5761 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     5821 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     5881 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     5941 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
     6001 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     6061 gactcttgtt ccaaactgga acaacactca accctatctc gggctattct tttgatttat
     6121 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     6181 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
     6241 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     6301 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     6361 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     6421 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     6481 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     6541 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     6601 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     6661 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     6721 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     6781 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     6841 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     6901 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     6961 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     7021 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     7081 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     7141 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     7201 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     7261 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     7321 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     7381 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     7441 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     7501 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     7561 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     7621 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     7681 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     7741 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     7801 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     7861 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     7921 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     7981 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     8041 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     8101 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     8161 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     8221 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     8281 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     8341 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     8401 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     8461 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     8521 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     8581 gaattgtgag cggataacaa tttcacacag gaaacagcta tgacatgatt acgaattaa
//
