LOCUS       sequence-392048-        7455 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient expression of LYCHOS (GPR155) Y551A mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7455)
  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 7455)
  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) Y551A-HA
FEATURES             Location/Qualifiers
     source          1..7455
                     /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             3576..3602
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(3615..3704)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     regulatory      3755..3764
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3768..3902
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3805..3824)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3859..3878
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3971..4328
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3971..4162
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3994..4014)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4179..4314
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4348..4365)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4348..4364)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4357..4379)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4577..5032
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4664..4683)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4874..4895
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5049..5068)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5168..5190
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5228..5246)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5314..5418
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5419..5487,5488..6279)
                     /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(5637..5656)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6450..7038
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6939..6958
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7192..7209
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7326..7347
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7362..7367,7368..7385,7386..7392)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7400..7416
                     /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     7405..7427
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7424..7440
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7424..7440
                     /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 tgcagagaat ctgactatcg
      961 ccgtaaacat gaccaagact ttgcctacag cagtaacgca tggatttaat tccactaatg
     1021 acccaccttc aatgtcaatt acaaggcttt 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 agcgctgaag
     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 accacgcgtc
     3541 ccgggagatc taatggtggt ggtggtggga tgggctaccc ctatgatgtg cctgactacg
     3601 cataaggatc cttatgcata atctgggaca tcgtatgggt aggagccagc gtaatctgga
     3661 acatcatagg gataacctgc atagtccggg acgtcatagg gataagaacc tccaccagat
     3721 cctccgcctg accctcctcc agatccccag cttggccgcc atggcccaac ttgtttattg
     3781 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     3841 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga
     3901 tcgggaatta attcggcgca gcaccatggc ctgaaataac ctctgaaaga ggaacttggt
     3961 taggtacctt ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa
     4021 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     4081 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     4141 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     4201 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     4261 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     4321 tttgcaaaaa gctgttaaca gcttggcact ggccgtcgtt ttacaacgtc gtgactggga
     4381 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     4441 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     4501 atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg
     4561 tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     4621 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     4681 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     4741 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat
     4801 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     4861 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcgggc
     4921 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     4981 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc
     5041 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     5101 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     5161 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     5221 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     5281 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     5341 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     5401 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     5461 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     5521 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     5581 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     5641 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     5701 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     5761 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     5821 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     5881 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     5941 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     6001 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     6061 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     6121 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     6181 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     6241 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     6301 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     6361 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     6421 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     6481 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     6541 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     6601 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     6661 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     6721 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     6781 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     6841 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     6901 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     6961 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     7021 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     7081 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     7141 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     7201 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     7261 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     7321 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
     7381 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
     7441 atgattacga attaa
//
