LOCUS       sequence-393246-        5895 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Leucine Zipper (LZ) LYCHOS LED-Flag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5895)
  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 5895)
  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: pET28a LZ LED WT-FLAG
FEATURES             Location/Qualifiers
     source          1..5895
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(176..199)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(224..235)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             complement(773..796)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(800..817)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             836..858
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    873..897
                     /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     complement(897..916)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(898..916)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1000..1019)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1225..1302
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1303..2385
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(1322..1341)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3194..3385
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3379..3401)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3487..3626
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3542..3561
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3641..3658)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3812..4400)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3892..3911)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4511..4530)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4522..5337
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             4522..5337
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4595..4614)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5429..5884)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5566..5587)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5778..5797
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcttacttgt
      181 catcatcatc cttataatcc tgtgcaagtt ctttttttaa agcctgcaac tcccatttta
      241 actgtgccaa ctctttttta ttcgcttgca gttccttttt cagggcgccg gacccacttc
      301 cgccgtggcg ggttaactgt tggtccccac tctgcagata ctgctcttcc tcttgtgcaa
      361 ggatacagga ctgcgaatta caacgtgaaa cgcagtgatt attcttttcg aatgaaggga
      421 tcaccggctc ggacgttgac gtattcgcaa tgggctcgat tgaggggcaa tgcagctctc
      481 cgtttcccat agagcaactg caacagctag tttctgggat cgacgaagtg aataattcgg
      541 gttcgttgac cggcgctgga ctctcttcga atgccgtgtt ccccggttct acaactttgt
      601 ggctttgaga ttggtcgaaa ccttcatagg accccgcctg tgcagtttga ttcataccgg
      661 aaccaccctg agctaatttc ttctccagag cttgattctt ccattccagt tgggctaatt
      721 ttttctcaag agcttgcaac tttttctcca attgttccga ggccatggat ccgggcccct
      781 ggaacagaac ttccaggctg tggtgatgat gatgatggct gctgcccatg gtatatctcc
      841 ttcttaaagt taaacaaaat tatttctaga ggggaattgt tatccgctca caattcccct
      901 atagtgagtc gtattaattt cgcgggatcg agatctcgat cctctacgcc ggacgcatcg
      961 tggccggcat caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg
     1021 atggggaaga tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg
     1081 tggcaggccc cgtggccggg ggactgttgg gcgccatctc cttgcatgca ccattccttg
     1141 cggcggcggt gctcaacggc ctcaacctac tactgggctg cttcctaatg caggagtcgc
     1201 ataagggaga gcgtcgagat cccggacacc atcgaatggc gcaaaacctt tcgcggtatg
     1261 gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc agtaacgtta
     1321 tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag
     1381 gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc ggagctgaat
     1441 tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt
     1501 gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc
     1561 gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc
     1621 tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat
     1681 ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta
     1741 tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt
     1801 acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg
     1861 ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact
     1921 cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt
     1981 caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac
     2041 gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg
     2101 gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat cccgccgtta
     2161 accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa
     2221 ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga
     2281 aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     2341 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     2401 tgtaagttag ctcactcatt aggcaccggg atctcgaccg atgcccttga gagccttcaa
     2461 cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac ttatgactgt
     2521 cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca ttttcggcga
     2581 ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat tcggaatctt
     2641 gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca
     2701 ggccattatc gccggcatgg cggccccacg ggtgcgcatg atcgtgctcc tgtcgttgag
     2761 gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga
     2821 gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt
     2881 cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct gcaccattat
     2941 gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta catctgtatt
     3001 aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca tccataccgc
     3061 cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag taacccgtat
     3121 cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta
     3181 cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag
     3241 aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga
     3301 catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc tcgcgcgttt
     3361 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     3421 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     3481 tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat
     3541 gcggcatcag agcagattgt actgagagtg caccattgcg gtgtgaaata ccgcacagat
     3601 gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc
     3661 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     3721 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     3781 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     3841 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     3901 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     3961 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     4021 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     4081 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     4141 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     4201 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     4261 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     4321 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     4381 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     4441 ggaacgaaaa ctcacgttaa gggattttgg tcatgaacaa taaaactgtc tgcttacata
     4501 aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcttg ctctaggccg
     4561 cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc
     4621 gggcaatcag gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc agagttgttt
     4681 ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac
     4741 tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat
     4801 gcatggttac tcaccactgc gatccccggg aaaacagcat tccaggtatt agaagaatat
     4861 cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg
     4921 attcctgttt gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa
     4981 tcacgaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg
     5041 cctgttgaac aagtctggaa agaaatgcat aaacttttgc cattctcacc ggattcagtc
     5101 gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt
     5161 tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg
     5221 aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt
     5281 gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaagaa
     5341 ttaattcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     5401 gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc
     5461 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     5521 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     5581 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     5641 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     5701 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     5761 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     5821 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     5881 gcgtcccatt cgcca
//
