LOCUS       sequence-474948-        6208 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient expression of N-terminally HA-tagged human Sestrin2 
            (E451Q).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6208)
  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 6208)
  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: HA-Sestrin2 (E451Q)
FEATURES             Location/Qualifiers
     source          1..6208
                     /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      963..972
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             975..1001
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      2508..2517
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2521..2655
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2558..2577)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2612..2631
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2724..3081
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2724..2915
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2747..2767)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2932..3067
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3101..3118)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3101..3117)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3110..3132)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3330..3785
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3417..3436)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3627..3648
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3802..3821)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3921..3943
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3981..3999)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4067..4171
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4172..4240,4241..5032)
                     /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(4390..4409)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5203..5791
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5692..5711
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5945..5962
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6079..6100
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6115..6120,6121..6138,6139..6145)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6153..6169
                     /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     6158..6180
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6177..6193
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6177..6193
                     /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 attcccgaac cgacagtcgg tctcttcacc aaggccattc
      961 gcgccaccat gggctacccc tatgatgtgc ctgactacgc agatctcaat ggtggtggtg
     1021 gtgggtcgac gatgatcgtg gcggactccg agtgccgcgc agagctcaag gactacctgc
     1081 ggttcgcccc gggcggcgtc ggcgactcgg gccccggaga ggagcagagg gagagccggg
     1141 ctcggcgagg ccctcgaggg cccagcgcct tcatccccgt ggaggaggtc cttcgggagg
     1201 gggctgagag cctcgagcag cacctggggc tggaggcact gatgtcctct gggcgagtag
     1261 acaacctggc agtggtgatg ggcctgcacc ctgactactt taccagcttc tggcgcctgc
     1321 actacctgct gctgcacacg gatggtccct tggccagctc ctggcgccac tacattgcca
     1381 tcatggctgc cgcccgccat cagtgttctt acctggtagg ctcccacatg gccgagtttc
     1441 tgcagactgg tggtgaccct gagtggctgc tgggcctcca ccgggccccc gagaagctgc
     1501 gcaaactcag cgagatcaac aagttgctgg cgcatcggcc atggctcatc accaaggaac
     1561 acatccaggc cttgctgaag accggcgagc acacttggtc cctggccgag ctcattcagg
     1621 ctctggtcct gctcacccac tgccactcgc tctcctcctt cgtgtttggc tgtggcatcc
     1681 tccctgaggg ggatgcagat ggcagccctg ccccccaggc acctacaccc cctagtgaac
     1741 agagcagccc cccaagcagg gacccgttga acaactctgg gggctttgag tctgcccgcg
     1801 acgtggaggc gctgatggag cgcatgcagc agctgcagga gagcctgctg cgggatgagg
     1861 ggacgtccca ggaggagatg gagagccgct ttgagctgga gaagtcagag agcctgctgg
     1921 tgaccccctc agctgacatc ctggagccct ctccacaccc agacatgctg tgctttgtgg
     1981 aagaccctac tttcggatat gaggacttca ctcggagagg ggctcaggca ccccctacct
     2041 tccgggccca ggattatacc tgggaagacc atggctactc gctgatccag cggctttacc
     2101 ctgagggtgg gcagctgctg gatgagaagt tccaggcagc ctatagcctc acctacaata
     2161 ccatcgccat gcacagtggt gtggacacct ccgtgctccg cagggccatc tggaactata
     2221 tccactgcgt ctttggcatc agatatgatg actatgatta tggggaggtg aaccagctcc
     2281 tggagcggaa cctcaaggtc tatatcaaga cagtggcctg ctacccagag aagaccaccc
     2341 gaagaatgta caacctcttc tggaggcact tccgccactc acagaaggtc cacgtgaact
     2401 tgctgctcct ggaggcgcgc atgcaagccg ctctgctgta cgccctccgt gccatcaccc
     2461 gctacatgac ctgagcggcc gctaagtaag taaggatccc cagcttggcc gccatggccc
     2521 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     2581 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     2641 tatcatgtct ggatcgggaa ttaattcggc gcagcaccat ggcctgaaat aacctctgaa
     2701 agaggaactt ggttaggtac cttctgaggc ggaaagaacc agctgtggaa tgtgtgtcag
     2761 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2821 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     2881 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     2941 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     3001 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     3061 ggaggcctag gcttttgcaa aaagctgtta acagcttggc actggccgtc gttttacaac
     3121 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     3181 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     3241 gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     3301 cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc
     3361 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     3421 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3481 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3541 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3601 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     3661 ccctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     3721 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
     3781 aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     3841 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     3901 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     3961 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     4021 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     4081 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     4141 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     4201 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     4261 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     4321 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     4381 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     4441 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     4501 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     4561 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     4621 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     4681 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     4741 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     4801 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     4861 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     4921 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     4981 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     5041 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     5101 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     5161 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     5221 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     5281 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     5341 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     5401 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     5461 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     5521 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     5581 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     5641 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     5701 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     5761 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     5821 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     5881 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     5941 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
     6001 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     6061 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
     6121 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
     6181 aaacagctat gacatgatta cgaattaa
//
