LOCUS       sequence-474949-        5692 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient expression of C-terminally HA-tagged human CASTOR1 (D304A 
            mutant).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5692)
  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 5692)
  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: CASTOR1-HA (D304A)
FEATURES             Location/Qualifiers
     source          1..5692
                     /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      931..940
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1945..1971
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      1992..2001
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2005..2139
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2042..2061)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2096..2115
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2208..2565
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2208..2399
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2231..2251)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2416..2551
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2585..2602)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2585..2601)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2594..2616)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2814..3269
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2901..2920)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3111..3132
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3286..3305)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3405..3427
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3465..3483)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3551..3655
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3656..3724,3725..4516)
                     /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(3874..3893)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4687..5275
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5176..5195
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5429..5446
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5563..5584
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5599..5604,5605..5622,5623..5629)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5637..5653
                     /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     5642..5664
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5661..5677
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5661..5677
                     /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 attcgtcgac gccaccatgg agctgcacat cctagaacac
      961 cgggtgcggg tgctgagcgt cgcccgtccc ggtctctggc tctacaccca cccgctcatc
     1021 aagctgctct tcctgccccg ccgcagccgg tgcaagttct tcagcctgac ggagacccct
     1081 gaggattaca cgcttatggt ggacgaggag ggctttaaag agctgccccc atctgagttc
     1141 ctgcaagtag ctgaggccac atggctggtg ctgaacgtgt cgtctcacag cggtgcggca
     1201 gtgcaggctg ctggggtcac caagatcgcc cgttcggtca tcgcgccact ggccgagcac
     1261 cacgtgtctg tgctgatgct gtccacttac cagacggact tcatcctggt gcgggagcag
     1321 gacctgtccg tggtgatcca cacgctggcc caggagttcg acatttaccg cgaggtgggc
     1381 ggagagcctg tgcctgtgac gagggatgat tccagcaatg gctttccccg cactcagcat
     1441 gggcccagcc ccacggtgca tcccatccag agcccacaga accgcttctg tgtcctcaca
     1501 ctggaccctg agacgcttcc agccatcgcc accaccctca tagatgtcct cttctactcg
     1561 cacagcaccc ccaaggaggc agcctctagc agtcctgaac ccagctccat cacgttcttt
     1621 gccttctctc tcatcgaggg ttatatctcc attgtcatgg atgctgaaac acagaaaaag
     1681 ttccccagtg acctcctgct gaccagctcc tcgggggagc tgtggaggat ggtgcgcatc
     1741 ggtggacagc ccctgggctt tgatgaatgt ggcatcgtgg cacagattgc aggtcccctg
     1801 gctgccgctg acatctctgc ctactacatc agcaccttca acttcgccca cgccctggtg
     1861 cccgaggacg gtatcggcag cgtcatcgag gtcctccagc ggcggcagga aggcctggct
     1921 tccgcggccg caggaggtgg aggttacccc tatgatgtgc ctgactacgc atgataagga
     1981 tccccagctt ggccgccatg gcccaacttg tttattgcag cttataatgg ttacaaataa
     2041 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     2101 ttgtccaaac tcatcaatgt atcttatcat gtctggatcg ggaattaatt cggcgcagca
     2161 ccatggcctg aaataacctc tgaaagagga acttggttag gtaccttctg aggcggaaag
     2221 aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     2281 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     2341 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     2401 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     2461 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     2521 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct gttaacagct
     2581 tggcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     2641 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     2701 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc
     2761 tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc
     2821 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2881 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2941 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     3001 acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc
     3061 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     3121 gttccaaact ggaacaacac tcaaccctat ctcgggctat tcttttgatt tataagggat
     3181 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     3241 ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga
     3301 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     3361 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     3421 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     3481 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     3541 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     3601 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     3661 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     3721 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     3781 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     3841 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     3901 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     3961 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     4021 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     4081 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     4141 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     4201 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     4261 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     4321 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     4381 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     4441 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     4501 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     4561 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     4621 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     4681 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     4741 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     4801 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     4861 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     4921 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     4981 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     5041 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     5101 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     5161 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     5221 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     5281 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     5341 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     5401 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     5461 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     5521 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
     5581 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     5641 gagcggataa caatttcaca caggaaacag ctatgacatg attacgaatt aa
//
