LOCUS       sequence-266498-        6203 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  fluorescent cholesterol-binding probe.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6203)
  AUTHORS   Lim CY, Davis OB, Shin HR, Zhang J, Berdan CA, Jiang X, Counihan JL,
            Ory DS, Nomura DK, Zoncu R
  TITLE     ER-lysosome contacts enable cholesterol sensing by mTORC1 and drive 
            aberrant growth signalling in Niemann-Pick type C.
  JOURNAL   Nat Cell Biol. 2019 Oct;21(10):1206-1218. doi: 
            10.1038/s41556-019-0391-5. Epub 2019 Sep 23.
  PUBMED    31548609
REFERENCE   2  (bases 1 to 6203)
  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: 
            "10.1038/s41556-019-0391-5"; journalName: "Nat Cell Biol"; date: 
            "2019-10"; volume: "21"; issue: "10"; pages: "1206-1218"
COMMENT     Sequence Label: pGEX-KG-D4H*-mCherry
FEATURES             Location/Qualifiers
     source          1..6203
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     51..70
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     304..321
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        394..471
                     /gene="lacI"
                     /label=lacI promoter
     CDS             472..1554
                     /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(491..510)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1603..1608,1609..1626,1627..1633)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1641..1657
                     /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     1665..1681
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1665..1681
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1677..2204
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(1697..1714)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1697..1713)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1706..1728)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(2306..2311,2312..2327,2328..2334)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2342..2358
                     /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)."
     CDS             2381..3034
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2992..3014
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             3041..3058
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3485..4192
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3630..3648)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3898..3917
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(4376..4398)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4543..4561)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4629..4733
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4734..4802,4803..5594)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4952..4971)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5765..6203,1..150)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
       61 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
      121 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
      181 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
      241 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
      301 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
      361 catctgtgcg gtatttcaca ccgcataaat tccgacacca tcgaatggcg caaaaccttt
      421 cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca
      481 gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg
      541 gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg
      601 gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg
      661 attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt
      721 aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc
      781 gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc
      841 attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt
      901 ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat
      961 gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg
     1021 ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa
     1081 tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg
     1141 tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg
     1201 gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc
     1261 gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc
     1321 ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc
     1381 ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg
     1441 gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     1501 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     1561 cgcaattaat gtaagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
     1621 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
     1681 ccatgattac ggattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
     1741 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
     1801 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgctttg
     1861 cctggtttcc ggcaccagaa gcggtgccgg aaagctggct ggagtgcgat cttcctgagg
     1921 ccgatactgt cgtcgtcccc tcaaactggc agatgcacgg ttacgatgcg cccatctaca
     1981 ccaacgtgac ctatcccatt acggtcaatc cgccgtttgt tcccacggag aatccgacgg
     2041 gttgttactc gctcacattt aatgttgatg aaagctggct acaggaaggc cagacgcgaa
     2101 ttatttttga tggcgttgga attagcttat cgactgcacg gtgcaccaat gcttctggcg
     2161 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg
     2221 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt
     2281 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga
     2341 attgtgagcg gataacaatt tcacacagga aacagtattc atgtccccta tactaggtta
     2401 ttggaaaatt aagggccttg tgcaacccac tcgacttctt ttggaatatc ttgaagaaaa
     2461 atatgaagag catttgtatg agcgcgatga aggtgataaa tggcgaaaca aaaagtttga
     2521 attgggtttg gagtttccca atcttcctta ttatattgat ggtgatgtta aattaacaca
     2581 gtctatggcc atcatacgtt atatagctga caagcacaac atgttgggtg gttgtccaaa
     2641 agagcgtgca gagatttcaa tgcttgaagg agcggttttg gatattagat acggtgtttc
     2701 gagaattgca tatagtaaag actttgaaac tctcaaagtt gattttctta gcaagctacc
     2761 tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa acatatttaa atggtgatca
     2821 tgtaacccat cctgacttca tgttgtatga cgctcttgat gttgttttat acatggaccc
     2881 aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa aaacgtattg aagctatccc
     2941 acaaattgat aagtacttga aatccagcaa gtatatagca tggcctttgc agggctggca
     3001 agccacgttt ggtggtggcg accatcctcc aaaatcggat ctggttccgc gtggatcccc
     3061 gggaatttcc ggtggtggtg gtggaattct agactccatg ggtcgactcg agatgaaggg
     3121 aaaaataaac ttagatcata gtggagccta tgttgcacag tttgaagtag cctgggatga
     3181 agtttcagcc gacaaagaag gaaatgaagt tttaactcat aaaacatggg atggaaatta
     3241 tcaatctaaa acagctcact attcaacagt aatacctctt gaagctaatg caagaaatat
     3301 aagaataaaa gcaagagagt gtacaggcct ttggtgggaa tggtggagag atgttataag
     3361 tgaatatgat gttccattaa caaataatat aaatgtttca atatggggaa caactttata
     3421 ccctggatct agtattactt acaatagaat tctgcagtcg acggtaccgc gggcccggga
     3481 tccgatggtg agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt
     3541 caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga
     3601 gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct
     3661 gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa
     3721 gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga
     3781 gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca
     3841 ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc
     3901 cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga
     3961 cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga
     4021 cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa
     4081 cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta
     4141 cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca aggcggccgc
     4201 gactctagat cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc
     4261 tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttagcttaa
     4321 ttcatcgtga ctgactgacg atctgcctcg cgcgtttcgg tgatgacggt gaaaacctct
     4381 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     4441 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt
     4501 cacgtagcga tagcggagtg tataattctt gaagacgaaa gggcctcgtg atacgcctat
     4561 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     4621 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     4681 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     4741 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     4801 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     4861 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     4921 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg
     4981 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     5041 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     5101 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     5161 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     5221 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag
     5281 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     5341 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     5401 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     5461 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     5521 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     5581 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     5641 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     5701 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     5761 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     5821 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     5881 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     5941 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     6001 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     6061 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     6121 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     6181 aagggagaaa ggcggacagg tat
//
