LOCUS       sequence-393288-        5544 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Leucine Zipper (LZ) control-Flag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5544)
  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 5544)
  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 control-FLAG
FEATURES             Location/Qualifiers
     source          1..5544
                     /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(422..445)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(449..466)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             485..507
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    522..546
                     /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(546..565)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(547..565)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(649..668)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        874..951
                     /gene="lacI"
                     /label=lacI promoter
     CDS             952..2034
                     /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(971..990)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2843..3034
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3028..3050)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3136..3275
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3191..3210
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3290..3307)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3461..4049)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3541..3560)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4160..4179)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4171..4986
                     /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             4171..4986
                     /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(4244..4263)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5078..5533)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5215..5236)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5427..5446
                     /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 cgccaccgga accaccctga gctaatttct tctccagagc ttgattcttc cattccagtt
      361 gggctaattt tttctcaaga gcttgcaact ttttctccaa ttgttccgag gccatggatc
      421 cgggcccctg gaacagaact tccaggctgt ggtgatgatg atgatggctg ctgcccatgg
      481 tatatctcct tcttaaagtt aaacaaaatt atttctagag gggaattgtt atccgctcac
      541 aattccccta tagtgagtcg tattaatttc gcgggatcga gatctcgatc ctctacgccg
      601 gacgcatcgt ggccggcatc accggcgcca caggtgcggt tgctggcgcc tatatcgccg
      661 acatcaccga tggggaagat cgggctcgcc acttcgggct catgagcgct tgtttcggcg
      721 tgggtatggt ggcaggcccc gtggccgggg gactgttggg cgccatctcc ttgcatgcac
      781 cattccttgc ggcggcggtg ctcaacggcc tcaacctact actgggctgc ttcctaatgc
      841 aggagtcgca taagggagag cgtcgagatc ccggacacca tcgaatggcg caaaaccttt
      901 cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca
      961 gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg
     1021 gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg
     1081 gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg
     1141 attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt
     1201 aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc
     1261 gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc
     1321 attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt
     1381 ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat
     1441 gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg
     1501 ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa
     1561 tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg
     1621 tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg
     1681 gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc
     1741 gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc
     1801 ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc
     1861 ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg
     1921 gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     1981 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     2041 cgcaattaat gtaagttagc tcactcatta ggcaccggga tctcgaccga tgcccttgag
     2101 agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact
     2161 tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat
     2221 tttcggcgag gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt
     2281 cggaatcttg cacgccctcg ctcaagcctt cgtcactggt cccgccacca aacgtttcgg
     2341 cgagaagcag gccattatcg ccggcatggc ggccccacgg gtgcgcatga tcgtgctcct
     2401 gtcgttgagg acccggctag gctggcgggg ttgccttact ggttagcaga atgaatcacc
     2461 gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac
     2521 atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt cagcgccctg
     2581 caccattatg ttccggatct gcatcgcagg atgctgctgg ctaccctgtg gaacacctac
     2641 atctgtatta acgaagcgct ggcattgacc ctgagtgatt tttctctggt cccgccgcat
     2701 ccataccgcc agttgtttac cctcacaacg ttccagtaac cgggcatgtt catcatcagt
     2761 aacccgtatc gtgagcatcc tctctcgttt catcggtatc attaccccca tgaacagaaa
     2821 tcccccttac acggaggcat cagtgaccaa acaggaaaaa accgccctta acatggcccg
     2881 ctttatcaga agccagacat taacgcttct ggagaaactc aacgagctgg acgcggatga
     2941 acaggcagac atctgtgaat cgcttcacga ccacgctgat gagctttacc gcagctgcct
     3001 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     3061 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     3121 tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg
     3181 cttaactatg cggcatcaga gcagattgta ctgagagtgc accattgcgg tgtgaaatac
     3241 cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg
     3301 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     3361 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     3421 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     3481 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     3541 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     3601 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     3661 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     3721 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     3781 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     3841 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     3901 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     3961 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4021 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4081 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgaacaat aaaactgtct
     4141 gcttacataa acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcttgc
     4201 tctaggccgc gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc
     4261 gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca
     4321 gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc
     4381 agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact
     4441 cctgatgatg catggttact caccactgcg atccccggga aaacagcatt ccaggtatta
     4501 gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg
     4561 ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct
     4621 caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt
     4681 aatggctggc ctgttgaaca agtctggaaa gaaatgcata aacttttgcc attctcaccg
     4741 gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa
     4801 ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc
     4861 atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa
     4921 tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt
     4981 ttctaagaat taattcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     5041 aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg
     5101 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     5161 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     5221 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     5281 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     5341 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     5401 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     5461 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     5521 ctacagggcg cgtcccattc gcca
//
