LOCUS       sequence-392053-        7308 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient expression of LYCHOS (GPR155) F43I mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7308)
  AUTHORS   Shin HR, Citron YR, Wang L, Tribouillard L, Goul CS, Stipp R, 
            Sugasawa Y, Jain A, Samson N, Lim CY, Davis OB, Castaneda-Carpio D, 
            Qian M, Nomura DK, Perera RM, Park E, Covey DF, Laplante M, Evers 
            AS, Zoncu R
  TITLE     Lysosomal GPCR-like protein LYCHOS signals cholesterol sufficiency 
            to mTORC1.
  JOURNAL   Science. 2022 Sep 16;377(6612):1290-1298. doi: 
            10.1126/science.abg6621. Epub 2022 Aug 25.
  PUBMED    36007018
REFERENCE   2  (bases 1 to 7308)
  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: 
            "10.1126/science.abg6621"; journalName: "Science"; date: 
            "2022-09-16- 16"; volume: "377"; issue: "6612"; pages: "1290-1298"
COMMENT     Sequence Label: pRK5-LYCHOS (GPR155) F43I-HA
FEATURES             Location/Qualifiers
     source          1..7308
                     /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             complement(2819..2830)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             3564..3590
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      3608..3617
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3621..3755
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3658..3677)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3712..3731
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3824..4181
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3824..4015
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3847..3867)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4032..4167
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4201..4218)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4201..4217)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4210..4232)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4430..4885
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4517..4536)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4727..4748
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4902..4921)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5021..5043
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5081..5099)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5167..5271
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5272..5340,5341..6132)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGESGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5490..5509)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6303..6891
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6792..6811
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7045..7062
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7179..7200
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7215..7220,7221..7238,7239..7245)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7253..7269
                     /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     7258..7280
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7277..7293
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7277..7293
                     /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 tatcgatgcc accatgaatt ctaatttacc tgcagagaac ttaaccattg
      961 cagtcaatat gaccaagact ttgcctacag cagtaacgca tggatttaat tccactaatg
     1021 acccaccttc aatgtcaatt acaaggctta ttccagcctt actggaatgc tttggcattg
     1081 tcctttgtgg ctacatagca ggaagggcca atgtcataac atcaacccag gccaaaggac
     1141 taggaaattt tgtctccaga tttgcacttc cagctttatt attcaaaaac atggttgtac
     1201 ttaatttttc caatgtggac tggtccttcc tatatagtat cttaattgcc aaagcttctg
     1261 tatttttcat tgtatgtgta ttaaccttat tggttgccag tcctgatagt cgatttagca
     1321 aagctggact attccctatt tttgctacac aaagtaatga ctttgcattg ggatacccta
     1381 tagttgaagc tttatatcaa actacatacc cagaatatct ccagtacatt tatttggtgg
     1441 caccaatatc tcttatgatg ttaaacccta tagggtttat tttctgtgaa atccaaaagt
     1501 ggaaagacac tcaaaatgct tctcaaaata aaataaaaat tgtgggactc ggactcctgc
     1561 gtgtattaca gaacccaata gtatttatgg tcttcattgg catcgccttc aattttattc
     1621 ttgatcgaaa ggtacctgta tatgtcgaaa attttcttga tggacttgga aattcttttt
     1681 ctggatcagc cctattttat cttggtctca cgatggtggg aaaaataaag agactgaaga
     1741 agtcggcatt tgtagtacta attcttctca tcacagctaa acttctggtg ctgccacttc
     1801 tgtgcagaga aatggtggaa ctcttggaca agggcgacag tgtggtgaac catacaagtt
     1861 tatcaaatta tgcatttctg tatggtgtat ttcctgtagc accaggagtg gctatctttg
     1921 caacacaatt caacatggaa gtagaaatta taacctcagg gatggtgata agcacatttg
     1981 tgtctgctcc catcatgtac gtttctgcct ggttactgac ctttcccact atggacccta
     2041 agccattggc atatgccatc cagaatgtta gttttgatat aagtattgtc agcctgatct
     2101 ccttgatctg gtctctggct attcttcttt tgagtaagaa atataaacag cttcctcata
     2161 tgcttacaac taatttactc attgctcagt ctattgtctg tgctggaatg atgatatgga
     2221 attttgttaa agaaaaaaat tttgttggac aaattttggt gtttgttcta ttgtacagct
     2281 ccctctatag cacctacctg tggacaggcc ttctagcaat ttctttgttt cttttgaaaa
     2341 agcgagagag ggtacaaatt cctgttggaa taatcataat atctggctgg ggaattcctg
     2401 ctctccttgt tggtgttctt ttgataactg gaaaacacaa tggagatagc attgactcag
     2461 ccttctttta tggaaaagaa cagatgatca ccacagcagt caccctgttc tgcagcatcc
     2521 tgatagctgg catatccctc atgtgcatga accagactgc ccaagcagga agctatgaag
     2581 gtttcgatca gtctcagagc cacaaagtgg tggagcctgg aaatactgct tttgaggaga
     2641 gtccagcacc agtaaatgaa ccagaacttt ttacaagctc tattccagaa acaagttgct
     2701 gctcctgctc catgggaaat ggtgaattac actgcccatc aatagagcca atagcaaaca
     2761 caagcaccag tgagcctgtg attccttcgt ttgagaaaaa caatcattgt gtgagtcgct
     2821 gtaactccca gagctgcata ttagcccagg aagaagaaca gtatctacag agtggagacc
     2881 agcaactgac ccgacatgtg ttgctgtgtt tacttctcat cattggcctg ttcgctaatc
     2941 tttccagttg tttatggtgg ctattcaacc aagagcctgg aagactttat gttgagttac
     3001 agtttttctg tgccgtgttt aactttggtc agggatttat ttcctttgga atctttggat
     3061 tagataaaca tttaatcatc ctgcctttca aaagaagact tgaattccta tggaacaata
     3121 aagacacagc agaaaacagg gattctcctg tttcagagga aataaaaatg acctgtcaac
     3181 aatttatcca ttatcaccgt gacctctgta tccgaaacat tgtcaaagaa agaaggtgtg
     3241 gtgcaaagac ttctgctgga actttctgtg gctgtgacct ggtgagctgg ctaattgaag
     3301 tcggccttgc ctccgaccgt ggtgaagctg tgatatacgg agacaggctg gtacaagggg
     3361 gagtcatcca acatattacc aatgagtatg aattccggga tgagtacttg ttttacagat
     3421 ttcttcaaaa gagtcctgaa cagagtcctc ctgctattaa tgcaaacact ctccaacagg
     3481 aaagatataa agaaattgag cattcatccc caccctcaca ttcccctaag accagatcta
     3541 atggtggtgg tggtgggatg ggctacccct atgatgtgcc tgactacgca taaggatccc
     3601 cagcttggcc gccatggccc aacttgttta ttgcagctta taatggttac aaataaagca
     3661 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     3721 ccaaactcat caatgtatct tatcatgtct ggatcgggaa ttaattcggc gcagcaccat
     3781 ggcctgaaat aacctctgaa agaggaactt ggttaggtac cttctgaggc ggaaagaacc
     3841 agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3901 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     3961 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     4021 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     4081 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     4141 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctgtta acagcttggc
     4201 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     4261 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     4321 cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct
     4381 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt
     4441 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     4501 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     4561 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     4621 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga
     4681 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     4741 caaactggaa caacactcaa ccctatctcg ggctattctt ttgatttata agggattttg
     4801 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     4861 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc
     4921 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     4981 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     5041 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     5101 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     5161 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     5221 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     5281 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     5341 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     5401 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     5461 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     5521 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     5581 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     5641 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     5701 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     5761 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     5821 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     5881 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     5941 ccggctggct ggtttattgc tgataaatct ggagccggtg agagtgggtc tcgcggtatc
     6001 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     6061 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     6121 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     6181 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     6241 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6301 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6361 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6421 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     6481 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6541 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6601 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6661 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6721 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     6781 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     6841 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     6901 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     6961 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     7021 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     7081 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     7141 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     7201 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     7261 ggataacaat ttcacacagg aaacagctat gacatgatta cgaattaa
//
