LOCUS       sequence-392098-        7308 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Transient expression of LYCHOS (GPR155) P44A 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 Apr 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) P44A-HA
FEATURES             Location/Qualifiers
     source          1..7308
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        2..381
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        382..585
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     536..556
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     582..606
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        816..834
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     816..833
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     847..866
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             complement(2807..2818)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             3552..3578
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      3596..3605
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3609..3743
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3646..3665)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3700..3719
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3812..4169
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3812..4003
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3835..3855)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4020..4155
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4189..4206)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4189..4205)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4198..4220)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4418..4873
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4505..4524)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4715..4736
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4890..4909)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5009..5031
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5069..5087)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5155..5259
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5260..5328,5329..6120)
                     /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(5478..5497)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6291..6879
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6780..6799
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7033..7050
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7167..7188
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7203..7208,7209..7226,7227..7233)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7241..7257
                     /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     7246..7268
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7265..7281
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7265..7281
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 cgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
       61 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
      121 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
      181 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
      241 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
      301 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
      361 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
      421 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
      481 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
      541 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt
      601 cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga
      661 tccagcctcc gcggccggga acggtgcatt ggaacgcgga ttccccgtgc caagagtgac
      721 gtaagtaccg cctatagagt ctataggccc acccccttgg cttcgttaga acgcggctac
      781 aattaataca taaccttatg tatcatacac atacgattta ggtgacacta tagaataaca
      841 tccactttgc ctttctctcc acaggtgtcc actcccaggt ccaactgcac ctcggttcta
      901 tcgatgccac catgaattct aatttacctg cagagaactt aaccattgca gtcaatatga
      961 ccaagacttt gcctacagca gtaacgcatg gatttaattc cactaatgac ccaccttcaa
     1021 tgtcaattac aaggcttttt gcagccttac tggaatgctt tggcattgtc ctttgtggct
     1081 acatagcagg aagggccaat gtcataacat caacccaggc caaaggacta ggaaattttg
     1141 tctccagatt tgcacttcca gctttattat tcaaaaacat ggttgtactt aatttttcca
     1201 atgtggactg gtccttccta tatagtatct taattgccaa agcttctgta tttttcattg
     1261 tatgtgtatt aaccttattg gttgccagtc ctgatagtcg atttagcaaa gctggactat
     1321 tccctatttt tgctacacaa agtaatgact ttgcattggg ataccctata gttgaagctt
     1381 tatatcaaac tacataccca gaatatctcc agtacattta tttggtggca ccaatatctc
     1441 ttatgatgtt aaaccctata gggtttattt tctgtgaaat ccaaaagtgg aaagacactc
     1501 aaaatgcttc tcaaaataaa ataaaaattg tgggactcgg actcctgcgt gtattacaga
     1561 acccaatagt atttatggtc ttcattggca tcgccttcaa ttttattctt gatcgaaagg
     1621 tacctgtata tgtcgaaaat tttcttgatg gacttggaaa ttctttttct ggatcagccc
     1681 tattttatct tggtctcacg atggtgggaa aaataaagag actgaagaag tcggcatttg
     1741 tagtactaat tcttctcatc acagctaaac ttctggtgct gccacttctg tgcagagaaa
     1801 tggtggaact cttggacaag ggcgacagtg tggtgaacca tacaagttta tcaaattatg
     1861 catttctgta tggtgtattt cctgtagcac caggagtggc tatctttgca acacaattca
     1921 acatggaagt agaaattata acctcaggga tggtgataag cacatttgtg tctgctccca
     1981 tcatgtacgt ttctgcctgg ttactgacct ttcccactat ggaccctaag ccattggcat
     2041 atgccatcca gaatgttagt tttgatataa gtattgtcag cctgatctcc ttgatctggt
     2101 ctctggctat tcttcttttg agtaagaaat ataaacagct tcctcatatg cttacaacta
     2161 atttactcat tgctcagtct attgtctgtg ctggaatgat gatatggaat tttgttaaag
     2221 aaaaaaattt tgttggacaa attttggtgt ttgttctatt gtacagctcc ctctatagca
     2281 cctacctgtg gacaggcctt ctagcaattt ctttgtttct tttgaaaaag cgagagaggg
     2341 tacaaattcc tgttggaata atcataatat ctggctgggg aattcctgct ctccttgttg
     2401 gtgttctttt gataactgga aaacacaatg gagatagcat tgactcagcc ttcttttatg
     2461 gaaaagaaca gatgatcacc acagcagtca ccctgttctg cagcatcctg atagctggca
     2521 tatccctcat gtgcatgaac cagactgccc aagcaggaag ctatgaaggt ttcgatcagt
     2581 ctcagagcca caaagtggtg gagcctggaa atactgcttt tgaggagagt ccagcaccag
     2641 taaatgaacc agaacttttt acaagctcta ttccagaaac aagttgctgc tcctgctcca
     2701 tgggaaatgg tgaattacac tgcccatcaa tagagccaat agcaaacaca agcaccagtg
     2761 agcctgtgat tccttcgttt gagaaaaaca atcattgtgt gagtcgctgt aactcccaga
     2821 gctgcatatt agcccaggaa gaagaacagt atctacagag tggagaccag caactgaccc
     2881 gacatgtgtt gctgtgttta cttctcatca ttggcctgtt cgctaatctt tccagttgtt
     2941 tatggtggct attcaaccaa gagcctggaa gactttatgt tgagttacag tttttctgtg
     3001 ccgtgtttaa ctttggtcag ggatttattt cctttggaat ctttggatta gataaacatt
     3061 taatcatcct gcctttcaaa agaagacttg aattcctatg gaacaataaa gacacagcag
     3121 aaaacaggga ttctcctgtt tcagaggaaa taaaaatgac ctgtcaacaa tttatccatt
     3181 atcaccgtga cctctgtatc cgaaacattg tcaaagaaag aaggtgtggt gcaaagactt
     3241 ctgctggaac tttctgtggc tgtgacctgg tgagctggct aattgaagtc ggccttgcct
     3301 ccgaccgtgg tgaagctgtg atatacggag acaggctggt acaaggggga gtcatccaac
     3361 atattaccaa tgagtatgaa ttccgggatg agtacttgtt ttacagattt cttcaaaaga
     3421 gtcctgaaca gagtcctcct gctattaatg caaacactct ccaacaggaa agatataaag
     3481 aaattgagca ttcatcccca ccctcacatt cccctaagac cagatctaat ggtggtggtg
     3541 gtgggatggg ctacccctat gatgtgcctg actacgcata aggatcccca gcttggccgc
     3601 catggcccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     3661 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     3721 atgtatctta tcatgtctgg atcgggaatt aattcggcgc agcaccatgg cctgaaataa
     3781 cctctgaaag aggaacttgg ttaggtacct tctgaggcgg aaagaaccag ctgtggaatg
     3841 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3901 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3961 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     4021 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     4081 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag
     4141 gcttttttgg aggcctaggc ttttgcaaaa agctgttaac agcttggcac tggccgtcgt
     4201 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     4261 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     4321 gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg
     4381 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     4441 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     4501 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     4561 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     4621 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     4681 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     4741 acactcaacc ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc
     4801 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     4861 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     4921 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     4981 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     5041 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     5101 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     5161 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     5221 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     5281 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     5341 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     5401 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     5461 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     5521 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     5581 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     5641 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     5701 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     5761 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     5821 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     5881 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     5941 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     6001 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     6061 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     6121 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     6181 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     6241 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     6301 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     6361 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     6421 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     6481 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     6541 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     6601 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     6661 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     6721 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     6781 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     6841 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     6901 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     6961 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     7021 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     7081 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     7141 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     7201 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     7261 cacacaggaa acagctatga catgattacg aattaattcg agctcgcc
//
